Targeted replacement of endogenous t cell receptors

Abstract

To provide methods of editing the genome of a human T cell.SOLUTION: The present disclosure provides a method of editing the genome of a human T cell, the method comprising inserting a nucleic acid sequence into a target region in exon 1 of a T cell receptor (TCR)-subunit constant gene in the human T cell, a nucleic acid sequence encoding from a N-terminus to a C-terminus, (i) a first self-cleaving peptide sequence; (ii) a first heterologous TCR subunit chain comprising the variable region and the constant region of the TCR subunit; (iii) a second self-cleaving peptide sequence; (iv) a variable region of a second heterologous TCR subunit chain; and (v) a portion of the N-terminus of the endogenous TCR subunit, wherein, if the endogenous TCR subunit is TCR-α, the first subunit is TCR-β, and the second subunit is TCR-α, and wherein if the endogenous TCR subunit is TCR-β, the first is TCR-α, and the second is TCR-β.SELECTED DRAWING: Figure 1a

Description

[Technical field]

[0001] Prior Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 578,153, filed October 27, 2017, which is incorporated by reference in its entirety. [Background technology]

[0002] 2. Background of the Invention T cells are the most actively studied cell type in the growing field of adoptive cell therapy. T cells interact specifically with targets of their T cell receptor (TCR), allowing for highly specific responses with minimal side effects. These highly effective and specific responses can be engineered toward novel antigens and targets by inserting new receptors with the desired specificity into T cells. However, the development of entirely new types of receptors is time-consuming, expensive, and fails to take advantage of the fact that the body, through the development of the endogenous T cell repertoire, naturally produces TCRs that bind to most possible antigen targets. The ability to obtain human T cells and replace their endogenous TCRs with TCRs with the desired antigen specificity could be novel in the development and application of adoptive T cell therapy. Summary of the Invention

[0003] The present disclosure is directed to compositions and methods for editing the genome of human T cells.The inventors have discovered that heterologous TCRs can be inserted into target regions in the genome of T cells, so that the heterologous TCRs are under the control of endogenous TCR promoters.The methods and compositions provided herein can be used to replace the endogenous TCRs in human T cells with heterologous TCRs with desired antigen specificity.In some embodiments, the target region in the genome of T cells is the natural T cell receptor locus.

[0004] In some embodiments, the present disclosure provides a method for editing the genome of a human T cell. In some embodiments, the method includes, from N-terminus to C-terminus, (i) a first self-cleaving peptide sequence; (ii) a first heterologous TCR subunit chain comprising a variable region and a constant region of a TCR subunit; (iii) a second self-cleaving peptide sequence; (iv) a variable region of a second heterologous TCR subunit chain; and (v) The method includes inserting a nucleic acid sequence encoding an N-terminal portion of an endogenous TCR subunit into a target region in exon 1 of a T cell receptor (TCR) subunit constant gene in a human T cell, wherein if the endogenous TCR subunit is a TCR-alpha (TCR-α) subunit, the first heterologous TCR subunit chain is a heterologous TCR-beta (TCR-β) subunit chain and the second heterologous TCR subunit chain is a heterologous TCR-α subunit chain, and if the endogenous TCR subunit is a TCR-β subunit, the first heterologous TCR subunit chain is a heterologous TCR-α subunit chain and the second heterologous TCR subunit chain is a heterologous TCR-β subunit chain. In some embodiments, the method includes inserting a nucleic acid sequence encoding, from N-terminus to C-terminus, (i) a first self-cleaving peptide sequence; (ii) a heterologous TCR-β chain; (iii) a second self-cleaving peptide sequence; (iv) a variable region of a heterologous TCR-α chain; and (v) an N-terminal portion of an endogenous TCR-α subunit into a target region in exon 1 of a TCR alpha subunit constant gene (TRAC) in a human T cell.

[0005] In some embodiments, the method includes inserting a nucleic acid sequence encoding, from N-terminus to C-terminus, (i) a first self-cleaving peptide sequence; (ii) a heterologous TCR-α chain; (iii) a second self-cleaving peptide sequence; (iv) a variable region of a heterologous TCR-β chain; and (v) an N-terminal portion of an endogenous TCR-β subunit into a target region in exon 1 of a TCR-beta subunit constant gene (TRBC) in a human T cell.

[0006] In some embodiments, the nucleic acid is inserted by introducing a viral vector containing the nucleic acid into the T cell. In some embodiments, the nucleic acid is inserted by introducing a non-viral vector containing the nucleic acid into the T cell. In some embodiments, the nucleic acid is inserted into the T cell by introducing a nucleic acid sequence into the T cell, which is (a) a targeted nuclease that cleaves a target region in exon 1 of the TCR-α subunit constant gene (TRAC) to create an insertion site in the genome of the T cell; and (b) a nucleic acid sequence that is integrated into the insertion site by homology-directed repair (HDR). In some embodiments, the nucleic acid is inserted into the T cell by introducing a targeted nuclease that cleaves a target region in exon 1 of the TCR-β subunit constant gene (TRBC) to create an insertion site in the genome of the T cell; and (b) a nucleic acid sequence that is integrated into the insertion site by homology-directed repair (HDR). In some embodiments, the 5' and 3' ends of the nucleic acid comprise nucleotide sequences that are homologous to genomic sequences adjacent to the target region. In some embodiments, the 5' and 3' ends of the nucleic acid comprise nucleotide sequences that are homologous to the genomic sequence adjacent to the insertion site. In some embodiments, the targeting nuclease introduces a double-stranded break at the insertion site. In some embodiments, the nucleic acid sequence is introduced into the cell as a double-stranded or single-stranded nucleic acid. In some embodiments, the nucleic acid is introduced into the cell as a double-stranded or single-stranded DNA template. In some embodiments, the nucleic acid sequence is introduced into the cell as a linear nucleic acid.

[0007] In some embodiments, the first self-cleaving peptide and the second self-cleaving peptide are the same or different viral 2A peptides.

[0008] In some embodiments, the targeting nuclease is selected from the group consisting of RNA-guided nuclease domain, transcription activator-like effector nuclease (TALEN), zinc finger nuclease (ZFN) and megaTAL.In some embodiments, the RNA-guided nuclease is Cpf1 nuclease or Cas9 nuclease, and the method further comprises introducing into the cell a guide RNA that specifically hybridizes to a target region in exon 1 of TRAC.In some embodiments, the Cpf1 nuclease or Cas9 nuclease, guide RNA and nucleic acid are introduced into the cell as a ribonucleoprotein complex (RNP)-DNA template complex, comprising (i) Cpf1 nuclease or Cas9 nuclease and guide RNA; and (ii) DNA template.

[0009] In some embodiments, the molar ratio of RNP to DNA template in the complex is about 3:1 to about 100:1. In some embodiments, the RNP-DNA template complex is formed by incubating the RNP with the DNA template at a temperature of about 20°C to 25°C for about 10 minutes to about 30 minutes. In some embodiments, the RNP-DNA template complex and the cell are mixed before introducing the RNP-DNA template complex into the cell. In some embodiments, the RNP-DNA template complex comprises at least two structurally distinct RNP complexes. In some embodiments, the at least two structurally distinct RNP complexes comprise structurally distinct guide RNAs. In some embodiments, each of the structurally distinct RNP complexes comprises a Cas9 nickase, and the structurally distinct guide RNAs hybridize to opposite strands of the target region.

[0010] In some embodiments, the introducing comprises electroporation. In some embodiments, the nucleic acid is about 1×10 5 ~Approx. 2×10 6 In some examples, the targeting nuclease and DNA template are introduced into a population of about 1×10 T cells. 5 ~Approx. 2×106 In some embodiments, at least two structurally distinct DNA templates are introduced into a population of T cells. In some embodiments, at least two structurally distinct DNA templates are non-viral templates. In some embodiments, each of the at least two structurally distinct DNA templates encodes a unique combination of a variable region of a heterologous TCR-β chain of an antigen-specific T cell receptor and a variable region of a heterologous TCR-α chain of an antigen-specific T cell receptor. In some embodiments, the T cells are regulatory T cells, effector T cells, or naive T cells. In some embodiments, the T cells are effector T cells, and the effector T cells are CD8 + In some embodiments, the T cell is an effector T cell, and the effector T cell is a CD4 + In some embodiments, effector T cells are CD4 + CD8 + T cells.

[0011] In some embodiments, the method further comprises culturing the T cells under conditions that allow expression of a heterologous TCR-β chain and a heterologous TCR-α chain to form an antigen-specific T cell receptor. In some embodiments, the method further comprises culturing the modified T cells under conditions effective to expand the population of modified cells. In some embodiments, the method further comprises purifying the T cells that express the antigen-specific T cell receptor.

[0012] In some embodiments, the disclosure provides a method of editing the genome of a human T cell, comprising: (a) inserting, from N-terminus to C-terminus, a first nucleic acid sequence encoding: (i) a self-cleaving peptide sequence; (ii) a heterologous TCR-α chain of an antigen-specific T cell receptor; and (iii) a portion of exon 1 N-terminal of an endogenous TCR alpha subunit, into a target region in exon 1 of a TCR alpha subunit constant gene (TRAC) in the human T cell; and (b) inserting, from N-terminus to C-terminus, a second nucleic acid sequence encoding: (i) a second self-cleaving peptide sequence; (ii) a heterologous TCR-β chain of an antigen-specific T cell receptor; and (iii) a portion of exon 1 N-terminal of an endogenous TCR beta subunit, into a target region in exon 1 of a TCR beta subunit constant gene (TRBC) in the human T cell.

[0013] In some embodiments, the first and / or second nucleic acid sequence is inserted by introducing a viral vector containing the first and / or second nucleic acid into the T cell. In some embodiments, the first and / or second nucleic acid sequence is inserted by introducing a non-viral vector containing the first and / or second nucleic acid into the T cell. In some embodiments, the first and second nucleic acid are inserted into the T cell by introducing (a) one or more targeting nucleases that create a first insertion site in exon 1 of TRAC and a second insertion site in exon 1 of TCR beta subunit constant gene (TRBC); and (b) a first nucleic acid sequence; and (c) a second nucleic acid sequence, where the first nucleic acid sequence is inserted into the first insertion site in exon 1 of TRAC by homology-directed repair (HDR) and the second nucleic acid sequence is inserted into the second insertion site in exon 1 of TRBC. In some embodiments, the nucleic acid sequence is introduced into the cell as a double-stranded or single-stranded DNA template. In some embodiments, the nucleic acid sequence is introduced into the cell as a linear DNA template.

[0014] In some embodiments, the 5' and 3' ends of the first nucleic acid sequence comprise a nucleotide sequence that is homologous to a genomic sequence adjacent to a target region in exon 1 of the TRAC gene. In some embodiments, the 5' and 3' ends of the first nucleic acid sequence comprise a nucleotide sequence that is homologous to a genomic sequence adjacent to a first insertion site in exon 1 of the TRAC gene. In some embodiments, the 5' and 3' ends of the second nucleic acid sequence comprise a nucleotide sequence that is homologous to a genomic sequence adjacent to a target region in exon 1 of the TRBC gene. In some embodiments, the 5' and 3' ends of the second nucleic acid sequence comprise a nucleotide sequence that is homologous to a genomic sequence adjacent to a second insertion in exon 1 of the TRBC gene.

[0015] In some embodiments, the one or more targeting nucleases introduce double-stranded breaks at the first and second insertion sites. In some embodiments, the first self-cleaving peptide and the second self-cleaving peptide are the same or different viral 2A peptides. In some embodiments, the one or more targeting nucleases are selected from the group consisting of RNA-guided nuclease domain, transcription activator-like effector nuclease (TALEN), zinc finger nuclease (ZFN), or megaTAL.

[0016] In some embodiments, the RNA-guided nuclease is Cpf1 nuclease or Cas9 nuclease, and the method further comprises introducing into the cell a first guide RNA that specifically hybridizes to a target region in exon 1 of TRAC, and a second guide RNA that specifically hybridizes to a target region in exon 1 of TRBC. In some embodiments, the Cpf1 nuclease or Cas9 nuclease, the first guide RNA, and the first nucleic acid are introduced into the cell as a ribonucleoprotein complex (RNP)-DNA template complex that includes (i) a ribonucleoprotein complex (RNP) comprising Cpf1 nuclease or Cas9 nuclease and the first guide RNA; and (ii) a first DNA template. In some embodiments, the Cpf1 nuclease or Cas9 nuclease, the second guide RNA, and the second nucleic acid are introduced into the cell as an RNP-DNA template complex that includes: (i) an RNP that includes the Cpf1 nuclease or Cas9 nuclease and the second guide RNA; and (ii) a second DNA template.

[0017] In some embodiments, the molar ratio of RNP to DNA template in the complex is about 3:1 to about 100:1. In some embodiments, the RNP-DNA template complex is formed by incubating the RNP with the DNA template at a temperature of about 20°C to 25°C for about 10 minutes to about 30 minutes. In some embodiments, the RNP-DNA template complex comprises at least two structurally distinct RNP complexes. In some embodiments, each of the structurally distinct RNP complexes comprises a Cas9 nickase, and the structurally distinct guide RNAs hybridize to opposite strands of the target region. In some embodiments, the introduction comprises electroporation.

[0018] In some embodiments, the first and second nucleic acids are about 1×10 5 ~about 2×10 6 In some embodiments, the one or more targeting nucleases and the first and second nucleic acids are introduced into about 1×10 T cells. 5 ~about 2×106 In some embodiments, at least two structurally different first DNA templates are introduced into the cell. In some embodiments, at least two structurally different first DNA templates comprise different variable regions of the TCR-α chain of the antigen-specific T cell receptor. In some embodiments, at least two structurally different second DNA templates are introduced into the cell. In some embodiments, at least two structurally different second DNA templates comprise different variable regions of the TCR-β chain of the antigen-specific T cell receptor.

[0019] In some embodiments, the T cells are regulatory T cells, effector T cells, or naive T cells. In some embodiments, the T cells are effector T cells, and the effector T cells are CD8 + In some embodiments, the T cell is an effector T cell, and the effector T cell is a CD4 + In some embodiments, effector T cells are CD4 + CD8 + T cells.

[0020] In some embodiments, the method further comprises culturing the T cells under conditions that allow expression of a heterologous TCR-β chain and a heterologous TCR-α chain to form an antigen-specific T cell receptor. In some embodiments, the method further comprises culturing the T cells under conditions that allow expression of a heterologous TCR-β chain and a heterologous TCR-α chain to form an antigen-specific T cell receptor. In some embodiments, the method further comprises culturing the modified T cells under conditions effective to expand the population of modified cells. In some embodiments, the method further comprises purifying the T cells expressing the antigen-specific T cell receptor.

[0021] In other aspects, the disclosure provides modified T cells comprising a nucleic acid sequence encoding, from N-terminus to C-terminus: (i) a first self-cleaving peptide sequence; (ii) a variable region of a heterologous T cell receptor (TCR)-β chain; (iii) a second self-cleaving peptide sequence; (iv) a variable region of a heterologous TCR-α chain; and (v) an N-terminal portion of an endogenous TCR alpha subunit, wherein the nucleic acid sequence is integrated into exon 1 of the TRAC gene.

[0022] In some embodiments, the disclosure provides modified T cells further comprising: (a) a first nucleic acid sequence encoding, from N-terminus to C-terminus, (i) a first self-cleaving sequence; (ii) a variable region of a heterologous TCR-α chain, and (iii) a portion of the N-terminus of an endogenous TCR-α chain, wherein the first nucleic acid sequence is integrated into exon 1 of the TRAC gene; and (b) a second nucleic acid sequence encoding, from N-terminus to C-terminus, (i) a first self-cleaving sequence; (ii) a variable region of a heterologous TCR-β chain, and (iii) a portion of the N-terminus of an endogenous TCR-β chain, wherein the second nucleic acid sequence is integrated into exon 1 of the TRAC gene.

[0023] In some embodiments, the disclosure provides methods of treating cancer in a human subject, further comprising: (a) obtaining T cells from a subject; (b) modifying the T cells to express a heterologous antigen-specific T cell receptor that recognizes a tumor-specific antigen; and (c) administering the modified T cells to the subject.

[0024] The methods and compositions described herein for modifying T cells to express heterologous TCR-α chains and heterologous TCR-β chains can also be used to edit human gamma delta (γδ) T cells. For example, in some embodiments, the method includes inserting a nucleic acid sequence encoding, from N-terminus to C-terminus, (i) a first self-cleaving peptide sequence; (ii) a variable region of a heterologous TCR-β chain; (iii) a second self-cleaving peptide sequence; (iv) a variable region of a heterologous TCR-α chain; and (v) a portion of the N-terminus of endogenous TCR-α into a target region in exon 1 of the T cell receptor gamma subunit constant (TRGC) gene in human T cells.

[0025] In other embodiments, the method includes inserting a nucleic acid sequence encoding, from N-terminus to C-terminus, (i) a first self-cleaving peptide sequence; (ii) a variable region of a heterologous TCR-β chain; (iii) a second self-cleaving peptide sequence; (iv) a full-length heterologous TCR-α chain; and (v) a stop codon into a target region in exon 1 of the T cell receptor gamma subunit constant (TRGC) gene in a human T cell such that when the nucleic acid is inserted, the nucleic acid encoding the heterologous TCR-β and TCR-α sequences is under the control of the endogenous TCR-γ promoter.

[0026] In another embodiment, the method includes inserting a nucleic acid sequence encoding, from N-terminus to C-terminus, (i) a first self-cleaving peptide sequence; (ii) a variable region of a heterologous TCR-δ chain; (iii) a second self-cleaving peptide sequence; (iv) a variable region of a heterologous TCR-γ chain; and (v) a portion of the N-terminus of an endogenous TCR-γ subunit into a target region in exon 1 of a T cell receptor gamma subunit constant (TRGC) gene in a human T cell. Also provided is a method of editing the genome of a human T cell, comprising inserting a nucleic acid sequence encoding, from N-terminus to C-terminus, (i) a first self-cleaving peptide sequence; (ii) a variable region of a heterologous TCR-γ chain; (iii) a second self-cleaving peptide sequence; (iv) a variable region of a heterologous TCR-δ chain; and (v) a portion of the N-terminus of an endogenous TCR-α subunit into a target region in exon 1 of a TRAC gene in a human T cell. In other embodiments, the method includes inserting a nucleic acid sequence encoding, from N-terminus to C-terminus, (i) a first self-cleaving peptide sequence; (ii) a variable region of a heterologous TCR-γ chain; (iii) a second self-cleaving peptide sequence; (iv) a full-length heterologous TCR-δ chain; and (v) a stop codon into a target region in exon 1 of the T cell receptor gamma subunit constant (TRAC) gene in a human T cell such that when the nucleic acid is inserted, the heterologous TCR-γ and TCR-δ sequences are under the control of the endogenous TCR-α promoter. [The present invention 1001] 1. A method for editing the genome of a human T cell, comprising inserting a nucleic acid sequence into a target region in exon 1 of a T cell receptor (TCR) subunit constant gene in the human T cell, The nucleic acid sequence, from the N-terminus to the C-terminus, is (i) a first self-cleaving peptide sequence, (ii) a first heterologous TCR subunit chain comprising a variable region and a constant region of a TCR subunit; (iii) a second self-cleaving peptide sequence; (iv) the variable region of a second heterologous TCR subunit chain, and (v) The N-terminal portion of an endogenous TCR subunit Code the following: When the endogenous TCR subunit is a TCR-alpha (TCR-α) subunit, the first heterologous TCR subunit chain is a heterologous TCR-beta (TCR-β) subunit chain and the second heterologous TCR subunit chain is a heterologous TCR-α subunit chain; when the endogenous TCR subunit is a TCR-β subunit, the first heterologous TCR subunit chain is a heterologous TCR-α subunit chain and the second heterologous TCR subunit chain is a heterologous TCR-β subunit chain. method. [The present invention 1002] From the N-terminus to the C-terminus, (i) a first self-cleaving peptide sequence, (ii) heterologous TCR-β chain; (iii) a second self-cleaving peptide sequence; (iv) the variable region of a heterologous TCR-α chain, and (v) The N-terminal portion of the endogenous TCR-α subunit The method of claim 1001, wherein a nucleic acid sequence encoding the above is inserted into exon 1 of the TCR-alpha subunit constant gene (TRAC) in human T cells. [The present invention 1003] From the N-terminus to the C-terminus, (i) a first self-cleaving peptide sequence, (ii) heterologous TCR-α chain; (iii) a second self-cleaving peptide sequence; (iv) a variable region of a heterologous TCR-β chain, and (v) the N-terminal portion of the endogenous TCR-β subunit The method of claim 10, wherein a nucleic acid sequence encoding the above is inserted into exon 1 of the TCR-beta subunit constant gene (TRBC) in human T cells. [The present invention 1004] The method according to any of claims 1001 to 1003, wherein the nucleic acid sequence is a double-stranded or single-stranded nucleic acid sequence, and the nucleic acid sequence is inserted by introducing the nucleic acid sequence into a T cell. [The present invention 1005] The method of any of claims 1001 to 1004, wherein the nucleic acid is inserted by introducing a viral vector containing the nucleic acid into a T cell. [The present invention 1006] The nucleic acid is (a) a targeted nuclease that cleaves a target region in exon 1 of the TRAC gene to create an insertion site in the genome of the T cell; and (b) a nucleic acid sequence to be inserted into the insertion site by homology-directed repair (HDR); The method of any of claims 1001, 1002, 1004 or 1005, wherein the antibody is inserted into the T cell by introducing into the T cell. [The present invention 1007] The nucleic acid is (a) a targeted nuclease that cleaves a target region in exon 1 of the TRBC gene to create an insertion site in the genome of the T cell; and (b) a nucleic acid sequence to be inserted into the insertion site by homology-directed repair (HDR); The method of any of claims 1001, 1003, 1004 or 1005, wherein the antibody is inserted into the T cell by introducing into the T cell. [The present invention 1008] The method of any of claims 1001 to 1007, wherein the 5' and 3' ends of the nucleic acid sequence comprise nucleotide sequences that are homologous to genomic sequences adjacent to the target region. [The present invention 1009] The method of claim 1008, wherein the 5' and 3' ends of the nucleic acid sequence comprise nucleotide sequences that are homologous to genomic sequences adjacent to the insertion site. [The present invention 1010] The method of any of claims 1006 to 1009, wherein the targeted nuclease introduces a double-stranded break at the insertion site. [The present invention 1011] The method of any one of claims 1001 to 1010, wherein the nucleic acid sequence is a single-stranded DNA template. [The present invention 1012] The method of any one of claims 1001 to 1011, wherein the nucleic acid sequence is a linear DNA template. [The present invention 1013] The method of any of claims 1001 to 1012, wherein the first self-cleaving peptide and the second self-cleaving peptide are the same or different viral 2A peptides. [The present invention 1014] The method of any of claims 1006 to 1013, wherein the targeting nuclease is selected from the group consisting of an RNA-guided nuclease domain, a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), and a megaTAL. [The present invention 1015] The method of claim 1014, wherein the RNA-guided nuclease is Cpf1 nuclease or Cas9 nuclease, and the method further comprises the step of introducing into the cell a guide RNA that specifically hybridizes to a target region in exon 1 of TRAC. [The present invention 1016] The method of claim 1014, wherein the RNA-guided nuclease is Cpf1 nuclease or Cas9 nuclease, and the method further comprises the step of introducing into the cell a guide RNA that specifically hybridizes to a target region in exon 1 of TRBC. [The present invention 1017] Cpf1 nuclease or Cas9 nuclease, guide RNA, and nucleic acid, (i) a ribonucleoprotein complex (RNP) comprising a Cpf1 nuclease or a Cas9 nuclease and a guide RNA; and (ii) DNA template The method of any one of claims 1015 to 1016, wherein the nucleic acid sequence is introduced into the cell as a ribonucleoprotein complex (RNP)-DNA template complex comprising: [The present invention 1018] The method of claim 10, wherein the molar ratio of RNP to DNA template in the complex is from about 3:1 to about 100:1. [The present invention 1019] The method of any one of claims 1017 to 1018, wherein the RNP-DNA template complex is formed by incubating the RNP with the DNA template at a temperature of about 20°C to 25°C for about 10 minutes to about 30 minutes. [The present invention 1020] The method of any of claims 1017 to 1019, wherein the RNP-DNA template complex and the cell are mixed prior to introducing the RNP-DNA template complex into the cell. [The present invention 1021] The method of any of claims 1017 to 1020, wherein the RNP-DNA template complex comprises at least two structurally distinct RNP complexes. [The present invention 1022] The method of claim 1021, wherein the at least two structurally distinct RNP complexes comprise structurally distinct guide RNAs. [The present invention 1023] The method of claim 1022, wherein each of the structurally distinct RNP complexes comprises a Cas9 nickase and the structurally distinct guide RNAs hybridize to opposing strands of the target region. [The present invention 1024] The method of any one of claims 1004 to 1023, wherein the introducing comprises electroporation. [The present invention 1025] The nucleic acid or RNP:DNA template complex is approximately 1 × 10 5 ~about 2×10 6 The method according to any one of claims 1004 to 1024, wherein the antibody is introduced into a population of T cells. [The present invention 1026] The method of claim 1025, wherein at least two structurally distinct DNA templates are introduced into the cell. [The present invention 1027] The method of the present invention, wherein each of the at least two structurally distinct DNA templates encodes a unique combination of a variable region of a heterologous TCR-β chain of an antigen-specific T cell receptor and a variable region of a heterologous TCR-α chain of an antigen-specific T cell receptor. [The present invention 1028] The method of any of claims 1001 to 1027, wherein the T cell is a regulatory T cell, an effector T cell, or a naive T cell. [The present invention 1029] The T cell is an effector T cell, and the effector T cell is CD8 + The method of claim 1028, wherein the cell is a T cell or a CD4+ cell. [The present invention 1030] Effector T cells are CD4 + CD8 + The method of claim 1029, wherein the cell is a T cell. [The present invention 1031] The method of any of claims 1001 to 1030, further comprising culturing the T cells under conditions allowing expression of a heterologous TCR-β chain and a heterologous TCR-α chain to form an antigen-specific T cell receptor. [The present invention 1032] The method of claim 1031, further comprising culturing the modified T cells under conditions effective to expand the population of the modified cells. [The present invention 1033] The method of claim 1031 or 1032, further comprising the step of purifying T cells expressing an antigen-specific T cell receptor. [The present invention 1034] A modified T cell produced by any one of the methods of the present invention 1001 to 1033. [The present invention 1035] 1. A modified T cell comprising a nucleic acid sequence, The nucleic acid sequence, from the N-terminus to the C-terminus, is (i) a first self-cleaving peptide sequence, (ii) a first heterologous TCR subunit chain; (iii) a second self-cleaving peptide sequence; (iv) the variable region of a second heterologous TCR subunit chain, and (v) The N-terminal portion of an endogenous TCR subunit Code the following: When the endogenous TCR subunit is a TCR-alpha (TCR-α) subunit, the first heterologous TCR subunit chain is a heterologous TCR-beta (TCR-β) subunit chain and the second heterologous TCR subunit chain is a heterologous TCR-α subunit chain; when the endogenous TCR subunit is a TCR-β subunit, the first heterologous TCR subunit chain is a heterologous TCR-α subunit chain and the second heterologous TCR subunit chain is a heterologous TCR-β subunit chain. Modified T cells. [The present invention 1036] The nucleic acid sequence, from the N-terminus to the C-terminus, is (i) a first self-cleaving peptide sequence, (ii) heterologous T cell receptor (TCR)-β chain; (iii) a second self-cleaving peptide sequence; (iv) the variable region of a heterologous TCR-α chain, and (v) the N-terminal portion of the endogenous TCR alpha subunit Code the following: The nucleic acid sequence is integrated into exon 1 of the TRAC gene. The modified T cells of the present invention. [The present invention 1037] The nucleic acid sequence, from the N-terminus to the C-terminus, is (i) a first self-cleaving peptide sequence, (ii) heterologous T cell receptor (TCR)-α chain; (iii) a second self-cleaving peptide sequence; (iv) The N-terminal portion of the endogenous TCR-β subunit Code the following: The nucleic acid sequence is integrated into exon 1 of the TRBC1 or TRBC2 gene; The modified T cells of the present invention. [The present invention 1038] From the N-terminus to the C-terminus (i) a first self-cleaving peptide sequence, (ii) a heterologous T cell receptor (TCR)-α chain variable region; (iii) a second self-cleaving peptide sequence; (iv) a variable region of a heterologous TCR-β chain, and (v) the N-terminal portion of the endogenous TCR beta subunit A nucleic acid sequence encoding the nucleic acid sequence A modified T cell comprising: [The present invention 1039] A method for editing the genome of a T cell, comprising the steps of: (a) From the N-terminus to the C-terminus, (i) a self-cleaving peptide sequence, (ii) a heterologous TCR-α chain of an antigen-specific T cell receptor, and (iii) the N-terminal portion of exon 1 of the endogenous TCR alpha subunit into a target region in exon 1 of a TCR alpha subunit constant gene (TRAC) in a human T cell; and (b) From the N-terminus to the C-terminus, (i) a second self-cleaving peptide sequence, (ii) a heterologous TCR-β chain of an antigen-specific T cell receptor, and (iii) the N-terminal portion of exon 1 of the endogenous TCR beta subunit Inserting a nucleic acid sequence encoding the above into a target region in exon 1 of the TCR beta subunit constant gene (TRBC) in human T cells. [The present invention 1040] The method of claim 1039, wherein the first and / or second nucleic acid sequence is a double-stranded or single-stranded nucleic acid, and the first and / or second nucleic acid is inserted by introducing the first and / or second nucleic acid into a T cell. [The present invention 1041] The method of claim 1040, wherein the first and / or second nucleic acid sequence is inserted by introducing a viral vector comprising the first and / or second nucleic acid into the T cell. [The present invention 1042] The first and second nucleic acids (a) one or more targeted nucleases that create a first insertion site in exon 1 of the TRAC gene and a second insertion site in exon 1 of the TRBC gene; (b) a first nucleic acid sequence, and (c) a second nucleic acid sequence. The method of any one of claims 1040 to 1041, wherein the antibody is inserted into the T cell by introducing into the T cell. [The present invention 1043] The method of any of claims 1039 to 1042, wherein the 5' and 3' ends of the first nucleic acid sequence comprise a nucleotide sequence that is homologous to a genomic sequence adjacent to a first target region in exon 1 of TRAC. [The present invention 1044] The method of claim 1043, wherein the 5' and 3' ends of the second DNA template comprise nucleotide sequences that are homologous to genomic sequences adjacent to the second target region in exon 1 of TRBC. [The present invention 1045] The method of any of claims 1042 to 1044, wherein the one or more targeted nucleases introduce a double-stranded break at the first and second insertion sites. [The present invention 1046] The method of any of claims 1039 to 1045, wherein the first and / or second nucleic acid sequence is a single-stranded DNA template. [The present invention 1047] The method of any of claims 1039 to 1046, wherein the first and / or second nucleic acid is a linear DNA template. [The present invention 1048] The method of any of claims 1039 to 1047, wherein the first self-cleaving peptide and the second self-cleaving peptide are the same or different viral 2A peptides. [The present invention 1049] Any of the methods of claims 1042 to 1048, wherein the one or more targeted nucleases are selected from the group consisting of an RNA-guided nuclease domain, a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), or a megaTAL. [The present invention 1050] The method of claim 1049, wherein the RNA-guided nuclease is Cpf1 nuclease or Cas9 nuclease, and the method further comprises the step of introducing into the cell a first guide RNA that specifically hybridizes to a target region in exon 1 of TRAC, and a second guide RNA that specifically hybridizes to a target region in exon 1 of TRBC. [The present invention 1051] The Cpf1 nuclease or Cas9 nuclease, the first guide RNA, and the nucleic acid sequence (i) a ribonucleoprotein complex (RNP) comprising a Cas9 nuclease or a Cpf1 nuclease and a first guide RNA; and (ii) A first DNA template The method of claim 10, wherein the nucleic acid sequence is introduced into the cell as a ribonucleoprotein complex (RNP)-DNA template complex comprising: [The present invention 1052] The Cpf1 nuclease or Cas9 nuclease, the second guide RNA, and the second nucleic acid sequence (i) an RNP comprising a Cpf1 nuclease or a Cas9 nuclease and a second guide RNA; and (ii) A second DNA template The method of claim 10, wherein the nucleic acid sequence is introduced into the cell as an RNP-DNA template complex comprising: [The present invention 1053] 13. The method of claim 1051 or 1052, wherein the molar ratio of RNP to DNA template in the complex is from about 3:1 to about 100:1. [The present invention 1054] The method of any of claims 1051 to 1053, wherein the RNP-DNA template complex is formed by incubating the RNP with the DNA template at a temperature of about 20°C to about 25°C for about 10 minutes to about 30 minutes. [The present invention 1055] The method of any of claims 1051 to 1054, wherein the RNP-DNA template complex comprises at least two structurally distinct RNP complexes. [The present invention 1056] The method of claim 1055, wherein each of the structurally distinct RNP complexes comprises a Cas9 nickase and the structurally distinct guide RNAs hybridize to opposing strands of the target region. [The present invention 1057] The method of any one of claims 1039 to 1056, wherein the introducing comprises electroporation. [The present invention 1058] The nucleic acid sequence or RNP:DNA template complex is approximately 1×105 ~Approx. 2×10 6 The method according to any one of claims 1039 to 1057, wherein the antibody is introduced into individual T cells. [The present invention 1059] The method of claim 1058, wherein at least two structurally distinct first DNA templates are introduced into the cell. [The present invention 1060] The method of claim 1059, wherein the at least two structurally distinct first DNA templates comprise different variable regions of the TCR-α chain of an antigen-specific T cell receptor. [The present invention 1061] The method of claim 1060, wherein at least two structurally distinct second DNA templates are introduced into the cell. [The present invention 1062] The method of claim 1061, wherein the at least two structurally distinct second DNA templates comprise different variable regions of TCR-β chains of an antigen-specific T cell receptor. [The present invention 1063] The method of any of claims 1039 to 1062, wherein the T cell is a regulatory T cell, an effector T cell, or a naive T cell. [The present invention 1064] The T cell is an effector T cell, and the effector T cell is CD8 + The method of claim 1063, wherein the cell is a T cell or a CD4+ cell. [The present invention 1065] Effector T cells are CD4 + CD8 + The method of claim 1064, wherein the T cell is a T cell. [The present invention 1066] Any of the methods of claims 1039 to 1065, further comprising culturing the T cells under conditions allowing expression of a heterologous TCR-β chain and a heterologous TCR-α chain to form an antigen-specific T cell receptor. [The present invention 1067] The method of claim 1066, further comprising culturing the T cells under conditions allowing expression of the heterologous TCR-β chain and the heterologous TCR-α chain to form an antigen-specific T cell receptor. [The present invention 1068] The method of claim 1067, further comprising culturing the modified T cells under conditions effective to expand the population of the modified cells. [The present invention 1069] The method of any one of claims 1067 to 1068, further comprising the step of purifying T cells expressing an antigen-specific T cell receptor. [The present invention 1070] A modified T cell produced by any one of the methods of the present invention 1039 to 1069. [The present invention 1071] (a) From the N-terminus to the C-terminus, (i) a first self-cleaving sequence, (ii) the variable region of a heterologous TCR-α chain, and (iii) The N-terminal portion of the endogenous TCR-α chain and a first nucleic acid sequence encoding the (b) From the N-terminus to the C-terminus, (i) a first self-cleaving sequence, (ii) a variable region of a heterologous TCR-β chain, and (iii) The N-terminal portion of the endogenous TCR-β chain and a second nucleic acid sequence integrated into exon 1 of the TRBC gene, A modified T cell comprising: [The present invention 1072] (a) modifying T cells of a subject using any of the methods of the present invention 1001-1033 or 1039-1069 so as to express an antigen-specific T cell receptor that recognizes a tumor-specific antigen in the subject; 23. A method of treating cancer in a human subject comprising: [The present invention 1073] The method of claim 1072, wherein the subject's T cells are modified in vivo. [The present invention 1074] (a) obtaining T cells from a subject; (b) modifying T cells using any of the methods of claims 1001-1033 or 1039-1069 to express an antigen-specific T cell receptor that recognizes a tumor-specific antigen in the subject; and (c) administering the modified T cells to a subject. The method of the present invention 1072, comprising: [Brief description of the drawings]

[0027] This application includes the following figures. The figures are intended to illustrate certain aspects and / or features of the compositions and methods, and to supplement any descriptions of the compositions and methods. The figures do not limit the scope of the compositions and methods, unless the description expressly indicates otherwise.

[0028] [Figure 1a] Schematic diagram depicting the insertion into T cells by homology-directed repair of a single non-viral DNA template that contains, from N- to C-terminus, a nucleic acid sequence encoding: (i) a T2A self-cleaving peptide sequence; (ii) a full-length (i.e., variable and constant regions) heterologous TCR-β chain (NYESO-β); (iii) a P2A self-cleaving peptide sequence; (iv) a variable region of a heterologous TCR-α chain (NYESO-α); and (v) a portion of the N-terminus of an endogenous TCR alpha subunit. Following insertion of the DNA template in exon 1 of the TRAC gene by homology-directed repair, the DNA template was transcribed and translated to produce a full-length NYESO-β chain and a full-length NYESO-α chain that form an antigen-specific TCR that recognizes the NY-ESO-1 melanoma neoantigen. [Figure 1b] 1 is a schematic depicting the insertion of a single non-viral DNA template at the TCR-β locus (TRBC1 or TRBC2). The template contains a nucleic acid sequence encoding, from N-terminus to C-terminus, (i) the T2A self-cleaving peptide sequence; (ii) the full-length (i.e., variable and constant regions) of a heterologous TCR-α chain (NYESO-α); (iii) the P2A self-cleaving peptide sequence; (iv) the variable region of a heterologous TCR-β chain (NYESO-β); and (v) a portion of the N-terminus of an endogenous TCR beta subunit, into a T cell by homologous recombination repair. [Diagram 2]1 is a schematic diagram depicting the simultaneous replacement of both endogenous T-cell α and T-cell β receptor chains by inserting (a) a non-viral DNA template comprising a nucleic acid sequence encoding, from N-terminus to C-terminus, (i) a P2A self-cleaving peptide sequence; (ii) the variable region of the TCR-α chain of an antigen-specific T-cell receptor (NYESO-α); and (iii) a portion of exon 1 at the N-terminus of the endogenous TCR-α subunit; and (b) a DNA template comprising a nucleic acid sequence encoding, from N-terminus to C-terminus, (i) a T2A self-cleaving peptide sequence; (ii) the variable region of the TCR-β chain of an antigen-specific T-cell receptor; and (iii) a portion of exon 1 at the N-terminus of the endogenous TCR-β subunit. [Figure 3a] 1 is a schematic depicting insertion of heterologous (TCR)-β and TCR-α chains in T cells to form a polyclonal library of T cell receptors. A plurality of different DNA templates, e.g., non-viral DNA templates, including a nucleic acid sequence encoding, from N-terminus to C-terminus, (i) a first self-cleaving peptide sequence; (ii) a full-length (e.g., variable and constant region) heterologous T cell receptor (TCR)-β chain; (iii) a second self-cleaving peptide sequence; (iv) a variable region of a heterologous TCR-α chain; and (v) a portion of the N-terminus of an endogenous TCR alpha subunit, were electroporated simultaneously to generate a population of T cells with a synthetic T cell repertoire of desired TCR sequences. [Figure 3b] 1 is a diagram showing the design of T cell repertoire from known TCR sequence or from natural repertoire in endogenous T cell population of interest, for example, from subject.For example, TCR can be, but is not limited to, the TCR expressed by tumor-infiltrating lymphocytes, the TCR expressed by autoreactive T cells in autoimmune disease sites, or the TCR from pathogen-responsive T cells. [Figure 4a]Figure 1 shows the FACS analysis of CD4+ and CD8+ T cells electroporated with heterologous NYESO TCR.As described herein, CD4+ and CD8+ T cells from two healthy human blood donors are electroporated with non-viral constructs that contain heterologous NYESO-α and heterologous NYESO-β.Four days after electroporation, cells are stained with fluorescently labeled MHC-dextramers that contain peptides recognized by the incorporated NYESO-specific TCR. [Figure 4b] Figure 1 shows the FACS analysis of CD8+ T cells electroporated with heterologous NYESO TCR.As described herein, CD8+ T cells from healthy human blood donors are electroporated with non-viral DNA constructs that contain heterologous NYESO-α and heterologous NYESO-β.Four days after electroporation, cells are stained with fluorescently labeled MHC-dextramers that contain peptides recognized by incorporated NYESO-specific TCR. [Diagram 5] FACS analysis of CD8+ cells electroporated with heterologous NYESO TCR. Cells were treated as in Figures 3 and 4 with the addition of staining for TCR expression (with an antibody that binds all potential human TCRs) versus antigen-specific staining with NYESO MHC-dextramer. In the majority of T cells that did not have homologous recombination replacement of the endogenous TCR, the endogenous TCR was knocked out by truncation with TRAC exon 1 gRNA and introduction of a small insertion deletion mutation (indel) by non-homologous end joining. As expected, almost all NYESO positive cells were also positive for TCR expression. [Figure 6a] 1 shows that primary human T cells containing the heterologous NYESO TCR kill cancer cells in an in vitro cell killing assay. [Figure 6b] 1 shows results from one time point of an in vitro cell killing assay using human T cells involving xenogeneic NYESO TCR killing. [Figure 7]Figure 7a-f shows in vivo functionality of T cells with non-viral TCR replacement. (a) Schematic of in vivo human antigen-specific tumor xenograft model. (b) Two days after transfer of 5x106 bulk non-viral targeted T cells (~10% TCR+ NYESO-1+, ~10% TCR+ NYESO-1-, and ~80% TCR- NYESO-1-), NY-ESO-1+ non-viral edited T cells selectively accumulated in tumors compared to spleens. n=5 mice per 4 human T cell donors. (c) Ten days after transfer of 5x106 bulk non-viral targeted CFSE-labeled T cells, NYESO-1 TCR+ cells showed higher proliferation than TCR- or TCR+ NYESO-1- T cells and higher proliferation (CFSE low) in tumors than in spleens. At 10 days post-transfer, TCR- and TCR+NYESO- T cells were difficult to find within the tumor. (d) Individual longitudinal tumor volume trajectories of the data summarized in Figure 8f. (e,f) In these experiments, 17 days after T cell transfer (d), non-viral TCR replaced cells appeared to exhibit higher NY-ESO-1 TCR expression and lower exhaustion marker expression. Transfer of both lentiviral transduced and non-viral TCR replaced cells showed a significant reduction in tumor burden at day 24. In this experimental model, non-viral TCR replacement showed a further reduction compared to lentiviral transduction (Figure 8f). [Figure 8a] Figure 2 shows antigen-specific cytokine production and degranulation in CD8+ T cells upon TCR replacement. [Figure 8b] 1 shows antigen-specific target cell killing by CD8+ T cells upon TCR replacement. [Figure 8c] Schematic representation of tumor mouse xenograft model. [Figure 8d] We demonstrate the scalable potential of non-viral replacement of endogenous TCRs for adoptive cell therapy. [Figure 8e] 1 shows selective in vivo localization of NY-ESO-1 TCR+ T cells to tumors. [Figure 8f]Tumor growth after adoptive transfer of NY-ESO-1 TCR+ non-viral or lentiviral modifications or vehicle only (saline) is shown. n=2 (a,b) independent healthy donors with mean and standard deviation (a,b) of technical triplicates. n=6 (d) or n=2 (e,f) individual healthy donors in 5 (e) or 7 (f) mice with mean and standard deviation (d-f). **P<0.01, ***P<0.001, ****P<0.0001 (Two Way ANOVA with Holm-Sidak's multiple comparison test). [Figure 9-1] Figure 9a shows TCR mispairing analysis after retroviral delivery of NY-ESO-1 specific TCR or non-viral TCR replacement in gated CD4+ or CD8+ T cells. Non-viral TCR replacement results in less TCR mispairing compared to retroviral TCR delivery. Viral introduction of new TCRs will result in infected cells expressing at least four different TCRs (new TCR-α + new TCR-β; new TCR-α + endogenous TCR-β; endogenous TCR-α and new TCR-β; endogenous TCR-α + endogenous TCR-β). Staining for the specific β-chain of the newly introduced TCR (VB13.1) together with the MHC-peptide multimer (NYESO) can provide a rough estimate of TCR mispairing by distinguishing between cells that predominantly expressed the introduced TCR (VB13.1+ NYESO+; new TCR-α + new TCR-β) versus cells that predominantly expressed one of the potential mispaired TCRs (VB13.1+ NYESO-; endogenous TCR-α + new TCR-β). [Figure 9-2] Figures 9b-9c show TCR replacement by targeting the whole new TCR to TRAC (Figure 9b, also possible with multiple knockouts of TCR-β), the whole new TCR to TRBC1 / 2 (Figure 9c), or multiple replacement with new TCR-α to TRAC and new TCR-β to TRBC1 / 2. [Figure 9-3]FIG. 9d shows that functional cytokine production was selectively observed following antigen exposure in gated CD4+ T cells as well as gated CD8+ T cells (FIG. 8a). [Figure 9-4] Figure 9e shows that non-viral TCR replacement was consistently observed in both CD8+ and CD4+ T cells across a cohort of six healthy blood donors at 4 days post-electroporation. Figure 9f shows that in a second cohort of six additional healthy blood donors, 100 million T cells from each donor were electroporated with the NY-ESO-1 TCR replacement HDR template and on-target gRNA / Cas9 (Figure 8d). The percentage of CD4+ and CD8+ T cells that were NY-ESO-1 TCR+ was consistent over the 10-day expansion post-electroporation. Figure 9g shows that CD8+ T cells showed a slight proliferation advantage over CD4+ T cells over the 10-day expansion post-non-viral genome targeting. [Figure 9-5] Figure 9h shows the results of co-incubation of the indicated melanoma cell lines with the indicated sorted T cell populations at a T cell to cancer cell ratio of 1:5. At 72 hours post-co-incubation, the percentage of cancer cell confluency (nuclear RFP marks cancer cells) was recorded by automated microscopy. T cells expressing the NY-ESO-1 antigen-specific TCR showed robust target cell killing only in target cancer cell lines expressing both NY-ESO-1 and HLA-A*0201 class I MHC alleles, either by retroviral transduction or non-viral knock-in endogenous TCR replacement. [Figure 9-6] Figure 9i shows the results of using a matrix of on / off-target gRNAs and on / off-target HDR templates for targeted cell killing of the NY-ESO-1+ HLA-A*0201+ A375 cancer cell line (off-target gRNAs and HDRTs were specific for RAB11A-GFP fusion protein knock-in). Only cells with both on-target gRNAs and on-target HDR templates demonstrated targeted cell killing. [Figure 9-7]Figure 9j shows that sorted NY-ESO-1+ TCR+ cells (on-target gRNA, on-target HDR template) from the bulk T cell edited population showed a strong dose-response effect on target cancer cell killing. T cell to cancer cell ratios of 2:1 and above showed near complete killing of target cancer cells within 48 hours. T cell to cancer cell ratios of less than 1:16 showed evidence of robust target cell killing by 144 hours. [Figure 9-8] Figure 9k shows that target cell killing by non-viral TCR-replaced T cells was specifically attributable to the NY-ESO-1-recognizing TCR+ cell population observed by flow cytometry after non-viral TCR replacement. Starting with the bulk-edited T cell population (all electroporated with on-target gRNA and HDR template), we separately sorted three cell populations: NY-ESO-1+TCR+ cells (non-viral-replaced TCR), NY-ESO-1-TCR- cells (TCR knockout) (gray), and NY-ESO-1-TCR+ cells (cells that retained their native TCR but did not have the NY-ESO-specific knock-in TCR). Only the sorted NY-ESO-1+ TCR+ population demonstrated target cell killing (T cell to cancer cell ratio of 4:1). One representative donor from n=2 (a,d) or n=3 (b,c) independent healthy donors with mean and standard deviation of technical triplicates (d). Means and standard deviations of four technical replicates from n=6 independent healthy donors (e,f) or from n=2 independent healthy donors (i–k) are shown. Mean and individual values ​​from n=2 independent healthy donors (h). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] definition As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0030] The term "nucleic acid" or "nucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in either single-stranded or double-stranded form and polymers thereof. Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0031] The term "gene" can refer to a segment of DNA involved in producing or encoding a polypeptide chain. It can include regions preceding and following the coding region (leader and trailer), as well as intervening sequences (introns) between individual coding segments (exons). Alternatively, the term "gene" can refer to a segment of DNA involved in producing or encoding a non-translated RNA, such as an rRNA, a tRNA, a guide RNA (e.g., a single guide RNA), or a microRNA.

[0032] "Treating" refers to any indication of success in treating or ameliorating or preventing a disease, condition, or disorder, including any objective or subjective parameter, such as diminishment; remission; reduction in symptoms or making the disease state more tolerable to the patient; slowing the rate of degeneration or decline; or lessening the debilitating end point of degeneration.

[0033] "Promoter" is defined as one or more nucleic acid control sequences that direct the transcription of nucleic acid.As used herein, promoter includes the necessary nucleic acid sequence near the start site of transcription, such as TATA element in case of polymerase II type promoter.Promoter may optionally include distal enhancer or repressor element, which may be located several thousand base pairs away from the start site of transcription.

[0034] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.

[0035] "Polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. As used herein, these terms encompass amino acid chains of any length, including full-length proteins, in which the amino acid residues are linked by covalent peptide bonds.

[0036] As used herein, the term "complementary" or "complementarity" refers to a specific base pairing between nucleotides or nucleic acids. In some embodiments, for example, but not limited to, base pairing between a target region in exon 1 of the TRAC gene and a guide RNA is described. Complementary nucleotides are generally A and T (or A and U), and G and C. The guide RNA described herein can include a sequence, e.g., a DNA targeting sequence, that is fully complementary or substantially complementary (e.g., has 1-4 mismatches) to a genomic sequence in exon 1 of the TRAC gene in a T cell.

[0037] As used throughout, subject means an individual.For example, subject is a mammal, such as a primate, more specifically, human.This term does not indicate a specific age or sex.Thus, adult and neonatal subjects, whether male or female, are intended to be covered.As used herein, patient or subject may be used interchangeably and may refer to a subject suffering from a disease or disorder.

[0038] "CRISPR / Cas" system refers to a broad class of bacterial systems for defense against foreign nucleic acids. CRISPR / Cas systems are found in a wide range of eubacterial and archaeal organisms. CRISPR / Cas systems include type I, type II, and type III subtypes. Wild-type type II CRISPR / Cas systems utilize an RNA-mediated nuclease, such as Cas9, complexed with guide and activator RNA to recognize and cleave foreign nucleic acids. Guide RNAs with both guide RNA and activator RNA activity are also known in the art. In some cases, such dual activity guide RNAs are referred to as single guide RNAs (sgRNAs).

[0039] Cas9 homologs are found in a wide variety of eubacteria, including, but not limited to, bacteria from the following taxa: Actinobacteria, Aquificae, Bacteroidetes-Chlorobi, Chlamydiae-Verrucomicrobia, Chloroflexi, Cyanobacteria, Firmicutes, Proteobacteria, Spirochaetes, and Thermotogae. An exemplary Cas9 protein is the Streptococcus pyogenes Cas9 protein. Additional Cas9 proteins and their homologues are described, for example, in Chylinksi, et al., RNA Biol. 2013 May 1; 10(5): 726-737; Nat. Rev. Microbiol. 2011 June; 9(6): 467-477; Hou, et al., Proc Natl Acad Sci US A. 2013 Sep 24; 110(39): 15644-9; Sampson et al., Nature. 2013 May 9; 497(7448): 254-7; and Jinek, et al., Science. 2012 Aug 17; 337(6096): 816-21. Any Cas9 nuclease variant provided herein can be optimized for efficient activity or enhanced stability in host cells. Thus, engineered Cas9 nucleases are also contemplated.

[0040] As used herein, the term "Cas9" refers to an RNA-mediated nuclease (e.g., of or derived from bacterial or archaeal origin). Exemplary RNA-mediated nucleases include the Cas9 protein and its homologs. Other RNA-mediated nucleases include Cpf1 (see, e.g., Zetsche et al., Cell, Volume 163, Issue 3, p759-771, 22 October 2015) and its homologs. Similarly, as used herein, terms such as "Cas9 ribonucleoprotein" complexes refer to a complex between a Cas9 protein and a crRNA (e.g., a guide RNA or a single guide RNA), a Cas9 protein and a trans-activating crRNA (tracrRNA), a Cas9 protein and a guide RNA, or a combination thereof (e.g., a complex comprising a Cas9 protein, a tracrRNA, and a crRNA guide RNA). It is understood that in any of the embodiments described herein, the Cas9 nuclease can be replaced with a Cpf1 nuclease.

[0041] As used herein, the phrase "edit" in the context of editing the genome of a cell refers to inducing structural changes in the sequence of the genome at a target genome region.For example, editing can take the form of inserting a nucleotide sequence into the genome of a cell.The nucleotide sequence can code for a polypeptide or a fragment thereof.Such editing can be performed, for example, by inducing a double-stranded break in the target genome region, or by inducing a pair of single-stranded nicks on opposite strands that flank the target genome region.Methods for inducing single-stranded or double-stranded breaks at or within the target genome region include the use of a Cas9 nuclease domain or its derivative and a guide RNA or a pair of guide RNAs directed to the target genome region.

[0042] As used herein, the phrase "introduce" in the context of introducing a nucleic acid or a complex containing a nucleic acid, such as an RNP-DNA template complex, refers to the transfer of a nucleic acid sequence or an RNP-DNA template complex from outside the cell to inside the cell.In some cases, introducing refers to the transfer of a nucleic acid or a complex from outside the cell to inside the nucleus of the cell.Various methods of such transfer are contemplated, including, but not limited to, electroporation, contact with nanowires or nanotubes, receptor-mediated internalization, cell-penetrating peptide-mediated transfer, liposome-mediated transfer, etc.

[0043] As used herein, the term "heterologous" refers to a nucleic acid sequence or polypeptide that is not naturally found in a human T cell. The term "heterologous sequence" refers to a sequence that is not normally found in a given T cell in nature. Thus, a heterologous nucleotide or protein sequence can be: (a) foreign to its host cell (i.e., exogenous to the cell); (b) naturally found in the host cell (i.e., endogenous), but present in the cell in an unnatural amount (i.e., greater or less than naturally found in the host cell); or (c) naturally found in the host cell, but located outside its natural locus.

[0044] As used herein, the term "primary" in the context of primary cells refers to cells that are not transformed or immortalized. Such primary cells can be cultured, subcultured, or passaged a limited number of times (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times). In some cases, primary cells are adapted to in vitro culture conditions. In some cases, primary cells can be isolated from organisms, systems, organs, or tissues, optionally selected, and directly utilized without culture or subculture. In some cases, primary cells are stimulated, activated, or differentiated. For example, primary T cells can be activated by contacting (e.g., culturing in the presence of) CD3, CD28 agonist, IL-2, IFN-γ, or a combination thereof.

[0045] As used herein, the phrase "T cells" refers to lymphoid cells that express T cell receptor molecules. T cells include human alpha beta (αβ) T cells and human gamma delta (γδ) T cells. T cells include, but are not limited to, naive T cells, stimulated T cells, primary T cells (e.g., uncultured), cultured T cells, immortalized T cells, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, combinations thereof, or subpopulations thereof. T cells are CD4 + , CD8 + , or CD4 + and CD8 + T cells can be helper cells, e.g., T H 1. T H 2. T H 3. T H 9. T H 17 or T FH The T cells can be cytotoxic T cells. The regulatory T cells can be FOXP3 + or FOXP3 - The T cells can be alpha / beta T cells or gamma / delta T cells. Optionally, the T cells are CD4 +CD25 高 CD127 低 In some cases, the T cells are type 1 regulatory (Tr1), T H 3, CD8+CD28-, Treg17, and Qa-1 restricted T cells, or a combination or subpopulation thereof. + T cells. In some cases, T cells are CD4 + CD25 低 CD127 高 effector T cells. In some cases, T cells are CD4 + CD25 低 CD127 高 CD45RA 高 CD45RO - The T cell is a naive T cell. The T cell may be a genetically engineered recombinant T cell. In some cases, the recombinant T cell has a modified (e.g., heterologous) T cell receptor.

[0046] As used herein, the term "TCR receptor" refers to a heterodimer consisting of two TCR subunit chains (e.g., TCR-α and TCR-β, TCRγ and TCRδ) that function in the activation of T cells in response to antigen. When expressed in a T cell, each TCR subunit chain of a TCR receptor contains a constant region that anchors the TCR subunit chain to the cell membrane and a variable region that functions in antigen recognition and binding, for example, when a first TCR subunit chain (e.g., TCR-α) and a second TCR subunit chain (e.g., TCR-β) chain form a heterodimeric TCR receptor.

[0047] As used herein, the term "non-homologous end joining" or NHEJ refers to the cellular process in which the cut or nicked ends of DNA strands are directly ligated without the need for homologous template nucleic acid.NHEJ can result in the addition, deletion, substitution, or combination of one or more nucleotides at the repair site.

[0048] As used herein, the term "homologous recombination repair" or HDR refers to the cellular process in which DNA strand breaks or nicked ends are repaired by polymerization from homologous template nucleic acid. Thus, the original sequence is replaced by the sequence of the template. The homologous template nucleic acid can be provided by a homologous sequence (sister chromatid, homologous chromosome, or repeated region on the same or different chromosome) elsewhere in the genome. Alternatively, an exogenous template nucleic acid can be introduced to obtain specific HDR-induced changes of sequence at the target site. In this way, specific mutations can be introduced at the break site.

[0049] As used herein, single-stranded DNA template or double-stranded DNA template refers to the DNA oligonucleotide that can be used by cell as a template for editing the genome of T cell, for example, by HDR.Generally, single-stranded DNA template or double-stranded DNA template has at least one homologous region to target site.In some cases, single-stranded DNA template or double-stranded DNA template has two homologous regions, for example, 5' end and 3' end, adjacent to the region containing the heterologous sequence that is inserted into the target cleavage site or insertion site.

[0050] Detailed Description of the Invention The following description lists various aspects and embodiments of the present compositions and methods.Specific embodiments are not intended to define the scope of the compositions and methods.Rather, the embodiments merely provide non-limiting examples of various compositions and methods that are at least included within the scope of the disclosed compositions and methods.The description should be read from the perspective of a person skilled in the art, and therefore does not necessarily include information that is known to a person skilled in the art.

[0051] Provided herein are compositions and methods for editing the genome of human T cells.The inventors have discovered that heterologous TCRs can be inserted into target regions in the genome of T cells, so that the heterologous TCRs are under the control of endogenous TCR promoters.The methods and compositions provided herein can be used to create modified T cells with desired antigen specificity.These modified T cells can be used, for example, to treat cancer, autoimmune disease, or infection in subjects.

[0052] In some embodiments, the nucleic acid sequence encoding the variable region of a heterologous TCR-β chain and the variable region of a heterologous TCR-α chain is inserted into exon 1 of the TRAC gene in the genome of the T cell. In some embodiments, the nucleic acid sequence encoding the variable region of a heterologous TCR-α chain and the variable region of a heterologous TCR-β chain is inserted into exon 1 of a TRBC gene, such as exon 1 of TRBC1 or TRBC2, in the genome of the T cell. In some embodiments, the nucleic acid sequence is introduced via homologous recombination repair or as otherwise described herein.

[0053] In some embodiments, (a) a first nucleic acid sequence encoding a variable region of a heterologous TCR-α chain of a T cell receptor; (b) a second nucleic acid sequence encoding a variable region of a heterologous TCR-β chain of an antigen-specific T cell receptor is inserted into exon 1 of the TRAC gene and exon 1 of the TRBC gene, respectively. In some embodiments, the nucleic acid sequences are introduced via homologous recombination repair or as otherwise described herein.

[0054] Methods for generating engineered human T cells Methods for editing the genome of a T cell include a method of editing the genome of a human T cell, comprising inserting a nucleic acid sequence or construct into a target region in exon 1 of a T cell receptor (TCR) subunit constant gene in a human T cell. The nucleic acid construct contiguously encodes, from N-terminus to C-terminus, a first heterologous TCR subunit chain, including a variable region and a constant region of the TCR subunit chain, and a variable region of a second heterologous TCR subunit chain. The construct further encodes a first self-cleaving peptide preceding the variable region of the first heterologous TCR subunit chain, and a second self-cleaving peptide between the first heterologous TCR subunit chain and the second heterologous TCR subunit chain. In some methods, when the endogenous TCR subunit is a TCR-alpha (TCR-α) subunit, the first heterologous TCR subunit chain is a heterologous TCR-beta (TCR-β) subunit chain, and the second heterologous TCR subunit chain is a heterologous TCR-α subunit chain. In some methods, when the endogenous TCR subunit is a TCR-β subunit, the first heterologous TCR subunit chain is a heterologous TCR-α subunit chain and the second heterologous TCR subunit chain is a heterologous TCR-β subunit chain.

[0055] In some embodiments, the nucleic acid construct or sequence encoding the variable region of a TCR subunit chain is a nucleic acid construct or sequence encoding a TCR subunit chain, i.e., a nucleic acid encoding the variable and constant regions of a TCR subunit chain, e.g., a full-length TCR subunit chain. In some examples, the nucleic acid encodes a full-length TCR-α, TCR-β, TCR-γ, or TCR-δ subunit chain. In some examples, the nucleic acid construct or sequence encodes the variable regions of a first heterologous TCR subunit chain (e.g., a full-length TCR subunit chain) and a second heterologous TCR subunit chain. In some examples, the first and second heterologous TCR subunit chains are different. In some examples, the nucleic acid construct encodes, from N-terminus to C-terminus, the variable regions of a heterologous full-length TCR-β subunit chain and a heterologous TCR-α subunit chain. In other examples, the nucleic acid construct encodes, from N-terminus to C-terminus, the variable regions of a heterologous full-length TCR-α subunit chain and a heterologous TCR-β subunit chain.

[0056] Methods for editing the genome of a T cell include a method of editing the genome of a human T cell, comprising inserting a nucleic acid sequence or construct into a target region in exon 1 of a TCR-α subunit (TRAC) gene in a human T cell. In some embodiments, the target region is in exon 1 of the constant domain of the TRAC gene. In other embodiments, the target region is in exon 1, exon 2, or exon 3, prior to the start of the sequence encoding the TCR-α transmembrane domain. The nucleic acid construct sequentially encodes, from N-terminus to C-terminus, a variable region of a heterologous T cell receptor (TCR)-β chain followed by a variable region of a heterologous TCR-α chain. The construct further encodes a first self-cleaving peptide preceding the variable region of the heterologous TCR-β chain and a second self-cleaving peptide between the variable region of the heterologous TCR-β chain and the variable region of the heterologous TCR-α chain. The construct further encodes a portion of the N-terminus of the endogenous TCR-α subunit following the variable region of the heterologous TCR-α chain. Depending on the insertion site in the TRAC gene, the size of the nucleic acid encoding the N-terminal portion of the endogenous TCR-α subunit can vary. The size of the nucleic acid encoding the N-terminal portion of the endogenous TCR-α subunit will depend on the number of nucleotides in the endogenous TRAC nucleic acid sequence between the start of TRAC exon 1 and the target insertion site. For example, see FIG. 1a, where the number of nucleotides between the start of TRAC exon 1 and the insertion site was 25 nucleotides, so 25 nucleotides encoding the N-terminus of the endogenous TCR-α subunit were included in the construct. Similarly, if the number of nucleotides between the start of TRAC exon 1 and the insertion site is less than or more than 25 nucleotides, there may be less than or more than 25 nucleotides of the nucleic acid encoding the N-terminal portion of the endogenous TCR-α subunit in the construct. In some examples, the nucleic acid construct encodes, from N-terminus to C-terminus, a first self-cleaving peptide sequence, a heterologous (i.e., variable region and constant region) TCR-β subunit chain (e.g., a full-length TCR-β subunit chain), a second self-cleaving peptide sequence, the variable region of a heterologous TCR-α subunit chain, and a portion of the N-terminus of an endogenous TCR-α subunit.Exemplary constructs include those depicted in FIG. 1a.

[0057] The method for editing the genome of a T cell also includes a method for editing the genome of a human T cell, comprising inserting a nucleic acid sequence or construct into a target region in exon 1 of a TCR-β subunit (TRBC) gene in a human T cell. In some embodiments, the target region is in exon 1 of the TRBC1 or TRBC2 gene. The nucleic acid construct sequentially encodes, from N-terminus to C-terminus, the variable region of a heterologous T cell receptor (TCR)-α chain followed by the variable region of a heterologous TCR-β chain. The construct further encodes a first self-cleaving peptide preceding the variable region of the heterologous TCR-α chain and a second self-cleaving peptide between the variable region of the heterologous TCR-α chain and the variable region of the heterologous TCR-β chain. The construct further encodes the N-terminal portion of the endogenous TCR-β subunit following the variable region of the heterologous TCR-β chain. Depending on the insertion site in TRBC1 or TRBC2, the size of the nucleic acid encoding the N-terminal portion of the endogenous TCR-β subunit can vary. The size of the nucleic acid encoding the N-terminal portion of the endogenous TCR-α subunit will depend on the number of nucleotides in the endogenous TRBC nucleic acid sequence between the start of exon 1 of TRBC1 or TRBC2 TRBC and the target insertion site. For example, the number of nucleotides between the start of TRBC exon 1 and the insertion site was 25 nucleotides, so 25 nucleotides encoding the N-terminus of the endogenous TCR-β subunit were included in the construct, see FIG. 1b. Similarly, if the number of nucleotides between the start of TRBC1 or TRBC2 exon 1 and the insertion site is less than or more than 25 nucleotides, there may be less than or more than 25 nucleotides of nucleic acid encoding the N-terminal portion of the endogenous TCR-β subunit in the construct. In some examples, the nucleic acid construct encodes, from N-terminus to C-terminus, a first self-cleaving peptide sequence, a heterologous (i.e., variable and constant region) TCR-α subunit chain (e.g., a full-length TCR-α subunit chain), a second self-cleaving peptide sequence, a variable region of a heterologous TCR-β subunit chain, and a portion of the N-terminus of an endogenous TCR-β subunit. Exemplary constructs include those depicted in FIG. 1b.

[0058] Examples of self-cleaving peptides include, but are not limited to, self-cleaving viral 2A peptides, such as porcine teschovirus-1 (P2A) peptide, Thosea asigna virus (T2A) peptide, equine rhinitis A virus (E2A) peptide, or foot and mouth disease virus (F2A) peptide. Self-cleaving 2A peptides allow expression of multiple gene products from a single construct. (See, for example, Chng et al. "Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells", MAbs 7(2): 403-412 (2015)). In some embodiments, the first and second self-cleaving peptides are the same. In other embodiments, the first and second self-cleaving peptides are different.

[0059] When inserted, the construct encoding the first self-cleaving peptide, the heterologous full-length TCR-β chain, the second self-cleaving peptide, the variable region of the TCR-α chain, and the N-terminal portion of the endogenous TCR-α subunit, in that order, is under the control of the endogenous TCR-α promoter and TCR-α regulatory elements. Once the construct is integrated into the genome of the T cell and is under the control of the endogenous TCR-α promoter, the T cell can be cultured under conditions that allow the transcription of a single mRNA sequence encoding the fusion polypeptide from the inserted construct. The fusion polypeptide comprises the first self-cleaving peptide, the heterologous full-length TCR-β chain, the second self-cleaving peptide, the heterologous full-length TCR-α chain, and the N-terminal portion of the endogenous TCR-α subunit, in that order.

[0060] Similarly, when inserted into TRBC1 or TRBC2, a construct encoding, in that order, a first self-cleaving peptide, a heterologous full-length TCR-α chain, a second self-cleaving peptide, a variable region of a TCR-β chain, and a portion of the N-terminus of an endogenous TCR-β subunit is under the control of an endogenous TCR-β promoter and TCR-β regulatory elements. Once the construct is integrated into the genome of a T cell and placed under the control of an endogenous TCR-β promoter, the T cell can be cultured under conditions that allow transcription of a single mRNA sequence encoding a fusion polypeptide from the inserted construct. The fusion polypeptide comprises, in that order, a first self-cleaving peptide, a heterologous full-length TCR-α chain, a second self-cleaving peptide, a heterologous full-length TCR-β chain, and a portion of the N-terminus of an endogenous TCR-β subunit.

[0061] By inserting the construct into exon 1 of the TRAC gene, the remaining exons (exons 2 and 3) of the TRAC gene are spliced ​​together with exon 1 into the final mRNA sequence. Translation of this mRNA sequence results in the expression of one protein that is self-cleaved into three separate polypeptide sequences: an inactive endogenous variable region peptide lacking a transmembrane domain (which can be degraded in the endoplasmic reticulum or secreted post-translationally), a full-length heterologous antigen-specific TCR-β chain, and a full-length heterologous antigen-specific TCR-α chain (see FIG. 1a). The full-length antigen-specific TCR-β chain and the full-length antigen-specific TCR-α chain form a TCR with the desired antigen specificity.

[0062] Similarly, by inserting the construct into exon 1 of the TRBC1 / 2 gene, the remaining exons (exons 2-4) of the TRBC1 / 2 gene are spliced ​​together with exon 1 into the final mRNA sequence. Translation of this mRNA sequence results in the expression of one protein that is self-cleaved into three separate polypeptide sequences: an inactive endogenous variable region peptide lacking a transmembrane domain (which can be degraded in the endoplasmic reticulum or secreted post-translationally), a full-length heterologous antigen-specific TCR-β chain, and a full-length heterologous antigen-specific TCR-α chain. The full-length antigen-specific TCR-β chain and the full-length antigen-specific TCR-α chain form a TCR with the desired antigen specificity.

[0063] Alternatively, a heterologous TCR-α chain coding sequence, e.g., a sequence encoding the variable region of the TCR-α chain, and a heterologous TCR-β chain coding sequence, e.g., a sequence encoding the variable region of the TCR-β chain, are inserted into the genome of a T cell, where the heterologous TCR-α chain is inserted into exon 1 of the TRAC gene and the heterologous TCR-β chain is inserted into exon 1 of the TRBC gene. In some embodiments, a first nucleic acid sequence or construct encoding a heterologous TCR-α chain and a second nucleic acid sequence or construct encoding a heterologous TCR-β chain are used to insert the heterologous TCR-α chain into exon 1 of the TRAC gene and the heterologous TCR-β chain into exon 1 of the TRBC gene, respectively.

[0064] In the method using the first and second nucleic acid constructs, the target region for insertion of the nucleic acid encoding the heterologous TCR-α chain is in exon 1 of the TRAC gene. In some embodiments, the target region for insertion of the nucleic acid encoding the heterologous TCR-β chain is downstream of the endogenous TRBC1 or TRBC2 promoter and located in exon 1 of the TRBC1 or TRBC2 gene. The first nucleic acid sequentially encodes, from N-terminus to C-terminus, a first self-cleaving peptide, followed by a heterologous TCR-α chain, followed by a portion of the N-terminus of the endogenous TCR-α subunit. The second nucleic acid construct sequentially encodes, from N-terminus to C-terminus, a second self-cleaving peptide, followed by a nucleic acid encoding the heterologous TCR-β chain, followed by a portion of the N-terminus of the endogenous TCR-β subunit. Exemplary constructs include those described in FIG. 2.

[0065] When inserted, the first construct, encoding the first self-cleaving peptide and the heterologous TCR-α chain in that order, is under the control of the endogenous TCR-α promoter and TCR-α regulatory elements. The second construct, encoding the second self-cleaving peptide and the heterologous TCR-β chain in that order, is under the control of the endogenous TCR-β promoter and TCR-β regulatory elements. Once the constructs are integrated into the genome of the T cell and are under the control of the endogenous TCR-α and TCR-β promoters, the T cell is cultured under conditions that allow transcription of separate mRNA sequences from the first and second constructs. By inserting the first construct into exon 1 of the TRAC gene, the remaining exons (exons 2 and 3) of the TRAC gene are spliced ​​together with exon 1 into the final mRNA sequence encoding the full-length heterologous TCR-α chain. Similarly, by inserting the second construct into exon 1 of the TRBC gene, the remaining exons of the TRBC gene (exons 2 and 3) are spliced ​​together with exon 1 into an mRNA sequence encoding a full-length heterologous TCR-β chain.

[0066] Translation of the mRNA sequence encoding the first self-cleaving peptide and the full-length xenogeneic TCR-α chain results in the expression of an inactive endogenous variable region peptide lacking a transmembrane domain (which can be degraded, for example, in the endoplasmic reticulum or secreted after translation) and a full-length xenogeneic antigen-specific TCR-α chain. Translation of the mRNA sequence encoding the second self-cleaving peptide and the full-length xenogeneic TCR-β chain results in the expression of an inactive endogenous variable region peptide lacking a transmembrane domain (which can be degraded, for example, in the endoplasmic reticulum or secreted after translation) and a full-length xenogeneic antigen-specific TCR-β chain. The full-length xenogeneic antigen-specific TCR-β chain and the full-length xenogeneic antigen-specific TCR-α chain form a TCR with the desired antigen specificity.

[0067] In the methods provided herein, the variable region of the heterologous TCR-β chain comprises variable (V), diversity (D), and joining (J) alleles. In the methods provided herein, the variable region of the heterologous TCR-α chain comprises V and J alleles. See, e.g., Kuby, J., Immunology, 7 th Ed., WH Freeman & Co., New York (2013).

[0068] In some embodiments, the nucleic acid sequence is inserted into the genome of T cell by introducing a vector, such as a viral vector, that contains the nucleic acid.Examples of viral vectors include, but are not limited to, adeno-associated virus (AAV) vector, retrovirus vector or lentivirus vector.In some embodiments, the lentivirus vector is an integrase-deficient lentivirus vector.

[0069] In some embodiments, the nucleic acid sequence is inserted into the genome of the T cell via non-viral delivery. In non-viral delivery methods, the nucleic acid may be naked DNA or in a non-viral plasmid or vector.

[0070] In some embodiments, the nucleic acid is inserted into the T cell by introducing into the T cell (a) a targeting nuclease that cleaves a target region in exon 1 of the TRAC gene to create an insertion site in the genome of the T cell; and (b) a nucleic acid sequence that is integrated into the insertion site by HDR. In some embodiments, the nucleic acid is inserted into the T cell by introducing into the T cell (a) a targeting nuclease that cleaves a target region in exon 1 of the TRBC gene to create an insertion site in the genome of the T cell; and (b) a nucleic acid sequence that is integrated into the insertion site by HDR.

[0071] In some embodiments, the method further comprises introducing into the cell a guide RNA that specifically hybridizes to a target region of exon 1 of the TRAC gene. In some embodiments, the method further comprises introducing into the cell a guide RNA that specifically hybridizes to a target region of exon 1 of the TRBC gene.

[0072] In embodiments in which the first and second nucleic acid sequences are used to insert the heterologous TCR-α chain and the heterologous TCR-β chain into exon 1 of the TRAC gene and exon 1 of the TRBC gene, respectively, the first and second nucleic acids are inserted into the T cell by introducing into the T cell: (a) one or more targeting nucleases that create a first insertion site in exon 1 of the TRAC gene and a second insertion site in exon 1 of the TRBC gene; (b) the first nucleic acid sequence; and (c) the second nucleic acid sequence. In some embodiments, the method further comprises introducing into the cell a guide RNA that specifically hybridizes to a target region in exon 1 of the TRAC gene and a guide RNA that specifically hybridizes to a target region in exon 1 of the TRBC gene.

[0073] In some embodiments, the 5' and 3' ends of the nucleic acid sequence each comprise a nucleotide sequence that is homologous to a genomic sequence adjacent to a target region in the genome of the T cell, for example, a target region in exon 1 of the TRAC gene or a target region in exon 1 of the TRBC gene. In some cases, the nucleotide sequence that is homologous to the genomic sequence is about 50-300 nucleotides in length. In some cases, the nucleotide sequence that is homologous to the genomic sequence or a portion thereof is at least 80%, 90%, 95% complementary to the genomic sequence. In some embodiments, the 5' and 3' ends of the nucleic acid sequence comprise a nucleotide sequence that is homologous to a genomic sequence at an insertion site in exon 1 of the TRAC gene. In some embodiments, the 5' and 3' ends of the nucleic acid sequence comprise a nucleotide sequence that is homologous to a genomic sequence adjacent to an insertion site in exon 1 of the TRBC gene.

[0074] In some cases, the nucleic acid sequence is introduced into the cell as a linear DNA template. In some cases, the nucleic acid sequence is introduced into the cell as a double-stranded DNA template. In some cases, the DNA template is a single-stranded DNA template. In some cases, the single-stranded DNA template is a pure single-stranded DNA template. As used herein, "pure single-stranded DNA" refers to single-stranded DNA that is substantially devoid of the other or opposite strand of DNA. By "substantially devoid," it is meant that the pure single-stranded DNA is at least 100 times more devoid of one strand than the other strand of DNA. In some cases, the DNA template is a double-stranded or single-stranded plasmid or minicircle.

[0075] In some embodiments, the targeting nuclease is selected from the group consisting of an RNA-guided nuclease domain, a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), and a megaTAL (see, e.g., Merkert and Martin, "Site-Specific Genome Engineering in Human Pluripotent Stem Cells," Int. J. Mol. Sci. 18(7): 1000 (2016)). In some embodiments, the RNA-guided nuclease is a Cas9 nuclease, and the method further comprises introducing into the cell a guide RNA that specifically hybridizes to a target region in the genome of the T cell, for example, a target region in exon 1 of the TRAC gene. In other embodiments, the RNA-guided nuclease is a Cas9 nuclease, and the method further comprises introducing into the cell a guide RNA that specifically hybridizes to a target region in exon 1 of the TRBC gene.

[0076] Guide RNA (gRNA) sequences, as used throughout, are sequences that interact with a site-specific or targeting nuclease and specifically bind or hybridize to a target nucleic acid in the genome of a cell, such that the gRNA and the targeting nuclease co-localize with the target nucleic acid in the genome of the cell. Each gRNA contains a DNA targeting sequence or protospacer sequence, approximately 10-50 nucleotides in length, that specifically binds or hybridizes to a target DNA sequence in the genome. For example, the DNA targeting sequence is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the gRNA comprises a crRNA sequence and a transactivating crRNA (tracrRNA) sequence. In some embodiments, the gRNA does not comprise a tracrRNA sequence.

[0077] Generally, the DNA targeting sequence is designed to be complementary (e.g., fully complementary) or substantially complementary to the target DNA sequence. In some cases, the DNA targeting sequence can incorporate wobble or degenerate bases to allow for multiple genetic elements to be attached. In some cases, the 19 nucleotides at the 3' or 5' end of the binding region are fully complementary to the targeted genetic element. In some cases, the binding region can be modified to increase stability. For example, non-natural nucleotides can be incorporated to increase RNA resistance to degradation. In some cases, the binding region can be modified or designed to avoid or reduce the formation of secondary structures in the binding region. In some cases, the binding region can be designed to optimize GC content. In some cases, the GC content is preferably about 40% to about 60% (e.g., 40%, 45%, 50%, 55%, 60%). In some embodiments, the Cas9 protein can be in an active endonuclease form, such that upon binding to the target nucleic acid as part of a complex with a guide RNA or as part of a complex with a DNA template, a double-stranded break is introduced into the target nucleic acid. In the methods provided herein, a Cas9 polypeptide or a nucleic acid encoding a Cas9 polypeptide can be introduced into a T cell. The double-stranded break can be repaired by HDR to insert a DNA template into the genome of the T cell. Various Cas9 nucleases can be utilized in the methods described herein. For example, a Cas9 nuclease can be utilized that requires a NGG protospacer adjacent motif (PAM) immediately 3' to the region targeted by the guide RNA. Such a Cas9 nuclease can target a region in exon 1 of TRAC or exon 1 of TRBC that contains a NGG sequence. As another example, a Cas9 protein with an orthogonal PAM motif requirement can be used to target a sequence that does not have an adjacent NGG PAM sequence. Exemplary Cas9 proteins with orthogonal PAM sequence specificity include, but are not limited to, those described in Esvelt et al., Nature Methods 10: 1116-1121 (2013).

[0078] In some cases, the Cas9 protein is a nickase, and thus when bound to a target nucleic acid as part of a complex with a guide RNA, a single-strand break or nick is introduced into the target nucleic acid. A pair of Cas9 nickases, each bound to a structurally distinct guide RNA, can target two proximal sites in a target genomic region, thus introducing a pair of proximal single-strand breaks in the target genomic region, for example, exon 1 of the TRAC gene or exon 1 of the TRBC gene. A nickase pair can provide enhanced specificity, since off-target effects are more likely to result in a single nick that is generally repaired without damage by base excision repair mechanisms. Exemplary Cas9 nickases include Cas9 nucleases with D10A or H840A mutations (see, e.g., Ran et al. "Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity", Cell 154(6): 1380-1389 (2013)).

[0079] In some embodiments, the Cas9 nuclease, the guide RNA, and the nucleic acid sequence are introduced into the cell as a ribonucleoprotein complex (RNP)-DNA template complex, where the RNP-DNA template complex comprises (i) an RNP comprising the Cas9 nuclease and the guide RNA; and (ii) a DNA template encoding a heterologous TCR-β chain and a heterologous TCR-α chain that is inserted into exon 1 of the TRAC gene by HDR. In some embodiments, the Cas9 nuclease, the guide RNA, and the nucleic acid sequence are introduced into the cell as a ribonucleoprotein complex (RNP)-DNA template complex, where the RNP-DNA template complex comprises (i) an RNP comprising the Cas9 nuclease and the guide RNA; and (ii) a DNA template encoding a heterologous TCR-β chain and a heterologous TCR-α chain that is inserted into exon 1 of the TRAC gene by HDR.

[0080] In some embodiments, when separate constructs are used to insert a heterologous TCR-α chain into exon 1 of the TRAC gene in a T cell and a heterologous TCR-β chain into exon 1 of the TRBC gene in a T cell, (a) a first (RNP)-DNA template complex comprising (i) a Cas9 nuclease and a first guide RNA that specifically hybridizes to a target region in exon 1 of the TRAC gene; and (ii) a DNA template encoding a heterologous TCR α subunit; and (b) a second (RNP)-DNA template complex comprising (i) a Cas9 nuclease and a second guide RNA that specifically hybridizes to a target region in exon 1 of the TRBC gene; and (ii) a DNA template encoding a heterologous TCR β subunit are introduced into the cell. In some embodiments, the Cas9 nuclease in the first RNP-DNA template complex and the Cas9 nuclease in the second RNP-DNA template complex are the same. In some embodiments, the Cas9 nuclease in the first RNP-DNA template complex and the Cas9 nuclease in the second RNP-DNA template complex are different.

[0081] In some embodiments, the molar ratio of RNP to DNA template can be about 3:1 to about 100:1. For example, the molar ratio can be about 5:1 to 10:1, about 5:1 to about 15:1, 5:1 to about 20:1, 5:1 to about 25:1, about 8:1 to about 12:1, about 8:1 to about 15:1, about 8:1 to about 20:1, or about 8:1 to about 25:1.

[0082] In some embodiments, the DNA template in the RNP-DNA template complex is at a concentration of about 2.5 pM to about 25 pM. In some embodiments, the amount of DNA template is about 1 μg to about 10 μg.

[0083] In some cases, the RNP-DNA template complex is formed by incubating the RNP with the DNA template for less than about 1 minute to about 30 minutes at a temperature of about 20° C. to about 25° C. In some embodiments, the RNP-DNA template complex and the cells are mixed prior to introducing the RNP-DNA template complex into the cells.

[0084] In some embodiments, the nucleic acid sequence or the RNP-DNA template complex is introduced into the T cell by electroporation. Methods, compositions, and devices for electroporating cells to introduce the RNP-DNA template complex can include those described in the Examples herein. Additional or alternative methods, compositions, and devices for electroporating cells to introduce the RNP-DNA template complex can include those described in WO / 2006 / 001614 or Kim, JA et al. Biosens. Bioelectron. 23, 1353-1360 (2008). Additional or alternative methods, compositions, and devices for electroporating cells to introduce the RNP-DNA template complex can include those described in U.S. Patent Application Publication Nos. 2006 / 0094095; 2005 / 0064596; or 2006 / 0087522. Additional or alternative methods, compositions, and devices for electroporating cells to introduce RNP-DNA template complexes can include those described in Li, LH et al. Cancer Res. Treat. 1, 341-350 (2002); U.S. Patent Nos. 6,773,669; 7,186,559; 7,771,984; 7,991,559; 6485961; 7029916; and U.S. Patent Application Publication Nos. 2014 / 0017213; and 2012 / 0088842. Additional or alternative methods, compositions, and devices for electroporating cells to introduce RNP-DNA template complexes can include those described in Geng, T. et al. J. Control Release 144, 91-100 (2010); and Wang, J., et al. Lab. Chip 10, 2057-2061 (2010).

[0085] In some embodiments, a plurality of RNP-DNA template complexes, comprising at least two structurally different RNP-DNA template complexes, are introduced into cells.The term "multiple" used throughout means two or more.In some embodiments, at least two structurally different RNP-DNA template complexes comprise structurally different guide RNAs.In some embodiments, where at least two structurally different RNP complexes comprise structurally different guide RNAs, each of the structurally different RNP complexes comprises Cas9 nickase, and the structurally different guide RNAs hybridize to opposite strands of target region.

[0086] In some embodiments, at least two structurally different nucleic acids encoding the variable region of a heterologous TCR-β chain and the variable region of a heterologous TCR-α chain are introduced into exon 1 of the TRAC gene in a population of T cells.In this way, multiple nucleic acid sequences can be introduced into a population of T cells, each nucleic acid sequence encoding a unique combination of a heterologous TCR-β chain and a heterologous TCR-α chain.Each of the nucleic acid sequences can be introduced into the cells as part of an RNP-DNA template complex.

[0087] For example, the targeted nuclease can be complexed with multiple (e.g., 2, 3, 4, 5 or more, e.g., 2-10, 5-100, 20-100) unique DNA templates encoding the variable regions of a heterologous TCR-β chain and the variable regions of a heterologous TCR-α chain. Multiple complexes can be introduced simultaneously into a population of T cells to insert a single DNA template encoding a unique combination of the variable regions of a heterologous TCR-β chain and the variable regions of a heterologous TCR-α chain into exon 1 of the TRAC gene in individual T cells. Each T cell will only acquire a single DNA template, but across the population of cells, many unique, structurally distinct DNA templates will be incorporated into the T cells, creating a library of heterologous TCR sequences.

[0088] In some embodiments where separate constructs are used to insert a heterologous TCR-α chain into exon 1 of the TRAC gene and a heterologous TCR-β chain into exon 1 of the TRBC gene, a first targeting nuclease can be complexed with a plurality of unique DNA templates encoding the variable regions of the heterologous TCR-β chain to form a plurality of first RNP-DNA template complexes, and a second targeting nuclease can be complexed with a plurality of unique DNA templates encoding the variable regions of the heterologous TCR-α chain to form a plurality of second RNP-DNA template complexes. The first and second plurality of complexes can be introduced simultaneously into a population of T cells to create a library of heterologous TCR sequences.

[0089] Heterologous T cell repertoires can be designed and created from known TCR sequences, as well as from natural repertoires found in the endogenous T cell population of interest. For example, the TCR sequences can be TCR sequences obtained from tumor-infiltrating lymphocytes, from autoreactive T cells at the site of autoimmune disease, or from pathogen-responsive T cells.

[0090] In some embodiments, the nucleic acid sequence or RNP-DNA template complex is about 1×10 5 ~about 2×10 6 For example, the nucleic acid sequence or RNP-DNA template complex is introduced into about 1×10 T cells. 5 ~ approx. 5 x 10 cells 5 cells, approximately 1 x 10 5 ~ approx. 1 x 10 cells 6 cells, 1 x 10 5 ~ approx. 1.5 x 10 cells 6 cells, 1 x 10 5 ~ approx. 2 x 10 cells 6 cells, approximately 1 x 10 6 ~ approx. 1.5 x 10 cells 6 cells, or approximately 1 x 10 6 ~ approx. 2 x 10 cells 6 It can be introduced into individual cells.

[0091] In the methods and compositions provided herein, the human T cells can be primary T cells. In some embodiments, the T cells are regulatory T cells, effector T cells, or naive T cells. In some embodiments, the effector T cells are CD8 + In some embodiments, the T cells are CD4 + CD8 + The T cell.Also provided is any population of cells modified by any of the methods described herein.The cell can be in vitro, ex vivo, or in vivo.In some cases, the T cell is taken out of the subject, modified by any of the methods described herein, and administered to the patient.

[0092] In some embodiments, the modified T cells are cultured under conditions that allow expression of a heterologous TCR-β chain and a heterologous TCR-α chain to form a heterologous antigen-specific T cell receptor. In other embodiments, the T cells are cultured under conditions effective to expand the population of modified cells. In some embodiments, the T cells expressing the antigen-specific T cell receptor are purified.

[0093] composition Also provided are human T cells produced by any of the methods provided herein. Also provided are populations of human T cells produced by any of the methods provided herein. Further provided are a plurality of human T cells, in which at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more of the genome of the cells comprises a targeted insertion of a heterologous nucleic acid inserted into exon 1 of TRAC or exon 1 of TRBC. In some embodiments, the T cells are regulatory T cells, effector T cells, or naive T cells. In some embodiments, the effector T cells are CD8 + In some embodiments, effector T cells are CD4 + CD8 + T cells.

[0094] Also provided is a T cell comprising a nucleic acid sequence that encodes, from N-terminus to C-terminus, (i) a first self-cleaving peptide sequence; (ii) a first heterologous TCR subunit chain comprising a constant region and a variable region of the TCR subunit chain; (iii) a second self-cleaving peptide sequence; (iv) a variable region of a second heterologous TCR subunit chain; and (v) a portion of the N-terminus of an endogenous TCR subunit, wherein if the endogenous TCR subunit is a TCR-alpha (TCR-α) subunit, the first heterologous TCR subunit chain is a heterologous TCR-beta (TCR-β) subunit chain and the second heterologous TCR subunit chain is a heterologous TCR-α subunit chain, and wherein if the endogenous TCR subunit is a TCR-β subunit, the first heterologous TCR subunit chain is a heterologous TCR-α subunit chain and the second heterologous TCR subunit chain is a heterologous TCR-β subunit chain. In some embodiments, the nucleic acid sequence encodes, from N-terminus to C-terminus, (i) a first self-cleaving peptide sequence, (ii) a heterologous TCR-β subunit chain, (iii) a second self-cleaving peptide sequence, (iv) a variable region of the heterologous TCR-α subunit chain, and (v) a portion of the N-terminus of the endogenous TCR-α subunit chain. In some examples, the nucleic acid encodes, from N-terminus to C-terminus, (i) a first self-cleaving peptide sequence, (ii) a heterologous TCR-α subunit chain, (iii) a second self-cleaving peptide sequence, (iv) a variable region of the heterologous TCR-β subunit chain, and (v) a portion of the N-terminus of the endogenous TCR-β subunit.

[0095] Also provided are modified T cells comprising a nucleic acid sequence that encodes, from N-terminus to C-terminus, (i) a first self-cleaving peptide sequence; (ii) a variable region of a heterologous TCR-β chain; (iii) a second self-cleaving peptide sequence; (iv) a variable region of a heterologous TCR-α chain, and (v) a portion of the N-terminus of an endogenous TCR-α subunit, wherein the nucleic acid sequence is integrated into exon 1 of the TRAC gene. In some examples, the nucleic acid encodes, from N-terminus to C-terminus, the first self-cleaving peptide sequence, the heterologous (e.g., full-length) TCR-β subunit chain, the second self-cleaving peptide sequence, and the variable region of the heterologous TCR-α subunit chain.

[0096] Also provided are modified T cells comprising a nucleic acid sequence encoding, from N-terminus to C-terminus, (i) a first self-cleaving peptide sequence; (ii) a variable region of a heterologous TCR-α chain; (iii) a second self-cleaving peptide sequence; (iv) a variable region of a heterologous TCR-β chain, and (v) a portion of the N-terminus of an endogenous TCR-β subunit, wherein the nucleic acid sequence is integrated into exon 1 of the TRBC gene. In other examples, the nucleic acid encodes, from N-terminus to C-terminus, the first self-cleaving peptide sequence, the heterologous (e.g., full-length) TCR-α subunit chain, the second self-cleaving peptide sequence, and the variable region of the heterologous TCR-β subunit chain.

[0097] Also provided is a modified T cell comprising a nucleic acid sequence encoding, from N-terminus to C-terminus, (i) a first self-cleaving peptide sequence; (ii) a variable region of a heterologous TCR-β chain; (iii) a second self-cleaving peptide sequence; (iv) a variable region of a heterologous TCR-α chain, and (v) an N-terminal portion of an endogenous TCR-γ subunit, wherein the nucleic acid sequence is integrated into exon 1 of the TRGC gene.

[0098] Further provided is a modified T cell comprising: (a) a first nucleic acid sequence encoding, from N-terminus to C-terminus, (i) a first self-cleaving sequence; (ii) a variable region of a heterologous TCR-α chain, and (iii) an N-terminal portion of an endogenous TCR-α chain, integrated into exon 1 of the TRAC gene; and (b) a second nucleic acid sequence encoding, from N-terminus to C-terminus, (i) a first self-cleaving sequence; (ii) a variable region of a heterologous TCR-β chain, and (iii) an N-terminal portion of an endogenous TCR-β chain, integrated into exon 1 of the TRAC gene.

[0099] Treatment method Any of the methods and compositions described herein can be used to treat or prevent disease (e.g., cancer, infectious disease, autoimmune disease, transplant rejection, graft-versus-host disease, or other inflammatory disorder in a subject). In the therapeutic methods provided herein, for example, the nucleic acid sequence comprising the TCR-α or TCR-β chain of the TCR in a subject with cancer, infectious disease, autoimmune disease, possible transplant rejection, graft-versus-host disease, or other inflammatory disorder can be obtained from T cells in the subject. For example, T cells from tumor-infiltrating lymphocytes, T cells from autoimmune sites, or pathogen-responsive lymphocytes can be isolated from the subject to obtain the nucleic acid sequence comprising the TCR-α or TCR-β chain of the TCR. In some embodiments, monoclonal or polyclonal TCR sequences identified from patient samples can be used. For example, TCR repertoires from tumors or inflammatory sites can be obtained, and cells with these antigen specificities can be created by synthesizing TCR sequences and using them as DNA templates. Alternatively, sequences can be amplified, for example, PCR amplified, from clones / single cells from a subject, and the amplified sequences can be used as DNA templates.Once the sequences of the TCR-α and TCR-β chains of TCR are obtained, these sequences can be inserted into a population of T cells in or from a subject, replacing the endogenous TCR of T cells in the subject with a heterologous TCR of desired antigen specificity.In some embodiments, the population of modified T cells can be administered to a subject to treat disease.See, for example, Figure 3B.

[0100] Provided herein are methods of treating cancer in a human subject, comprising modifying the subject's T cells using any of the methods described herein to express an antigen-specific T cell receptor that recognizes a tumor-specific antigen in the subject.

[0101] In some embodiments, the cancer to be treated is selected from cancer of B-cell origin, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, colon cancer, chronic myeloid cancer, leukemia (e.g., acute myeloid leukemia, chronic lymphocytic leukemia (CLL) or acute lymphocytic leukemia (ALL)), prostate cancer, colon cancer, renal cell carcinoma, liver cancer, kidney cancer, ovarian cancer, gastric cancer, testicular cancer, rhabdomyosarcoma, and Hodgkin's lymphoma. In some embodiments, the cancer of B-cell origin is selected from the group consisting of B-lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, and B-cell non-Hodgkin's lymphoma.

[0102] In some embodiments of the methods of treatment described herein, such as cancer, infectious disease, or autoimmune disorder, T cells are modified in vivo. Any of the constructs described herein are delivered to patients in vivo. See, for example, U.S. Patent No. 9,737,604 and Zhang et al. "Lipid nanoparticle-mediated efficient delivery of CRISPR / Cas9 for tumor therapy", NPG Asia Materials Volume 9, page e441 (2017).

[0103] In some embodiments, the method of treating cancer in a human subject includes: (a) obtaining T cells from the subject; (b) modifying the T cells using any of the methods provided herein to express a heterologous antigen-specific T cell receptor that recognizes a tumor-specific antigen in the subject; and (c) administering the modified T cells to the subject. The phrase "tumor-specific antigen" as used throughout refers to an antigen that is unique to cancer cells or is more abundantly expressed in cancer cells than in non-cancer cells.

[0104] Further provided herein is a method of treating an autoimmune disease in a human subject, comprising modifying the subject's T cells, using any of the methods described herein, to express a heterologous antigen-specific T cell receptor that recognizes an antigen associated with the autoimmune disorder.

[0105] In some embodiments, a method for treating an autoimmune disease in a human subject includes (a) obtaining T cells from the subject; (b) modifying the T cells using any of the methods provided herein to express a heterologous antigen-specific T cell receptor that recognizes an antigen associated with an autoimmune disorder in the subject; and (c) administering the modified T cells to the subject. In some embodiments, the T cells are regulatory T cells.

[0106] Further provided herein is a method of treating an infection in a human subject comprising modifying the subject's T cells using any of the methods described herein to express a heterologous, antigen-specific T cell receptor, where the T cell receptor recognizes an antigen associated with the infection in the subject.

[0107] In some embodiments, a method of treating an infection in a human subject includes: (a) obtaining T cells from the subject; (b) modifying the T cells using any of the methods provided herein to express a heterologous antigen-specific T cell receptor that recognizes an antigen associated with the infection in the subject; and (c) administering the modified T cells to the subject.

[0108] Any of the therapeutic methods provided herein can further include a step of expanding the population of T cells before the endogenous TCR is replaced with the heterologous TCR. Any of the therapeutic methods provided herein can further include a step of expanding the population of T cells after the endogenous TCR is replaced with the heterologous TCR and prior to administration to the subject.

[0109] Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions.When these and other materials are disclosed herein, and when combinations, subsets, interactions, groups, etc. of these materials are disclosed, it should be understood that each of the various individual and group combinations and permutations of these compounds is specifically contemplated and described herein, even if they are not expressly disclosed.For example, when a method is disclosed and discussed, and several modifications that can be made to one or more molecules, including in the method, any and all combinations and permutations of the method, and possible modifications, are specifically contemplated unless otherwise indicated to the contrary.Similarly, any subset or combination of these is specifically contemplated and disclosed.This concept applies to all aspects of the present disclosure, including but not limited to the steps in the method of using the disclosed compositions. Thus, if there are various additional steps that can be performed, it is to be understood that each of these additional steps can be performed at any particular method step or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered as disclosed.

[0110] Publications cited herein and the material for which they are cited are specifically incorporated herein by reference in their entireties. EXAMPLES

[0111] The following examples are offered by way of illustration only and not by way of limitation. Those of ordinary skill in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially the same or similar results.

[0112] Isolation of human primary T cells for gene targeting Primary human T cells were isolated from healthy human donors, either from fresh whole blood samples, residues from leukapheresis chambers after Trima Apheresis (Blood Centers of the Pacific) or leukapheresis transfusion products (StemCell). Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood samples by Ficoll centrifugation using SepMate tubes (STEMCELL, according to manufacturer's instructions). T cells were isolated from whole cell source PBMCs by magnetic negative selection using the EasySep Human T Cell Isolation Kit (STEMCELL, according to manufacturer's instructions). Unless otherwise stated, isolated T cells were stimulated and used directly (fresh). When frozen cells were used, previously isolated T cells that had been frozen in Bambanker freezing medium (Bulldog Bio) according to manufacturer's instructions were thawed and cultured for 1 day in unstimulated medium before stimulation and treatment as described for freshly isolated samples. Fresh healthy human blood donors were consented under a protocol approved by the UCSF Committee on Human Research (CHR). Patient samples for gene editing were obtained under a protocol approved by the Yale Internal Review Board (IRB).

[0113] Primary T cell culture Unless otherwise stated, bulk T cells were cultured in XVivo™ 15 medium (STEMCELL) with 5% fetal bovine serum, 50 mM 2-mercaptoethanol, and 10 mM N-acetyl L-cystine. Serum-free medium without supplements (ImmunoCult XF T cell expansion medium, STEMCELL) and RPMI + 10% FBS were used in the indicated experiments (Figure 15). Immediately after isolation, T cells were stimulated with anti-human CD3 / CD28 magnetic Dynabeads (ThermoFisher) at a bead-to-cell concentration of 1:1 with a cytokine cocktail of 200 U / mL IL-2 (UCSF Pharmacy), 5 ng / mL IL-7 (ThermoFisher), and 5 ng / mL IL-15 (Life Tech) for 2 days. Following electroporation, T cells were cultured in medium with 500 U / mL IL-2. Throughout the culture, T cells were maintained at a density of approximately 1 million cells per mL of medium. Every 2–3 days after electroporation, additional medium was added along with additional fresh IL-2 to a final concentration of 500 U / mL, and cells were transferred to larger culture vessels as needed to maintain a density of 1 million cells / mL.

[0114] RNP production RNPs were generated by annealing two-component gRNA to Cas9 as previously described (Schumann et al. PNAS 112: 10437-10442 (2015); and Hultquist et al. Cell Rep. 17: 1438-1452 (2016)). Briefly, crRNAs and tracrRNA were chemically synthesized (Dharmacon, IDT), and recombinant Cas9-NLS, D10A-NLS, or dCas9-NLS were recombinantly produced and purified (QB3 Macrolab). Lyophilized RNA was resuspended in Tris-HCL (pH 7.4) with 150 mM KCl at a concentration of 160 μM and stored in aliquots at -80°C. crRNA and tracrRNA aliquots were thawed, mixed 1:1 by volume, and incubated at 37° C. for 30 min to form an 80 μM gRNA solution. Recombinant Cas9 and variants, stored at 40 uM in 20 mM HEPES-KOH pH 7.5, 150 mM KCl, 10% glycerol, 1 mM DTT, were then mixed 1:1 by volume (2:1 gRNA to Cas9 molar ratio) with 80 μM gRNA for 15 min at 37° C. to form 20 μM RNPs. RNPs were typically electroporated immediately after complexation.

[0115] dsDNA HDRT production Double-stranded DNA HDRT sequences were generated from PCR products. Novel HDR sequences were constructed using Gibson Assemblies, and HDR template sequences consisting of homology arms (usually synthesized as gBlocks from IDT) and the desired insert (such as GFP) were placed into a cloning vector for sequence verification and subsequent propagation. These plasmids were used as templates for high-throughput PCR amplification (Kapa Hotstart polymerase). PCR amplicons (dsDNA HDRT) were SPRI purified (1.0x) and eluted in a final volume of 3 μL H2O per 100 μL PCR reaction input. The concentration of HDRT was analyzed by nanodrop at a 1:20 dilution. The size of the amplified HDRT was confirmed by gel electrophoresis on a 1.0% agarose gel.

[0116] ssDNA HDRT production by exonuclease digestion To generate long ssDNA as HDR donor, DNA of interest was amplified by PCR using one normal unmodified PCR primer and another phosphorylated PCR primer. The DNA strand amplified using the phosphorylated primer will be the strand degraded using this method. This allows for the preparation of single-stranded sense DNA or single-stranded antisense DNA using each phosphorylated PCR primer. To generate ssDNA strand of interest, the phosphorylated strand of PCR product was degraded by subsequent treatment with two enzymes Strandase Mix A and Strandase Mix B at 37°C for 5 min each (per kb). The enzymes were inactivated by incubation at 80°C for 5 min. The resulting ssDNA HDR template was SPRI purified (1.0x) and eluted in H2O. A more detailed protocol of Guide-it™ Long ssDNA Production System (Takara Bio USA, Inc. #632644) can be found on the manufacturer's website.

[0117] ssDNA HDRT Production by Retrosynthesis The ssDNA donors were synthesized by reverse transcription of an RNA intermediate followed by hydrolysis of the RNA strand in the resulting RNA:DNA hybrid product as described by Leonetti et al. (http: / / www.biorxiv.org / content / early / 2017 / 08 / 21 / 178905). Briefly, the desired HDR donor was first cloned downstream of a T7 promoter and the T7-HDR donor sequence was amplified by PCR. RNA was synthesized by in vitro transcription using HiScribe T7 RNA polymerase (New England Biolabs) and reverse transcribed using TGIRT-III (InGex). After reverse transcription, NaOH and EDTA were added to 0.2 M and 0.1 M, respectively, and RNA hydrolysis was carried out at 95°C for 10 min. The reaction was quenched with HCl and the final ssDNA product was purified using Ampure XP magnetic beads (Beckman Coulter) and eluted in sterile RNAse-free HO. The quality of ssDNA was analyzed by capillary electrophoresis (Bioanalyzer, Agilent).

[0118] Primary T cell electroporation RNP and HDR templates were electroporated 2 days after the first T cell stimulation. T cells were collected from their culture vessels and magnetic CD3 / CD28 Dynabeads were removed by placing the cells on a magnet for 2 min. Immediately prior to electroporation, bead-removed cells were centrifuged at 90 x g for 10 min, aspirated, and resuspended in Lonza electroporation buffer P3 at 20 µL of buffer per million cells. For optimal editing, 1 million T cells were electroporated per well using a Lonza 4D 96-well electroporation system with pulse code EH115. Alternative cell concentrations from 200,000 up to 2 million cells per well showed lower efficiency. Alternative electroporation buffers were used as indicated, but with different optimal pulse settings (EO155 for OMEM buffer). Unless otherwise indicated, 2.5 μL of RNP (50 pmol total) was electroporated with 2 μL of 2 μg / μL HDR template (4 μg HDR template total).

[0119] For 96-well experiments, HDRT was first aliquoted into the wells of a 96-well polypropylene V-bottom plate. RNP was then added to the HDRT and allowed to incubate together for at least 30 seconds at RT. Finally, cells resuspended in electroporation buffer were added, briefly mixed with HDRT and RNP by pipetting, and a total volume of 24 μL (cells + RNP + HDRT) was transferred to a 96-well electroporation cuvette plate. Immediately after electroporation, 80 μL of pre-warmed medium (without cytokines) was added to each well, and the cells were left in the electroporation cuvette for 15 minutes at 37°C in a cell culture incubator. After 15 minutes, the cells were transferred to the final culture vessel.

[0120] Non-viral replacement of endogenous T cell receptors with a single HDR template Both TCR-α and TCR-β chains can be knocked in simultaneously in a single multiple round editing (Figure 2). This is similar to the targeting strategy in Figures 1a and 1b, except that only the variable regions of the desired antigen-specific TCR are inserted in place of both the TCR-α and TCR-β constant loci. This has the advantage that the total size of both insertions is reduced (from one 1.5 kbp insertion to two 500 bp insertions), but it also means that any T cell expressing both chains of the desired antigen-specific TCR has both its endogenous TCR-α and TCR-β chains knocked out that were previously recombined, e.g., preventing potentially undesirable pairing of the inserted antigen-specific TCR-α chain with the endogenous TCR-β chain.

[0121] Replacement of endogenous TCRs with a polyclonal library of T cell receptors Because the gRNA and homology arms are the same for replacing the endogenous TCR with any desired new heterologous TCR sequence, multiple different DNA templates containing various desired TCRs can be electroporated simultaneously. While any particular T cell will only acquire a single TCR from the various desired TCR panels, many of the TCR DNA templates will be incorporated into the entire population of electroporated cells, thus creating a synthetic T cell repertoire of desired sequences (Figure 3a).

[0122] Synthetic T cell repertoires can be designed and created from known TCR sequences or from natural repertoires found in endogenous T cell populations of interest (Figure 3b). Some examples include TCRs expressed by tumor-infiltrating lymphocytes (inserted into new populations of CD8+ or CD4+ T cell effectors), TCRs expressed by autoreactive T cells at sites of autoimmune disease (inserted into populations of regulatory T cells), or TCRs from pathogen-responsive T cells (inserted into CD8+ or CD4+ T cell effectors).

[0123] Non-viral replacement of endogenous T cell receptors with a single HDR template The genomic locus of the T cell receptor is extremely complex, with a wide variety of variable alleles (called V and J alleles for the TCR-α chain and V, D, and J alleles for the TCR-β chain) that undergo somatic genetic rearrangement during T cell development to produce functional T cell receptors. Although important for the diversity of the TCR repertoire, these recombined sequences differ across the polyclonal population of T cells, making targeted genome editing at the TCR locus (whether knock-out or knock-in) difficult. For targeting applications for both the TCR-α and TCR-β chains, there is a constant domain at the C-terminus of the protein that is shared by all T cells, regardless of which VJ or VDJ segments are rearranged. As shown in Figure 1b, the TCR-β locus has two constant regions (TRBC1 and TRBC2) that can be targeted. This constant sequence (constant exons labeled TRAC or TRBC exon 1, exon 2, etc.) allows a single set of genomic targeting reagents (in this application the CRISPR / Cas9 system, i.e., a single gRNA sequence) to be used to modify any T cell, whatever the rearranged TCR they express.

[0124] Using the above method, a DNA template encoding a heterologous TCR that specifically binds to a tumor antigen was created and used to replace endogenous TCR in human T cells. The homology-directed repair template (dsDNA produced by PCR or ssDNA produced by various methods described herein) used to replace endogenous TCR is about 2.1 kb long, containing 5' and 3' homology arms (about 300 bp) that are homologous to genomic sequences adjacent to the gRNA cleavage site (Figure 1a). Between these homology arms is about 1.5 kb of sequence inserted into the gRNA cleavage site by homology-directed repair. This inserted sequence begins with a multicistronic element (T2A self-excision peptide) followed by the full-length sequence of the TCR-β chain of the desired antigen-specific T cell receptor (in this example, the TCR specific for NY-ESO-1 melanoma neoantigen). A second multicistronic element (P2A self-excising peptide) follows the TCR-β chain, separating it from the variable (recombined V and J allele) sequences of the desired antigen-specific TCR-α chain. The remaining TRAC exons were spliced ​​together into the final mRNA sequence, so that only the variable regions of the TCR-α chain and the sequence of TRAC exon 1 up to the gRNA cleavage site (5' end) were inserted. The DNA template was introduced into T cells as part of the RNP:DNA template complex described above.

[0125] Transcription in cells where HDR was successful resulted in a polycistronic mRNA encoding both TCR-β and TCR-α. This targeting strategy yielded three peptide chains - (1) the remaining endogenous variable region peptide that does not possess a transmembrane pathway (and therefore would not be expressed on the cell surface) and that was either degraded in the endoplasmic reticulum or secreted post-transcriptionally; (2) the full-length desired antigen-specific TCR-β chain; and (3) the full-length desired antigen-specific TCR-α chain. The result was a T cell expressing both chains of the desired antigen-specific TCR under the control of the endogenous TCR promoter. Both chains were expressed by the T cell to form a xenogeneic TCR that specifically recognized the NY-ESO-1 melanoma neoantigen.

[0126] As shown in Figure 4a, 4 days after electroporation of the above constructs, CD4+ and CD8+ T cells from two healthy human blood donors were stained with fluorescently labeled MHC-dextramers containing peptides recognized by the incorporated NY-ESO-1 specific TCR (NYESO). Figure 4b shows a second experiment showing similar results in another healthy human donor, thus demonstrating the robustness and reproducibility of non-viral endogenous TCR replacement.

[0127] TCR replacement was also achieved at the TCR-β locus with a similar strategy to targeting TCR-α, although the β locus is more complex as there are two constant regions (TRBC1 and TRBC2) that are highly homologous to each other. The HDR template inserted a new full-length TCR-α and a new TCR-β VDJ region at the 5' end of the first TRBC1 exon using a gRNA targeting a sequence found in both TRBC1 and TRBC2. Due to the sequence similarity between the genomic regions of TRBC1 and TRBC2, the 3' homology arm of this construct was almost perfectly homologous to the equivalent region in TRBC2, while the 5' homology arm had approximately 85% homology to the TRBC2 genomic region in the 150 bp closest to the insertion site. Thus, insertion is likely to be predominant at the TRBC1 locus, but it may also be possible for it to occur at the TRBC2 locus or if there is an intervening deletion between TRBC1 and TRBC2. Instead of a gRNA that targets both, a gRNA that cleaves specifically at the position of TRBC1 or TRBC2 can also be used.

[0128] Figure 9a shows TCR mispairing analysis after retroviral delivery or non-viral TCR replacement of NY-ESO-1 specific TCR in gated CD4+ or CD8+ T cells. Non-viral TCR replacement results in less TCR mispairing compared to retroviral TCR delivery. Multiple replacement of TCRs with new TCR-α VJ domains targeted to TRAC and new TRB-β VDJ domains targeted to TRBC1 was also possible, offering a strategy to further reduce TCR mispairing (Figure 9b-c).

[0129] TCR replacement with dextramer and TCR staining CD4+ and CD8+ T cells from two healthy human blood donors were electroporated with single constructs as described above. Four days after electroporation, staining for TCR expression (with an antibody that binds all potential human TCRs) was performed against antigen-specific staining with NYESO MHC-dextramer. In the majority of T cells lacking homology directed replacement of the endogenous TCR (Figure 5), the endogenous TCR is knocked out by truncation with TRAC exon 1 gRNA and introduction of a small indel by non-homologous end joining. As expected, almost all NYESO positive cells are also positive for TCR expression.

[0130] In vitro cancer cell killing by TCR knock-in primary human T cells In vitro cancer cell killing assays demonstrated the functional target cell killing ability of the TCR knock-in cells. As described above, the 1G4 (NYESO-specific) TCR sequence was inserted into the endogenous TCRα locus in CD3+ primary T cells from healthy human donors. Seven days after electroporation, cells were stained with fluorescent NYESO peptide-MHC dextramer and NYESO+ cells were selected by fluorescence-activated cell sorting to achieve a pure population. This population was further cultured for another 5 days in standard T cell culture conditions (medium and IL-2). Twelve days after electroporation, the sorted T cells, in which the endogenous TCR had been replaced by the 1G4 TCR, were co-cultured with a cancer cell line presenting the NYESO antigen on the MHC (A375 cells, derived from melanoma and endogenously expressing the NYESO antigen and the MHC allele MHC-A2 recognized by the 1G4 TCR). Every 12 hours, the number of cancer cells was counted by quantitative fluorescence imaging (A375 cells express red fluorescent protein and therefore can be clearly counted separately from T cells). Fluorescence counting was automated using an Incucyte automated fluorescence microscope. Cultures were seeded with 3,000 cancer cells and 1G4+ T cells at the indicated ratios (see Figure 4a). Robust dose- and time-dependent killing of targeted cancer cells was observed in T cells edited from two healthy human donors, indicating functionality of the 1G4 TCR knock-in T cells. The percentage of cancer cell killing was determined by dividing the number of viable cancer cells in a given well by the number of viable cancer cells in control wells in which no T cells were present. A high ratio of T cells to cancer cells (~10:1) showed rapid (<24 hours) killing of nearly all cancer cells, whereas even a very low ratio of T cells to cancer cells (~0.1:1) showed robust killing with additional culture time (see Figure 6a).

[0131] In vitro cancer killing by 1G4 TCR knock-in T cells at a single time point was demonstrated (see FIG. 6b). Summary data was obtained from a single time point (36 hours after co-incubation) in the cell killing assay described above. NYESO dextramer+ (1G4+) T cells were sorted as described above to generate a 100% 1G4 positive population, which was then diluted with NYESO dextramer- (1G4-) T cells from the same donor to obtain the indicated percentage of NYESO+ stained T cells (10%, 1%, or 0%). Each of these populations was then co-cultured with cancer cells in an in vitro cancer killing assay as described above at a ratio of 8 T cells to 1 cancer cell (yielding 1G4+ T cell:cancer cell ratios of 8:1, 0.8:1, 0.08:1, and 0:1 for the 100%, 10%, 1%, and 0% NYESO+ T cell populations, respectively). As a positive control for cancer cell killing, cells from an additional healthy donor were transduced with a lentivirus that randomly integrated a 1G4 TCR expression cassette into the genome of the T cells (note that in this case the TCR is randomly integrated and expressed, as opposed to 1G4 TCR knock-in T cells, where the 1G4 TCR is expressed from the endogenous TCR promoter and replaces the endogenous TCR locus, simultaneously knocking out the endogenous TCR). Lentiviral-transduced T cells from this additional donor were similarly sorted to obtain a 100% 1G4+ population and similarly co-cultured with the target A375 cancer cell line at a T cell to cancer cell ratio of 8:1.

[0132] In vivo mouse solid tumor models All mouse experiments were completed under UCSF Institutional Animal Care and Use Committee protocols. All experiments used 8-12 week old NOD / SCID / IL-2Rγ null (NSG) male mice (Jackson Laboratory). 1 × 10 6 Mice were inoculated with tumors by subcutaneous injection of A375 human melanoma cells (ATCC CRL-1619). Seven days after tumor inoculation, tumor size was assessed and ranged from 15 to 30 mm. 3Mice with tumor volumes of 100 μl were randomly assigned to experimental and control treatment groups. The indicated numbers of T cells were resuspended in 100 μl of serum-free RPMI and injected retroorbitally. For tumor size measurement experiments, the length and width of the tumor were measured using electronic calipers and the volume was calculated using v = 1 / 6 * π * length * width * Size was calculated as (length + width) / 2. Investigators were blinded to the experimental treatment groups during size measurements. As indicated in the figures and legends, bulk edited T cell populations (5 × 10 6 ) or sorted NY-ESO-1 TCR+ population (3 × 10 6 ) were transferred. For bulk edited T cell transfer, because lentivirally edited cells generally had a higher percentage of NY-ESO-1 positive cells, mock infected cells were added to normalize the percentage of total T cells NY-ESO-1+ to be equal to that of the bulk population of non-virally edited T cells (~10% NY-ESO-1+). For sorted T cell transfer, NY-ESO-1+ T cells were FACS sorted 8 days after electroporation, expanded for an additional 2 days, and frozen (Bambanker freezing medium, Bulldog Bio). Non-virally or lentivirally modified human T cells were then thawed and placed in medium overnight prior to adoptive transfer. For flow cytometry analysis of adoptively transferred T cells, single cell suspensions from tumors and spleens were created by mechanical dissociation of tissues through 70 μm filters. All animal experiments were performed according to an approved IACUC protocol (UCSF) and in compliance with relevant ethical regulations, including a tumor size limit of 2.0 cm in any dimension.

[0133] In vivo functionality of T cells with non-viral TCR replacement A human antigen-specific tumor xenograft model (Figure 7a) was used to evaluate the in vivo functionality of T cells with non-viral TCR replacement. 1 × 10 6 A375 cells (human melanoma cell line; NY-ESO-1 antigen + and HLA-A *0201+) were inoculated subcutaneously into the shaved flank. Primary human T cells edited to express the NY-ESO-1 antigen-specific TCR were created (either through lentiviral transduction or non-viral TCR replacement), expanded for 10 days after transduction or electroporation, and frozen. Bulk edited populations were used (Figure 7b-c), or NY-ESO-1 TCR+ sorted populations (Figure 7d-f). Seven days after tumor inoculation, T cells were thawed and adoptively transferred via retro-orbital injection. As shown in Figure 7B, 5 × 10 6 Two days after transfer of bulk non-viral targeted T cells (~10% TCR+ NYESO-1+ (red), ~10% TCR+ NYESO-1- (orange), and ~80% TCR- NYESO-1- (green), see Fig. 7b), NY-ESO-1+ non-viral edited T cells selectively accumulated in the tumor compared to the spleen. n=5 mice per 4 human T cell donors. 5x10 6 Ten days after transfer of bulk non-viral targeted CFSE-labeled T cells, NYESO-1 TCR+ cells showed higher proliferation than TCR- or TCR+ NYESO-1- T cells and higher proliferation (CFSE low) in the tumor than in the spleen (Figure 7c). At 10 days post-transfer, TCR- and TCR+ NYESO- T cells were difficult to find in the tumor (Figure 7d). Figure 7d shows the longitudinal tumor volume trajectory of the data summarized in Figure 8f. 3 × 10 induced by either lentiviral transduction or non-viral TCR replacement. 6Sorted NY-ESO-1 TCR+ T cells were transferred 7 days after tumor seeding and compared to vehicle-only injections up to 24 days after tumor seeding. Note that the same data as vehicle control data is shown per donor compared to lentiviral delivery (top) and non-viral TCR replacement (bottom). In these experiments (Figure 7e-f), 17 days after T cell transfer, non-viral TCR replaced cells appeared to exhibit higher NY-ESO-1 TCR expression and lower exhaustion marker expression. Transfer of both lentiviral transduced and non-viral TCR replaced cells showed a significant reduction in tumor burden at day 24. Non-viral TCR replacement showed a further reduction compared to lentiviral transduction in this experimental model (Figure 8f). n=4 (Fig. 7b), n=2 (Fig. 7d-f), or n=1 (Fig. 7c) independent healthy donors with 5 (Fig. 7b, 7c) or 7 (Fig. 7d-f) mice per donor with mean (Fig. 7b, 7e, 7f) and standard deviation (Fig. 7b).

[0134] Tumor antigen-specific functions The tumor antigen-specific function of targeted human T cells was also evaluated. When targeted T cells were co-cultured with two different NY-ESO-1+ melanoma cell lines, M257 and M407, the engineered T cells potently and specifically produced IFN-γ and TNF-α and induced T cell degranulation (measured by CD107a surface expression) (Figure 8a). Cytokine production and degranulation were associated with the appropriate HLA-A expression, which is required for NY-ESO-1 TCR T cells to present the cognate NY-ESO-1 peptide. * NY-ESO-1 TCR knock-in T cells expressed the target M257-HLA-A in vitro at a rate similar to that of T cells transduced with a positive control retrovirus. *0201 cancer cells were rapidly killed (Figure 8b). The killing was mediated by the NY-ESO-1 antigen and HLA-A * It was selective for target cells expressing the 0201 allele, consistent across donors, and dependent on T cells being engineered with both the correct gRNA and HDR template (Figure 9h-k).

[0135] Finally, it was confirmed that non-viral genome targeting could be used to generate NY-ESO-1 TCR cells at a large scale, and that these cells had in vivo anti-tumor function (Figure 8c and Figure 7a). Given that knock-in efficiency was lower with non-viral targeting than with AAV templates of comparable size, we confirmed whether the number of NY-ESO-1 positive cells was sufficient for adoptive cell therapy. 100 million T cells from six healthy donors yielded an average of 385 million NY-ESO-1 TCR T cells per donor after 10 days of expansion (Figure 8d). NY-ESO-1 TCR knock-in T cells selectively localized, persisted, and expanded in the tumor rather than the spleen, similar to positive control lentiviral-transduced T cells (Figure 8e and Figure 7b-f). Adoptive transfer of sorted NY-ESO-1 TCR T cells also reduced tumor burden in treated animals (Figure 8f).

Claims

[Claim 1] The invention described in the specification or drawings of this application.

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