RU2835579C2 - Methods of producing cells expressing recombinant receptor and related compositions - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed are methods of producing genetically engineered T-cells, involving: introduction into T-cells of a CRISPR agent containing a guide RNA (nRNA) having a targeting domain which is complementary to at least one target site within exon 1 of the T-cell receptor alpha constant (TRAC) gene and/or a T-cell receptor beta constant (TRBC) gene, wherein said CRISPR agent is capable of causing genetic destruction of at least one target site, which leads to effective endogenous T-cell receptor (TCR) knockout. and introducing a template polynucleotide into said T cells, wherein said template polynucleotide comprises a [5' homology arm]-[transgene]-[3' homology arm]. Also disclosed is a method of producing genetically engineered T-cells, comprising introducing into T-cells having genetic destruction, at least one target site within exon 1 of the alpha T-cell receptor constant (TRAC) gene, leading to an effective endogenous T-cell receptor (TCR) knockout, a template polynucleotide, wherein said template polynucleotide contains a structure [5' homology arm]-[transgene]-[3' homology arm], where said transgene codes a recombinant TCR containing alpha (TCRα) chain and beta (TCRβ) chain, wherein said transgene comprises a nucleic acid sequence coding TCRα chain, and nucleic acid sequence coding TCRβ circuit and kit for implementation of the presented methods.

EFFECT: invention can be used for engineering of immune cells, in particular T-cells.

69 cl, 48 dwg, 10 tbl, 6 ex

Description

Cross-reference to related applications

This application claims priority to U.S. Provisional Patent Application No. 61/653,522, filed April 5, 2018, entitled “METHODS OF PRODUCING CELLS EXPRESSING A RECOMBINANT RECEPTOR AND RELATED COMPOSITIONS,” the contents of which are incorporated herein by reference in their entirety.

Including a list of sequences as a reference

This application is submitted together with a sequence listing in electronic format. The sequence listing is provided as a file entitled 735042012740SeqList.txt, created on April 3, 2019, and is 179 kilobytes in size. The information in the electronic sequence listing format is incorporated herein by reference in its entirety.

Field of technology

The present invention relates to methods for engineering immune cells, cellular compositions containing engineered immune cells, kits and finished products for targeted action on a nucleic acid sequence encoding a recombinant receptor to a specific genomic locus and/or for modulating gene expression at a genomic locus, and their use in connection with anti-cancer immunotherapy, including adoptive transfer of engineered T cells.

State of the art

Adoptive cell therapies that utilize recombinantly expressed T cell receptors (TCRs) or other antigen receptors ( e.g., chimeric antigen receptors (CARs)) to recognize tumor antigens are attractive therapeutic approaches for the treatment of cancers and other diseases. The expression and function of recombinant TCRs or other antigen receptors may be limited and/or heterogeneous within a cell population. Improved strategies are needed to achieve high and/or homogeneous levels of expression and function of recombinant receptors. These strategies can enable the generation of cells that exhibit desired expression levels and/or properties for use in adoptive immunotherapy, such as the treatment of cancer, infectious diseases, and autoimmune diseases. Methods, cells, compositions, and kits for use in methods that address such needs are provided.

The essence of the invention

Provided are genetically engineered cells that comprise a genetic disruption of at least one target site in a T cell receptor constant alpha ( TRAC ) gene and/or a T cell receptor constant beta ( TRBC ) gene, and a transgene encoding a recombinant receptor, such as a T cell receptor (TCR) or a chimeric antigen receptor (CAR), that is integrated, through homology-directed repair (HDR), at or near one or more target sites, and a composition comprising the engineered cells, methods for producing the engineered cells, and related methods and uses. In some aspects, by genetic disruption and targeted integration of transgene sequences, one or more endogenous TCR chains are reduced or inactivated in expression. In some of any such embodiments, the recombinant receptor may bind to an antigen that is associated with a cell or tissue of a disease, disorder, or condition. In some of any such embodiments, the recombinant receptor may bind to an antigen that is specific to a cell or tissue of the disease, disorder, or condition. In some of any such embodiments, the recombinant receptor may bind to an antigen that is expressed on a cell or tissue associated with the disease, disorder, or condition.

Also provided is a composition comprising an engineered cell or a plurality of engineered cells as described herein. In particular embodiments, at least or more than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or 90% of the cells in the composition comprise a genetic disruption of at least one target site in a gene encoding a domain or region of the T cell receptor constant alpha ( TRAC ) gene and/or the T cell receptor constant beta ( TRBC ) gene. In certain embodiments, at least or more than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the cells in the composition express the recombinant receptor or antigen-binding fragment thereof and/or exhibit antigen binding. In some of any such embodiments, at least or more than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the cells in the composition express the recombinant receptor or antigen-binding fragment thereof and/or exhibit antigen binding.

Compositions comprising a plurality of engineered T cells are provided. In some of any such embodiments, a composition comprising a plurality of engineered T cells comprising a recombinant receptor, or an antigen-binding fragment or chain thereof, encoded by a transgene, and a genetic disruption of at least one target site in a T cell receptor constant alpha ( TRAC ) gene and/or a T cell receptor constant beta ( TRBC ) gene, wherein the recombinant receptor is capable of binding to an antigen that is associated with, is specific for, and/or expressed on a cell or tissue of a disease, disorder, or condition, and wherein at least or more than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the cells in the composition comprise a genetic disruption of at least one target site in the TRAC gene and/or the TRBC gene; and/or at least or more than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or 90% of the cells in the composition express the recombinant receptor or antigen-binding fragment or chain thereof, and/or exhibit binding to the antigen; and the transgene encoding the recombinant receptor or antigen-binding fragment or chain thereof is integrated at or near one of the at least one target sites via homology-directed repair (HDR).

In some embodiments, the coefficient of variation of the expression and/or antigen binding of the recombinant receptor or antigen-binding fragment or chain thereof among the plurality of cells is lower than 0.70, 0.65, 0.60, 0.55, 0.50, 0.45, 0.40, 0.35, or 0.30 or less. In particular embodiments, the coefficient of variation of the expression and/or antigen binding of the recombinant receptor or antigen-binding fragment or chain thereof among the plurality of cells is at least 100%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% lower than the coefficient of variation of the expression and/or antigen binding of the same recombinant receptor that is integrated into the genome by random integration. In some of any such embodiments, the recombinant receptor is capable of binding to an antigen that is associated with, specific for, and/or expressed on a cell or tissue of the disease, disorder, or condition.

In certain embodiments, expression and/or antigen binding of the recombinant receptor or antigen-binding fragment thereof is assessed by contacting cells in the composition with a binding reagent specific for the TCRα chain or TCRβ chain and assessing binding of the reagent to the cells. In some embodiments, the binding reagent is an anti-TCR Vβ antibody or an anti-TCR Vα antibody that specifically recognizes a particular family of Vβ or Vα chains.

In particular embodiments, the binding agent is a peptide antigen-MHC complex, which is optionally a tetramer. In certain embodiments, the composition described herein further comprises a pharmaceutically acceptable carrier. A composition is provided comprising a plurality of engineered T cells comprising a recombinant receptor or antigen binding fragment or chain thereof encoded by a transgene and a genetic disruption of at least one target site in a T cell receptor constant alpha ( TRAC ) gene and/or a T cell receptor constant beta ( TRBC ) gene, wherein at least or more than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the cells in the composition comprise a genetic disruption of at least one target site in a TRAC gene and/or a TRBC gene; and a transgene encoding a recombinant TCR or antigen-binding fragment or chain thereof targeted for integration into or near at least one target site via homology-directed repair (HDR).

Also provided herein is a composition comprising a plurality of engineered T cells comprising a recombinant receptor or antigen-binding fragment or chain thereof encoded by a transgene and a genetic disruption of at least one target site in the T cell receptor constant alpha ( TRAC ) gene and/or the T cell receptor constant beta ( TRBC ) gene, wherein at least or more than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or 90% of the cells in the composition express the recombinant receptor or antigen-binding fragment thereof and/or exhibit antigen binding; and the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof is targeted for integration at or near the at least one target site via homology-directed repair (HDR).

Also provided is a composition comprising a plurality of engineered T cells comprising a recombinant receptor or antigen-binding fragment thereof encoded by a transgene, and genetic disruption of at least one target site in the T-cell receptor constant alpha gene (TRAC) and/or the T-cell receptor constant beta gene (TRBC), wherein the coefficient of variation of expression and/or antigen binding of the recombinant receptor among the plurality of cells is lower than 0.70, 0.65, 0.60, 0.55, 0.50, 0.45, 0.40, 0.35 or 0.30 or less.

Also provided herein is a composition comprising a plurality of engineered T cells comprising a recombinant receptor or antigen-binding fragment thereof encoded by a transgene and a genetic disruption of at least one target site in the T-cell receptor constant alpha (TRAC) gene and/or the T-cell receptor constant beta (TRBC) gene, wherein the coefficient of variation of expression and/or antigen binding of the recombinant receptor among the plurality of cells is at least 100%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% lower than the coefficient of variation of expression and/or antigen binding of the same recombinant receptor that is integrated into the genome by random integration.

In some embodiments, the composition is prepared by: (a) administering to a plurality of T cells one or more agents, wherein each of the one or more agents is independently capable of causing genetic disruption of a target site in the T cell receptor constant alpha (TRAC) gene and/or the T cell receptor constant beta (TRBC) gene, thereby causing genetic disruption of at least one target site; and (b) administering to a plurality of T cells a template polynucleotide comprising a transgene encoding a recombinant T cell receptor (TCR) or an antigen-binding fragment or chain thereof, wherein the transgene encoding the recombinant TCR or an antigen-binding fragment or chain thereof is targeted for integration at or near the at least one target site via homology-directed repair (HDR). In specific embodiments, expression and/or antigen binding of the recombinant receptor or antigen-binding fragment thereof is assessed by contacting cells in the composition with a binding reagent specific for the TCRα chain or TCRβ chain and assessing binding of the reagent to the cells.

In some of any such embodiments, the engineered T cells comprise at least one genetic disruption in the TRAC gene. In some of any such embodiments, the engineered T cells comprise at least one genetic disruption in the TRBC gene. In some of any such embodiments, the engineered T cells comprise at least one target site genetic disruption in the TRAC gene and at least one target site genetic disruption in the TRBC gene.

In certain embodiments, the binding reagent is an anti-TCR Vβ antibody or is an anti-TCR Vα antibody that specifically recognizes a particular family of Vβ or Vα chains. In some embodiments, the binding agent is a peptide antigen-MHC complex, which is optionally a tetramer. In certain embodiments, at least one of the one or more agents is capable of causing genetic disruption of a target site in a TRAC gene. In certain embodiments, at least one of the one or more agents is capable of causing genetic disruption of a target site in a TRBC gene. In some embodiments, the one or more agents comprise at least one agent that is capable of causing genetic disruption of a target site in a TRAC gene and at least one agent that is capable of causing genetic disruption of a target site in a TRBC gene. In specific embodiments, the TRBC gene is one or both of the T cell receptor beta constant 1 ( TRBC1 ) or T cell receptor beta constant 2 ( TRBC2 ) genes. In certain embodiments, the one or more agents capable of causing genetic disruption comprise a DNA-binding protein or a DNA-binding nucleic acid that specifically binds to or hybridizes to a target site. In some embodiments, the one or more agents capable of causing genetic disruption comprise (a) a fusion protein comprising a DNA-directed protein and a nuclease or (b) an RNA-directed nuclease.

In particular embodiments, the DNA-directed protein or RNA-directed nuclease comprises a zinc finger protein (ZFP), a TAL protein, or a clustered regularly intervening short palindromic nucleic acid (CRISPR)-related nuclease (Cas) specific for a target site. In certain embodiments, one or more agents comprises a zinc finger nuclease, a zinc finger nuclease (ZFN), a TAL effector nuclease (TALEN), and/or a CRISPR-Cas9 combination that specifically bind to, recognize, or hybridize to a target site. In some embodiments, each of the one or more agents comprises a guide RNA (gRNA) having a targeting domain that is complementary to at least one target site. In particular embodiments, one or more agents are administered as a ribonucleoprotein (RNP) complex comprising the gRNA and the Cas9 protein. In certain embodiments, the RNP is administered via electroporation, gene gun, calcium phosphate transfection, cell condensation, or cell squeezing. In some embodiments, the RNP is administered by electroporation. In specific embodiments, one or more agents are administered as one or more polynucleotides encoding gRNA and/or Cas9 protein. In certain embodiments, at least one target site is within an exon of a TRAC , TRBC1 , and/or TRBC2 gene.

In some of any such embodiments, the genetic disruption is performed by a zinc finger nuclease (ZFN), a TAL effector nuclease (TALEN), or a CRISPR-Cas9 combination that specifically binds to, recognizes, or hybridizes to a target site. In some of any such embodiments, the genetic disruption by the CRISPR-Cas9 combination comprises a guide RNA (gRNA) having a targeting domain that is complementary to at least one target site. In some of any such embodiments, the CRISPR-Cas9 combination is a ribonucleoprotein (RNP) complex comprising a gRNA and a Cas9 protein. In some of any such embodiments, the RNP is introduced via electroporation. In some of any such embodiments, the at least one target site is within an exon of a TRAC , TRBC1 , and/or TRBC2 gene.

In some embodiments, the gRNA has a targeting domain that is complementary to a target site in the TRAC gene and comprises a sequence selected from the group consisting of UCUCUCAGCUGGUACACGGC (SEQ ID NO:28), UGGAUUUAGAGUCUCUCAGC (SEQ ID NO:29), ACACGGCAGGGUCAGGGUUC (SEQ ID NO:30), GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:31), GCUGGUACACGGCAGGGUCA (SEQ ID NO:32), CUCAGCUGGUACACGGC (SEQ ID NO:33), UGGUACACGGCAGGGUC (SEQ ID NO:34), GCUAGACAUGAGGUCUA (SEQ ID NO:35), GUCAGAUUUGUUGCUCC (SEQ ID NO:36), UCAGCUGGUACACGGCA (SEQ ID NO:37), GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:38), GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:39), GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:40), GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:41), GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:42), GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:43), GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:44), GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:45), GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:46), GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:47), GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:48), GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:49), GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:50), GAGAAUCAAAAUCGGUGAAU (SEQ ID NO NO:37), GCAGACAGACUUGUCAC (SEQ ID NO:38), GGUACACGGCAGGGUCA (SEQ ID NO:39), CUUCAAGAGCAACAGUGCUG (SEQ ID NO:40), AGAGCAACAGUGCUGUGGCC (SEQ ID NO:41), AAAGUCAGAUUUGUUGCUCC (SEQ ID NO:42), ACAAAACUGUGCUAGACAUG (SEQ ID NO:43), AAACUGUGCUAGACAUG (SEQ ID NO:44), UGUGCUAGACAUGAGGUCUA (SEQ ID NO:45), GGCUGGGGAAGAAGGUGUCUUC (SEQ ID NO:46), GCUGGGGAAGAAGGUGUCUUC (SEQ ID NO:47), GGGGAAGAAGGUGUCUUC (SEQ ID NO:48), GUUUUGUCUGUGAUAUACACAU (SEQ ID NO:49), GGCAGACAGACUUGUCACUGGAUU (SEQ ID NO:50), GCAGACAGACUUGUCACUGGAUU (SEQ ID NO:51), GACAGACUUGUCACUGGAUU (SEQ ID NO:52), GUGAAUAGGCAGACAGACUUGUCA (SEQ ID NO:53), GAAUAGGCAGACAGACUUGUCA (SEQ ID NO:54), GAGUCUCUCAGCUGGUACACGG (SEQ ID NO:55), GUCUCUCAGCUGGUACACGG (SEQ ID NO:56), GGUACACGGCAGGGUCAGGGUU (SEQ ID NO:57), and GUACACGGCAGGGUCAGGGUU (SEQ ID NO:58). In particular embodiments, the gRNA has a targeting domain comprising the sequence GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:31).

In certain embodiments, the gRNA has a targeting domain that is complementary to a target site in one or both of the TRBC1 and TRBC2 genes and comprises a sequence selected from the group consisting of CACCCAGAUCGUCAGCGCCG (SEQ ID NO:59), CAAACACAGCGACCUCGGGU (SEQ ID NO:60), UGACGAGUGGACCCAGGAUA (SEQ ID NO:61), GGCUCUCGGAGAAUGACGAG (SEQ ID NO:62), GGCCUCGGCGCUGACGAUCU (SEQ ID NO:63), GAAAAACGUGUUCCCACCCG (SEQ ID NO:64), AUGACGAGUGGACCCAGGAU (SEQ ID NO:65), AGUCCAGUUCUACGGGCUCU (SEQ ID NO:66), CGCUGUCAAGUCCAGUUCUA (SEQ ID NO:67), AUCGUCAGCGCCGAGGCCUG (SEQ ID NO:68), UCAAACACAGCGACCUCGGG (SEQ ID NO:69), CGUAGAACUGGACUUGACAG (SEQ ID NO:70), AGGCCUCGGCGCUGACGAUC (SEQ ID NO:71), UGACAGCGGAAGUGGUUGCG (SEQ ID NO:72), UUGACAGCGGAAGUGGUUGC (SEQ ID NO:73), UCUCCGAGAGCCCGUAGAAC (SEQ ID NO:74), CGGGUGGGAACACGUUUUUC (SEQ ID NO:75), GACAGGUUUGGCCCUAUCCU (SEQ ID NO:76), GAUCGUCAGCGCCGAGGCCU (SEQ ID NO:77), GGCUCAAACACAGCGACCUC (SEQ ID NO:78), UGAGGUCUCGGCCACCUUC (SEQ ID NO:79) AGGCUUCUACCCCGACCACG (SEQ ID NO:80), CCGACCACGUGGAGCUGAGC (SEQ ID NO:81), UGACAGGUUUGGCCCUAUCC (SEQ ID NO:82), CUUGACAGCGGAAGUGGUUG (SEQ ID NO:83), AGAUCGUCAGCGCCGAGGCC (SEQ ID NO:84), GCGCUGACGAUCUGGGUGAC (SEQ ID NO:85), UGAGGGCGGGGCUGCUCCUUG (SEQ ID NO:86), GUUGCGGGGGUUCUGCCAGA (SEQ ID NO:87), AGCUCAGCUCCACGUGGUCG (SEQ ID NO:88), GCGGCUGCUCAGGCAGUAUC (SEQ ID NO:89), GCGGGGGUUCUGCCAGAAGG (SEQ ID NO:90), UGGCUCAAACACAGCGACCU (SEQ ID NO:91), ACUGGACUUGACAGCGGAAG (SEQ ID NO:92), GACAGCGGAAGUGGUUGCGG (SEQ ID NO:93), GCUGUCAAGUCCAGUUCUAC (SEQ ID NO:94), GUAUCUGGAGUCAUUGAGGG (SEQ ID NO:95), CUCGGCGCUGACGAUCU (SEQ ID NO:96), CCUCGGCGCUGACGAUC (SEQ ID NO:97), CCGAGAGCCCGUAGAAC (SEQ ID NO:98), CCAGAUCGUCAGCGCCG (SEQ ID NO:99), GAAUGACGAGUGGACCC (SEQ ID NO:100), GGGUGACAGGUUUGGCCCUAUC (SEQ ID NO:101), GGUGACAGGUUUGGCCCUAUC (SEQ ID NO:102), GUGACAGGUUUGGCCCUAUC (SEQ ID NO:103), GACAGGUUUGGCCCUAUC (SEQ ID NO:104), GAUACUGCCUGAGCAGCCGCCU (SEQ ID NO:105), GACCACGUGGAGCUGAGCUGGUGG (SEQ ID NO:106), GUGGAGCUGAGCUGGUGG (SEQ ID NO:107), GGGCGGGCUGCUCCUUGAGGGGCU (SEQ ID NO:108), GGCGGGCUGCUCCUUGAGGGGCU (SEQ ID NO:109), GCGGGCUGCUCCUUGAGGGGCU (SEQ ID NO:110), GGGCUGCUCCUUGAGGGGCU (SEQ ID NO:111), GGCUGCUCCUUGAGGGGCU (SEQ ID NO:112), GCUGCUCCUUGAGGGGCU (SEQ ID NO:113), GGUGAAUGGGAAGGAGGUGCACAG (SEQ ID NO:114), GUGAAUGGGAAGGAGGUGCACAG (SEQ ID NO:115), and GAAUGGGAAGGAGGUGCACAG (SEQ ID NO:116). In some embodiments, the gRNA has a targeting domain comprising the sequence GGCCUCGGCGCUGACGAUCU (SEQ ID NO:63).

In some of any such embodiments, the transgene is integrated by a template polynucleotide introduced into each of the plurality of T cells. In particular embodiments, the template polynucleotide comprises the structure [5' homology arm]-[transgene]-[3' homology arm]. In certain embodiments, the 5' homology arm and the 3' homology arm comprise nucleic acid sequences homologous to nucleic acid sequences surrounding at least one target site. In some embodiments, the 5' homology arm comprises nucleic acid sequences that are homologous to nucleic acid sequences 5' of the target site. In particular embodiments, the 3' homology arm comprises nucleic acid sequences that are homologous to nucleic acid sequences 3' of the target site. In certain embodiments, the 5' homology arm and the 3' homology arm independently have at least, or at least about, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides, or less, or less than about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides. In some embodiments, the 5' homology arm and the 3' homology arm are independently between about 50 and 100, 100 and 250, 250 and 500, 500 and 750, 750 and 1000, 1000 and 2000 nucleotides. In some of any such embodiments, the 5' homology arm and the 3' homology arm are independently between or about 50 and between or about 100 nucleotides in length, between or about 100 and between or about 250 nucleotides in length, between or about 250 and between or about 500 nucleotides in length, between or about 500 and between or about 750 nucleotides in length, between or about 750 and between or about 1000 nucleotides in length, or between or about 1000 and between or about 2000 nucleotides in length.

In particular embodiments, the 5' homology arm and the 3' homology arm independently have from or about 100 to 1000 nucleotides, from 100 to 750 nucleotides, from 100 to 600 nucleotides, from 100 to 400 nucleotides, from 100 to 300 nucleotides, from 100 to 200 nucleotides, from 200 to 1000 nucleotides, from 200 to 750 nucleotides, from 200 to 600 nucleotides, from 200 to 400 nucleotides, from 200 to 300 nucleotides, from 300 to 1000 nucleotides, from 300 to 750 nucleotides, from 300 to 600 nucleotides, from 300 to 400 nucleotides, from 400 to 1000 nucleotides, 400 to 750 nucleotides, 400 to 600 nucleotides, 600 to 1000 nucleotides, 600 to 750 nucleotides, or 750 to 1000 nucleotides. In particular embodiments, the 5' homology arm and the 3' homology arm independently have from or about 100 to from or about 1000 nucleotides, from 100 to 750 nucleotides, from 100 to 600 nucleotides, from 100 to 400 nucleotides, from 100 to 300 nucleotides, from 100 to 200 nucleotides, from 200 to 1000 nucleotides, from 200 to 750 nucleotides, from 200 to 600 nucleotides, from 200 to 400 nucleotides, from 200 to 300 nucleotides, from 300 to 1000 nucleotides, from 300 to 750 nucleotides, from 300 to 600 nucleotides, from 300 to 400 nucleotides, from 400 to 1000 nucleotides, 400 to 750 nucleotides, 400 to 600 nucleotides, 600 to 1000 nucleotides, 600 to 750 nucleotides, or 750 to 1000 nucleotides in length.

In some of any such embodiments, the 5' homology arm and the 3' homology arm are independently about or about 200, 300, 400, 500, 600, 700, or 800 nucleotides in length, or any value between any of the foregoing. In some of any such embodiments, the 5' homology arm and the 3' homology arm are independently greater than about or about 300 nucleotides in length. In some of any such embodiments, the 5' homology arm and the 3' homology arm are independently about or about 400, 500, or 600 nucleotides in length, or any value between any of the foregoing. In some of any such embodiments, the 5' homology arm and the 3' homology arm are independently about or about 500 and about or about 600 nucleotides in length. In some of any such variants, the 5' homology arm and the 3' homology arm are independently more than or about 300 nucleotides in length.

In some of any such embodiments, the transgene encoding the recombinant receptor or antigen-binding fragment or chain thereof is integrated at or near a target site in the TRAC gene. In some embodiments, the transgene encoding the recombinant receptor or antigen-binding fragment or chain thereof is integrated at or near a target site in one or both of the TRBC1 and TRBC2 genes.

In some of any such embodiments, the recombinant receptor is a chimeric antigen receptor (CAR). In some of any such embodiments, the CAR comprises an extracellular domain comprising an antigen-binding domain specific for an antigen. In some of any such embodiments, the antigen-binding domain is an scFv; a transmembrane domain; a cytoplasmic signaling domain derived from a costimulatory molecule; and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule. In some of any such embodiments, the CAR further comprises a spacer between the transmembrane domain and the antigen-binding domain. In some of any such embodiments, the costimulatory molecule is or comprises 4-1BB, optionally human 4-1BB. In some of any such embodiments, the ITAM-containing molecule is or comprises a CD3zeta signaling domain. In some of any such embodiments, the ITAM-containing molecule is a human CD3zeta signaling domain.

In some of any such embodiments, the recombinant receptor is a recombinant TCR or an antigen-binding fragment or chain thereof. In some of any such embodiments, the recombinant receptor is a recombinant TCR comprising an alpha (TCRα) chain and a beta (TCRβ) chain, and the transgene encoding the recombinant TCR or an antigen-binding fragment or chain thereof comprises a nucleic acid sequence encoding a TCRα chain and a nucleic acid sequence encoding a TCRβ chain. In some of any such embodiments, the transgene further comprises one or more multicistronic elements, and the multicistronic elements are located between the nucleic acid sequence encoding TCRα or a portion thereof and the nucleic acid sequence encoding TCRβ or a portion thereof. In some of any such variants, the multicistronic elements comprise a sequence encoding a ribosome skip element selected from T2A, P2A, E2A, or F2A or an internal ribosome entry site (IRES).

In some of any such embodiments, the engineered cells further comprise one or more second transgenes, wherein the second transgene is integrated at or near at least one target site via homology-directed repair (HDR). In some of any such embodiments, the recombinant receptor is a recombinant TCR, and the transgene encoding the recombinant TCR or an antigen-binding fragment or chain thereof comprises a nucleic acid sequence encoding one chain of the recombinant TCR, and the second transgene comprises a nucleic acid sequence encoding another chain of the recombinant TCR. In some of any such embodiments, the transgene encoding the recombinant TCR or an antigen-binding fragment or chain thereof comprises a nucleic acid sequence encoding a TCRα chain, and the second transgene comprises a nucleic acid sequence encoding a TCRβ chain or a portion thereof. In some of any such embodiments, the integration of the second transgene is accomplished by a second template polynucleotide introduced into each of the plurality of T cells, wherein said second template polynucleotide comprises the structure [second 5' homology arm]-[one or more second transgenes]-[second 3' homology arm].

In certain embodiments, the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof is targeted for integration at or near a target site in the TRAC gene. In some embodiments, the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof is targeted for integration at or near a target site in one or both of the TRBC1 and TRBC2 genes . In particular embodiments, the composition is prepared by further introducing into an immune cell one or more second template polynucleotides comprising one or more second transgenes, wherein the second transgene is targeted for integration at or near one of the at least one target site via homology-directed repair (HDR).

In certain embodiments, the second template polynucleotide comprises a structure of [second 5' homology arm]-[one or more second transgenes]-[second 3' homology arm]. In some embodiments, the second 5' homology arm and the second 3' homology arm comprise nucleic acid sequences that are homologous to nucleic acid sequences surrounding at least one target site. In particular embodiments, the second 5' homology arm comprises nucleic acid sequences that are homologous to nucleic acid sequences of a second 5' target site. In certain embodiments, the second 3' homology arm comprises nucleic acid sequences that are homologous to nucleic acid sequences of a second 3' target site.

In some embodiments, the second 5' homology arm and the second 3' homology arm independently have at least or at least about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500 or 2000 nucleotides or less or less than about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500 or 2000 nucleotides. In particular embodiments, the second 5' homology arm and the second 3' homology arm independently have between about 50 and 100, 100 and 250, 250 and 500, 500 and 750, 750 and 1000, 1000 and 2000 nucleotides. In certain embodiments, the second 5' homology arm and the second 3' homology arm independently have from or about 100 to 1000 nucleotides, from 100 to 750 nucleotides, from 100 to 600 nucleotides, from 100 to 400 nucleotides, from 100 to 300 nucleotides, from 100 to 200 nucleotides, from 200 to 1000 nucleotides, from 200 to 750 nucleotides, from 200 to 600 nucleotides, from 200 to 400 nucleotides, from 200 to 300 nucleotides, from 300 to 1000 nucleotides, from 300 to 750 nucleotides, from 300 to 600 nucleotides, from 300 to 400 nucleotides, from 400 to 1000 nucleotides, 400 to 750 nucleotides, 400 to 600 nucleotides, 600 to 1000 nucleotides, 600 to 750 nucleotides, or 750 to 1000 nucleotides.

In some embodiments, one or more second transgenes are targeted for integration at or near a target site in a TRAC gene. In particular embodiments, one or more second transgenes are targeted for integration at or near a target site in a TRBC1 or TRBC2 gene. In certain embodiments, a transgene encoding a recombinant TCR or antigen-binding fragment or chain thereof is targeted for integration at or near a target site in a TRAC gene, a TRBC1 gene, or a TRBC2 gene, and one or more second transgenes are targeted for integration at or near one or more target sites that are not targeted by the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof.

In some embodiments, a transgene encoding a recombinant TCR or antigen-binding fragment or chain thereof is targeted for integration at or near a target site in a TRAC gene, and one or more second transgenes are targeted for integration at or near one or more target sites in a TRBC1 gene and/or a TRBC2 gene. In particular embodiments, one or more second transgenes encode a molecule selected from a costimulatory ligand, a cytokine, a soluble single-strand variable fragment (scFv), an immunomodulatory fusion protein, a chimeric switch receptor (CSR), or a coreceptor. In certain embodiments, the encoded molecule is a costimulatory ligand, optionally selected from a tumor necrosis factor (TNF) ligand selected from 4-1BBL, OX40L, CD70, LIGHT, and CD30L, or an immunoglobulin (Ig) superfamily ligand selected from CD80 and CD86.

In some of any such embodiments, a transgene encoding a recombinant receptor or an antigen-binding fragment or chain thereof is integrated at or near a target site in a TRAC gene, and one or more second transgenes are integrated at or near one or more other target sites among the TRAC gene, the TRBC1 gene, or the TRBC2 gene, and which is not integrated by a transgene encoding a recombinant receptor or an antigen-binding fragment or chain thereof. In some of any such embodiments, a transgene encoding a recombinant receptor or an antigen-binding fragment or chain thereof is integrated at or near a target site in a TRAC gene, and one or more second transgenes are integrated at or near one or more target sites in the TRBC1 gene and/or the TRBC2 gene. In some of any such embodiments, one or more second transgenes encode molecules selected from a costimulatory ligand, a cytokine, a soluble single-stranded variable fragment (scFv), an immunomodulatory fusion protein, a chimeric switch receptor (CSR), or a coreceptor.

In some embodiments, the encoded molecule is a cytokine, optionally selected from IL-2, IL-3, IL-6, IL-11, IL-12, IL-7, IL-15, IL-21, granulocyte-monocyte colony-stimulating factor (GM-CSF), interferon alpha (IFN-α), interferon beta (IFN-β) or interferon gamma (IFN-γ) and erythropoietin. In specific embodiments, the encoded molecule is a soluble single-stranded variable fragment (scFv), which optionally binds a polypeptide that has an immunosuppressive effect or an immunostimulatory effect, selected from CD47, PD-1, CTLA-4 and their ligands or CD28, OX-40, 4-1BB and their ligands.

In certain embodiments, the encoded molecule is an immunomodulatory fusion protein optionally comprising: (a) an extracellular binding domain that specifically binds an antigen derived from CD200R, SIRPα, CD279 (PD-1), CD2, CD95 (Fas), CD152 (CTLA4), CD223 (LAG3), CD272 (BTLA), A2aR, KIR, TIM3, CD300, or LPA5; (b) an intracellular binding domain derived from CD3ε, CD3δ, CD3ζ, CD25, CD27, CD28, CD40, CD47, CD79A, CD79B, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD278 (ICOS), CD357 (GITR), XAPD11, DAP10, DAP12, FcRα, FcRβ, FcRγ, Fyn, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, ROR2, Ryk, Slp76, pTα, TCRα, TCRβ, TRFM, Zap70, PTCH2, or any combination thereof; and (c) a hydrophobic transmembrane domain derived from CD2, CD3ε, CD3δ, CD3ζ, CD25, CD27, CD28, CD40, CD79A, CD79B, CD80, CD86, CD95 (Fas), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD200R, CD223 (LAG3), CD270 (HVEM), CD272 (BTLA), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), CD279 (PD-1), CD300, CD357 (GITR), A2aR, DAP10, FcRα, FcRβ, FcRγ, Fyn, GAL9, KIR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPα, pTα, TCRα, TCRβ, TIM3, TRIM, LPA5 or Zap70. In some embodiments, the encoded molecule is a chimeric switch receptor (CSR), which optionally comprises a truncated extracellular domain of PD1 and the transmembrane and cytoplasmic signaling domains of CD28. In particular embodiments, the encoded molecule is a co-receptor, optionally selected from CD4 or CD8.

In certain embodiments, the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof encodes one chain of the recombinant TCR, and the second transgene encodes another chain of the recombinant TCR. In some embodiments, the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof encodes an alpha (TCRα) chain of the recombinant TCR, and the second transgene encodes a beta (TCRβ) chain of the recombinant TCR. In particular embodiments, the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof and/or one or more second transgenes independently further comprise a regulatory or control element.

In some of any such embodiments, the transgene encoding the recombinant receptor or antigen-binding fragment or chain thereof further comprises a heterologous regulatory or control element. In some of any such embodiments, the transgene encoding the recombinant receptor or antigen-binding fragment or chain thereof and/or one or more second transgenes independently further comprise a heterologous regulatory or control element. In some of any such embodiments, the heterologous regulatory or control element comprises a heterologous promoter. In some of any such embodiments, the heterologous promoter is or comprises the human elongation factor 1 alpha (EF1α) promoter or the MND promoter or a variant thereof. In some of any such embodiments, the heterologous promoter is an inducible promoter or a repressible promoter.

In certain embodiments, the regulatory or control element comprises a promoter, an enhancer, an intron, a polyadenylation signal, a Kozak consensus sequence, a splice acceptor sequence, or a splice donor sequence. In some embodiments, the regulatory or control element comprises a promoter. In particular embodiments, the promoter is selected from a constitutive promoter, an inducible promoter, a repressible promoter, and/or a tissue-specific promoter. In certain embodiments, the promoter is selected from an RNA pol I, pol II, or pol III promoter. In some embodiments, the promoter is selected from: a pol III promoter that is a U6 or H1 promoter; or a pol II promoter that is a CMV, SV40 early region, or adenovirus major late promoter. In particular embodiments, the promoter is or comprises the human elongation factor 1 alpha (EF1α) or MND promoter or a variant thereof. In certain embodiments, the promoter is an inducible promoter or a repressible promoter. In some embodiments, the promoter comprises a Lac operator sequence, a tetracycline operator sequence, a galactose operator sequence, or a doxycycline operator sequence, or an analog thereof, or is capable of binding to or being recognized by a Lac repressor or a tetracycline repressor, or an analog thereof. In particular embodiments, the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof, and/or one or more second transgenes independently comprise one or more multicistronic elements.

In certain embodiments, one or more multicistronic elements are located upstream of the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof and/or one or more second transgenes. In some embodiments, the multicistronic elements are located between the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof and the one or more second transgenes. In particular embodiments, the multicistronic elements are located between the nucleic acid sequence encoding TCRα or a portion thereof and the nucleic acid sequence encoding TCRβ or a portion thereof. In certain embodiments, the multicistronic elements comprise a sequence encoding a riboparticular skip element selected from T2A, P2A, E2A or F2A or an internal ribosome entry site (IRES).

In some of any such embodiments, the TCRα chain comprises a constant region (Cα) comprising an introduction of one or more cysteine residues and/or the TCRβ chain comprises a Cβ region comprising an introduction of one or more cysteine residues, wherein the one or more introduced cysteine residues are capable of forming one or more non-native disulfide bridges between the alpha chain and the beta chain. In some of any such embodiments, the introduction of the one or more cysteine residues comprises substitution of a non-cysteine residue with a cysteine residue. In some of any such embodiments, the Cα region comprises a cysteine at a position corresponding to position 48 with the numbering as set forth in any of SEQ ID NO: 24; and/or the Cβ region comprises a cysteine at a position corresponding to position 57 with the numbering as set forth in SEQ ID NO: 20.

In certain embodiments, the riboparticle skip element encoding sequence is targeted to be in-frame with a gene at the target site. In some embodiments, in HDR, the transgene encoding the recombinant TCR, or antigen-binding fragment or chain thereof, and/or one or more second transgenes are independently operably linked to an endogenous promoter of the gene at the target site. In certain embodiments, the recombinant TCR is capable of binding to an antigen that is associated with, specific for, and/or expressed on a cell or tissue of a disease, disorder, or condition. In particular embodiments, the disease, disorder, or condition is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, or a tumor or cancer. In particular embodiments, the antigen is a tumor antigen or a pathogenic antigen. In certain embodiments, the pathogenic antigen is a bacterial antigen or a viral antigen.

In some embodiments, the antigen is a viral antigen, and the viral antigen is selected from hepatitis A virus, hepatitis B virus, hepatitis C virus (HCV), human papillomavirus (HPV), hepatitis B virus infection, Epstein-Barr virus (EBV), human herpesvirus 8 (HHV-8), human T-cell leukemia virus-1 (HTLV-1), human T-cell leukemia virus-2 (HTLV-2), or cytomegalovirus (CMV). In specific embodiments, the antigen is an HPV antigen selected from HPV-16, HPV-18, HPV-31, HPV-33, and HPV-35. In certain embodiments, the antigen is an HPV-16 antigen, which is an HPV-16 E6 or HPV-16 E7 antigen. In some embodiments, the viral antigen is an EBV antigen selected from Epstein-Barr nuclear antigen (EBNA)-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA leader protein (EBNA-LP), latent membrane proteins LMP-1, LMP-2A and LMP-2B, EBV-EA, EBV-MA and EBV-VCA. In specific embodiments, the viral antigen is an HTLV antigen that is TAX. In certain embodiments, the viral antigen is an HBV antigen that is a hepatitis B core antigen or a hepatitis B envelope antigen. In some embodiments, the antigen is a tumor antigen.

In specific embodiments, the antigen is selected from glioma-associated antigen, β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, Melanin-A/MART-1, WT-1, S-100, MBP, CD63, MUC1 ( e.g., MUC1-8), p53, Ras, cyclin B1, HER-2/neu, carcinoembryonic antigen (CEA), gp100, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A11, MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-C1, BAGE, GAGE-1, GAGE-2, pl5, tyrosinase, tyrosinase-related protein 1 (TRP-1), tyrosinase-related protein 2 (TRP-2), β-catenin, NY-ESO-1, LAGE-1a, PP1, MDM2, MDM4, EGVFvIII, Tax, SSX2, telomerase, TARP, pp65, CDK4, vimentin, S100, eIF-4A1, IFN-inducible p78, melanotransferrin (p97), Uroplakin II, prostate-specific antigen (PSA), human kallikrein (huK2), prostate-specific membrane antigen (PSM) and prostatic acid phosphatase (PAP), neutrophil elastase, ephrin B2, BA-46, Bcr-abl, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Caspase 8, FRα, CD24, CD44, CD133, CD 166, epCAM, CA-125, HE4, Oval, estrogen receptor, progesterone receptor, uPA, PAI-1, CD19, CD20, CD22, ROR1, CD33/IL3Ra, c-Met, PSMA, Glycolipid F77, GD-2, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin.

In certain embodiments, the T cell is a CD8+ T cell or subtypes thereof. In some embodiments, the T cell is a CD4+ T cell or subtypes thereof. In particular embodiments, the T cell is autologous to the subject. In certain embodiments, the T cell is allogeneic to the subject. In some embodiments, the first template polynucleotide, one or more second template polynucleotides, and/or one or more polynucleotides encoding gRNA and/or Cas9 protein are contained in one or more vectors, which are optionally viral vectors. In particular embodiments, the vector is an AAV vector. In certain embodiments, the AAV vector is selected from an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, or AAV8 vector. In some embodiments, the AAV vector is an AAV2 or AAV6 vector. In particular embodiments, the viral vector is a retroviral vector. In certain embodiments, the viral vector is a lentiviral vector.

In some of any such embodiments, the T cells comprise a CD8+ T cell and/or CD4+ T cells or subtypes thereof. In some of any such embodiments, the T cells are autologous to the subject. In some of any such embodiments, the T cells are allogeneic to the subject. In some of any such embodiments, the composition described herein further comprises a pharmaceutically acceptable carrier.

In some embodiments, the administration of one or more agents capable of causing genetic destruction and the administration of the template polynucleotide are carried out simultaneously or sequentially, in any order. In particular embodiments, the administration of the template polynucleotide is carried out after the administration of one or more agents capable of causing genetic destruction. In certain embodiments, the template polynucleotide is administered immediately after or within about 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 6 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, or 4 hours after the administration of one or more agents capable of causing genetic destruction.

In some embodiments, the administration of the template polynucleotide and the administration of one or more second template polynucleotides are carried out simultaneously or sequentially, in any order. In specific embodiments, the administration of one or more agents capable of causing genetic destruction and the administration of the template polynucleotide are carried out in a single experimental reaction. In certain embodiments, the administration of one or more agents capable of causing genetic destruction and the administration of the template polynucleotide and the second template polynucleotides are carried out in a single experimental reaction. In some embodiments, the composition described herein further comprises a pharmaceutically acceptable carrier.

In some embodiments, methods for producing a genetically engineered immune cell are provided, which comprise (a) introducing into an immune cell one or more agents, wherein each of the one or more agents is independently capable of causing genetic disruption of a target site in a T cell receptor constant alpha ( TRAC ) gene and/or a T cell receptor constant beta ( TRBC ) gene, thereby causing genetic disruption of at least one target site; and (b) introducing into the immune cell a template polynucleotide comprising a transgene encoding a recombinant T cell receptor (TCR) or an antigen-binding fragment or chain thereof, wherein the transgene encoding the recombinant TCR or an antigen-binding fragment or chain thereof is targeted for integration at or near one of the at least one target site via homology-directed repair (HDR).

In some of any such embodiments, methods for producing a genetically engineered immune cell are provided, which comprise (a) introducing into the immune cell one or more agents, wherein each of the one or more agents is independently capable of causing genetic disruption of a target site in the T cell receptor alpha constant (TRAC) gene and/or the T cell receptor beta constant (TRBC) gene, thereby causing genetic disruption of at least one target site; and (b) introducing into an immune cell a template polynucleotide comprising a transgene encoding a recombinant receptor or an antigen-binding fragment or chain thereof, wherein said recombinant receptor is capable of binding to an antigen that is associated with, is specific for and/or is expressed on a cell or tissue of a disease, disorder or condition, wherein the transgene encoding the recombinant receptor or an antigen-binding fragment or chain thereof is targeted for integration into or near one of at least one target site via homology-directed repair (HDR), wherein the introduction of the template polynucleotide is carried out after the introduction of one or more agents capable of causing genetic destruction.

In some embodiments, methods for producing a genetically engineered immune cell are also provided, which comprise introducing into an immune cell having a genetic disruption of at least one target site in a T cell receptor constant alpha (TRAC) gene and/or a T cell receptor constant beta (TRBC) gene, a template polynucleotide comprising a transgene encoding a recombinant T cell receptor (TCR) or an antigen-binding fragment or chain thereof, wherein the genetic disruption is caused by one or more agents, wherein each of the one or more agents is independently capable of causing the genetic disruption, and the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof is targeted for integration at or near one of the at least one target position via homology-directed repair (HDR).

In some of any such embodiments, methods for producing a genetically engineered immune cell are also provided, which comprise introducing into an immune cell having a genetic disruption of at least one target site in a T cell receptor constant alpha (TRAC) gene and/or a T cell receptor constant beta (TRBC) gene, a matrix polynucleotide comprising a transgene encoding a recombinant receptor or an antigen-binding fragment or chain thereof, wherein said recombinant receptor is capable of binding to an antigen that is associated with, is specific for, and/or expressed on a cell or tissue of a disease, disorder, or condition, wherein the genetic disruption is caused by one or more agents, wherein each of the one or more agents is independently capable of causing the genetic disruption, and the transgene encoding the recombinant receptor or antigen-binding fragment or chain thereof is targeted for integration at or near one of the at least one target site via homology-directed repair (HDR).

In some of any such embodiments, the template polynucleotide is administered immediately after or within or about 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 6 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, or 4 hours after administration of one or more agents capable of causing genetic destruction. In some of any such embodiments, the template polynucleotide is administered at or about 2 hours after administration of one or more agents.

In some of any such embodiments, the one or more immune cells comprise T cells. In some of any such embodiments, the T cells comprise CD4+ T cells, CD8+ T cells, or CD4+ and CD8+ T cells. In some of any such embodiments, the T cells comprise CD4+ and CD8+ T cells, and the ratio of CD4+ to CD8+ T cells is from about 1:3 to about 3:1. In some of any such embodiments, optionally from about 1:2 to about 2:1, the ratio of CD4+ to CD8+ T cells is about 1:1.

In some of any such embodiments, one or more agents comprise a CRISPR-Cas9 combination, and the CRISPR-Cas9 combination comprises a guide RNA (gRNA) having a targeting domain that is complementary to at least one target site. In some of any such embodiments, the CRISPR-Cas9 combination is a ribonucleoprotein (RNP) complex comprising gRNA and a Cas9 protein. In some of any such embodiments, the concentration of RNP is from about 1 μM to about 5 μM. In some of any such embodiments, the concentration of RNP is about 2 μM.

In some of any such embodiments, one or more agents are introduced by electroporation. In some of any such embodiments, the template polynucleotide is contained in viral vectors, and the introduction of the template polynucleotide is carried out by transduction. In some of any such embodiments, the vector is an AAV vector.

In some of any such embodiments, the method comprises incubating the cells in vitro with stimulatory agents under conditions for stimulating or activating one or more immune cells and prior to administering the one or more agents. In some of any such embodiments, the stimulatory agents comprise both anti-CD3 and/or anti-CD28 antibodies, optionally anti-CD3/anti-CD28 beads. In some of any such embodiments, the ratio of beads to cells is about 1:1. In some of any such embodiments, the stimulatory agents are removed from the immune cell prior to administering the one or more agents.

In some of any such embodiments, the method further comprises incubating the cells prior to, during, or following administration of the one or more agents and/or administration of the template polynucleotide with one or more recombinant cytokines. In some of any such embodiments, the one or more recombinant cytokines are selected from the group consisting of IL-2, IL-7, and IL-15. In some of any such embodiments, the one or more recombinant cytokines are added at a concentration selected from an IL-2 concentration of about 10 U/mL to about 200 U/mL. In some of any such embodiments, the concentration is from about 50 IU/mL to about 100 U/mL; IL-7 at a concentration of from 0.5 ng/mL to 50 ng/mL. In some of any such embodiments, the concentration is from about 5 ng/mL to about 10 ng/mL and/or IL-15 at a concentration of from 0.1 ng/mL to 20 ng/mL, optionally from about 0.5 ng/mL to about 5 ng/mL. In some of any such embodiments, the incubation is carried out after administration of one or more agents and administration of the template polynucleotide for up to or about 24 hours, 36 hours, 48 hours, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days, optionally up to or about 7 days.

In some of any such embodiments, the recombinant receptor is a chimeric antigen receptor (CAR). In some of any such embodiments, the CAR comprises an extracellular domain comprising an antigen-binding domain specific for an antigen; a transmembrane domain; a cytoplasmic signaling domain derived from a costimulatory molecule; and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule. In some of any such embodiments, the CAR further comprises a spacer between the transmembrane domain and the antigen-binding domain. In some of any such embodiments, the antigen-binding domain is an scFv. In some of any such embodiments, the costimulatory molecule is or comprises 4-1BB. In some of any such embodiments, the costimulatory molecule is human 4-1BB. In some of any such embodiments, the ITAM-containing molecule is or comprises a CD3zeta signaling domain. In some of these variants, the ITAM-containing molecule is the human CD3zeta signaling domain.

In some of any such embodiments, the recombinant receptor is a recombinant TCR or an antigen-binding fragment or chain thereof. In some of any such embodiments, the recombinant receptor is a recombinant TCR comprising an alpha (TCRα) chain and a beta (TCRβ) chain, and the transgene encoding the recombinant TCR or an antigen-binding fragment or chain thereof comprises a nucleic acid sequence encoding a TCRα chain and a nucleic acid sequence encoding a TCRβ chain.

In some of any such embodiments, methods for producing a genetically engineered immune cell are provided, comprising (a) introducing into an immune cell one or more agents, wherein each of the one or more agents is independently capable of causing genetic disruption of a target site in a T cell receptor constant alpha (TRAC) gene and/or a T cell receptor constant beta (TRBC) gene, thereby causing genetic disruption of at least one target site; and (b) introducing into the immune cell a template polynucleotide comprising a transgene encoding a recombinant receptor, which is a recombinant T cell receptor (TCR) or an antigen binding fragment or chain thereof, wherein said transgene comprises a heterologous promoter, and wherein the transgene is targeted for integration at or near one of the at least one target site via homology directed repair (HDR).

In some of any such embodiments, methods for producing a genetically engineered immune cell are provided, comprising introducing into an immune cell having a genetic disruption of at least one target site in a T cell receptor constant alpha (TRAC) gene and/or a T cell receptor constant beta (TRBC) gene, a template polynucleotide comprising a transgene encoding a recombinant receptor, which is a recombinant T cell receptor (TCR) or an antigen-binding fragment or chain thereof, wherein said transgene comprises a heterologous promoter, wherein the genetic disruption is caused by one or more agents, wherein each of the one or more agents is independently capable of causing the genetic disruption, and the transgene encoding the recombinant receptor or antigen-binding fragment or chain thereof is targeted for integration at or near one of the at least one target site via homology-directed repair (HDR).

In some embodiments, at least one of the one or more agents is capable of causing genetic disruption of a target site in a TRAC gene. In particular embodiments, at least one of the one or more agents is capable of causing genetic disruption of a target site in a TRBC gene. In certain embodiments, the one or more agents comprise at least one agent that is capable of causing genetic disruption of a target site in a TRAC gene and at least one agent that is capable of causing genetic disruption of a target site in a TRBC gene. In some embodiments, the TRBC gene is one or both of the T cell receptor beta constant 1 ( TRBC1 ) or T cell receptor beta constant 2 ( TRBC2 ) genes.

Methods for producing a genetically engineered immune cell are provided, comprising: (a) introducing into an immune cell one or more agents, wherein each of the one or more agents is independently capable of causing genetic disruption of a target site in a T-cell receptor constant alpha (TRAC) gene and/or a T-cell receptor constant beta (TRBC) gene, thereby causing genetic disruption of at least one target site; and (b) introducing into the immune cell a matrix polynucleotide comprising a transgene encoding a recombinant T-cell receptor (TCR) or an antigen-binding fragment or chain thereof, wherein the transgene encoding the recombinant TCR or an antigen-binding fragment or chain thereof is targeted for integration at or near the target site via homology-directed repair (HDR).

Also provided are methods for producing a genetically engineered immune cell, comprising: introducing into an immune cell having a genetic disruption of at least one target site in the T-cell receptor constant alpha (TRAC) gene and the T-cell receptor constant beta (TRBC) gene, a matrix polynucleotide comprising a transgene encoding a recombinant T-cell receptor (TCR) or an antigen-binding fragment or chain thereof, wherein the genetic disruption is caused by one or more agents, wherein each of the one or more agents is independently capable of causing the genetic disruption, and the transgene encoding the recombinant TCR or an antigen-binding fragment or chain thereof is targeted for integration into or near one of the at least one target site via homology-directed repair (HDR). In specific embodiments, the TRBC gene is one or both of the T cell receptor beta constant 1 ( TRBC1 ) or T cell receptor beta constant 2 ( TRBC2 ) gene.

In some of any such embodiments, methods for producing a genetically engineered immune cell are also provided, comprising (a) introducing into an immune cell at least one agent that is capable of causing genetic disruption of a target site in a T cell receptor constant alpha (TRAC) gene and at least one agent that is capable of causing genetic disruption of a target site in a T cell receptor constant beta (TRBC) gene, thereby causing genetic disruption of the target sites; and (b) introducing into the immune cell a template polynucleotide comprising a transgene encoding a recombinant receptor, which is a recombinant T cell receptor (TCR) or an antigen-binding fragment or chain thereof, wherein the transgene encoding the recombinant receptor or antigen-binding fragment or chain thereof is targeted for integration at or near one of the at least one target site via homology-directed repair (HDR).

In some of any such embodiments, also provided are methods for producing a genetically engineered immune cell comprising introducing into an immune cell having a genetic disruption of at least one target site in a T cell receptor alpha constant (TRAC) gene and a genetic disruption of at least one target site in a T cell receptor beta constant (TRBC) gene, a template polynucleotide comprising a transgene encoding a recombinant receptor, which is a recombinant T cell receptor (TCR) or an antigen-binding fragment or chain thereof, wherein the genetic disruptions are caused by at least one agent that is capable of causing a genetic disruption of a target site in a TRAC gene and at least one agent that is capable of causing a genetic disruption in a TRBC gene, and the transgene encoding the recombinant receptor or antigen-binding fragment or chain thereof is targeted for integration into or near one of the at least one target site via homology-directed repair (HDR). In some of any such variants, the TRBC gene is one or both of the T cell receptor beta constant 1 ( TRBC1 ) or T cell receptor beta constant 2 ( TRBC2 ) genes.

In certain embodiments, the one or more agents capable of causing genetic disruption comprise a DNA-binding protein or a DNA-binding nucleic acid that specifically binds to or hybridizes to a target site. In some embodiments, the one or more agents capable of causing genetic disruption comprise (a) a fusion protein comprising a DNA-targeting protein and a nuclease, or (b) an RNA-guided nuclease. In particular embodiments, the DNA-targeting protein or RNA-guided nuclease comprises a zinc finger protein (ZFP), a TAL protein, or a clustered regularly intervening short padlingromic nucleic acid (CRISPR)-related nuclease (Cas) specific for a target site.

In certain embodiments, one or more agents comprise a zinc finger nuclease (ZFN), a TAL effector nuclease (TALEN), and/or a CRISPR-Cas9 combination that specifically bind to, recognize, or hybridize to a target site. In some embodiments, each of the one or more agents comprises a guide RNA (gRNA) having a targeting domain that is complementary to at least one target site. In some of any such embodiments, each of the one or more agents comprises a CRISPR-Cas9 combination, and the CRISPR-Cas9 combination comprises a guide RNA (gRNA) having a targeting domain that is complementary to at least one target site. In particular embodiments, one or more agents are administered as a ribonucleoprotein (RNP) complex comprising the gRNA and the Cas9 protein. In some of any such variants, the CRISPR-Cas9 combination is a ribonucleoprotein (RNP) complex containing gRNA and Cas9 protein. In some of any such variants, the RNP concentration is from about 1 μM to about 5 μM. In some of any such variants, the RNP concentration is about 2 μM.

In certain embodiments, the RNP is administered via electroporation, gene gun, calcium phosphate transfection, cell condensation or cell squeezing. In some embodiments, the RNP is administered via electroporation. In particular embodiments, one or more agents are administered as one or more polynucleotides encoding gRNA and/or Cas9 protein. In certain embodiments, at least one target site is within an exon of a TRAC , TRBC1 and/or TRBC2 gene. In some of any such embodiments, at least one target site is within an exon of a TRAC and an exon of a TRBC1 or TRBC2 gene.

In some embodiments, the gRNA has a targeting domain that is complementary to a target site in the TRAC gene and comprises a sequence selected from the group consisting of UCUCUCAGCUGGUACACGGC (SEQ ID NO:28), UGGAUUUAGAGUCUCUCAGC (SEQ ID NO:29), ACACGGCAGGGUCAGGGUUC (SEQ ID NO:30), GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:31), GCUGGUACACGGCAGGGUCA (SEQ ID NO:32), CUCAGCUGGUACACGGC (SEQ ID NO:33), UGGUACACGGCAGGGUC (SEQ ID NO:34), GCUAGACAUGAGGUCUA (SEQ ID NO:35), GUCAGAUUUGUUGCUCC (SEQ ID NO:36), UCAGCUGGUACACGGCA (SEQ ID NO:37), GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:38), GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:39), GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:40), GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:41), GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:42), GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:43), GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:44), GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:45), GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:46), GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:47), GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:48), GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:49), GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:50), GAGAAUCAAAAUCGGUGAAU (SEQ ID NO NO:37), GCAGACAGACUUGUCAC (SEQ ID NO:38), GGUACACGGCAGGGUCA (SEQ ID NO:39), CUUCAAGAGCAACAGUGCUG (SEQ ID NO:40), AGAGCAACAGUGCUGUGGCC (SEQ ID NO:41), AAAGUCAGAUUUGUUGCUCC (SEQ ID NO:42), ACAAAACUGUGCUAGACAUG (SEQ ID NO:43), AAACUGUGCUAGACAUG (SEQ ID NO:44), UGUGCUAGACAUGAGGUCUA (SEQ ID NO:45), GGCUGGGGAAGAAGGUGUCUUC (SEQ ID NO:46), GCUGGGGAAGAAGGUGUCUUC (SEQ ID NO:47), GGGGAAGAAGGUGUCUUC (SEQ ID NO:48), GUUUUGUCUGUGAUAUACACAU (SEQ ID NO:49), GGCAGACAGACUUGUCACUGGAUU (SEQ ID NO:50), GCAGACAGACUUGUCACUGGAUU (SEQ ID NO:51), GACAGACUUGUCACUGGAUU (SEQ ID NO:52), GUGAAUAGGCAGACAGACUUGUCA (SEQ ID NO:53), GAAUAGGCAGACAGACUUGUCA (SEQ ID NO:54), GAGUCUCUCAGCUGGUACACGG (SEQ ID NO:55), GUCUCUCAGCUGGUACACGG (SEQ ID NO:56), GGUACACGGCAGGGUCAGGGUU (SEQ ID NO:57), and GUACACGGCAGGGUCAGGGUU (SEQ ID NO:58). In particular embodiments, the gRNA has a targeting domain comprising the sequence GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:31).

In certain embodiments, the gRNA has a targeting domain that is complementary to a target site in one or both of the TRBC1 and TRBC2 genes and comprises a sequence selected from the group consisting of CACCCAGAUCGUCAGCGCCG (SEQ ID NO:59), CAAACACAGCGACCUCGGGU (SEQ ID NO:60), UGACGAGUGGACCCAGGAUA (SEQ ID NO:61), GGCUCUCGGAGAAUGACGAG (SEQ ID NO:62), GGCCUCGGCGCUGACGAUCU (SEQ ID NO:63), GAAAAACGUGUUCCCACCCG (SEQ ID NO:64), AUGACGAGUGGACCCAGGAU (SEQ ID NO:65), AGUCCAGUUCUACGGGCUCU (SEQ ID NO:66), CGCUGUCAAGUCCAGUUCUA (SEQ ID NO:67), AUCGUCAGCGCCGAGGCCUG (SEQ ID NO:68), UCAAACACAGCGACCUCGGG (SEQ ID NO:69), CGUAGAACUGGACUUGACAG (SEQ ID NO:70), AGGCCUCGGCGCUGACGAUC (SEQ ID NO:71), UGACAGCGGAAGUGGUUGCG (SEQ ID NO:72), UUGACAGCGGAAGUGGUUGC (SEQ ID NO:73), UCUCCGAGAGCCCGUAGAAC (SEQ ID NO:74), CGGGUGGGAACACGUUUUUC (SEQ ID NO:75), GACAGGUUUGGCCCUAUCCU (SEQ ID NO:76), GAUCGUCAGCGCCGAGGCCU (SEQ ID NO:77), GGCUCAAACACAGCGACCUC (SEQ ID NO:78), UGAGGUCUCGGCCACCUUC (SEQ ID NO:79) AGGCUUCUACCCCGACCACG (SEQ ID NO:80), CCGACCACGUGGAGCUGAGC (SEQ ID NO:81), UGACAGGUUUGGCCCUAUCC (SEQ ID NO:82), CUUGACAGCGGAAGUGGUUG (SEQ ID NO:83), AGAUCGUCAGCGCCGAGGCC (SEQ ID NO:84), GCGCUGACGAUCUGGGUGAC (SEQ ID NO:85), UGAGGGCGGGGCUGCUCCUUG (SEQ ID NO:86), GUUGCGGGGGUUCUGCCAGA (SEQ ID NO:87), AGCUCAGCUCCACGUGGUCG (SEQ ID NO:88), GCGGCUGCUCAGGCAGUAUC (SEQ ID NO:89), GCGGGGGUUCUGCCAGAAGG (SEQ ID NO:90), UGGCUCAAACACAGCGACCU (SEQ ID NO:91), ACUGGACUUGACAGCGGAAG (SEQ ID NO:92), GACAGCGGAAGUGGUUGCGG (SEQ ID NO:93), GCUGUCAAGUCCAGUUCUAC (SEQ ID NO:94), GUAUCUGGAGUCAUUGAGGG (SEQ ID NO:95), CUCGGCGCUGACGAUCU (SEQ ID NO:96), CCUCGGCGCUGACGAUC (SEQ ID NO:97), CCGAGAGCCCGUAGAAC (SEQ ID NO:98), CCAGAUCGUCAGCGCCG (SEQ ID NO:99), GAAUGACGAGUGGACCC (SEQ ID NO:100), GGGUGACAGGUUUGGCCCUAUC (SEQ ID NO:101), GGUGACAGGUUUGGCCCUAUC (SEQ ID NO:102), GUGACAGGUUUGGCCCUAUC (SEQ ID NO:103), GACAGGUUUGGCCCUAUC (SEQ ID NO:104), GAUACUGCCUGAGCAGCCGCCU (SEQ ID NO:105), GACCACGUGGAGCUGAGCUGGUGG (SEQ ID NO:106), GUGGAGCUGAGCUGGUGG (SEQ ID NO:107), GGGCGGGCUGCUCCUUGAGGGGCU (SEQ ID NO:108), GGCGGGCUGCUCCUUGAGGGGCU (SEQ ID NO:109), GCGGGCUGCUCCUUGAGGGGCU (SEQ ID NO:110), GGGCUGCUCCUUGAGGGGCU (SEQ ID NO:111), GGCUGCUCCUUGAGGGGCU (SEQ ID NO:112), GCUGCUCCUUGAGGGGCU (SEQ ID NO:113), GGUGAAUGGGAAGGAGGUGCACAG (SEQ ID NO:114), GUGAAUGGGAAGGAGGUGCACAG (SEQ ID NO:115), and GAAUGGGAAGGAGGUGCACAG (SEQ ID NO:116). In some embodiments, the gRNA has a targeting domain comprising the sequence GGCCUCGGCGCUGACGAUCU (SEQ ID NO:63).

In particular embodiments, the template polynucleotide comprises a [5' homology arm]-[transgene]-[3' homology arm] structure. In certain embodiments, the 5' homology arm and the 3' homology arm comprise nucleic acid sequences that are homologous to nucleic acid sequences surrounding at least one target site. In some embodiments, the 5' homology arm comprises nucleic acid sequences that are homologous to nucleic acid sequences 5' of the target site. In particular embodiments, the 3' homology arm comprises nucleic acid sequences that are homologous to nucleic acid sequences 3' of the target site.

In certain embodiments, the 5' homology arm and the 3' homology arm independently have at least, or at least about, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides, or less, or less than about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides. In some embodiments, the 5' homology arm and the 3' homology arm are independently between about 50 and about 100, 100 and 250, 250 and 500, 500 and 750, 750 and 1000, 1000 and 2000 nucleotides. In some of any such embodiments, the 5' homology arm and the 3' homology arm are independently between about 50 and about 100 nucleotides in length, between about 100 and about 250 nucleotides in length, between about 250 and about 500 nucleotides in length, between about 500 and about 750 nucleotides in length, between about 750 and about 1000 nucleotides in length, or between about 1000 and about 2000 nucleotides in length.

In particular embodiments, the 5' homology arm and the 3' homology arm independently have from or about 100 to 1000 nucleotides, from 100 to 750 nucleotides, from 100 to 600 nucleotides, from 100 to 400 nucleotides, from 100 to 300 nucleotides, from 100 to 200 nucleotides, from 200 to 1000 nucleotides, from 200 to 750 nucleotides, from 200 to 600 nucleotides, from 200 to 400 nucleotides, from 200 to 300 nucleotides, from 300 to 1000 nucleotides, from 300 to 750 nucleotides, from 300 to 600 nucleotides, from 300 to 400 nucleotides, from 400 to 1000 nucleotides, 400 to 750 nucleotides, 400 to 600 nucleotides, 600 to 1000 nucleotides, 600 to 750 nucleotides, or 750 to 1000 nucleotides. In some of any such embodiments, the 5' homology arm and the 3' homology arm are independently from about 100 to about 1000 nucleotides, from 100 to 750 nucleotides, from 100 to 600 nucleotides, from 100 to 400 nucleotides, from 100 to 300 nucleotides, from 100 to 200 nucleotides, from 200 to 1000 nucleotides, from 200 to 750 nucleotides, from 200 to 600 nucleotides, from 200 to 400 nucleotides, from 200 to 300 nucleotides, from 300 to 1000 nucleotides, from 300 to 750 nucleotides, from 300 to 600 nucleotides, from 300 to 400 nucleotides, from 400 to 1000 nucleotides, 400 to 750 nucleotides, 400 to 600 nucleotides, 600 to 1000 nucleotides, 600 to 750 nucleotides, or 750 to 1000 nucleotides in length.

In some of any such embodiments, the 5' homology arm and the 3' homology arm are independently about 200, 300, 400, 500, 600, 700, or 800 nucleotides in length, or any value between any of the foregoing. In some of any such embodiments, the 5' homology arm and the 3' homology arm are independently greater than about 300 nucleotides in length, optionally wherein the 5' homology arm and the 3' homology arm are independently about 400, 500, or 600 nucleotides in length, or any value between any of the foregoing. In some of any such embodiments, the 5' homology arm and the 3' homology arm are independently greater than about 300 nucleotides in length.

In some of any such embodiments, the transgene encoding the recombinant receptor, or antigen-binding fragment or chain thereof, is targeted for integration at or near a target site in the TRAC gene. In some of any such embodiments, the transgene encoding the recombinant receptor, or antigen-binding fragment or chain thereof, is targeted for integration at or near a target site in one or both of the TRBC1 and TRBC2 genes. In some of any such embodiments, the recombinant receptor is a recombinant TCR comprising an alpha (TCRα) chain and a beta (TCRβ) chain, and the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof comprises a nucleic acid sequence encoding a TCRα chain and a nucleic acid sequence encoding a TCRβ chain. In some of any such variants, the transgene further comprises one or more multicistronic elements, and the multicistronic elements are located between a nucleic acid sequence encoding TCRα or a portion thereof and a nucleic acid sequence encoding TCRβ or a portion thereof. In some of any such variants, the multicistronic elements comprise a sequence encoding a ribosome skip element selected from T2A, P2A, E2A, or F2A or an internal ribosome entry site (IRES).

In some of any such embodiments, the recombinant receptor is a recombinant TCR and the transgene encoding the recombinant TCR or an antigen-binding fragment or chain thereof comprises a nucleic acid sequence encoding one chain of the recombinant TCR and the second transgene comprises a nucleic acid sequence encoding the other chain of the recombinant TCR. In some of any such embodiments, the transgene encoding the recombinant TCR or an antigen-binding fragment or chain thereof comprises a nucleic acid sequence encoding a TCRα chain and the second transgene comprises a nucleic acid sequence encoding a TCRβ chain or a portion thereof.

In certain embodiments, a transgene encoding a recombinant TCR or antigen-binding fragment or chain thereof is targeted for integration at or near a target site in a TRAC gene. In some embodiments, a transgene encoding a recombinant TCR or antigen-binding fragment or chain thereof is targeted for integration at or near a target site in one or both of the TRBC1 and TRBC2 genes. In particular embodiments, comprising introducing into an immune cell one or more second template polynucleotides comprising one or more second transgenes, wherein the second transgene is targeted for integration at or near one of the at least one target site via homology-directed repair (HDR).

In certain embodiments, the second template polynucleotide comprises a structure of [second 5' homology arm]-[one or more second transgenes]-[second 3' homology arm]. In some embodiments, the second 5' homology arm and the second 3' homology arm comprise nucleic acid sequences that are homologous to nucleic acid sequences surrounding at least one target site. In particular embodiments, the second 5' homology arm comprises nucleic acid sequences that are homologous to nucleic acid sequences of a second 5' target site.

In certain embodiments, the second 3' homology arm comprises nucleic acid sequences that are homologous to nucleic acid sequences of the second 3' target site. In some embodiments, the second 5' homology arm and the second 3' homology arm independently have at least or at least about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500 or 2000 nucleotides or less or less than about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500 or 2000 nucleotides. In particular embodiments, the second 5' homology arm and the second 3' homology arm independently have between about 50 and 100, 100 and 250, 250 and 500, 500 and 750, 750 and 1000, 1000 and 2000 nucleotides. In certain embodiments, the second 5' homology arm and the second 3' homology arm independently have from or about 100 to 1000 nucleotides, from 100 to 750 nucleotides, from 100 to 600 nucleotides, from 100 to 400 nucleotides, from 100 to 300 nucleotides, from 100 to 200 nucleotides, from 200 to 1000 nucleotides, from 200 to 750 nucleotides, from 200 to 600 nucleotides, from 200 to 400 nucleotides, from 200 to 300 nucleotides, from 300 to 1000 nucleotides, from 300 to 750 nucleotides, from 300 to 600 nucleotides, from 300 to 400 nucleotides, from 400 to 1000 nucleotides, 400 to 750 nucleotides, 400 to 600 nucleotides, 600 to 1000 nucleotides, 600 to 750 nucleotides, or 750 to 1000 nucleotides.

In some embodiments, one or more second transgenes are targeted for integration at or near a target site in a TRAC gene. In particular embodiments, one or more second transgenes are targeted for integration at or near a target site in a TRBC1 or TRBC2 gene. In certain embodiments, a transgene encoding a recombinant TCR or antigen-binding fragment or chain thereof is targeted for integration at or near a target site in a TRAC gene, a TRBC1 gene, or a TRBC2 gene, and one or more second transgenes are targeted for integration at or near one or more target sites that are not targeted by the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof. In some of any such embodiments, the transgene encoding the recombinant receptor or antigen-binding fragment or chain thereof is targeted for integration at or near a target site in the TRAC gene, the TRBC1 gene, or the TRBC2 gene, and one or more second transgenes are targeted for integration at or near one or more other target sites among the TRAC gene, the TRBC1 gene, or the TRBC2 gene, and which are not targeted by the transgene encoding the recombinant receptor or antigen-binding fragment or chain thereof. In some embodiments, the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof is targeted for integration at or near a target site in the TRAC gene, and one or more second transgenes are targeted for integration at or near one or more target sites in the TRBC1 gene and/or the TRBC2 gene. In some of any such embodiments, a transgene encoding a recombinant receptor or antigen-binding fragment or chain thereof is targeted for integration at or near a target site in a TRAC gene, and one or more second transgenes are targeted for integration at or near one or more target sites in a TRBC1 gene and/or a TRBC2 gene.

In particular embodiments, the one or more second transgenes encode a molecule selected from a costimulatory ligand, a cytokine, a soluble single-stranded variable fragment (scFv), an immunomodulatory fusion protein, a chimeric switch receptor (CSR), or a coreceptor. In certain embodiments, the encoded molecule is a costimulatory ligand, optionally selected from a tumor necrosis factor (TNF) ligand selected from 4-1BBL, OX40L, CD70, LIGHT, and CD30L, or an immunoglobulin (Ig) superfamily ligand selected from CD80 and CD86. In some embodiments, the encoded molecule is a cytokine, optionally selected from IL-2, IL-3, IL-6, IL-11, IL-12, IL-7, IL-15, IL-21, granulocyte-monocyte colony-stimulating factor (GM-CSF), interferon alpha (IFN-α), interferon beta (IFN-β) or interferon gamma (IFN-γ) and erythropoietin. In specific embodiments, the encoded molecule is a soluble single-stranded variable fragment (scFv), which optionally binds a polypeptide that has an immunosuppressive effect or an immunostimulatory effect, selected from CD47, PD-1, CTLA-4 and their ligands or CD28, OX-40, 4-1BB and their ligands.

In certain embodiments, the encoded molecule is an immunomodulatory fusion protein optionally comprising: (a) an extracellular binding domain that specifically binds an antigen derived from CD200R, SIRPα, CD279 (PD-1), CD2, CD95 (Fas), CD152 (CTLA4), CD223 (LAG3), CD272 (BTLA), A2aR, KIR, TIM3, CD300, or LPA5; (b) an intracellular binding domain derived from CD3ε, CD3δ, CD3ζ, CD25, CD27, CD28, CD40, CD47, CD79A, CD79B, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD278 (ICOS), CD357 (GITR), XAPD11, DAP10, DAP12, FcRα, FcRβ, FcRγ, Fyn, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, ROR2, Ryk, Slp76, pTα, TCRα, TCRβ, TRFM, Zap70, PTCH2, or any combination thereof; and (c) a hydrophobic transmembrane domain derived from CD2, CD3ε, CD3δ, CD3ζ, CD25, CD27, CD28, CD40, CD79A, CD79B, CD80, CD86, CD95 (Fas), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD200R, CD223 (LAG3), CD270 (HVEM), CD272 (BTLA), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), CD279 (PD-1), CD300, CD357 (GITR), A2aR, DAP10, FcRα, FcRβ, FcRγ, Fyn, GAL9, KIR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPα, pTα, TCRα, TCRβ, TIM3, TRIM, LPA5 or Zap70. In some embodiments, the encoded molecule is a chimeric switch receptor (CSR), which optionally comprises a truncated extracellular domain of PD1 and the transmembrane and cytoplasmic signaling domains of CD28. In particular embodiments, the encoded molecule is a co-receptor, optionally selected from CD4 or CD8.

In certain embodiments, the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof encodes one chain of the recombinant TCR and the second transgene encodes another chain of the recombinant TCR. In some embodiments, the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof encodes an alpha (TCRα) chain of the recombinant TCR and the second transgene encodes a beta (TCRβ) chain of the recombinant TCR. In particular embodiments, the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof and/or one or more second transgenes independently further comprise a regulatory or control element. In certain embodiments, the regulatory or control element comprises a promoter, an enhancer, an intron, a polyadenylation signal, a Kozak consensus sequence, a splice acceptor sequence, or a splice donor sequence. In some embodiments, the regulatory or control element comprises a promoter. In particular embodiments, the promoter is selected from a constitutive promoter, an inducible promoter, a repressible promoter, and/or a tissue-specific promoter. In some embodiments, the promoter is selected from an RNA pol I, pol II, or pol III promoter.

In some of any such embodiments, the transgene encoding the recombinant receptor or antigen-binding fragment or chain thereof further comprises a regulatory or control element. In some of any such embodiments, the transgene encoding the recombinant receptor or antigen-binding fragment or chain thereof and/or one or more second transgenes independently further comprise a heterologous regulatory or control element. In some of any such embodiments, the heterologous regulatory or control element comprises a heterologous promoter. In some of any such embodiments, the heterologous promoter is or comprises the human elongation factor 1 alpha (EF1α) promoter or the MND promoter or a variant thereof. In some of any such embodiments, the heterologous promoter is an inducible promoter or a repressible promoter.

In particular embodiments, the promoter is selected from: a pol III promoter that is a U6 or H1 promoter; or a pol II promoter that is a CMV, SV40 early region, or adenovirus major late region promoter. In certain embodiments, the promoter is or comprises a human elongation factor 1 alpha (EF1α) promoter or a MND promoter or a variant thereof. In some embodiments, the promoter is an inducible promoter or a repressible promoter. In particular embodiments, the promoter comprises a Lac operator sequence, a tetracycline operator sequence, a galactose operator sequence, or a doxycycline operator sequence, or an analog thereof, or is capable of binding to or being recognized by a Lac repressor or a tetracycline repressor or an analog thereof.

In certain embodiments, the transgene encoding the recombinant TCR or antigen-binding fragment or chain and/or one or more second transgenes independently comprise one or more multicistronic elements. In some embodiments, the one or more multicistronic elements are located upstream of the transgene encoding the recombinant TCR or antigen-binding fragment or chain and/or one or more second transgenes. In particular embodiments, the multicistronic elements are located between the transgene encoding the recombinant TCR or antigen-binding fragment or chain and the one or more second transgenes. In certain embodiments, the multicistronic elements are located between the nucleic acid sequence encoding TCRα or a portion thereof and the nucleic acid sequence encoding TCRβ or a portion thereof. In some embodiments, the multicistronic elements comprise a sequence encoding a riboparticle skipping element selected from T2A, P2A, E2A, or F2A or an internal ribosome entry site (IRES). In some embodiments, the sequence encoding the riboparticle skipping element is targeted to be in frame with the gene at the target site.

In certain embodiments, a transgene encoding a recombinant TCR or antigen-binding fragment or chain thereof and/or one or more second transgenes are independently operably linked to an endogenous gene promoter at a target site.

In some of any such embodiments, the TCRα chain comprises a constant region (Cα) comprising an introduction of one or more cysteine residues and/or the TCRβ chain comprises a Cβ region comprising an introduction of one or more cysteine residues, wherein the one or more introduced cysteine residues are capable of forming one or more non-native disulfide bridges between the alpha chain and the beta chain. In some of any such embodiments, the introduction of the one or more cysteine residues comprises a substitution of a non-cysteine residue with a cysteine residue. In some of any such embodiments, the Cα region comprises a cysteine at a position corresponding to position 48 as numbered in any of SEQ ID NO: 24; and/or the Cβ region comprises a cysteine at a position corresponding to position 57 as numbered in SEQ ID NO: 20.

In some embodiments, the recombinant TCR is capable of binding to an antigen that is associated with, specific for, and/or expressed on a cell or tissue of a disease, disorder, or condition. In specific embodiments, the disease, disorder, or condition is an infectious disease, an autoimmune disease, an inflammatory disease, or a tumor or cancer. In certain embodiments, the antigen is a tumor antigen or a pathogenic antigen. In some embodiments, the pathogenic antigen is a bacterial antigen or a viral antigen.

In specific embodiments, the antigen is a viral antigen, and the viral antigen is hepatitis A virus, hepatitis B virus, hepatitis C virus (HCV), human papillomavirus (HPV), hepatitis B virus infections, Epstein-Barr virus (EBV), human herpes virus 8 (HHV-8), human T-cell leukemia virus-1 (HTLV-1), human T-cell leukemia virus-2 (HTLV-2), or cytomegalovirus (CMV). In certain embodiments, the antigen is an HPV antigen selected from HPV-16, HPV-18, HPV-31, HPV-33, and HPV-35. In some embodiments, the antigen is an HPV-16 antigen, which is an HPV-16 E6 or HPV-16 E7 antigen.

In specific embodiments, the viral antigen is an EBV antigen selected from Epstein-Barr nuclear antigen (EBNA)-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA leader protein (EBNA-LP), latent membrane proteins LMP-1, LMP-2A and LMP-2B, EBV-EA, EBV-MA and EBV-VCA. In certain embodiments, the viral antigen is an HTLV antigen that is TAX. In some embodiments, the viral antigen is an HBV antigen that is a hepatitis B core antigen or a hepatitis B envelope antigen.

In specific embodiments, the antigen is a tumor antigen. In certain embodiments, the antigen is selected from glioma-associated antigen, β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, Melanin-A/MART-1, WT-1, S-100, MBP, CD63, MUC1 ( e.g., MUC1-8), p53, Ras, cyclin B1, HER-2/neu, carcinoembryonic antigen (CEA), gp100, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A11, MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-C1, BAGE, GAGE-1, GAGE-2, pl5, tyrosinase, tyrosinase-related protein 1 (TRP-1), tyrosinase-related protein 2 (TRP-2), β-catenin, NY-ESO-1, LAGE-1a, PP1, MDM2, MDM4, EGVFvIII, Tax, SSX2, telomerase, TARP, pp65, CDK4, vimentin, S100, eIF-4A1, IFN-inducible p78, melanotransferrin (p97), Uroplakin II, prostate-specific antigen (PSA), human kallikrein (huK2), prostate specific membrane antigen (PSM) and prostatic acid phosphatase (PAP), neutrophil elastase, ephrin B2, BA-46, Bcr-abl, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Caspase 8, FRα, CD24, CD44, CD133, CD 166, epCAM, CA-125, HE4, Oval, estrogen receptor, progesterone receptor, uPA, PAI-1, CD19, CD20, CD22, ROR1, CD33/IL3Ra, c-Met, PSMA, Glycolipid F77, GD-2, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin.

In some of any such embodiments, the immune cells comprise or are enriched in T cells. In some of any such embodiments, the T cells comprise CD8+ T cells or subtypes thereof. In some of any such embodiments, the T cells comprise CD4+ T cells or subtypes thereof. In some of any such embodiments, the T cells comprise CD4+ T cells or subtypes thereof and CD8+ T cells or subtypes thereof. In some of any such embodiments, the T cells comprise CD4+ and CD8+ T cells and the ratio of CD4+ to CD8+ T cells is from about 1:3 to about 3:1. In some of any such embodiments, the ratio is from about 1:2 to about 2:1. In some of any such embodiments, the ratio is from about 1:1. In some embodiments, the immune cell is a T cell. In particular embodiments, the T cell is a CD8+ T cell or subtypes thereof. In certain embodiments, the T cell is a CD4+ T cell or subtypes thereof.

In some embodiments, the immune cell is derived from a multipotent or pluripotent cell, which is not necessarily an iPSC. In some of any such embodiments, the immune cell is a primary cell from a subject. In some of any such embodiments, the subject has or is suspected of having a disease or disorder. In some of any such embodiments, the subject is or is suspected of being healthy. In some of any such embodiments, the immune cell is autologous to the subject. In some of any such embodiments, the immune cell is allogeneic to the subject. In particular embodiments, the immune cell comprises a T cell that is autologous to the subject. In certain embodiments, the immune cell comprises a T cell that is autologous to the subject.

In some of any such embodiments, the template polynucleotides comprise one or more vectors, which are optionally viral vectors. In some of any such embodiments, the vector is a viral vector, and the viral vector is an AAV vector. In some of any such embodiments, the AAV vector is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and AAV8 vectors. In some of any such embodiments, the AAV vector is an AAV2 or AAV6 vector. In some of any such embodiments, the vector is a viral vector, and the viral vector is a retroviral vector. In some of any such embodiments, the viral vector is a lentiviral vector.

In some embodiments, the first template polynucleotide, one or more second template polynucleotides, and/or one or more polynucleotides encoding gRNA and/or Cas9 protein are contained in one or more vectors, which are not necessarily viral vectors. In particular embodiments, the vector is an AAV vector. In certain embodiments, the AAV vector is selected from an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, or AAV8 vector. In some embodiments, the AAV vector is an AAV2 or AAV6 vector. In particular embodiments, the viral vector is a retroviral vector. In certain embodiments, the viral vector is a lentiviral vector.

In some of any such embodiments, the template polynucleotide is at least about 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4760, 5000, 5250, 5500, 5750, 6000, 7000, 7500, 8000, 9000, or 10000 nucleotides in length, or any value between any of the foregoing. In some of any such embodiments, the polynucleotide is between about 2500 and about 5000 nucleotides, between about 3500 and about 4500 nucleotides, or between about 3750 nucleotides and about 4250 nucleotides in length.

In some embodiments, the administration of one or more agents capable of causing genetic destruction and the administration of the template polynucleotide are carried out simultaneously or sequentially, in any order. In particular embodiments, the administration of the template polynucleotide is carried out after the administration of one or more agents capable of causing genetic destruction. In certain embodiments, the template polynucleotide is administered immediately after or within about 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 6 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, or 4 hours after the administration of one or more agents capable of causing genetic destruction. In some of any such embodiments, the template nucleotide is administered within about 2 hours after the administration of the one or more agents.

In some of any such embodiments, the administration of one or more agents capable of causing genetic disruption and the administration of the template polynucleotide are carried out in a single experimental reaction. In some of any such embodiments, prior to administration of the one or more agents, the method comprises incubating cells, in vitro , with stimulatory agents under conditions of stimulation or activation of one or more immune cells. In some of any such embodiments, the stimulatory agents comprise both anti-CD3 and/or anti-CD28 antibodies, optionally anti-CD3/anti-CD28 beads. In some of any such embodiments, the ratio of beads to cells is about 1:1. In some of any such embodiments, the stimulatory agents are removed from the one or more immune cells prior to administration of the one or more agents.

In some of any such embodiments, the method further comprises incubating the cells before, during or after the administration of the one or more agents and/or the administration of the template polynucleotide with one or more recombinant cytokines. In some of any such embodiments, the one or more recombinant cytokines are selected from the group consisting of IL-2, IL-7 and IL-15. In some of any such embodiments, the one or more recombinant cytokines are added at a concentration selected from IL-2 at a concentration of about 10 U/ml to about 200 U/ml, optionally at a concentration of about 50 IU/ml to about 100 U/ml; IL-7 at a concentration of 0.5 ng/ml to 50 ng/ml. In some of any such embodiments, the concentration is from about 5 ng/mL to about 10 ng/mL, and/or IL-15 at a concentration of from 0.1 ng/mL to 20 ng/mL. In some of any such embodiments, the concentration is from about 0.5 ng/mL to about 5 ng/mL. In some of any such embodiments, the incubation is carried out after administration of one or more agents and administration of the template polynucleotide for up to about 24 hours, 36 hours, 48 hours, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days. In any of such embodiments, the administration is carried out for up to about 7 days.

In some embodiments, the administration of the template polynucleotide and the administration of one or more second template polynucleotides are carried out simultaneously or sequentially, in any order. In specific embodiments, the administration of one or more agents capable of causing genetic destruction and the administration of the template polynucleotide are carried out in a single experimental reaction. In certain embodiments, the administration of one or more agents capable of causing genetic destruction and the administration of the template polynucleotide and the second template polynucleotides are carried out in a single experimental reaction.

In some embodiments, at least or more than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the cells in the plurality of engineered cells comprise a genetic disruption of at least one target site in a gene encoding a domain or region of a T cell receptor constant alpha (TRAC) gene and/or a T cell receptor constant beta (TRBC) gene. In particular embodiments, at least or more than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the cells in the plurality of engineered cells express the recombinant receptor or antigen-binding fragment thereof and/or exhibit antigen binding or binding to an antigen. In certain embodiments, the coefficient of variation of the expression and/or antigen binding of the recombinant receptor or antigen-binding fragment thereof among the plurality of engineered cells is lower than 0.70, 0.65, 0.60, 0.55, 0.50, 0.45, 0.40, 0.35, or 0.30 or less. In some embodiments, the coefficient of variation of the expression and/or antigen binding of the recombinant receptor or antigen-binding fragment thereof among the plurality of engineered cells is at least 100%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% lower than the coefficient of variation of the expression and/or antigen binding of the same recombinant receptor that is integrated into the genome by random integration.

In specific embodiments, expression and/or antigen binding of the recombinant receptor or antigen-binding fragment thereof is assessed by contacting cells in the composition with a binding reagent specific for the TCRα chain or TCRβ chain and assessing binding of the reagent to the cells. In certain embodiments, the binding reagent is an anti-TCR Vβ antibody or is an anti-TCR Vα antibody that specifically recognizes a particular Vβ or Vα chain family. In some embodiments, the binding agent is a peptide antigen-MHC complex, which is optionally a tetramer.

Presented herein is an engineered cell or a plurality of engineered cells created using the method described herein.

In some of any such embodiments, provided herein is a method of treatment comprising administering an engineered cell, a plurality of engineered cells, or a composition to a subject in need thereof. In some of any such embodiments, the subject has a disease, disorder, or condition. In some of any such embodiments, the disease, disorder, or condition is cancer.

In some of any such embodiments, provided herein is the use of an engineered cell, a plurality of engineered cells, or a composition for treating a cancerous disease, disorder, or condition. In some of any such embodiments, the disease, disorder, or condition is cancer.

In some of any such embodiments, provided herein is the use of an engineered cell, a plurality of engineered cells, or a composition for the manufacture of a medicament for the treatment of a disease, disorder, or condition. In some of any such embodiments, the disease, disorder, or condition is cancer.

In some of any such embodiments, provided herein is the use of an engineered cell, a plurality of engineered cells, or a composition for use in the treatment of a cancerous disease, disorder, or condition. In some of any such embodiments, the disease, disorder, or condition is cancer.

Provided herein is a method of treatment comprising administering an engineered cell, a plurality of engineered cells, or a composition described herein to a subject. Provided herein is the use of an engineered cell, a plurality of engineered cells, or a composition described herein for treating cancer. Provided herein is the use of an engineered cell, a plurality of engineered cells, or a composition described herein in the manufacture of a medicament for treating cancer. In certain embodiments, an engineered cell, a plurality of engineered cells, or a composition described herein is provided for use in treating cancer.

In some of any such embodiments, provided herein is a kit comprising: one or more agents, wherein each of the one or more agents is independently capable of causing genetic disruption of a target site in the T cell receptor constant alpha (TRAC) gene and/or the T cell receptor constant beta (TRBC) gene; and a template polynucleotide comprising a transgene encoding a recombinant receptor or an antigen-binding fragment or chain thereof, wherein the transgene encoding the recombinant receptor or an antigen-binding fragment or chain thereof is targeted for integration at or near the target site via homology-directed repair (HDR), and instructions for performing the method of any of the embodiments described herein.

Also provided is a kit comprising: one or more agents, wherein each of the one or more agents is independently capable of causing genetic disruption of a target site in the T cell receptor constant alpha (TRAC) gene and/or the T cell receptor constant beta (TRBC) gene; and a template polynucleotide comprising a transgene encoding a recombinant TCR or an antigen-binding fragment or chain thereof, wherein the transgene encoding the recombinant TCR or an antigen-binding fragment or chain thereof is targeted for integration into or near the target site via homology-directed repair (HDR). In particular embodiments, the one or more agents capable of causing genetic disruption comprise a DNA binding protein or a DNA binding nucleic acid that specifically binds to or hybridizes to the target site.

In certain embodiments, the one or more agents capable of causing genetic disruption comprise (a) a fusion protein comprising a DNA-directed protein and a nuclease or (b) an RNA-directed nuclease. In some embodiments, the DNA-directed protein or RNA-directed nuclease comprises a zinc finger protein (ZFP), a TAL protein, or a clustered regular intermediate short padildromic nucleic acid (CRISPR)-related nuclease (Cas) specific for a target site. In particular embodiments, the one or more agents comprise a zinc finger nuclease (ZFN), a TAL effector nuclease (TALEN), and/or a CRISPR-Cas9 combination that specifically bind to, recognize, or hybridize to a target site. In certain embodiments, each of the one or more agents comprises a guide RNA (gRNA) having a targeting domain that is complementary to at least one target site.

In some embodiments, one or more agents are administered as a ribonucleoprotein (RNP) complex comprising gRNA and Cas9 protein. In particular embodiments, the RNP is administered via electroporation, gene gun, calcium phosphate transfection, cell condensation or cell compression. In certain embodiments, the RNP is administered via electroporation. In some embodiments, one or more agents are administered as one or more polynucleotides encoding gRNA and/or Cas9 protein. In particular embodiments, at least one target site is within an exon of a TRAC , TRBC1 and/or TRBC2 gene.

In certain embodiments, the gRNA has a targeting domain that is complementary to a target site in the TRAC gene and comprises a sequence selected from UCUCUCAGCUGGUACACGGC (SEQ ID NO:28), UGGAUUUAGAGUCUCUCAGC (SEQ ID NO:29), ACACGGCAGGGUCAGGGUUC (SEQ ID NO:30), GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:31), GCUGGUACACGGCAGGGUCA (SEQ ID NO:32), CUCAGCUGGUACACGGC (SEQ ID NO:33), UGGUACACGGCAGGGUC (SEQ ID NO:34), GCUAGACAUGAGGUCUA (SEQ ID NO:35), GUCAGAUUUGUUGCUCC (SEQ ID NO:36), UCAGCUGGUACACGGCA (SEQ ID NO:37), GCAGACAGACUUGUCAC (SEQ ID NO:38), GGUACACGGCAGGGUCA (SEQ ID NO:39), CUUCAAGAGCAACAGUGCUG (SEQ ID NO:40), AGAGCAACAGUGCUGUGGCC (SEQ ID NO:41), AAAGUCAGAUUUGUUGCUCC (SEQ ID NO:42), ACAAAACUGUGCUAGACAUG (SEQ ID NO:43), AAACUGUGCUAGACAUG (SEQ ID NO:44), UGUGCUAGACAUGAGGUCUA (SEQ ID NO:45), GGCUGGGGAAGAAGGUGUCUUC (SEQ ID NO:46), GCUGGGGAAGAAGGUGUCUUC (SEQ ID NO:47), GGGGAAGAAGGUGUCUUC (SEQ ID NO:48), GUUUUGUCUGUGAUAUACACAU (SEQ ID NO:49) GGCAGACAGACUUGUCACUGGAUU (SEQ ID NO:50), GCAGACAGACUUGUCACUGGAUU (SEQ ID NO:51), GACAGACUUGUCACUGGAUU (SEQ ID NO:52), GUGAAUAGGCAGACAGACUUGUCA (SEQ ID NO:53), GAAUAGGCAGACAGACUUGUCA (SEQ ID NO:54), GAGUCUCUCAGCUGGUACACGG (SEQ ID NO:55), GUCUCUCAGCUGGUACACGG (SEQ ID NO:56), GGUACACGGCAGGGUCAGGGUU (SEQ ID NO:57), and GUACACGGCAGGGUCAGGGUU (SEQ ID NO:58). In some embodiments, the gRNA has a targeting domain comprising the sequence GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:31).

In particular embodiments, the gRNA has a targeting domain that is complementary to a target site in one or more of the TRBC1 and TRBC2 genes and comprises a sequence selected from CACCCAGAUCGUCAGCGCCG (SEQ ID NO:59), CAAACACAGCGACCUCGGGU (SEQ ID NO:60), UGACGAGUGGACCCAGGAUA (SEQ ID NO:61), GGCUCUCGGAGAAUGACGAG (SEQ ID NO:62), GGCCUCGGCGCUGACGAUCU (SEQ ID NO:63), GAAAAACGUGUUCCCACCCG (SEQ ID NO:64), AUGACGAGUGGACCCAGGAU (SEQ ID NO:65), AGUCCAGUUCUACGGGCUCU (SEQ ID NO:66), CGCUGUCAAGUCCAGUUCUA (SEQ ID NO:67), AUCGUCAGCGCCGAGGCCUG (SEQ ID NO:68), AGUCCAGUUCUACGGGCUCU (SEQ ID NO:69), AGUCCAGUUCUACGGGCUCU (SEQ ID NO:70), AGUCCAGUUCUACGGGCUCU (SEQ ID NO:71), AGUCCAGUUCUACGGGCUCU (SEQ ID NO:72), AGUCCAGUUCUACGGGCUCU (SEQ ID NO:73), AGUCCAGUUCUACGGGCUCU (SEQ ID NO:74), AGUCCAGUUCUACGGGCUCU (SEQ ID NO:75), AGUCCAGUUCUACGGGCUCU (SEQ ID NO:76), AGUCCAGUUCUACGGGCUCU (SEQ ID NO:77), AGUCCAGUUCCAGUUCUACGGGCUCU (SEQ ID NO:78), AGUCCAGUUCCAGUUCUACGGGCUCU (SEQ ID NO:79), AGUCCAGUUCCAGUUCUACGGGCUCU (SEQ ID NO:80), AGUCCAGUUCCAGU ID NO:68), UCAAACACAGCGACCUCGGG (SEQ ID NO:69), CGUAGAACUGGACUUGACAG (SEQ ID NO:70), AGGCCUCGGCGCUGACGAUC (SEQ ID NO:71), UGACAGCGGAAGUGGUUGCG (SEQ ID NO:72), UUGACAGCGGAAGUGGUUGC (SEQ ID NO:73), UCUCCGAGAGCCCGUAGAAC (SEQ ID NO:74), CGGGUGGGAACACGUUUUUC (SEQ ID NO:75), GACAGGUUUGGCCCUAUCCU (SEQ ID NO:76), GAUCGUCAGCGCCGAGGCCU (SEQ ID NO:77), GGCUCAAACACAGCGACCUC (SEQ ID NO:78), UGAGGGUCUCGGCCACCUUC (SEQ ID NO:79), AGGCUUCUACCCCGACCACG (SEQ ID NO:80), CCGACCACGUGGAGCUGAGC (SEQ ID NO:81), UGACAGGUUUGGCCCUAUCC (SEQ ID NO:82), CUUGACAGCGGAAGUGGUUG (SEQ ID NO:83), AGAUCGUCAGCGCCGAGGCC (SEQ ID NO:84), GCGCUGACGAUCUGGGUGAC (SEQ ID NO:85), UGAGGGCGGGCUGCUCCUUG (SEQ ID NO:86), GUUGCGGGGGUUCUGCCAGA (SEQ ID NO:87), AGCUCAGCUCCACGUGGUCG (SEQ ID NO:88), GCGGCUGCUCAGGCAGUAUC (SEQ ID NO:89), GCGGGGGUUCUGCCAGAAGG (SEQ ID NO:90), UGGCUCAAACACAGCGACCU (SEQ ID NO:91), ACUGGACUUGACAGCGGAAG (SEQ ID NO:92) (SEQ ID NO:98), CCAGAUCGUCAGCGCCG (SEQ ID NO:99), GAAUGACGAGUGGACCC (SEQ ID NO:100), GGGUGACAGGUUUGGCCCUAUC (SEQ ID NO:101), GGUGACAGGUUUGGCCCUAUC (SEQ ID NO:102), GUGACAGGUUUGGCCCUAUC (SEQ ID NO:103), GACAGGUUUGGCCCUAUC (SEQ ID NO:104), GAUACUGCCUGAGCAGCCGCCU (SEQ ID NO:105), GACCACGUGGAGCUGAGCUGGUGG (SEQ ID NO:106), GUGGAGCUGAGCUGGUGG (SEQ ID NO:107), GGGCGGGCUGCUCCUUGAGGGGCU (SEQ ID NO:108), GGCGGGCUGCUCCUUGAGGGGCU (SEQ ID NO:109), GCGGGCUGCUCCUUGAGGGGCU (SEQ ID NO:110), GGGCUGCUCCUUGAGGGGCU (SEQ ID NO:111), GGCUGCUCCUUGAGGGGCU (SEQ ID NO:112), GCUGCUCCUUGAGGGGCU (SEQ ID NO:113), GGUGAAUGGGAAGGAGGUGCACAG (SEQ ID NO:114), GUGAAUGGGAAGGAGGUGCACAG (SEQ ID NO:115) and GAAUGGGAAGGAGGUGCACAG (SEQ ID NO:116). In certain embodiments, the gRNA has a targeting domain comprising the sequence GGCCUCGGCGCUGACGAUCU (SEQ ID NO:63).

In some embodiments, the template polynucleotide comprises a [5' homology arm]-[transgene]-[3' homology arm] structure. In particular embodiments, the 5' homology arm and the 3' homology arm comprise nucleic acid sequences that are homologous to nucleic acid sequences surrounding at least one target site. In certain embodiments, the 5' homology arm comprises nucleic acid sequences that are homologous to nucleic acid sequences 5' of a target site. In some embodiments, the 3' homology arm comprises nucleic acid sequences that are homologous to nucleic acid sequences 3' of a target site. In particular embodiments, the 5' homology arm and the 3' homology arm independently have at least or at least about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides, or less or less than about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides. In certain embodiments, the 5' homology arm and the 3' homology arm independently have between about 50 and 100, 100 and 250, 250 and 500, 500 and 750, 750 and 1000, 1000 and 2000 nucleotides.

In some embodiments, the 5' homology arm and the 3' homology arm are independently from or about 100 to 1000 nucleotides, 100 to 750 nucleotides, 100 to 600 nucleotides, 100 to 400 nucleotides, 100 to 300 nucleotides, 100 to 200 nucleotides, 200 to 1000 nucleotides, 200 to 750 nucleotides, 200 to 600 nucleotides, 200 to 400 nucleotides, 200 to 300 nucleotides, 300 to 1000 nucleotides, 300 to 750 nucleotides, 300 to 600 nucleotides, 300 to 400 nucleotides, 400 to 1000 nucleotides, 400 to 750 nucleotides, 400 to 600 nucleotides, 600 to 1000 nucleotides, 600 to 750 nucleotides, or 750 to 1000 nucleotides. In particular embodiments, the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof is targeted for integration at or near a target site in the TRAC gene. In certain embodiments, the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof is targeted for integration at or near a target site in one or both of the TRBC1 and TRBC2 genes . In some aspects, the kit further comprises one or more second template polynucleotides comprising one or more second transgenes, wherein the second transgene is targeted for integration at or near one of the at least one target site via homology-directed repair (HDR).

In particular embodiments, the second template polynucleotide comprises a [second 5' homology arm]-[one or more second transgenes]-[second 3' homology arm] structure. In certain embodiments, the second 5' homology arm and the second 3' homology arm comprise nucleic acid sequences that are homologous to nucleic acid sequences surrounding at least one target site. In some embodiments, the second 5' homology arm comprises nucleic acid sequences that are homologous to nucleic acid sequences of a second 5' target site. In particular embodiments, the second 3' homology arm comprises nucleic acid sequences that are homologous to nucleic acid sequences of a second 3' target site. In certain embodiments, the second 5' homology arm and the second 3' homology arm independently have at least or at least about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500 or 2000 nucleotides, or less or less than about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500 or 2000 nucleotides. In some embodiments, the second 5' homology arm and the second 3' homology arm independently have between about 50 and 100, 100 and 250, 250 and 500, 500 and 750, 750 and 1000, 1000 and 2000 nucleotides.

In particular embodiments, the second 5' homology arm and the second 3' homology arm independently have from or about 100 to 1000 nucleotides, from 100 to 750 nucleotides, from 100 to 600 nucleotides, from 100 to 400 nucleotides, from 100 to 300 nucleotides, from 100 to 200 nucleotides, from 200 to 1000 nucleotides, from 200 to 750 nucleotides, from 200 to 600 nucleotides, from 200 to 400 nucleotides, from 200 to 300 nucleotides, from 300 to 1000 nucleotides, from 300 to 750 nucleotides, from 300 to 600 nucleotides, from 300 to 400 nucleotides, from 400 to 1000 nucleotides, 400 to 750 nucleotides, 400 to 600 nucleotides, 600 to 1000 nucleotides, 600 to 750 nucleotides, or 750 to 1000 nucleotides.

In certain embodiments, one or more second transgenes are targeted for integration at or near a target site in a TRAC gene. In some embodiments, one or more second transgenes are targeted for integration at or near a target site in a TRBC1 or TRBC2 gene. In particular embodiments, a transgene encoding a recombinant TCR or antigen-binding fragment or chain thereof is targeted for integration at or near a target site in a TRAC gene, a TRBC1 gene, or a TRBC2 gene, and one or more second transgenes are targeted for integration at or near one or more target sites that are not targeted by the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof.

In certain embodiments, a transgene encoding a recombinant TCR or antigen-binding fragment or chain thereof is targeted for integration at or near a target site in a TRAC gene, and one or more second transgenes are targeted for integration at or near one or more target sites in a TRBC1 gene and/or a TRBC2 gene. In some embodiments, one or more second transgenes encode a molecule selected from a costimulatory ligand, a cytokine, a soluble single-strand variable fragment (scFv), an immunomodulatory fusion protein, a chimeric switch receptor (CSR), or a coreceptor. In particular embodiments, the encoded molecule is a costimulatory ligand optionally selected from tumor necrosis factor (TNF), a ligand selected from 4-1BBL, OX40L, CD70, LIGHT and CD30L, or an immunoglobulin (Ig) superfamily ligand selected from CD80 and CD86. In certain embodiments, the encoded molecule is a cytokine optionally selected from IL-2, IL-3, IL-6, IL-11, IL-30, IL-7, IL-24, IL-30, granulocyte monocyte colony stimulating factor (GM-CSF), interferon alpha (IFN-α), interferon beta (IFN-β) or interferon gamma (IFN-γ), and erythropoietin.

In some embodiments, the encoded molecule is a soluble single-stranded variable fragment (scFv) that optionally binds a polypeptide that has an immunosuppressive effect or an immunostimulatory effect selected from CD47, PD-1, CTLA-4 and their ligands, or CD28, OX-40, 4-1BB and their ligands. In particular embodiments, the encoded molecule is an immunomodulatory fusion protein optionally comprising: (a) an extracellular binding domain that specifically binds an antigen derived from CD290R, SIRPα, CD279 (PD-1), CD2, CD95 (Fas), CD242 (CTLA4), CD223 (LAG3), CD272 (BTLA), A2aR, KIR, TIM3, CD300, or LPA5; (b) an intracellular binding domain derived from CD3ε, CD3δ, CD3ζ, CD25, CD27, CD28, CD40, CD47, CD79A, CD79B, CD224 (OX40), CD227 (4-1BB), CD240 (SLAMF1), CD278 (ICOS), CD357 (GITR), XAPD11, DAP10, DAP30, FcRα, FcRβ, FcRγ, Fyn, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, ROR2, Ryk, Slp76, pTα, TCRα, TCRβ, TRFM, Zap70, PTCH2, or any combination thereof; and (c) a hydrophobic transmembrane domain derived from CD2, CD3ε, CD3δ, CD3ζ, CD25, CD27, CD28, CD40, CD79A, CD79B, CD80, CD86, CD95 (Fas), CD224 (OX40), CD227 (4-1BB), CD240 (SLAMF1), CD242 (CTLA4), CD290R, CD223 (LAG3), CD270 (HVEM), CD272 (BTLA), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), CD279 (PD-1), CD300, CD357 (GITR), A2aR, DAP10, FcRα, FcRβ, FcRγ, Fyn, GAL9, KIR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPα, pTα, TCRα, TCRβ, TIM3, TRIM, LPA5, or Zap70. In certain embodiments, the encoded molecule is a chimeric switch receptor (CSR) that optionally comprises a truncated extracellular domain of PD1 and transmembrane and cytoplasmic signaling domains of CD28.

In some embodiments, the encoded molecule is a co-receptor, optionally selected from CD4 or CD8. In particular embodiments, the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof encodes one chain of the recombinant TCR and the second transgene encodes another chain of the recombinant TCR. In certain embodiments, the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof encodes the alpha (TCRα) chain of the recombinant TCR and the second transgene encodes the beta (TCRβ) chain of the recombinant TCR. In some embodiments, the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof and/or one or more second transgenes independently further comprise a regulatory or control element.

In particular embodiments, the regulatory or control element comprises a promoter, an enhancer, an intron, a polyadenylation signal, a Kozak consensus sequence, a splice acceptor sequence, or a splice donor sequence. In certain embodiments, the regulatory or control element comprises a promoter. In some embodiments, the promoter is selected from a constitutive promoter, an inducible promoter, a repressible promoter, and/or a tissue-specific promoter. In particular embodiments, the promoter is selected from an RNA pol I, pol II, or pol III promoter. In certain embodiments, the promoter is selected from: a pol III promoter that is a U6 or H1 promoter; or a pol II promoter that is a CMV, SV40 early region, or adenovirus major late region promoter. In some embodiments, the promoter is or comprises the human elongation factor 1 alpha (EF1α) promoter or the MND promoter, or a variant thereof. In particular embodiments, the promoter is an inducible promoter or a repressible promoter. In certain embodiments, the promoter comprises a Lac operator sequence, a tetracycline operator sequence, a galactose operator sequence, or a doxycycline operator sequence, or is an analog thereof, or is capable of binding to or being recognized by a Lac repressor or a tetracycline repressor, or an analog thereof.

In some embodiments, the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof and/or one or more second transgenes independently comprises one or more multicistronic elements. In particular embodiments, the one or more multicistronic elements are located upstream of the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof and/or one or more second transgenes. In certain embodiments, the multicistronic elements are located between the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof and the one or more second transgenes. In some embodiments, the multicistronic elements are located between the nucleic acid sequence encoding TCRα or a portion thereof and the nucleic acid sequence encoding TCRβ or a portion thereof. In particular embodiments, the multicistronic elements comprise a sequence encoding a ribosome-defined skip-over element selected from T2A, P2A, E2A, or F2A, or an internal ribosome entry site (IRES). In particular embodiments, the sequence encoding the ribosome-defined skip-over element is targeted to be in-frame with the gene at the target site.

In some embodiments, in HDR, the transgene encoding the recombinant TCR or an antigen-binding fragment or chain thereof, and/or one or more second transgenes are independently operably linked to an endogenous gene promoter at a target site. In particular embodiments, the recombinant TCR is capable of binding to an antigen that is associated with, specific for, and/or expressed on a cell or tissue of a disease, disorder, or condition. In certain embodiments, the disease, disorder, or condition is an infectious disease, an autoimmune disease, an inflammatory disease, or a tumor or cancer. In some embodiments, the antigen is a tumor antigen or a pathogenic antigen. In particular embodiments, the pathogenic antigen is a bacterial antigen or a viral antigen. In certain embodiments, the antigen is a viral antigen, and the viral antigen is hepatitis A virus, hepatitis B virus, hepatitis C virus (HCV), human papillomavirus (HPV), hepatitis B virus infections, Epstein-Barr virus (EBV), human herpes virus 8 (HHV-8), human T-cell leukemia virus-1 (HTLV-1), human T-cell leukemia virus-2 (HTLV-2), or cytomegalovirus (CMV). In some embodiments, the antigen is an antigen from HPV selected from HPV-25, HPV-27, HPV-31, HPV-33, and HPV-35.

In specific embodiments, the antigen is an HPV-25 antigen that is an HPV-25 E6 or HPV-25 E7 antigen. In certain embodiments, the viral antigen is an EBV antigen selected from Epstein-Barr nuclear antigen (EBNA)-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA leader protein (EBNA-LP), latent membrane proteins LMP-1, LMP-2A and LMP-2B, EBV-EA, EBV-MA and EBV-VCA. In some embodiments, the viral antigen is an HTLV antigen that is TAX. In specific embodiments, the viral antigen is an HBV antigen that is a hepatitis B core antigen or a hepatitis B envelope antigen. In certain embodiments, the antigen is a tumor antigen.

In some embodiments, the antigen is selected from glioma-associated antigen, β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, Melanin-A/MART-1, WT-1, S-100, MBP, CD63, MUC1 ( e.g., MUC1-8), p53, Ras, cyclin B1, HER-2/neu, carcinoembryonic antigen (CEA), gp100, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A11, MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-C1, BAGE, GAGE-1, GAGE-2, pl5, tyrosinase, tyrosinase-related protein 1 (TRP-1), tyrosinase-related protein 2 (TRP-2), β-catenin, NY-ESO-1, LAGE-1a, PP1, MDM2, MDM4, EGVFvIII, Tax, SSX2, telomerase, TARP, pp65, CDK4, vimentin, S100, eIF-4A1, IFN-inducible p78, melanotransferrin (p97), Uroplakin II, prostate-specific antigen (PSA), human kallikrein (huK2), prostate specific membrane antigen (PSM), and prostatic acid phosphatase (PAP), neutrophil elastase, ephrin B2, BA-46, Bcr-abl, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Caspase 8, FRα, CD24, CD44, CD223, CD 256, epCAM, CA-224, HE4, Oval, estrogen receptor, progesterone receptor, uPA, PAI-1, CD28, CD29, CD22, ROR1, CD33/IL3Ra, c-Met, PSMA, Glycolipid F77, GD-2, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin.

In particular embodiments, the first template polynucleotide, one or more second template polynucleotides, and/or one or more polynucleotides encoding gRNA and/or Cas9 protein are contained in one or more vectors, which are not necessarily viral vectors. In certain embodiments, the vector is an AAV vector. In some embodiments, the AAV vector is selected from an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, or AAV8 vector. In particular embodiments, the AAV vector is an AAV2 or AAV6 vector. In certain embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector.

Brief description of the drawings

FIG. 1A depicts the surface expression of CD8 and a peptide-MHC tetramer complexed with an antigen recognized by a typical recombinant TCR (TCR #1), as assessed by flow cytometry, for T cells subjected to knockout of endogenous TCR encoding genes engineered to express TCR #1 using different expression methods: cells subjected to lentiviral transduction to randomly integrate recombinant TCR encoding sequences (“TCR #1 Lenti”), cells subjected to random integration and CRISPR/Cas9 mediated knockout (KO) of TRAC (“TCR #1 Lenti KO”); or cells subjected to targeted integration via HDR at the TRAC locus of recombinant TCR encoding sequences under the control of the human EF1α promoter (TCR #1 HDR KO). FIG. 1B and 1C show the mean fluorescence intensity (MFI; FIG. 1B ) and coefficient of variation (standard deviation of signal in a cell population divided by the mean signal in the corresponding population; FIG. 1C ) of cell surface expression of peptide-MHC tetramer binding in CD8+ T cells engineered to express TCR #1.

FIG. 2A depicts the surface expression of CD8 and a peptide-MHC tetramer complexed with an antigen recognized by a typical recombinant TCR (TCR #2), as assessed by flow cytometry, for T cells subjected to knockout of endogenous TCR encoding genes engineered to express TCR #2 using different expression methods: cells subjected to lentiviral transduction to randomly integrate recombinant TCR encoding sequences (“TCR #2 Lenti”), cells subjected to random integration and CRISPR/Cas9 mediated knockout (KO) of TRAC (“TCR #2 Lenti KO”); or cells subjected to targeted integration via HDR at the TRAC locus of recombinant TCR encoding sequences under the control of the human EF1α promoter (TCR #2 HDR KO). FIG. 2B shows the mean fluorescence intensity (MFI) of cell surface expression of peptide-MHC tetramer binding in CD8+ T and CD4+ T cells engineered to express TCR #2.

FIG. 3A depicts the average cytolytic activity of various CD8+ T cells expressing recombinant TCR #1 as described above, generated from 2 donors, represented by the area under the curve (AUC) % killing, compared to mock-transduced controls and normalized to Vbeta expression (recombinant TCR specific staining) for each group described above, after incubation of effector cells as described above with target cells expressing HPV 16 E7 at effector to target (E:T) ratios of 10:1, 5:1, and 2.5:1. CD8+ cells transduced with a lentivirus encoding a reference TCR capable of binding to HPV 16 E7 but containing murine Cα and Cβ regions were assessed as a control (“Lenti Ref”). FIG. 3B shows the mean IFNγ secretion (pg/ml) by various CD8+ T cells expressing recombinant TCR #1 as described above.

FIG. 4A depicts the average cytolytic activity of various CD8+ T cells expressing recombinant TCR #2 as described above, generated from 2 donors, represented by the area under the curve (AUC) % killing, compared to mock-transduced controls and normalized to Vbeta expression (recombinant TCR specific staining) for each group described above, after incubation of effector cells as described above with target cells expressing HPV 16 E7 at effector to target (E:T) ratios of 10:1, 5:1, and 2.5:1. CD8+ cells transduced with a lentivirus encoding a reference TCR capable of binding to HPV 16 E7 but containing murine Cα and Cβ regions were evaluated as a control (“Lenti Ref”). FIG. 4B and 4C depict the mean secretion of IFNγ (pg/ml; FIG. 4B ) and IL-2 (pg/ml; FIG. 4C ) by various CD8+ T cells expressing recombinant TCR #2 as described above. FIGS. 4D and 4E depict the cytolytic activity of various CD8+ ( FIG. 4D ) or CD4+ ( FIG. 4E ) T cells expressing recombinant TCR #2 as shown by a plurality of viable target cells over time. FIGS. 4F and 4G depict IFNγ secretion by various cells expressing recombinant TCR #2 at an E:T ratio of 2.5:1 ( FIG. 4F ) or 10:1 ( FIG. 4G ).

FIGS. 5A and 5B show the viability, as determined by % of cells stained with acridine orange (AO) and propidium iodide (PI), during cryopreservation (freezing) or after thawing after cryopreservation (thawing) in various CD4+ ( FIG. 5A ) or CD8+ ( FIG. 5B ) cells engineered to express recombinant TCR #2.

FIGS. 6A and 6B depict the surface expression of CD8, CD3, Vbeta (by recombinant TCR specific staining), and peptide-MHC tetramer in complex with the antigen recognized by the recombinant TCR, as assessed by flow cytometry, for T cells subjected to knockout of the genes encoding the endogenous TCR, engineered to express recombinant T cell receptor (TCR) using different expression modes: cells subjected to CRISPR/Cas9 mediated knockout (KO) of TRAC and TRBC (“TCRαβ KO”) or retaining endogenous TCR expression (“TCRαβ WT”); cells subjected to targeted integration of HDR at the TRAC locus of recombinant TCR encoding sequences linked to the EF1α or MND promoter (“HDR EF1α” or “HDR MND”); cells subjected to lentiviral transduction for random integration of recombinant TCR encoding sequences (“lenti human”) or recombinant TCR encoding sequences containing the murine constant domain (“lenti mouse”) or mock transduction as a control (“mock transd”).

FIGS. 6C and 6D depict the geometric mean fluorescence intensity (gMFI) of cell surface Vbeta expression and peptide-MHC tetramer binding in CD8+ ( FIG. 6C ) or CD4+ ( FIG. 6D ) T cells engineered to express recombinant T cell receptor (TCR) using different expression methods as described above.

FIGS. 6E and 6F show the coefficient of variation (standard deviation of signal in a population of cells divided by the mean signal in the respective population) in CD8+ T cells engineered to express recombinant T cell receptor (TCR) using different expression methods as described above for Vbeta expression ( FIG. 6F ) and peptide-MHC tetramer binding ( FIG. 6E ).

FIGS. 7A-7C depict surface expression of CD3 and CD8, as assessed by flow cytometry, for T cells subjected to knockout of genes encoding the endogenous TCR, engineered to express a recombinant T cell receptor (TCR) using different expression methods: cells subjected to CRISPR/Cas9-mediated knockout (KO) of TRAC, TRBC , or both TRAC and TRBC ; cells subjected to targeted integration of HDR at the TRAC locus of recombinant TCR encoding sequences linked to the EF1α promoter, MND promoter, or alpha promoter of the endogenous TCR using the P2A ribosome skip sequence (“HDR EF1α,” “HDR MND,” or “HDR P2A,” respectively), or cells subjected to mock transduction as a control (“mock transd”) ( FIG. 7A ); cells that retain endogenous TCR expression and are lentivirally transduced to randomly integrate recombinant TCR encoding sequences linked to the EF1α promoter (“lenti EF1α”) or the MND promoter (“lenti MND”), or linked to the EF1α promoter with sequences encoding a truncated receptor as a surrogate marker (“lenti EF1α/tReceptor”), or are mock transduced as a control (“mock”) ( FIG. 7B ). FIG. 7C depicts the percentage of CD3+CD8+ cells among CD8+ cells in each of the groups described above.

FIGS. 8A-8C depict peptide-MHC tetramer binding and CD8 surface expression, as assessed by flow cytometry, for T cells subject to knockout of genes encoding endogenous TCRs engineered to express recombinant T cell receptor (TCR) using different expression methods: cells subject to CRISPR/Cas9 mediated knockout (KO) of TRAC, TRBC , or both TRAC and TRBC ; cells subjected to targeted integration of HDR at the TRAC locus of recombinant TCR encoding sequences linked to the EF1α promoter, MND promoter, or endogenous TCR alpha promoter using the P2A ribosome skip sequence (“HDR EF1α,” “HDR MND,” or “HDR P2A,” respectively) or cells subjected to mock transduction as a control (“mock transd”) ( FIG. 8A ); cells that retain endogenous TCR expression and were lentivirally transduced to randomly integrate recombinant TCR encoding sequences linked to the EF1α promoter (“lenti EF1α”) or the MND promoter (“lenti MND”), or linked to the EF1α promoter with truncated receptor encoding sequences as a surrogate marker (“lenti EF1α/tReceptor”), or were mock transduced as a control (“mock”) ( FIG. 8B ). FIG. 8C depicts the proportion of tetramer+CD8+ cells among CD8+ cells in each of the groups described above at day 7 and day 13.

FIGS. 9A-9D depict surface expression of Vbeta (specific recombinant TCR staining) and CD8, as assessed by flow cytometry, for T cells subjected to knockout of genes encoding endogenous TCR, engineered to express recombinant T cell receptor (TCR) using different expression methods: cells subjected to CRISPR/Cas9-mediated knockout (KO) of TRAC, TRBC , or both TRAC and TRBC ; cells subjected to targeted integration of HDR at the TRAC locus of recombinant TCR encoding sequences linked to the EF1α promoter, MND promoter, or endogenous TCR alpha promoter using the P2A ribosome skip sequence (“HDR EF1α,” “HDR MND,” or “HDR P2A,” respectively) or cells subjected to mock transduction as a control (“mock transd”) ( FIG. 9A ); cells that retain endogenous TCR expression and were lentivirally transduced to randomly integrate recombinant TCR encoding sequences linked to the EF1α promoter (“lenti EF1α”) or the MND promoter (“lenti MND”), or linked to the EF1α promoter with sequences encoding a truncated receptor as a surrogate marker (“lenti EF1α/Receptor”), or were mock transduced as a control (“mock”) ( FIG. 9B ). FIGS. 9C and 9D depict the proportion of Vbeta+CD8+ cells among CD8+ cells ( FIG. 9C ) and the proportion of Vbeta+CD4+ cells among CD4+ cells ( FIG. 9D ) in each of the groups described above at day 7 and day 13.

FIG. 10 depicts the cytolytic activity of various CD8+ T cells expressing the recombinant TCR as described above, represented by the area under the curve (AUC) of % kill compared to mock-transduced controls and normalized to Vbeta expression for each group, from incubation of effector cells as described above with target cells expressing HPV 16 E7 at effector to target (E:T) ratios of 10:1, 5:1, and 2.5:1. CD8+ cells transduced with a lentivirus encoding the reference TCR capable of binding to HPV 16 E7 but containing the murine Cα and Cβ regions were evaluated as a control (“lenti mouse E7 ref”).

FIG. 11 shows IFNγ secretion (pg/ml) by various CD8+ T cells expressing the recombinant TCR as described above from incubation of effector cells as described above with target cells expressing HPV 16 E7 at effector to target (E:T) ratios of 10:1 and 2.5:1. CD8+ cells transduced with a lentivirus encoding the reference TCR capable of binding to HPV 16 E7 but containing murine Cα and Cβ regions were evaluated as a control (“lenti mouse E7 ref”).

FIG. 12 depicts a heat map showing the relative activity of different T cells expressing the recombinant TCR as described above in different functional assays: AUC % killing at E:T ratios of 10:1, 5:1, and 2.5:1 (“AUC”), tetramer binding in CD8+ cells at days 7 and 13 (“CD8 tetramer”), a proliferation assay (“CTV pulse”) using SCC152 cells or T2 target cells and injected with antigen peptide, and IFNγ secretion from CD8+ cells (“IFNγ-secreting CD8”).

FIGS. 13A-13B depict the results of changes in tumor volume over time in the UPCI:SCC152 squamous cell carcinoma mouse model injected with CD4+ and CD8+ cells engineered to express recombinant TCR#2 type I elicited by different pathways: TCR#2 driven by the human elongation factor 1 alpha (EF1α) promoter and targeted for integration at the TRAC locus via HDR (TCR#2 HDR KO EF1α); TCR#2 driven by the endogenous TRAC (upstream inserted in-frame ribosome skip element P2A) promoter and targeted for integration at the TRAC locus via HDR (TCR#2 HDR KO P2A); TCR#2 randomly integrated using a lentiviral construct (TCR#2 Lenti); TCR#2 randomly integrated using a lentiviral construct into cells containing a knockout of endogenous TRAC (TCR#2 Lenti KO); and a reference TCR capable of binding to HPV 16 E7 but containing murine Cα and Cβ regions randomly integrated using a lentiviral construct (Lenti ref), compared to mice that did not receive the engineered cells (tumor only) or that received cells processed under the same conditions as those used for electroporation but without the addition of RNP (mock KO), at a dose of 6×10 ⁶ ( FIG. 13A ) or 3×10 ⁶ ( FIG. 13B ) TCR-expressing cells.

FIGS. 14A-14B depict the survival curve of mice from each group described above for mice receiving a dose of 6×10 ⁶ ( FIG. 14A ) or 3×10 ⁶ ( FIG. 14B ) cells expressing recombinant TCR.

FIGS. 15A-15B depict the % change in body weight over time in mice in each group described above for mice receiving a dose of 6×10 ⁶ ( FIG. 15A ) or 3×10 ⁶ ( FIG. 15B ) cells expressing recombinant TCR.

FIGS. 16A-16B depict the results of integration at various time points for the different homology arm lengths tested, as assessed by changes in GFP templates at 24, 48, and 72 hours (FIG. 16A) , and at 96 hours or 7 days (FIG. 16B) after transduction with AAV preparations containing HDR template polynucleotides .

FIGS. 17A-17B show the changes in integration ratio for HDR using different homology arm lengths, after 24, 48, 72, and 96 hours or 7 days for four different donors, Donors 1 and 2 (FIG. 17A) and Donors 3 and 4 (FIG. 17B) .

FIGS. 18A-18B depict the results of evaluating the expression and activity of exemplary anti-CD19 CARs in cells engineered by integrating a nucleic acid sequence into the endogenous TRAC locus. FIG. 18A depicts surface expression of CD3 and anti-CD19 CAR (as determined by staining with an anti-idiotypic (anti-ID) antibody that specifically recognizes the CAR) as assessed by flow cytometry for T cells subjected to knockout of the genes encoding the endogenous TCR, engineered to express the anti-CD19 CAR using different expression modes: cells subjected to retroviral transduction to randomly integrate recombinant TCR encoding sequences (“Retrovirus Only”), cells subjected to targeted integration via HDR into the TRAC locus of recombinant TCR encoding sequences under the control of the human EF1α promoter (EF1α) or the endogenous TRAC promoter, using the P2A ribosome skip sequence (P2A). In FIG. 18B depicts the expression, as assessed by flow cytometry, of typical T cells expressing anti-CD19 CAR for the different expression routes described above, electroporated with ribonucleoprotein (RNP) complexes containing TRAC -targeted or TRBC -targeted gRNA.

FIGS. 19A-19C depict the expression and antigen-specific function of cells expressing an exemplary anti-CD19 CAR engineered using different expression methods after repeated rounds of antigen stimulation with target cells. FIG. 19A depicts the proportion of cells expressing the CAR observed after 3 rounds of stimulation with target cells. FIG. 19B depicts the mean fluorescence intensity (MFI) and FIG. 19C depicts the coefficient of variation (the standard deviation of the signal in a population of cells divided by the mean signal in the respective population) for T cells engineered to express the anti-CD19 CAR after 3 rounds of stimulation.

FIGS. 20A-20B depict IFNγ secretion ( FIG. 20A; pg/ml) and cytolytic activity ( FIG. 20B) of cells expressing a typical anti-CD19 CAR using different engineering methods incubated with K562 target cells engineered to express CD19 (K562-CD19) or non-K562 engineered (parental) at an effector to target (E:T) ratio of 2:1.

FIGS. 21A-21B depict the results of assessing the expression and activity of a representative anti-BCMA CAR in cells engineered by integrating nucleic acid sequences into the endogenous TRAC locus. FIG. 21A depicts the surface expression of CD3 and anti-BCMA CAR (recognized by the BCMA-Fc fusion protein), as assessed by flow cytometry, for T cells engineered to express the anti-BCMA CAR using different expression methods: cells retrovirally transduced to randomly integrate recombinant TCR encoding sequences (“Lentivirus Only”), cells targeted by HDR integration into the TRAC locus of recombinant TCR encoding sequences under the control of the human EF1α promoter (EF1α) or the endogenous TCR alpha promoter, using the P2A ribosome skip sequence (P2A). FIG. 21B depicts the expression, as assessed by flow cytometry, of typical T cells expressing anti-BCMA CAR for the different expression routes described above, electroporated with ribonucleoprotein (RNP) complexes containing TRAC -targeted or TRBC -targeted gRNA.

FIGS. 22A-22B depict the expression and antigen-specific function of cells expressing a representative anti-BCMA CAR engineered using different expression methods after repeated rounds of antigen stimulation with target cells. FIG. 22A depicts the proportion of cells expressing the CAR observed after 3 rounds of stimulation with target cells. FIG. 22B shows the level of IFNγ (upper panel, pg/mL) and interleukin-2 (IL-2; lower panel) secretion.

Detailed description

Provided herein are methods for producing genetically engineered immune cells expressing a recombinant receptor, such as a recombinant T cell receptor (TCR). Also provided are genetically engineered immune cells expressing a recombinant receptor, such as a recombinant T cell receptor (TCR), and compositions comprising such cells. The embodiments provided include specifically targeting nucleic acid sequences encoding the recombinant receptor to a specific locus, such as one or more endogenous TCR gene loci. In some contexts, the present embodiments include causing targeted genetic disruption, such as creating a DNA break, using genetic engineering and homology-directed repair (HDR) techniques to target nucleic acids encoding a recombinant receptor at endogenous TCR gene loci, thereby reducing or eliminating expression of endogenous TCR genes and promoting uniform or homogeneous expression of the recombinant receptor in a cell population. Related cellular compositions, nucleic acids, and kits for use in the methods presented herein are also provided.

T cell-based therapies, such as adoptive T cell therapies (including those involving the use of engineered cells expressing recombinant receptors specific for the disease or disorder in question, such as TCR, CAR and/or other recombinant antigen receptors) may be effective in the treatment of cancer and other diseases and disorders. In certain contexts, available approaches to generating engineered cells for adoptive cell therapy may not always be entirely satisfactory. In some contexts, optimal efficacy may depend on the ability of the administered cells to express the recombinant receptor, and for the recombinant receptor, the ability to recognize and bind to a target, such as a target antigen, in the subject, tumors and their environments, and the ability to uniformly, homogeneously and/or stably express receptors between cells, such as a population of immune cells and/or cells in a therapeutic cell composition.

In some cases, currently available methods, such as random integration of recombinant receptor encoding sequences, are not entirely satisfactory in one or more of these aspects. In some aspects, variable integration of recombinant receptor encoding sequences may result in unstable expression, variable nucleic acid copy number, possible insertional mutagenesis and/or variability in receptor expression and/or genetic disruption in a cellular composition, such as a therapeutic cellular composition. In some aspects, the use of particular random integration vectors, such as certain lentiviral vectors, requires replication competent lentivirus (RCL) analysis.

In some cases, the stability and/or efficiency of expression of the recombinant receptor is limited in certain cells or certain cell populations engineered using currently available methods. In some embodiments, the recombinant receptor is expressed only in certain cells, and the level of expression or antigen binding by the recombinant receptor varies widely among cells in the population. In certain aspects, the level of expression of the recombinant receptor may be difficult to predict, control and/or regulate. In some cases, semi-random or random integration of a transgene encoding a receptor into the genome of a cell may, in some cases, cause side and/or unwanted effects due to integration of the nucleic acid sequence into an unwanted location in the genome, such as an essential gene or a gene critical for regulating cell activity. In some cases, random integration of a receptor-encoding nucleic acid sequence may result in unstable, unregulated, uncontrolled, and/or suboptimal expression or antigen binding, oncogenic transformation, and transcriptional silencing of the nucleic acid sequence, depending on the site of integration and/or copy number of the nucleic acid sequence. In other cases, particularly for recombinant TCRs, suboptimal expression of an engineered or recombinant TCR, which may result from expression of one or more endogenous TCR chains in engineered cells, may result in a mismatch between the recombinant TCRα or β chain and the endogenous TCRα or β chain. In some aspects, mispaired TCRs may result in undesired cell targeting and potential side effects. In some aspects, mispaired TCRs may compete for invariant CD3 signaling molecules that are involved in allowing expression of the recombinant TCR complex on the cell surface, thereby reducing recombinant TCR expression on the cell surface and/or the ability to recognize and bind to a target, such as a target antigen.

In some embodiments, targeted genetic disruption of one or more endogenous TCR gene loci may result in a reduced risk or potential for mismatch between the engineered or recombinant TCR chains and the endogenous TCR. Mismatched TCRs may, in some aspects, create new TCRs that may potentially cause a greater risk of unwanted or unintended antigen recognition and/or side effects, and/or may reduce expression levels of desired engineered or recombinant TCRs. In some aspects, reducing or preventing expression of the endogenous TCR may increase expression of the engineered or recombinant TCR in T cells or T cell compositions, compared to cells in which TCR expression is not reduced or prevented. In some embodiments, expression of the recombinant TCR may be increased by 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or more. For example, in some cases, suboptimal expression of an engineered or recombinant TCR may occur due to competition with endogenous TCR and/or TCRs having mismatched chains for signaling molecules and/or domains, such as invariant CD3 signaling molecules (e.g., availability of co-expressed CD3 δ, ε, γ, and ζ chains), that are involved in permitting expression of the complex on the cell surface. In some aspects, available CD3ζ molecules may limit TCR expression and function in cells. In some aspects, currently available transgene delivery methods, such as encoding recombinant receptors, such as recombinant TCRs, may exhibit insufficient integration and/or reduced expression of the recombinant receptors. In some aspects, the efficiency of integration and/or expression of the recombinant receptor in a population may be low and/or variable.

In some aspects, the development of humanized and/or fully human recombinant TCRs presents technical challenges. For example, in some aspects, the receptor of the humanized and/or fully human recombinant TCR competes with endogenous TCR complexes and may form a mismatch with endogenous TCRα and/or TCRβ chains, which may, in certain aspects, reduce the signaling, activity and/or expression of the recombinant TCR, and ultimately cause a decrease in the activity of the engineered cells. One way to address these challenges is to engineer recombinant TCRs with murine constant domains to prevent a mismatch with endogenous human TCRα or TCRβ chains. However, the use of recombinant TCRs with murine sequences may, in some aspects, pose a risk of immune response. The disclosed polynucleotides, reagents, articles of manufacture, kits and methods address these issues by inserting sequences encoding all or part of a recombinant TCR into an endogenous gene encoding one or more TCR chains. In particular aspects, such insertion serves to disrupt expression of the endogenous TCR gene while allowing expression of a fully humanized and/or human recombinant TCR, reducing the likelihood of competition from or miscomplementation with endogenous TCR chains or the use of murine sequences that may potentially be immunogenic.

In some contexts, the available approaches to engineering a plurality and/or population of cells yield heterogeneous, non-uniform and/or uneven expression of the recombinant receptor due to differences in nucleic acid delivery efficiency, differences in genomic integration location and/or copy number, disruption of complementarity and/or competition with endogenous TCR chains and/or other factors. In some contexts, the available approaches to engineering yield a population of cells that is heterogeneous in terms of expression of the recombinant receptor and/or knockout of specific loci. In some aspects, heterogeneous and non-uniform expression in a population of cells may result in decreased overall expression levels, stability of expression and/or antigen binding by the recombinant receptor, decreased function of the engineered cells and/or a non-uniform drug product, thereby reducing the efficacy of the engineered cells.

In some embodiments, provided herein are methods for generating or making genetically engineered cells that comprise a TRAC and/or TRBC locus comprising nucleic acid sequences encoding a recombinant TCR or a fragment thereof. In some aspects, the TRAC and/or TRBC locus in the genetically engineered cells comprises a transgene sequence (also referred to herein as an exogenous or heterologous nucleic acid sequence) encoding all or a portion of a recombinant TCR integrated into an endogenous TRAC and/or TRBC locus that typically encodes TCRα or TCRβ constant domains. In some embodiments, the methods comprise initiating targeted homology-directed disruption and repair (HDR) using one or more template polynucleotides comprising a transgene encoding all or a portion of a recombinant TCR, thereby targeting the integration of the transgene into the TRAC and/or TRBC locus. Also provided are cells with cellular compositions created by the methods. In some aspects, eliminating expression of endogenous TCRα and/or TCRβ chains may reduce the disruption of complementarity between endogenous and engineered or recombinant chains.

In some embodiments, the present polynucleotides, transgenes and/or vectors, when delivered to immune cells, cause expression of recombinant receptors, such as TCR, which can modulate T cell activity and, in some cases, can modulate T cell differentiation or homeostasis. The resulting genetically engineered cells or cellular compositions can be used in adoptive cell therapy methods.

In some aspects, compared to conventional methods for producing genetically engineered immune cells expressing a recombinant receptor, such as a recombinant T cell receptor (TCR) or a chimeric antigen receptor (CAR), the present methods allow for higher, more stable and/or much more uniform or homogeneous expression of the recombinant receptor. In some aspects, the present embodiments offer advantages in producing engineered T cells with improved, uniform, homogeneous, robust and/or stable expression of the recombinant receptor, while minimizing potential for complementation failure, off-targeting, semi-random or random transgene integration and/or competition from endogenous TCRs. In some aspects, the present variants allow predictable and stable integration at a single target gene locus or multiple target gene loci, provide a stable copy number (typically 1 or 2) of the nucleic acids, have a reduced, low, or absent potential for insertional mutagenesis, provide stability in recombinant receptor expression and endogenous receptor gene expression in a cell population, and eliminate the need for RCL analysis. In some aspects, the present variants are based on the observations that targeted knockout of nucleic acids encoding the recombinant receptor at one or more endogenous TCR gene loci reduces or eliminates endogenous TCR gene expression, resulting in higher overall expression levels, more uniform and stable expression and/or antigen binding, and improved function of the engineered cells, including improved antitumor effects.

The disclosed embodiments also offer advantages in producing engineered T cells wherein all cells that express the recombinant receptor also have the expression of one or more endogenous TCR gene loci (such as endogenous genes encoding TCRα and/or TCRβ chains) knocked out, reduced, and/or eliminated via genome editing and HDR. Compared to approaches that can produce a heterogeneous mixture, wherein some cells that express the recombinant receptor may be knocked out for endogenous TCR gene loci, while other cells that express the recombinant receptor may retain the endogenous TCR gene locus, the disclosed embodiments can be used to create a substantially more homogeneous and uniform population of cells, such as wherein all cells that express the recombinant receptor contain a knockout of one or more endogenous TCR gene loci.

In some aspects, the present embodiments are based on the observations of improved efficiency of TCR integration and expression and antigen binding using a targeted knockin approach. Targeted knockout of one or more endogenous TCR gene loci (such as endogenous genes encoding TCRα and/or TCRβ chains) via genome editing, combined with a targeted knockin of nucleic acids encoding a recombinant receptor (such as a recombinant TCR or CAR) via homology-directed repair (HDR), can result in engineered T cells that have improved expression, function, and expression uniformity and/or other desirable characteristics or properties, and are extremely efficient.

Also provided are methods for constructing, preparing and obtaining engineered cells and kits and devices for creating or obtaining engineered cells. Polynucleotides, such as viral vectors, that contain a nucleic acid sequence encoding a recombinant receptor or a portion thereof, and methods for introducing such polynucleotides into cells, such as transduction, or physical delivery, such as electroporation, are also provided. Compositions containing engineered cells, methods, kits and devices for introducing cells and compositions into subjects, such as for adoptive cell therapy, are also provided.

All publications, including patent documents, scientific articles, and databases, referenced in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication was individually incorporated by reference. If a definition given herein conflicts with or is otherwise inconsistent with a definition given in patents, applications, published applications, and other publications that are incorporated by reference, the definition set forth herein controls over the definition that is incorporated by reference.

The section headings used here are for organizational purposes only and should not be construed as limiting the subject matter described.

I. METHODS FOR OBTAINING CELLS EXPRESSING A RECOMBINANT RECEPTOR BY HOMOLOGY-DIRECTED REPAIR (HDR)

Provided herein are methods for producing a genetically engineered immune cell, such as a genetically engineered T cell, for adoptive cell therapy, related compositions, methods, uses, and kits and articles of manufacture for performing the methods. Immune cells are typically engineered to express a recombinant molecule, such as a recombinant receptor, such as a recombinant T cell receptor (TCR) or a chimeric antigen receptor (CAR). In some embodiments, also provided are compositions comprising a population of cells that are engineered to express a recombinant receptor, such as a TCR or CAR, such as a population of cells that exhibits improved, uniform, homogeneous, and/or stable expression and/or antigen binding by the recombinant receptor, including genetically engineered immune cells produced by any of the provided methods. In some embodiments, the present compositions exhibit a reduced coefficient of variation in expression and/or antigen binding compared to that of a cell population and/or compositions created by conventional methods. In some embodiments, methods and uses of the composition and/or cells for therapy are also provided, including those involving administration of the composition and/or cells.

In some embodiments, methods for producing a genetically engineered immune cell, such as a genetically engineered T cell, for adoptive cell therapy are provided. In some embodiments, the provided methods comprise introducing into an immune cell one or more agents capable of causing genetic disruption of one or more target sites (also known as a "target position," "target DNA sequence," or "target location") in a gene encoding a T cell receptor alpha (TCRα) chain domain or region and/or one or more genes encoding a T cell receptor beta (TCRβ) chain domain or region (also referred to herein as "one or more agents" or "agents" with reference to aspects of the provided methods); and introducing into an immune cell a polynucleotide, such as a template polynucleotide, comprising a transgene encoding a recombinant receptor or chain thereof, wherein the transgene encoding the recombinant receptor or chain thereof is targeted to or near one of at least one target site via homology-directed repair (HDR).

In some embodiments, methods are provided for creating or producing genetically engineered cells that comprise a TRAC and/or TRBC locus comprising nucleic acid sequences encoding a recombinant TCR or a fragment thereof. In some aspects, the TRAC and/or TRBC locus in the genetically engineered cells comprises a transgene sequence (also referred to herein as exogenous or heterologous nucleic acid sequences) encoding all or a portion of a recombinant TCR integrated into an endogenous TRAC and/or TRBC locus that typically encodes TCRα or TCRβ constant domains. In some embodiments, the methods comprise initiating targeted homology-directed disruption and repair (HDR) using one or more template polynucleotides comprising a transgene encoding all or a portion of a recombinant TCR, thereby targeting the integration of the transgene into the TRAC and/or TRBC locus. Also presented are cells and cell compositions created by the methods.

In particular embodiments, a transgene sequence encoding all or a portion of a recombinant TCR comprises a nucleotide sequence encoding a TCRα chain and/or a TCRβ chain. In some embodiments, one or more polynucleotides, such as template polynucleotides, may be used. In some embodiments, each polynucleotide, such as a template polynucleotide, may comprise a nucleotide sequence encoding either a TCRα chain or a TCRβ chain. In some embodiments, a polynucleotide, such as a template polynucleotide, comprises a nucleic acid sequence encoding all or a portion of a recombinant receptor or chain, such as a recombinant TCR or chain. In certain embodiments, the nucleic acid sequence is targeted to a target site that is located in a locus of a gene that encodes an endogenous receptor, such as one or more genes encoding an endogenous TCR chain or a portion thereof. In certain embodiments, the nucleic acid sequence is targeted for integration into an endogenous gene locus. In certain embodiments, the integration genetically disrupts expression of the endogenous receptor encoded by the gene at the target site. In certain embodiments, a transgene encoding a portion of a recombinant receptor is targeted to the gene locus via HDR.

In some embodiments, the provided methods comprise administering to an immune cell one or more agents, wherein each of the one or more agents is independently capable of causing genetic disruption of a target site in a T cell receptor constant alpha (TRAC) gene and/or a T cell receptor constant beta (TRBC) gene, thereby causing genetic disruption of at least one target site; and administering to the immune cell a template polynucleotide comprising a transgene encoding a recombinant T cell receptor (TCR) or an antigen-binding fragment or chain thereof, wherein the transgene encoding the recombinant TCR or an antigen-binding fragment or chain thereof is targeted for integration at or near one of the at least one target site via homology-directed repair (HDR). In certain embodiments, integration at or near the target site is into a portion of the coding sequence of a TRAC and/or TRBC gene, such as a portion of the coding sequence downstream, or 3', of the target site.

In some embodiments, one of at least one of the target sites is in the T cell receptor constant alpha (TRAC) gene. In some embodiments, one of at least one of the target sites is in the T cell receptor constant beta 1 ( TRBC1 ) or T cell receptor constant beta 2 ( TRBC2 ) gene. In some embodiments, one or more target sites are in the TRAC gene and one or both of the TRBC1 and TRBC2 genes.

In some embodiments, the present methods comprise introducing into an immune cell having a genetic disruption of one or more target sites in a gene encoding a domain or region of a T cell receptor alpha (TCRα) chain and/or one or more genes encoding a domain or region of a T cell receptor beta (TCRβ) chain, a template polynucleotide comprising a transgene encoding a recombinant receptor, wherein the transgene encoding the recombinant receptor or chain thereof is targeted to or near at least one target site via HDR.

In the present embodiments, the term "introduction" encompasses a variety of methods for introducing DNA into a cell, either in vitro or in vivo, wherein such methods include conversion, transduction, transfection (e.g., electroporation), and infection. Vectors are used to introduce DNA encoding molecules into cells. Possible vectors include plasmid vectors and viral vectors. Viral vectors include retroviral vectors, lentiviral vectors, and other vectors such as adenoviral vectors or adeno-associated vectors. Methods such as electroporation can also be used to introduce or deliver a protein or ribonucleoprotein (RNP), such as one containing the Cas9 protein in complex with a target gRNA, into the cells in question.

In some cases, the embodiments provided herein comprise one or more targeted genetic disruptions, such as DNA breaks, at one or more endogenous TCR gene loci (such as genes encoding TCRα and/or TCRβ chains) by genome editing techniques combined with targeted knock-in of nucleic acids encoding a recombinant receptor (such as a recombinant TCR or CAR), homology-directed repair (HDR). In some embodiments, the HDR step requires a break, such as a double-strand break, in DNA at a target genomic location. In some embodiments, the DNA break is produced by a genome editing step, such as DNA breaks created by targeted nucleases used in genome editing.

In some embodiments, the embodiments include creating a targeted DNA break using genome editing techniques and/or targeted nucleases, followed by HDR based on one or more template polynucleotides, such as template polynucleotides that contain homologous sequences and one or more transgenes, such as nucleic acids encoding a recombinant receptor or chain thereof and/or other exogenous or recombinant nucleic acids, to specifically target and integrate nucleic acid sequences encoding a recombinant receptor or chain thereof and/or other exogenous or recombinant nucleic acids at or near the DNA break.

In some embodiments, targeted genetic disruption and targeted integration of nucleic acids encoding a recombinant receptor via HDR occurs at one or more target sites (also referred to as a “target position,” “target DNA sequence,” or “target location”) of endogenous genes that encode one or more domains, regions, and/or chains of an endogenous T cell receptor (TCR). In some embodiments, targeted genetic disruption is caused in a TCRα gene. In some embodiments, targeted genetic disruption is caused in a TCRβ gene. In some embodiments, targeted genetic disruption is caused in an endogenous TCRα gene and an endogenous TCRβ gene. The endogenous TCR genes may include one or more genes encoding a TCRα constant domain (encoded by TRAC in humans) and/or a TCRβ constant domain (encoded by TRBC1 or TRBC2 in humans).

In some embodiments, targeted genetic disruption of one or more endogenous TCR gene loci may result in a reduced risk or likelihood of mismatch between the engineered or recombinant TCR chains and the endogenous TCR. Mismatched TCRs may create a new TCR that may potentially cause an increased risk of unwanted or unintended antigen recognition and/or adverse effects, and/or may reduce expression levels of desired engineered or recombinant TCRs. In some aspects, reducing or preventing expression of the endogenous TCR may increase expression of the engineered or recombinant TCR in T cells or T cell compositions compared to cells in which TCR expression is not reduced or prevented. In some embodiments, expression of the recombinant TCR may be increased by 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or more. For example, in some cases, suboptimal expression of engineered or recombinant TCRs may occur due to competition with endogenous TCRs and/or mismatched TCRs for signaling domains, such as invariant CD3 signaling molecules, that are involved in permitting cell surface expression of the complex.

In some embodiments, the template polynucleotide is introduced into the engineered cells before, simultaneously with, or after the introduction of agents capable of causing one or more targeted genetic disruptions. In the presence of one or more targeted genetic disruptions, such as a DNA break, the template polynucleotide can be used as a DNA repair template to efficiently copy and integrate transgenes, such as a nucleic acid sequence encoding a recombinant receptor, near the site of targeted genetic disruption via HDR based on homology between the endogenous gene sequence surrounding the target site and the 5' and/or 3' homology arms included in the template polynucleotide.

In some embodiments, the genome editing and HDR steps are performed simultaneously and/or in a single experimental reaction. In some embodiments, the genome editing and HDR steps are performed continuously or sequentially, in a single or sequential experimental reactions. In some embodiments, the genome editing and HDR steps are performed in separate experimental reactions, simultaneously or at different times.

The immune cells may comprise a population of cells comprising T cells. Such cells may be cells obtained from a subject, such as those obtained from a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a white blood cell sample, an apheresis product, or a leukapheresis product. In some embodiments, the T cells may be separated or selected to enrich the population of T cells using positive selection or negative selection and enrichment methods. In some embodiments, the population comprises CD4+, CD8+, or CD4+ and CD8+ T cells. In some embodiments, the step of introducing the polynucleotide array and the step of introducing the agent (e.g., Cas9/nRNA RNP) may be performed simultaneously or sequentially in any order. In particular embodiments, the polynucleotide array is introduced into the immune cells after the step of introducing the agents (e.g., Cas9/nRNA RNP) has caused genetic disruption. In some embodiments, prior to, during, or following administration of the polynucleotide array and one or more agents (e.g., Cas9/nRNA RNP), the cells are cultured or incubated under conditions that promote cell multiplication and/or proliferation.

In particular embodiments of the present methods, the administration of the template polynucleotide is carried out after the administration of one or more agents capable of causing genetic disruption. Any described method of administering one or more agents may be used, depending on the particular agent used to cause genetic disruption. In some aspects, the disruption is carried out by genome editing, for example, using an RNA-guided nuclease, such as a clustered regularly interspaced short palindromic nucleic acids (CRISPR)-Cas system, such as the CRISPR-Cas9 system, specific for the TRAC or TRBC locus to be disrupted. In some embodiments, an agent comprising Cas9 and a guide RNA (gRNA) comprising a targeting domain that targets a region of the TRAC or TRBC locus is administered to a cell. In some embodiments, the agent is or comprises a ribonucleoprotein (RNP) complex of Cas9 and gRNA comprising a TRAC/TRBC targeting domain (Cas9/gRNA RNP). In some embodiments, the administering comprises contacting the agent or a portion thereof with the cells in vitro, which may comprise culturing or incubating the cell and the agent for up to 24, 36, or 48 hours, or 3, 4, 5, 6, 7, or 8 days. In some embodiments, the administering may further comprise delivering the agent to the cells. In various embodiments, the methods, compositions, and cells of the present invention employ direct delivery of Cas9 ribonucleoprotein (RNP) complexes and gRNA to the cells, such as by electroporation. In some embodiments, the RNP complexes comprise gRNA that has been modified to comprise a 3' poly-A tail and a 5' Anti-Reverse Cap Analog (ARCA) cap. In some cases, electroporation of the modified cells comprises cold shocking the cells, such as at 32°C, followed by electroporation of the cells and prior to plating.

In such aspects of the present methods, the template polynucleotide is introduced into the cells after the introduction of one or more agents, such as Cas9/nRNA RNP, for example, which have been introduced by electroporation. In some embodiments, the template polynucleotide is introduced immediately after the introduction of one or more agents capable of causing genetic disruption. In some embodiments, the template polynucleotide is introduced into the cells within or about 30 seconds, within or about 1 minute, within or about 2 minutes, within or about 3 minutes, within or about 4 minutes, within or about 5 minutes, within or about 6 minutes, within or about 6 minutes, within or about 8 minutes, within or about 9 minutes, within or about 10 minutes, within or about 15 minutes, within or about 20 minutes, within or about 30 minutes, within or about 40 minutes, within or about 50 minutes, within or about 60 minutes, within or about 90 minutes, within or about 2 hours, within or about 3 hours, or within or about 4 hours after administration of one or more agents capable of causing genetic destruction. In some embodiments, the template polynucleotide is administered to the cells within a time period of about 15 minutes to about 4 hours after administration of one or more agents, such as within about 15 minutes to about 3 hours, about 15 minutes to about 2 hours, about 15 minutes to about 1 hour, about 15 minutes to about 30 minutes, about 30 minutes to about 4 hours, about 30 minutes to about 3 hours, about 30 minutes to about 2 hours, about 30 minutes to about 1 hour, about 1 hour to about 4 hours, about 1 hour to about 3 hours, about 1 hour to about 2 hours, about 2 hours to about 4 hours, about 2 hours to about 3 hours, or about 3 hours to about 4 hours. In some embodiments, the template polynucleotide is administered to the cells within about 2 hours after administration of one or more agents, such as Cas9/nRNA RNP, for example, which have been administered by electroporation.

Any method of introducing the template polynucleotide may be used, depending on the specific methods used to deliver the template polynucleotide into the cells. Typical methods include methods for transferring nucleic acids encoding receptors, including viral, such as retroviral or lentiviral, transduction, transposons, and electroporation. In specific embodiments, viral transduction methods are used. In some embodiments, the template polynucleotides may be transferred or introduced into the cells in particles of a recombinant infectious virus, such as, for example, vectors derived from simian virus 40 (SV40), adenoviruses, adeno-associated virus (AAV). In some embodiments, the recombinant nucleic acids are transferred into T cells using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors (see, e.g., Koste et al. (2014) Gene Therapy 2014 Apr 3. doi: 10,1038/gt,2014,25; Carlens et al. (2000) Exp Hematol 28(10): 1137–46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. 2011 November 29(11): 550–557. In particular embodiments, the viral vector is an AAV, such as AAV2 or AAV6.

In some embodiments, before, during, or after contacting the agent with the cells and/or before, during, or after delivery (e.g., electroporation), the present methods include incubating the cells in the presence of a cytokine, a stimulatory agent, and/or an agent that is capable of causing proliferation, stimulation, or activation of immune cells (e.g., T cells). In some embodiments, at least a portion of the incubation is carried out in the presence of a stimulatory agent that is or comprises an antibody specific for CD3, an antibody specific for CD28, and/or a cytokine such as anti-CD3/anti-CD28 beads. In some embodiments, at least a portion of the incubation is carried out in the presence of a cytokine such as one or more of recombinant IL-2, recombinant IL-7, and/or recombinant IL-15. In some embodiments, the incubation is carried out for up to 8 days hours before or after the administration of one or more agents, such as Cas9/nRNA RNP, such as via electroporation, and a template polynucleotide, such as up to 24 hours, 36 hours, or 48 hours, or 3, 4, 5, 6, 7, or 8 days.

In some embodiments, the method comprises activating or stimulating the cells with a stimulating agent (e.g., anti-CD3/anti-CD28 antibodies) prior to administering the agent, e.g., Cas9/nRNA RNP, and the polynucleotide array. In some embodiments, incubation in the presence of a stimulating agent (e.g., anti-CD3/anti-CD28) is carried out for 6 hours to 96 hours, such as 24-48 hours or 24-36 hours prior to administering one or more agents, such as Cas9/nRNA RNP, such as via electroporation. In some embodiments, incubation with stimulating agents may further comprise the presence of a cytokine, such as one or more of recombinant IL-2, recombinant IL-7, and/or recombinant IL-15. In some embodiments, the incubation is carried out in the presence of a recombinant cytokine, such as IL-2 (e.g., from 1 U/ml to 500 U/ml, such as from 10 U/ml to 200 U/ml, such as at least or about 50 U/ml or 100 U/ml), IL-7 (e.g., from 0.5 ng/ml to 50 ng/ml, such as from 1 ng/ml to 20 ng/ml, such as at least or about 5 ng/ml or 10 ng/ml), or IL-15 (e.g., from 0.1 ng/ml to 50 ng/ml, such as from 0.5 ng/ml to 25 ng/ml, such as at least or about 1 ng/ml or 5 ng/ml). In some embodiments, the stimulating agent (e.g., anti-CD3/anti-CD28 antibodies) is washed or removed from the cells prior to the introduction or delivery to the cells of agents capable of causing genetic disruption of the Cas9/nRNA RNP and/or polynucleotide template. In some embodiments, prior to the introduction of the agents, the cells are allowed to rest, such as by removing any stimulating or activating agent. In some embodiments, prior to the introduction of the agents, the stimulating or activating agent and/or cytokines are not removed.

In some embodiments, after administration of agents, such as Cas9/nRNA, and/or a polynucleotide array, the cells are incubated, cultured, or grown in a nutrient medium in the presence of a recombinant cytokine, such as one or more of recombinant IL-2, recombinant IL-7, and/or recombinant IL-15. In some embodiments, the incubation is carried out in the presence of a recombinant cytokine, such as IL-2 (e.g., from 1 U/ml to 500 U/ml, such as from 10 U/ml to 200 U/ml, such as at least or about 50 U/ml or 100 U/ml), IL-7 (e.g., from 0.5 ng/ml to 50 ng/ml, such as from 1 ng/ml to 20 ng/ml, such as at least or about 5 ng/ml or 10 ng/ml), or IL-15 (e.g., from 0.1 ng/ml to 50 ng/ml, such as from 0.5 ng/ml to 25 ng/ml, such as at least or about 1 ng/ml or 5 ng/ml). The cells may be incubated or cultured under conditions that cause the cells to proliferate or multiply. In some embodiments, the cells may be incubated or cultured until a threshold number of cells for harvesting, such as a therapeutically effective dose, is reached.

In some embodiments, the incubation during any step of the method or the entire method can be carried out at a temperature from 30°C±2°C to 39°C±2°C, such as at least or about at least 30°C±2°C, 32°C±2°C, 34°C±2°C, or 37°C±2°C. In some embodiments, at least a portion of the incubation is carried out at 30°C±2°C and at least a portion of the incubation is carried out at 37°C±2°C.

A. Genetic destruction

In some embodiments, one or more targeted genetic disruptions are caused at an endogenous TCRα gene and/or an endogenous TCRβ gene. In some embodiments, the targeted genetic disruption is caused at one or more genes encoding the constant domain of TCRα (also known as the constant region of TCRα; encoded by TRAC in humans) and/or the constant domain of TCRβ (also known as the constant region of TCRβ; encoded by TRBC1 or TRBC2 in humans). In some embodiments, the targeted genetic disruption is caused at the TRAC, TRBC1 , and TRBC2 loci.

In some embodiments, the targeted genetic disruption produces a break or nick in DNA. In some embodiments, at the site of the DNA break, the action of cellular DNA repair mechanisms can produce a knockout, insertion, missense or sign mutation, such as a biallelic sign mutation, deletion of all or part of a gene. In some embodiments, the genetic disruption can be directed to one or more exons of a gene or a portion thereof, such as the first or second exon. In some embodiments, a DNA binding protein or DNA binding nucleic acid that specifically binds to or hybridizes to sequences in a region near one of at least one target site is used for the targeted disruption. In some aspects, in the absence of exogenous template polynucleotides for HDR, the disruption, the targeted genetic disruption causes a deletion, mutation or insertion in an exon of a gene. In some embodiments, template polynucleotides, such as template polynucleotides that include nucleic acid sequences encoding a recombinant receptor and homologous sequences, can be introduced to target the integration of the recombinant receptor encoding sequences at or near the site of genetic disruption via HDR (see Section I.B. herein).

In some embodiments, the genetic disruption is carried out by administering one or more agents capable of causing genetic disruption. In some embodiments, such agents comprise a DNA binding protein or DNA binding nucleic acid that specifically binds to or hybridizes with a gene. In some embodiments, the agent comprises different components, such as a fusion protein comprising a DNA-targeting protein and a nuclease or RNA-directed nuclease. In some embodiments, the agents can be directed to one or more target locations, such as the TRAC gene and one or both of the TRBC1 and TRBC2 genes.

In some embodiments, the genetic disruption occurs at a target site (also referred to and/or known as a “target position,” “target DNA sequence,” or “target location”). In some embodiments, the target site is or includes a site on a target DNA (e.g., genomic DNA) that is modified by one or more agents capable of causing genetic disruption, such as a Cas9 molecule in complex with a gRNA that defines a target site. For example, in some embodiments, the target site may include locations on DNA, such as the endogenous TRAC , TRBC1 , and/or TRBC2 locus, where DNA cleavage or breaks occur. In some aspects, the integration of a nucleic acid sequence via HDR may occur at or near a target site or target sequence. In some embodiments, the target site may be a site between two nucleotides, such as adjacent nucleotides, on DNA to which one or more nucleotides are added. The target site may comprise one or more nucleotides that are altered by the template polynucleotide. In some embodiments, the target site is in a target sequence (e.g., a sequence to which the gRNA binds). In some embodiments, the target site is upstream or downstream of the target sequence.

1. Target sites on genes encoding endogenous T-cell receptor (TCR)

In some embodiments, the targeted genetic disruption occurs at endogenous genes that encode one or more domains, regions, and/or chains of an endogenous T cell receptor (TCR). In some embodiments, the genetic disruption is directed at an endogenous gene locus that encodes TCRα and/or TCRβ. In some embodiments, the genetic disruption is directed at a gene encoding a TCRα constant domain ( TRAC in humans) and/or a TCRβ constant domain ( TRBC1 or TRBC2 in humans).

In some embodiments, a “T cell receptor” or “TCR,” including endogenous TCRs, is a molecule that contains alternating α and β chains (also known as TCRα and TCRβ, respectively) or alternating γ and δ chains (also known as TCRγ and TCRδ, respectively), or antigen-binding portions thereof, and that is capable of specifically binding to a peptide bond on an MHC molecule. In some embodiments, the TCR is the αβ form. Typically, TCRs that exist in the αβ and γδ forms are genetically structurally similar, but the T cells expressing them may have different anatomical locations or functions. Typically, one T cell expresses one type of TCR. A TCR may be found on the cell surface or in a soluble form. Typically, a TCR is found on the surface of a T cell (or T lymphocyte), where it is typically responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules.

In some embodiments, the TCR may comprise a variable domain and a constant domain (also known as a constant region), a transmembrane domain, and/or a short cytoplasmic tail (see, e.g., Janeway et al. , Immunobiology: The Immune System in Health and Disease , ^3rd Ed., Current Biology Publications, p. 4:33, 1997). In some embodiments, the TCR chain comprises one or more constant domains. For example, the extracellular portion of a given TCR chain ( e.g., the TCRα chain or the TCRβ chain) may contain two immunoglobulin-like domains, such as variable domains ( e.g., Vα or Vβ; typically amino acids 1-116 based on Kabat nomenclature, Kabat et al. , “Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5 ^th ed.) and a constant domain ( e.g., the constant domain of the α chain or TCR Cα, typically positions 117-259 of the chain based on Kabat nomenclature, or the constant domain of the β chain or TCR Cβ, typically positions 117-295 of the chain based on Kabat nomenclature) located near the cell membrane. For example, in some cases, the extracellular portion of the TCR, formed by two chains, contains two membrane-proximal constant domains and two membrane-distal variable domains.

In some embodiments, the endogenous TCR Cα is encoded by the TRAC gene (IMGT nomenclature). An exemplary sequence of the human T cell receptor alpha chain constant domain gene locus ( TRAC ) is set forth in SEQ ID NO: 1 (NCBI Reference Sequence: NG_001332.3, TRAC ). In some embodiments, the encoded endogenous Cα comprises the amino acid sequence set forth in SEQ ID NO: 19 or 24 (UniProtKB Accession No. P01848 or Genbank Accession No. CAA26636.1). In certain embodiments, genetic disruption is directed to, adjacent to, or within the TRAC locus. In particular embodiments, genetic disruption is directed to, adjacent to, or within an open reading frame of the TRAC locus. In certain embodiments, genetic disruption is directed to, adjacent to, or within an open reading frame that encodes the TCRα constant domain. In some embodiments, the genetic disruption is directed to, adjacent to, or at a locus having a nucleic acid sequence as set forth in SEQ ID NO: 1, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to all or a portion, such as at least 500, 1000, 1500, 2000, 2500, 3000, 3500, or 4000 contiguous nucleotides, of the nucleic acid sequence as set forth in SEQ ID NO: 1.

In humans, the typical genomic TRAC locus contains an open reading frame that contains 4 exons and 3 introns. The typical TRAC mRNA transcript may span the sequence corresponding to Chromosome 14 coordinates: 22,547,506–22,552,154, on the forward strand, referring to the human genome version GRCh38 (UCSC Genome Browser on Human Dec. 2013 (GRCh38/hg38) Assembly). Table 1 below provides the exon and intron coordinates of the open reading frames and untranslated regions of the transcript of the typical human TRAC locus.

Table 1. Coordinates of exons and introns of a typical human TRAC locus (GRCh38, chromosome 14, straight strand).

Beginning (GrCh38) The End (GrCh38) Length 5' UTR and Exon 1 22,547,506 22,547,778 273 Intron 1-2 22,547,779 22,549,637 1,859 Exon 2 22,549,638 22,549,682 45 Intron 2-3 22,549,683 22,550,556 874 Exon 3 22,550,557 22,550,664 108 Intron 3-4 22,550,665 22,551,604 940 Exon 4 and 3' UTR 22,551,605 22,552,154 550

In some embodiments, the endogenous TCR Cβ is encoded by the TRBC1 or TRBC2 genes (IMGT nomenclature). An exemplary sequence of a human T cell receptor beta chain constant domain 1 gene locus ( TRBC1 ) is set forth in SEQ ID NO: 2 (NCBI Reference Sequence: NG_001333.2, TRBC1 ); and an exemplary sequence of a human T cell receptor beta chain constant domain 2 gene locus ( TRBC2 ) is set forth in SEQ ID NO: 3 (NCBI Reference Sequence: NG_001333.2, TRBC2 ). In some embodiments, the encoded Cβ has or comprises the amino acid sequence set forth in SEQ ID NO: 20, 21, or 25 (Uniprot Accession No. P01850, A0A5B9, or A0A0G2JNG9). In some embodiments, the genetic disruption is directed to, adjacent to, or within the TRBC1 gene locus. In certain embodiments, the genetic disruption is directed to, adjacent to, or within an open reading frame of the TRBC1 locus. In certain embodiments, the genetic disruption is directed to, adjacent to, or within an open reading frame that encodes the TCRβ constant domain. In some embodiments, the genetic disruption is directed to, adjacent to, or at a locus having a nucleic acid sequence as set forth in SEQ ID NO: 2 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to all or a portion of, such as at least 500, 1000, 1500, 2000, 2500, 3000, 3500, or 4000 contiguous nucleotides of the nucleic acid sequence as set forth in SEQ ID NO: 2.

In humans, the typical genomic locus of TRBC1 contains an open reading frame that contains 4 exons and 3 introns. The typical mRNA transcript of TRBC1 can span the sequence corresponding to the coordinates of Chromosome 7: 142,791,694-142,793,368, on the forward strand, referring to the human genome version GRCh38 (UCSC Genome Browser on Human Dec. 2013 (GRCh38/hg38) Assembly). Table 2 below presents the coordinates of the exons and introns of the open reading frames and untranslated regions of the transcript of the typical human TRBC1 locus.

Table 2. Coordinates of exons and introns of a typical human TRBC1 locus (GRCh38, chromosome 7, straight strand).

Beginning (GrCh38) The End (GrCh38) Length 5' UTR and Exon 1 142,791,694 142,792,080 387 Intron 1-2 142,792,081 142,792,521 441 Exon 2 142,792,522 142,792,539 18 Intron 2-3 142,792,540 142,792,691 152 Exon 3 142,792,692 142,792,798 107 Intron 3-4 142,792,799 142,793,120 322 Exon 4 and 3' UTR 142,793,121 142,793,368 248

In certain embodiments, the genetic disruption is directed to, adjacent to, or within the TRBC2 locus. In certain embodiments, the genetic disruption is directed to, adjacent to, or within an open reading frame of the TRBC2 locus. In certain embodiments, the genetic disruption is directed to, adjacent to, or within an open reading frame that encodes the TCRβ constant domain. In some embodiments, the genetic disruption is directed to, adjacent to, or at a locus having a nucleic acid sequence as set forth in SEQ ID NO: 3 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to all or a portion of, such as at least 500, 1000, 1500, 2000, 2500, 3000, 3500, or 4000 contiguous nucleotides of the nucleic acid sequence as set forth in SEQ ID NO: 3.

In humans, the typical genomic locus of TRBC2 contains an open reading frame that contains 4 exons and 3 introns. The typical TRBC2 mRNA transcript can span the sequence corresponding to chromosome 7 coordinates: 142,801,041-142,802,748, on the forward strand, referring to the human genome version GRCh38 (UCSC Genome Browser on Human Dec. 2013 (GRCh38/hg38) Assembly). Table 3 below presents the coordinates of the exons and introns of the open reading frames and untranslated regions of the transcript of the typical human TRBC2 locus.

Table 3. Coordinates of exons and introns of a typical human TRBC2 locus (GRCh38, chromosome 7, straight strand).

Beginning (GrCh38) The End (GrCh38) Length 5' UTR and Exon 1 142,801,041 142,801,427 387 Intron 1-2 142,801,428 142,801,943 516 Exon 2 142,801,944 142,801,961 18 Intron 2-3 142,801,962 142,802,104 143 Exon 3 142,802,105 142,802,211 107 Intron 3-4 142,802,212 142,802,502 291 Exon 4 and 3' UTR 142,802,503 142,802,748 246

In some aspects, a transgene (e.g., exogenous nucleic acid sequences) in a template polynucleotide can be used to determine the location of target sites and/or homology arms. In some aspects, a target site of genetic disruption can be used as a guide for creating template polynucleotides and/or homology arms used for HDR. In some embodiments, genetic disruption can be directed near the desired site of targeted integration of transgene sequences (e.g., encoding a recombinant TCR or portions thereof). In some aspects, the target site is within an exon of the TRAC, TRBC1 and/or TRBC2 open reading frame of the locus. In some aspects, the target site is within an intron of the TRAC, TRBC1 and/or TRBC2 open reading frame of the locus.

In some embodiments, the genetic disruption, e.g., DNA break, is targeted in close proximity to the start of a coding region (e.g., an early coding region, e.g., within 500 bp of the start codon or the remaining coding sequence, e.g., downstream of the first 500 bp of the start codon). In some embodiments, the genetic disruption, e.g., DNA break, is targeted to an early coding region of the gene in question, e.g., TRAC, TRBC1 and/or TRBC2 , including a sequence located immediately downstream of the transcription start site, within the first exon of the coding sequence, or within 500 bp of the transcription start site (e.g., less than 500, 450, 400, 350, 300, 250, 200, 150, 100, or 50 bp) or within 500 bp of the transcription start site. from the initiation codon (e.g., less than 500, 450, 400, 350, 300, 250, 200, 150, 100, or 50 bp).

In some embodiments, the target site is within an exon of an endogenous TRAC , TRBC1 , and/or TRBC2 locus. In certain embodiments, the target site is within an intron of an endogenous TRAC , TRBC1 , and/or TRBC2 locus. In some aspects, the target site is within a regulatory or control element, such as a promoter, 5' untranslated region (UTR), or 3' UTR of a TRAC , TRBC1 , and/or TRBC2 locus. In certain embodiments, the target site is within an open reading frame of an endogenous TRAC , TRBC1 , and/or TRBC2 locus. In particular embodiments, the target site is within an exon in an open reading frame of a TRAC , TRBC1 , and/or TRBC2 locus.

In particular embodiments, the genetic disruption, such as a DNA break, is directed at or within an open reading frame of the gene or locus in question, such as TRAC , TRBC1 and/or TRBC2 . In some embodiments, the genetic disruption is directed at or within an intron in the open reading frame of the gene or locus in question. In some embodiments, the genetic disruption is directed within an exon in the open reading frame of the gene or locus in question.

In certain embodiments, the genetic disruption, such as a DNA break, is directed at or within an intron. In certain embodiments, the genetic disruption, such as a DNA break, is directed at or within an exon. In some embodiments, the genetic disruption, such as a DNA break, is directed at or within an exon of the gene in question, such as TRAC, TRBC1 , and/or TRBC2 .

In some embodiments, the genetic disruption, such as a DNA break, is directed within a TRAC gene exon, open reading frame, or locus. In certain embodiments, the genetic disruption is in the first exon, second exon, third exon, or fourth exon of the TRAC gene, open reading frame, or locus. In particular embodiments, the genetic disruption is in the first exon of the TRAC gene, open reading frame, or locus. In some embodiments, the genetic disruption is within 500 base pairs (bp) downstream of the 5' end of the first exon in the TRAC gene, open reading frame, or locus. In particular embodiments, the genetic disruption is between the largest 5' nucleotide of exon 1 and upstream of the largest 3' nucleotide of exon 1. In certain embodiments, the genetic disruption is within 400 bp, 350 bp, 300 bp, 250 bp, 200 bp, 150 bp, 100 bp, or 50 bp downstream of the 5' end of the first TRAC gene exon, open reading frame, or locus. In particular embodiments, the genetic disruption is between 1 bp and 400 bp, between 50 bp and 300 bp, between 100 bp and 200 bp, or between 100 bp and 150 bp. downstream of the 5' end of the first TRAC gene exon, open reading frame, or locus, all inclusive. In certain embodiments, the genetic disruption is between 100 bp and 150 bp downstream of the 5' end of the first TRAC gene exon, open reading frame, or locus, all inclusive.

In certain embodiments, the genetic disruption, such as a DNA break, is directed within a TRBC gene exon, open reading frame, or locus, such as TRBC1 and/or TRBC2 . In certain embodiments, the genetic disruption is in the first exon, second exon, third exon, or fourth exon of the TRBC1 and/or TRBC2 gene, open reading frame, or locus. In some embodiments, the genetic disruption is in the first exon of the TRBC1 and/or TRBC2 gene, open reading frame, or locus. In certain embodiments, the genetic disruption is in the first exon, second exon, third exon, or fourth exon of the TRBC1 and/or TRBC2 gene, open reading frame, or locus. In some embodiments, the genetic disruption is between the largest 5' nucleotide of exon 1 and upstream of the largest 3' nucleotide of exon 1. In particular embodiments, the genetic disruption is within the first exon of the TRBC gene, open reading frame, or locus. In some embodiments, the genetic disruption is within 400 bp, 350 bp, 300 bp, 250 bp, 200 bp, 150 bp, 100 bp, or 50 bp downstream of the 5' end of the first exon of the TRBC1 and/or TRBC2 gene, open reading frame, or locus. In particular embodiments, the genetic disruption is between 1 bp and 400 bp, between 50 bp and 300 bp, between 100 bp and 200 bp, or between 100 bp and 200 bp. or between 100 bp and 150 bp downstream of the 5' end of the first TRBC1 and/or TRBC2 gene exon, open reading frame, or locus, all inclusive. In certain embodiments, the genetic disruption is between 100 bp and 150 bp downstream of the 5' end of the first TRBC1 and/or TRBC2 gene exon, open reading frame, or locus, inclusive.

2. Methods of genetic destruction

Methods for creating genetic disruption, including those described herein, can include the use of one or more agents capable of causing genetic disruption, such as engineered systems for causing genetic disruption, cleavage and/or a double-strand break (DSB) or nick at a target site or target position in endogenous DNA, such that repair of the break by an erroneous process, such as non-homologous end joining (NHEJ) or repair using an HDR repair template, can result in a gene knockout and/or insertion of the sequence in question (e.g., exogenous nucleic acid sequences or a transgene encoding part of a chimeric receptor) near the target site or position. Also provided are one or more agents capable of causing genetic disruption for use in the methods provided herein. In some aspects, one or more agents can be used in combination with the template nucleotides provided herein to mediate homology-directed repair (HDR) directed integration of transgene sequences (e.g., those described herein in Section I.B).

In some embodiments, one or more agents capable of causing genetic disruption comprise a DNA binding protein or a DNA binding nucleic acid that specifically binds to or hybridizes to a particular site or location in the genome, such as a target site or target location. In some aspects, targeted genetic disruption, such as DNA nicking or cleavage, of endogenous TCR encoding genes is achieved using a protein or nucleic acid that is combined or complexed with a genome editing nuclease, such as a chimeric or fusion protein. In some embodiments, one or more agents capable of causing genetic disruption comprise an RNA-directed nuclease or a fusion protein comprising a DNA-directed protein and a nuclease.

In some embodiments, the agent comprises different components, such as an RNA-directed nuclease or a fusion protein comprising a DNA-directed protein and a nuclease. In some embodiments, the targeted genetic disruption is performed using a DNA targeting molecule that comprises a DNA binding protein, such as one or more zinc finger proteins (ZFPs) or transcription activator-like effectors (TALEs), fused to a nuclease, such as an endonuclease. In some embodiments, the targeted genetic disruption is performed using RNA-directed nucleases, such as the clustered regularly intervening short paddledromic nucleic acid (CRISPR)-related (Cas) system (including Cas and/or Cfp1). In some embodiments, targeted genetic disruption is carried out using agents capable of causing genetic disruption, such as sequence-specific or targeted nucleases, including DNA-binding targeted nucleases and genome-editing nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) and RNA-guided nucleases such as the CRISPR-associated nuclease (Cas) system, specifically designed to target at least one target site, gene sequence, or portions thereof. Exemplary ZFNs, TALEs, and TALENs are described in, for example, Lloyd et al. , Frontiers in Immunology, 4(221): 1-7 (2013).

Zinc finger proteins (ZFPs), transcription activator-like effectors (TALEs), and CRISPR binding domains can be “engineered” to bind to a specific nucleotide sequence, such as by engineering (changing one or more amino acids) the recognition helix region of a naturally occurring ZFP or TALE protein. Engineered DNA binding proteins (ZFPs or TALEs) are proteins that do not exist in nature. Reasonable criteria for design include the use of substitution rules and computerized algorithms to process information in a database storing information on the designs and binding data of existing ZFPs and/or TALEs. See, e.g., U.S. Patent Nos. 6,140,081; 6,453,242; and 6,534,261; see also WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496 and US Publication No. 20110301073.

In some embodiments, one or more agents are specifically directed to at least one target site, such as near a gene of interest, such as TRAC, TRBC1 and/or TRBC2 . In some embodiments, the agent comprises a ZFN, TALEN or CRISPR/Cas9 combination that specifically binds to, recognizes or hybridizes to target sites. In some embodiments, the CRISPR/Cas9 system comprises an engineered crRNA/AKTRr RNA (“single guide RNA”) to effect specific cleavage. In some embodiments, the agent comprises nucleases based on the Argonaute system (e.g., from T. thermophilus , known as 'TtAgo', (Swarts et al. (2014) Nature 507(7491): 258-261). Targeted cleavage using any of the nuclease systems described herein can be used to insert transgene sequences, e.g., nucleic acid sequences encoding a recombinant receptor, into a specific target location, e.g., on endogenous TCR genes, using either HDR or NHEJ-mediated methods.

In some embodiments, a “zinc finger DNA binding protein” (or binding domain) is a protein or domain within a larger protein that binds DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within a binding domain whose structure is stabilized through the coordination of a zinc ion. The term zinc finger DNA binding protein is often shortened to zinc finger protein or ZFP. Among the ZFPs are artificial ZFP domains that target specific DNA sequences, typically 9-18 nucleotides in length, created by assembling individual fingers. ZFPs include those in which an individual finger domain is approximately 30 amino acids in length and contains an alpha helix containing two invariant histidine residues coordinated through zinc to two cysteines of a single beta turn, and having two, three, four, or five fingers. In general, the sequence specificity of a ZFP can be altered by making amino acid substitutions at four helix positions (−1, 2, 3, and 6) on the zinc finger recognition helix. Thus, for example, a ZFP or ZFP-containing molecule that does not exist in nature, for example, is engineered to bind to a selected target site.

In some cases, the DNA-targeting molecule is or comprises a zinc finger DNA binding domain fused to a DNA cleavage domain to form a zinc finger nuclease (ZFN). For example, fusion proteins comprise a cleavage domain (or cleavage half-domain) from at least one type IIS restriction nuclease and one or more zinc finger binding domains, which may or may not be engineered. In some cases, the cleavage domain is taken from the type IIS restriction endonuclease FokI, which typically catalyzes double-stranded DNA cleavage, 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other. See, e.g., U.S. Patent Nos. 5,356,802; and 5,487,994; Li et al. (1992) Proc. Natl. Acad. Sci. USA 89:4275–4279; Li et al. (1993) Proc. Natl. Acad. Sci. USA 90:2764–2768; Kim et al. (1994a) Proc. Natl. Acad. Sci. USA 91:883–887; Kim et al. (1994b) J. Biol. Chem. 269:978–982. Some gene-specific engineered zinc fingers are available commercially. For example, a zinc finger engineering platform called CompoZr is available that yields specifically targeted zinc fingers for thousands of targets. See, for example, Gaj et al. , Trends in Biotechnology , 2013, 31(7), 397–405. In some cases, commercially available zinc fingers are used or custom-made ones are created.

In some embodiments, one or more target sites, such as within the TRAC , TRBC1 and/or TRBC2 genes, can be directed to genetic disruption by the engineered ZFNs. Exemplary ZFNs that target endogenous T cell receptor (TCR) genes include those described in, for example, US 2015/0164954, US 2011/0158957, US 2015/0056705, US 8956828 and Torikawa et al. (2012) Blood 119:5697-5705, the disclosures of which are incorporated by reference in their entireties, or those set forth in any of SEQ ID NOS:213-224 ( TRAC ) or SEQ ID NOS:225 and 226 ( TRBC ).

Transcription activator-like effector (TALE) is a protein from the bacterial species Xanthomonas that contains multiple repeat sequences, where each repeat contains di-residues at position 12 and 13 (RVD) that are specific to each nucleotide base of the targeting nucleic acid sequence. Binding domains with similar modular base-to-base nucleic acid binding properties (MBBBD) can also be derived from different bacterial species. The new modular proteins have the advantage of exhibiting greater sequence variability than TAL repeats. In some embodiments, the RVDs associated with recognition of different nucleotides are HD for recognition of C, NG for recognition of T, NI for recognition of A, NN for recognition of G or A, NS for recognition of A, C, G or T, HG for recognition of T, IG for recognition of T, NK for recognition of G, HA for recognition of C, ND for recognition of C, HI for recognition of C, HN for recognition of G, NA for recognition of G, SN for recognition of G or A and YG for recognition of T, TL for recognition of A, VT for recognition of A or G and SW for recognition of A. In some embodiments, critical amino acids 12 and 13 can be mutated to other amino acid residues to modulate their specificity for nucleotides A, T, C and G, and in particular to improve such specificity.

In some embodiments, a “TALE DNA binding domain” or “TALE” is a polypeptide comprising one or more TALE repeat domains/units. The repeat domains, each comprising a pair of variable amino acid residues (RVDs), are involved in binding the TALE to its cognate target DNA sequence. An individual “repeat unit” (also referred to as a “repeat”) is typically 33-35 amino acids in length and exhibits at least some sequence homology to other TALE repeat sequences in a naturally occurring TALE protein. TALE proteins can be engineered to bind to a target site using canonical or non-canonical RVDs in the repeat units. See, e.g., U.S. Patent Nos. 8,586,526 and 9,458,205.

In some embodiments, a "TALE nuclease" (TALEN) is a fusion protein comprising a nucleic acid binding domain, typically derived from a transcription activator-like effector (TALE), and a catalytic domain of a nuclease that cleaves a target nucleic acid sequence. The catalytic domain comprises a nuclease domain or a domain having endonuclease activity, such as, for example, I-TevI, ColE7, NucA and Fok-I. In a particular embodiment, the TALE domain can be fused to a meganuclease, such as, for example, I-CreI and I-OnuI, or a functional variant thereof. In some embodiments, the TALEN is a monomeric TALEN. A monomeric TALEN is a TALEN that does not require dimerization for specific recognition and cleavage, such as the fusion of engineered TAL repeats to the catalytic domain of I-TevI described in WO2012138927. TALENs have been described and applied for gene targeting and gene modification (see, e.g., Boch et al. (2009) Science 326(5959): 1509–12; Moscou and Bogdanove (2009) Science 326(5959): 1501; Christian et al. (2010) Genetics 186(2): 757–61; Li et al. (2011) Nucleic Acids Res 39(1): 359–72).

In some embodiments, the TRAC , TRBC1 , and/or TRBC2 genes may be targeted for genetic disruption by an engineered TALEN. Exemplary TALENs that target endogenous T cell receptor (TCR) genes include those described in, for example, WO 2017/070429, WO 2015/136001, US20170016025, and US20150203817, the disclosures of which are incorporated herein by reference in their entirety.

In some embodiments, “TtAgo” is a prokaryotic Argonaute protein thought to be involved in gene silencing. TtAgo is derived from the bacterium Thermus thermophilus . See, e.g., Swarts et al, ( 2014) Nature 507(7491): 258–261 , Sheng et al., (2013) Proc. Natl. Acad. Sci. USA 111, 652). The “TtAgo system” includes all components that require activation, such as guide DNA for cleavage by the TtAgo enzyme.

In some embodiments, the engineered zinc finger protein, TALE protein, or CRISPR/Cas system does not exist in nature and is primarily produced by an empirical process such as phage display, interaction trap, or hybrid selection. See, e.g., U.S. Patent No. 5,789,538; U.S. Patent No. 5,925,523; U.S. Patent No. 6,007,988; U.S. Patent No. 6,013,453; U.S. Patent No. 6,200,759; WO 95/19431; WO 96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO 01/60970; WO 01/88197 and WO 02/099084.

Zinc finger and TALE DNA binding domains can be engineered to bind a specific nucleotide sequence, such as by engineering (changing one or more amino acids) the recognition helix region of a natural zinc finger protein or by engineering the amino acids involved in DNA binding (variable amino acid residue pairs or RVD regions). Therefore, engineered zinc finger proteins or TALE proteins are proteins that do not exist in nature. Non-limiting examples of methods for engineering zinc finger proteins and TALEs include design and selection. An engineered protein is a protein that does not exist in nature, the design/composition of which is the result of substantially reasonable criteria. Reasonable criteria for design include the application of substitution rules and computerized algorithms to process information in a database storing information on designs and binding data of existing ZFP or TALE designs (canonical and non-canonical RVDs) and binding data. See, for example, U.S. Patent Nos. 9,458,205; 8,586,526; 6,140,081; 6,453,242; and 6,534,261; see also WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536; and WO 03/016496.

Various methods and compositions for targeted cleavage can be used, for example, to cause targeted mutagenesis, cause targeted deletions of cellular DNA sequences, and promote targeted recombination at a specific chromosomal locus. See, for example, U.S. Patent Nos. 9,255,250; 9,200,266; 9,045,763; 9,005,973; 9,150,847; 8,956,828; 8,945,868; 8,703,489; 8,586,526; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,067,317; 7,262,054; 7,888,121; 7,972,854; 7,914,796; 7,951,925; 8,110,379; 8,409,861; U.S. Patent Publications 20030232410; 20050208489; 20050026157; 20050064474; 20060063231; 20080159996; 201000218264; 20120017290; 20110265198; 20130137104; 20130122591; 20130177983; 20130196373; 20140120622; 20150056705; 20150335708; 20160030477 and 20160024474, the disclosures of which are incorporated herein by reference in their entireties. Also provided are one or more agents capable of causing genetic destruction. Also provided are polynucleotides (e.g., nucleic acid molecules) encoding one or more components of one or more agents capable of causing genetic destruction.

a. Crispr/Cas9

In some embodiments, targeted genetic disruption, such as DNA nicking, of endogenous TCR-encoding genes such as TRAC and TRBC1 or TRBC2 in humans is achieved using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins. See Sander and Joung, (2014) Nature Biotechnology , 32(4): 347–355.

In general, “CRISPR system” refers collectively to transcripts and other elements involved in the expression or direction of activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence ( e.g., tracrRNA or active partial tracrRNA), a tracr-mate sequence (encompassing the “direct repeat” and tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also called a “spacer” in the context of an endogenous CRISPR system), and/or other sequences and transcripts from a CRISPR locus.

In some aspects, a CRISPR/Cas nuclease or CRISPR/Cas nuclease system comprises a noncoding guide RNA (gRNA) that binds DNA in a sequence-specific manner and a Cas protein ( e.g., Cas9) with nuclease functionality.

1) Guide RNA (gRNA)

In some embodiments, one or more agents comprise at least one of: a guide RNA (gRNA) having a targeting domain that is complementary to a target site of a TRAC gene; a gRNA having a targeting domain that is complementary to a target site of one or both of the TRBC1 and TRBC2 genes; or at least one nucleic acid encoding a gRNA.

In some aspects, a "gRNA molecule" refers to a nucleic acid that facilitates the specific targeting or homing of a gRNA molecule/Cas9 molecule complex to a target nucleic acid, such as a locus on the genomic DNA of a cell. gRNA molecules can be monomolecular (having one RNA molecule), sometimes referred to herein as "chimeric" gRNAs, or modular (containing more than one, and typically two, separate RNA molecules). In general, a guide sequence, such as a guide RNA, is any polynucleotide sequence that contains at least a portion of a sequence that has sufficient complementarity with a target polynucleotide sequence, such as the TRAC , TRBC1 , and/or TRBC2 genes in humans, to hybridize to the target sequence at a target site and direct sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, in the context of forming a CRISPR complex, a “target sequence” generally refers to a sequence to which a designed guide sequence has complementarity, wherein hybridization between the target sequence and a domain, such as a targeting domain, of the guide RNA facilitates formation of the CRISPR complex. Perfect complementarity is not necessarily required, so long as there is sufficient complementarity for hybridization to facilitate formation of the CRISPR complex. In general, the guide sequence is selected to reduce the degree of secondary structure in the guide sequence. The secondary structure may be determined by any suitable polynucleotide prediction algorithm.

In some embodiments, a guide RNA (gRNA) specific to the locus in question ( e.g., at the TRAC, TRBC1 , and/or TRBC2 loci in humans) is used for RNA-guided nucleases, such as Cas, to cause a DNA break at the target site or position. Methods for designing gRNAs and exemplary targeting domains may include those described in, for example, WO2015/161276, WO2017/193107, WO2017/093969, US2016/272999, and US2015/056705.

Several exemplary gRNA structures with the domains identified herein are described in WO2015/161276, such as in FIGS. 1A-1G herein. Without wishing to be bound by theory, with respect to the three-dimensional shape or intra- and interstrand interactions of the active form of the gRNA, regions of high complementarity are sometimes shown as duplexes in WO2015/161276, such as in FIGS. 1A-1G herein and other depictions provided herein.

In some cases, the gRNA is a monomolecular or chimeric gRNA comprising from 5' to 3': a targeting domain that is directed to a target site or position, such as in a sequence from the TRAC locus (an exemplary nucleotide sequence of the human TRAC gene locus is set forth in SEQ ID NO:1; NCBI Reference Sequence: NG_001332.3, TRAC ; an exemplary genomic sequence is described in Table 1 herein); a first complementarity domain; a binding domain; a second complementarity domain (which is complementary to the first complementarity domain); a proximal domain; and, optionally, a tail domain. In some cases, the gRNA is a monomolecular or chimeric gRNA comprising, from 5' to 3': a targeting domain that targets a target site or position, such as in a sequence from a TRBC1 or TRBC2 locus (an exemplary nucleotide sequence of the human TRBC1 gene locus is set forth in SEQ ID NO:2; NCBI Reference Sequence: NG_001333,2, TRBC1 ; an exemplary genomic sequence described in Table 2 herein; an exemplary nucleotide sequence of the human TRBC2 gene locus is set forth in SEQ ID NO:3; NCBI Reference Sequence: NG_001333,2, TRBC2 ; an exemplary genomic sequence described in Table 3 herein); a first complementarity domain; a binding domain; a second complementarity domain (which is complementary to the first complementarity domain); a proximal domain; and, optionally, a tail domain.

In other cases, the gRNA is a modular gRNA containing a first and second strand. In these cases, the first strand preferably comprises from 5' to 3': a targeting domain (which targets a target site or position, such as in a sequence from the TRAC locus (an exemplary nucleotide sequence of the human TRAC gene locus is set forth in SEQ ID NO:1; NCBI Reference Sequence: NG_001332.3, TRAC ; an exemplary genomic sequence described in Table 1 herein) or the TRBC1 or TRBC2 locus (an exemplary nucleotide sequence of the human TRBC1 gene locus is set forth in SEQ ID NO:2; NCBI Reference Sequence: NG_001333.2, BCTP11 ; an exemplary genomic sequence described in Table 2 herein; an exemplary nucleotide sequence of the human TRBC2 gene locus is set forth in SEQ ID NO:3; NCBI Reference Sequence: NG_001333.2, TRBC2 ); and a first complementarity domain. The second strand typically includes, from 5' to 3': optionally, a 5' extension domain; a second complementarity domain; a proximal domain; and optionally, a tail domain.

A) Domain of targeted impact

Examples of targeting domain arrangements include those described in WO2015/161276, such as in FIGS. 1A-1G herein. The targeting domain comprises a nucleotide sequence that is complementary, such as at least 80, 85, 90, 95, 98 or 99% complementary, such as fully complementary, to a target sequence on a target nucleic acid. The strand of a target nucleic acid comprising the target sequence is referred to herein as a "complementary strand" of the target nucleic acid. Guidance on the selection of targeting domains can be found, for example, in Fu Y et al ., Nat Biotechnol 2014 (doi: 10.1038/nbt.2808) and Sternberg SH et al ., Nature 2014 (doi: 10.1038/nature13011).

The targeting domain is part of the RNA molecule and therefore contains a basic uracil (U), whereas any DNA encoding a gRNA molecule will contain a basic thymine (T). Without wishing to be bound by theory, in some embodiments, it is believed that the complementarity of the targeting domain to the target sequence plays a role in the specificity of the interaction of the gRNA molecule/Cas9 molecule complex with the target nucleic acid. It is understood that in a pair of the targeting domain and the target sequence, the uracil bases in the targeting domain will pair with the adenine bases in the target sequence. In some embodiments, the target domain itself contains, in the 5'-3' direction, an optional second domain and a core domain. In some embodiments, the core domain is fully complementary to the target sequence. In some embodiments, the targeting domain is 5-50 nucleotides in length. The strand of the target nucleic acid to which the targeting domain is complementary is referred to herein as the complementary strand. Some or all of the nucleotides of the domain may have a modification, such as to make it less susceptible to degradation, improve biocompatibility, etc. As a non-limiting example, the backbone of the targeting domain may be modified with phosphorothioate or other modifications. In some cases, the nucleotide of the targeting domain may contain a 2' modification, such as 2-acetylation, such as 2' methylation, or other modifications.

In different variants, the targeting domain is 16-26 nucleotides in length (i.e. it is 16 nucleotides in length or 17 nucleotides in length or 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length).

B) Typical domains of targeted influence

Exemplary targeting domains contained in gRNA for targeted genetic disruption of human TRAC, TRBC1 , or TRBC2 include those described in, for example, WO2015/161276, WO2017/193107, WO2017/093969, US2016/272999, and US2015/056705, or a targeting domain that can bind to target sequences described below. Exemplary targeting domains contained in gRNA for targeted genetic disruption of the human TRAC locus using S. pyogenes or S. aureus Cas9 may include any of those described in

Table 4. Typical sequences of the TRAC nRNA targeting domain

Name of nRNA Domain of targeted influence Types of Cas9 SEQ ID NO: TRAC -10 UCUCUCAGCUGGUACACGGC S.pyogenes 28 TRAC -110 UGGAUUUAGAGUCUCUCAGC S.pyogenes 29 TRAC -116 ACACGGCAGGGUCAGGGUUC S.pyogenes 30 TRAC -16 GAGAAUCAAAAUCGGUGAAU S.pyogenes 31 TRAC -4 GCUGGUACACGGCAGGGUCA S.pyogenes 32 TRAC -49 CUCAGCUGGUACACGGC S.pyogenes 33 TRAC -2 UGGUACACGGCAGGGUC S.pyogenes 34 TRAC -30 GCUAGACAUGAGGUCUA S.pyogenes 35 TRAC -43 GUCAGAUUUGUUGCUCC S.pyogenes 36 TRAC -23 UCAGCUGGUACACGGCA S.pyogenes 37 TRAC -34 GCAGACAGACUUGUCAC S.pyogenes 38 TRAC -25 GGUACACGGCAGGGUCA S.pyogenes 39 TRAC -128 CUUCAAGAGCAACAGUGCUG S.pyogenes 40 TRAC -105 AGAGCAACAGUGCUGUGGCC S.pyogenes 41 TRAC -106 AAAGUCAGAUUUGUUGCUCC S.pyogenes 42 TRAC -123 ACAAACUGUGCUAGACAUG S.pyogenes 43 TRAC -64 AAACUGUGCUAGACAUG S.pyogenes 44 TRAC -97 UGUGCUAGACAUGAGGUCUA S.pyogenes 45 TRAC -148 GGCUGGGGAAGAAGGUGUCUUC S. aureus 46 TRAC -147 GCGGGGAAGAAGGUGUCUUC S. aureus 47 TRAC -234 GGGGAAGAAGGUGUCUUC S. aureus 48 TRAC -167 GUUUUGUCUGUGAUAUACACAU S. aureus 49 TRAC -177 GGCAGACAGACUUGUCACUGGAUU S. aureus 50 TRAC -176 GCAGACAGACUUGUCACUGGAUU S. aureus 51 TRAC -257 GACAGACUUGUCACUGGAUU S. aureus 52 TRAC -233 GUGAAUAGGCAGACAGACUUGUCA S. aureus 53 TRAC -231 GAAUAGGCAGACAGACUUGUCA S. aureus 54 TRAC -163 GAGUCUCAGCUGGUACACGG S. aureus 55 TRAC -241 GUCUCUCAGCUGGUACACGG S. aureus 56 TRAC -179 GGUACACGGCAGGGUCAGGGUU S. aureus 57 TRAC -178 GUACACGGCAGGGUCAGGGUU S. aureus 58

Exemplary targeting domains contained in gRNA for targeted genetic disruption of the human TRBC1 or TRBC2 locus using S. pyogenes or S. aureus Cas9 may include any of those shown in Table 5 .

Table 5. Typical targeting domain sequences of TRBC1 or TRBC2 nRNA

Name of nRNA Domain of targeted influence Types of Cas9 SEQ ID NO: TRBC -40 CACCCAGAUCGUCAGCGCCG S.pyogenes 59 TRBC -52 CAAACACAGCGACCUCGGGU S.pyogenes 60 TRBC -25 UGACGAGUGGACCCAGGAUA S.pyogenes 61 TRBC -35 GGCUCUCGGAGAAUGACGAG S.pyogenes 62 TRBC -50 GGCCUCGGCGCUGACGAUCU S.pyogenes 63 TRBC -39 GAAAAACGUGUUCCCACCCG S.pyogenes 64 TRBC -49 AUGACGAGUGGACCCAGGAU S.pyogenes 65 TRBC -51 AGUCCAGUUCUACGGGCUCU S.pyogenes 66 TRBC -26 CGCUGUCAAGUCCAGUUCUA S.pyogenes 67 TRBC -47 AUCGUCAGCGCCGAGGCCUG S.pyogenes 68 TRBC -45 UCAAACACAGCGACCUCGGG S.pyogenes 69 TRBC -34 CGUAGAACUGGACUUGACAG S.pyogenes 70 TRBC -227 AGGCCUCGGCGCUGACGAUC S.pyogenes 71 TRBC -41 UGACAGCGGAAGUGGUUGCG S.pyogenes 72 TRBC -30 UUGACAGCGGAAGUGGUUGC S.pyogenes 73 TRBC -206 UCUCCGAGAGCCCGUAGAAC S.pyogenes 74 TRBC -32 CGGGUGGGAACACGUUUUUC S.pyogenes 75 TRBC -276 GACAGGUUUGGCCCUAUCCU S.pyogenes 76 TRBC -274 GAUCGUCAGCGCCGAGGCCU S.pyogenes 77 TRBC -230 GGCUCAAACACAGCGACCUC S.pyogenes 78 TRBC -235 UGAGGGUCUCGGCCACCUUC S.pyogenes 79 TRBC -38 AGGCUUCUACCCCGACCACG S.pyogenes 80 TRBC -223 CCGACCACGUGGAGCUGAGC S.pyogenes 81 TRBC -221 UGACAGGUUUGGCCCUAUCC S.pyogenes 82 TRBC -48 CUUGACAGCGGAAGUGGUUG S.pyogenes 83 TRBC -216 AGAUCGUCAGCGCCGAGGCC S.pyogenes 84 TRBC -210 GCGCGUGACGAUCUGGGUGAC S.pyogenes 85 TRBC -268 UGAGGGCGGGCUGCUCCUUG S.pyogenes 86 TRBC -193 GUUGCGGGGGUUCUGCCAGA S.pyogenes 87 TRBC -246 AGCUCAGCUCCACGUGGUCG S.pyogenes 88 TRBC -228 GCGGCUGCUCAGGCAGUAUC S.pyogenes 89 TRBC -43 GCGGGGGUUCUGCCAGAAGG S.pyogenes 90 TRBC -272 UGGCUCAAACACAGCGACCU S.pyogenes 91 TRBC -33 ACUCGACUGACCGGAAG S.pyogenes 92 TRBC -44 GACAGCGGAAGUGGUUGCGG S.pyogenes 93 TRBC -211 GCUGUCAAGUCCAGUUCUAC S.pyogenes 94 TRBC -253 GUAUCUGGAGUCAUUGAGGG S.pyogenes 95 TRBC -18 CUCGGCCGCUGACGAUCU S.pyogenes 96 TRBC -6 CCUCGGCGCUGACGAUC S.pyogenes 97 TRBC -85 CCGAGAGCCCGUAGAAC S.pyogenes 98 TRBC -129 CCAGAUCGUCAGCGCCG S.pyogenes 99 TRBC -93 GAUGAGAGUGGACCC S.pyogenes 100 TRBC -415 GGGUGACAGGUUUGGCCCUAUC S. aureus 101 TRBC -414 GGUGACAGGUUUGGCCCUAUC S. aureus 102 TRBC -310 GUGACAGGUUUGGCCCUAUC S. aureus 103 TRBC -308 GACAGGUUUGGCCCUAUC S. aureus 104 TRBC -401 GAUACUGCCUGAGCAGCCGCCU S. aureus 105 TRBC -468 GACCACGUGGAGCUGAGCUGGUGG S. aureus 106 TRBC -462 GUGGAGCUGAGCUGGUGG S. aureus 107 TRBC -424 GGGCGGGCUGCUCCUUGAGGGGCU S. aureus 108 TRBC -423 GGCGGGCUGCUCCUUGAGGGGCU S. aureus 109 TRBC -422 GCGGGCUGCUCCUUGAGGGGCU S. aureus 110 TRBC -420 GGGCUGCUCCUUGAGGGGCU S. aureus 111 TRBC -419 GGCUGCUCCUUGAGGGGCU S. aureus 112 TRBC -418 GCUGCUCCUUGAGGGGCU S. aureus 113 TRBC -445 GGUGAAUGGGAAGGAGGUGCACAG S. aureus 114 TRBC -444 GUGAAUGGGAAGGAGGUGCACAG S. aureus 115 TRBC -442 GAAUGGGAAGGAGGUGCACAG S. aureus 116

In some embodiments, the gRNA for targeting TRAC, TRBC1 and/or TRBC2 can be any of those described herein or elsewhere, such as in WO2015/161276, WO2017/193107, WO2017/093969, US2016/272999 and US2015/056705, or a targeting domain that binds to the targeting sequences described below. In some embodiments, the sequence targeted by the CRISPR/Cas9 gRNA in the TRAC gene locus is shown in SEQ ID NOS: 117, 163 and 165-211, such as GAGAATCAAAATCGGTGAAT (SEQ ID NO:163) or ATTCACCGATTTTGATTCTC (SEQ ID NO:117). In some embodiments, the sequence targeted by the CRISPR/Cas9 gRNA in the TRBC1 and/or TRBC2 gene loci is shown in SEQ ID NOS: 118, 164, and 212, such as GGCCTCGGCGCTGACGATCT (SEQ ID NO:164) or AGATCGTCAGCGCCGAGGCC (SEQ ID NO:118). In some embodiments, the gRNA targeting domain sequence for targeting a target site in the TRAC gene locus is GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:31). In some embodiments, the gRNA targeting domain sequence for targeting a target site in the TRBC1 and/or TRBC2 gene loci is GGCCUCGGCGCUGACGAUCU (SEQ ID NO:63).

In some embodiments, the gRNA for targeting the TRAC gene locus can be produced in vitro by transcription of the sequence AGCGCTCTCGTACAGAGTTGGCATTATAATACGACTCACTATAGGG GAGAATCAAAATCGGTGAAT GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT (shown in SEQ ID NO:26; the bold underlined portion is complementary to the target site in the TRAC locus) or by chemical synthesis, wherein the gRNA has the sequence 5'- GAG AAU CAA AAU CGG UGA AU G UUU UAG AGC UAG AAA UAG CAA GUU AAA AUA AGG CUA GUC CGU UAU CAA CUU GAA AAA GUG GCA CCG AGU CGG UGC UUU U -3' (as set forth in SEQ ID NO:27; see Osborn et al. , Mol Ther. 24(3):570-581 (2016)). Other exemplary gRNA sequences for producing genetic disruption of endogenous genes encoding TCR domains or regions, e.g., TRAC , TRBC1 , and/or TRBC2, are described, e.g., in International Patent Application Publication No. WO2015/161276. Exemplary methods for genome editing of endogenous TCR loci include those described in, e.g., U.S. Publication Nos. US2011/0158957, US2014/0301990, US2015/0098954, US2016/0208243; US2016/272999, and US2015/056705; International PCT Publication Nos. WO2014/191128, WO2015/136001, WO2015/161276, WO2016/069283, WO2016/016341, WO2017/193107, and WO2017/093969; and Osborn et al. (2016) Mol. Ther. 24(3):570-581. Any of the known methods that can be used to achieve genetic disruption of endogenous genes encoding TCR domains or regions can be used in the embodiments provided herein.

In some embodiments, the targeting domains comprise domains for introducing genetic disruption at the TRAC, TRBC1 , and/or TRBC2 loci using S. pyogenes Cas9 or using N. meningitidis Cas9. In some embodiments, the targeting domains comprise domains for introducing genetic disruption at the TRAC, TRBC1 , and/or TRBC2 loci using S. pyogenes Cas9. Either of the targeting domains can be used with a S. pyogenes Cas9 molecule that creates a double-strand break (Cas9 nuclease) or a single-strand break (Cas9 nickase).

In some embodiments, dual targeting is used to create two nicks on opposite strands of DNA using S. pyogenes Cas9 nicases with two targeting domains that are complementary to opposite strands of DNA, e.g., a gRNA containing any minus strand targeting domain can be paired with any gRNA containing a plus strand targeting domain. In some embodiments, the two gRNAs are targeted to DNA such as a PAM facing outward and the distance between the 5' ends of the gRNAs is 0-50 bp. In some embodiments, the two gRNAs are used to target two Cas9 nucleases or two Cas9 nicases, e.g., using a Cas9 molecule/gRNA molecule complex pair directed by two different gRNA molecules to cleave a target domain with two single-strand breaks on opposite strands of the target domain. In some embodiments, the two Cas9 nickases may comprise a molecule having HNH activity, e.g., a Cas9 molecule having RuvC activity that is inactivated, e.g., by a Cas9 molecule having a mutation at D10, e.g., a D10A mutation, a molecule having RuvC activity, e.g., a Cas9 molecule having HNH activity that is inactivated, e.g., by a Cas9 molecule having a mutation at H840, e.g., H840A, or a molecule having RuvC activity, e.g., a Cas9 molecule having HNH activity that is inactivated, e.g., by a Cas9 molecule having a mutation at N863, e.g., N863A. In some embodiments, each of the two gRNAs forms a complex with a D10A Cas9 nickase.

In some embodiments, the target sequence (target domain) is located adjacent to a TRAC, TRBC1 and/or TRBC2 locus, such as any portion of the TRAC, TRBC1 and/or TRBC2 coding sequence set forth in SEQ ID NOs: 1-3 or described in Tables 1-3 herein. In some embodiments, a target nucleic acid complementary to the targeting domain is located in an early coding region of a subject gene, such as TRAC, TRBC1 and/or TRBC2. Targeting an early coding region can be used to genetically disrupt (i.e., eliminate expression of) a subject gene. In some embodiments, the early coding region of a subject gene includes a sequence located immediately following an initiation codon (e.g., ATG) or within 500 bp of the initiation codon. from the initiation codon (e.g., less than 500, 450, 400, 350, 300, 250, 200, 150, 100, 50 bp, 40 bp, 30 bp, 20 bp, or 10 bp). In particular examples, the target nucleic acid is within 200 bp, 150 bp, 100 bp, 50 bp, 40 bp, 30 bp, 20 bp, or 10 bp of the initiation codon. In some examples, the gRNA targeting domain is complementary, such as at least 80, 85, 90, 95, 98, or 99% complementarity, such as fully complementary, to a target sequence on a target nucleic acid, such as a target nucleic acid in the TRAC, TRBC1 , and/or TRBC2 locus.

In some aspects, the gRNA can be targeted to a site within an exon of an open reading frame of an endogenous TRAC, TRBC1 , and/or TRBC2 locus. In some aspects, the gRNA can be targeted to a site within an intron of an open reading frame of a TRAC, TRBC1 , and/or TRBC2 locus. In some aspects, the gRNA can be targeted to a site within a regulatory or control element, such as a promoter, of a TRAC, TRBC1 , and/or TRBC2 locus. In some aspects, the target site on a TRAC , TRBC1 , and/or TRBC2 locus to which the gRNA is targeted can be any target site described herein, such as in Section IA1. In some embodiments, the gRNA may be targeted to a site within or close to exons corresponding to an early coding region, such as exon 1, 2, or 3 of the open reading frame of an endogenous TRAC, TRBC1 , and/or TRBC2 locus, or including a sequence immediately downstream of the transcription start site, within exon 1, 2, or 3, or within less than 500, 450, 400, 350, 300, 250, 200, 150, 100, or 50 bp. from exon 1, 2, or 3. In some embodiments, the gRNA may be targeted to a site near exon 2 of the endogenous TRAC, TRBC1 , and/or TRBC2 locus or within less than 500, 450, 400, 350, 300, 250, 200, 150, 100, or 50 bp of exon 2.

C) First complementarity domain

Examples of the first complementarity domain include those described in WO2015/161276, such as in FIGS. 1A-1G herein. The first complementarity domain is complementary to the second complementarity domain described herein, and typically has sufficient complementarity with the second complementarity domain to form a duplex region under at least some physiological conditions. The first complementarity domain is typically 5-30 nucleotides in length, and may be 5-25 nucleotides in length, 7-25 nucleotides in length, 7-22 nucleotides in length, 7-18 nucleotides in length, or 7-15 nucleotides in length. In different variants, the first complementarity domain is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length.

Typically, the first complementarity domain does not have exact complementarity with the target of the second complementarity domain. In some embodiments, the first complementarity domain may have 1, 2, 3, 4, or 5 nucleotides that are not complementary to the corresponding nucleotide of the second complementarity domain. For example, a segment of 1, 2, 3, 4, 5, or 6 (e.g., 3) nucleotides of the first complementarity domain may not pair into a duplex, and may form a non-duplex or off-loop region. In some cases, an unpaired or off-loop region, such as 3 nucleotides outside the loop, is present on the second complementarity domain. This unpaired region optionally begins 1, 2, 3, 4, 5, or 6, such as 4, nucleotides from the 5' end of the second complementarity domain.

The first complementarity domain may include 3 subdomains, which in the 5'-3' direction are: a 5' subdomain, a central subdomain and a 3' subdomain. In some embodiments, the 5' subdomain is 4-9, such as 4, 5, 6, 7, 8 or 9 nucleotides in length. In some embodiments, the central subdomain is 1, 2 or 3, such as 1, nucleotide in length. In some embodiments, the 3' subdomain is 3-25, such as 4-22, 4-18, or 4-10, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.

In some embodiments, the first and second complementarity domains, when formed into a duplex, comprise 11 base pairs, such as in the gRNA sequence (one base pair underlined, one in bold): NNNNNNNNNNNNNNNNNNNN GUUUUAG A GCUA GAAA UAGC AAG UUAAAAU AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:142).

In some embodiments, the first and second complementarity domains, when formed into a duplex, comprise 15 base pairs, such as in the gRNA sequence (one base pair underlined, one in bold): NNNNNNNNNNNNNNNNNNNN GUUUUAG A GCUAUGCU GAAA AGCAUAGC AAG UUAAAAU AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:143).

In some embodiments, the first and second complementarity domains, when formed into a duplex, comprise 16 base pairs, such as in the gRNA sequence (one base pair underlined, one in bold): NNNNNNNNNNNNNNNNNNNN GUUUUAG A GCUAUGCUG GAAA CAGCAUAGC AAG UUAAAAU AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:144).

In some embodiments, the first and second complementarity domains, when formed into a duplex, comprise 21 base pairs, such as in the gRNA sequence (one base pair underlined, one in bold): NNNNNNNNNNNNNNNNNNNN GUUUUAG A GCUAUGCUGUUUUG GAAA CAAAACAGCAUAGC AAG UUAAAAU AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:145).

In some embodiments, nucleotides are substituted to remove the poly-U ACTPt, such as in the gRNA sequences (substituted nucleotides are underlined): NNNNNNNNNNNNNNNNNNNNGU A UUAGAGCUAGAAAUAGCAAGUUAA U AUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:146); NNNNNNNNNNNNNNNNNNNNGUUU A AGAGCUAGAAAUAGCAAGUU U AAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:147); and NNNNNNNNNNNNNNNNNNNNNNGU A UUAGAGCUAUGCUGU A UUGGAAACAA U ACAGCAUAGCAAGUUAA U AUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:148).

The first complementarity domain may share homology with, or be derived from, a naturally occurring first complementarity domain. In some embodiments, it has at least 50% homology to a first complementarity domain described herein, such as a S. pyogenes , S. aureus, N. meningtidis, or S. thermophilus first complementarity domain.

It should be noted that one or more, or even all, of the nucleotides of the first complementarity domain may have a modification along the lines described here for the targeting domain.

D) Binding domain

Examples of binding domains include those described in WO2015/161276, such as in FIGS. 1A-1G herein. In a monomolecular or chimeric gRNA, the binding domain serves to link a first complementarity domain to a second complementarity domain of the monomolecular gRNA. The binding domain may link the first and second complementarity domains covalently or non-covalently. In some embodiments, the linkage is covalent. In some embodiments, the binding domain covalently links the first and second complementarity domains, see, such as WO2015/161276, such as in FIGS. 1B-1E herein. In some embodiments, the binding domain is or comprises a covalent linkage located between the first complementarity domain and the second complementarity domain. Typically, the binding domain comprises one or more, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides, but in different embodiments the linker may be 20, 30, 40, 50 or even 100 nucleotides in length.

In modular gRNA molecules, the two molecules linked by hybridization of the complementarity domains and the binding domain may not be present. See, for example, WO2015/161276, for example, in FIG. 1A herein.

A plurality of binding domains are suitable for use in monomolecular gRNA molecules. The binding domains may consist of a covalent bond or be short, i.e., one or more nucleotides, such as 1, 2, 3, 4, or 5 nucleotides in length. In some embodiments, the binding domain is 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides in length. In some embodiments, the binding domain is 2-50, 2-40, 2-30, 2-20, 2-10, or 2-5 nucleotides in length. In some embodiments, the binding domain shares homology with, or is derived from, a natural sequence, such as a tracrRNA sequence, that is 5' to the second complementarity domain. In some embodiments, the binding domain has at least 50% homology to a binding domain described herein.

As described here for the first complementarity domain, some or all of the nucleotides of the binding domain may include a modification.

E) 5’ Extension domain

In some cases, the modular gRNA may comprise an additional sequence, 5' to the second complementarity domain, referred to herein as a 5' extension domain, WO2015/161276, for example, in FIG. 1A herein. In some embodiments, the 5' extension domain is 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, or 2-4 nucleotides in length. In some embodiments, the 5' extension domain is 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides in length.

F) Second complementarity domain

Examples of a second complementarity domain include those described in WO2015/161276, such as in FIGS. 1A-1G herein. The second complementarity domain is complementary to the first complementarity domain, and typically has sufficient complementarity with the second complementarity domain to form a duplex region under at least some physiological conditions. In some cases, such as shown in WO2015/161276, such as in FIGS. 1A-1B herein, the second complementarity domain may include a sequence that does not have complementarity with the first complementarity domain, such as a sequence that falls outside the duplex region.

The second complementarity domain may be 5-27 nucleotides in length, and in some cases may be longer than the first region of complementarity. For example, the second complementarity domain may be 7-27 nucleotides in length, 7-25 nucleotides in length, 7-20 nucleotides in length, or 7-17 nucleotides in length. More specifically, the complementarity domain may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides in length.

In some embodiments, the second complementarity domain comprises 3 subdomains that, in the 5'-3' direction, include: a 5' subdomain, a central subdomain, and a 3' subdomain. In some embodiments, the 5' subdomain is 3-25, such as 4-22, 4-18, or 4-10, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the central subdomain is 1, 2, 3, 4, or 5, such as 3, nucleotides in length. In some embodiments, the 3' subdomain is 4-9, such as 4, 5, 6, 7, 8, or 9 nucleotides in length.

In some embodiments, the 5' subdomain and the 3' subdomain of the first complementarity domain, respectively, are complementary, such as fully complementary, with the 3' subdomain and the 5' subdomain of the second complementarity domain.

The second complementarity domain may share homology with or be derived from a naturally occurring second complementarity domain. In some embodiments, it has at least 50% homology to a second complementarity domain described herein, such as S. pyogenes , S. aureus, N. meningtidis , or S. thermophilus, the first complementarity domain.

Some or all of the nucleotides of the second complementarity domain may have a modification, such as the modification described herein.

G) Proximal domain

Examples of proximal domains include those described in WO2015/161276, such as in FIGS. 1A-1G herein. In some embodiments, the proximal domain is 5-20 nucleotides in length. In some embodiments, the proximal domain may share homology with or be derived from a natural proximal domain. In some embodiments, it has at least 50% homology to a proximal domain described herein, such as a S. pyogenes , S. aureus, N. meningtidis, or S. thermophilus proximal domain.

Some or all of the nucleotides of the proximal domain may have modifications along the lines described here.

H) Tail domain

Examples of tail domains include those described in WO2015/161276, such as in FIGS. 1A-1G herein. As can be seen by examining the tail domains in WO2015/161276, such as in FIGS. 1A and FIGS. 1B-1F herein, a wide variety of tail domains are suitable for use in gRNA molecules. In various embodiments, the tail domain is 0 (none), 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides in length. In certain embodiments, the nucleotides of the tail domain are derived from or share homology with a sequence from the 5' end of a natural tail domain, see, for example, WO2015/161276, such as in FIGS. 1D or 1E herein. The tail domain also optionally includes sequences that are complementary to each other and that, at least under some physiological conditions, form a duplex region.

Tail domains may share homology with or be derived from natural proximal tail domains. As a non-limiting example, a given tail domain according to various embodiments of the present invention may share at least 50% homology with a natural tail domain disclosed herein, such as a S. pyogenes , S. aureus, N. meningtidis, or S. thermophilus tail domain.

In certain cases, the tail domain includes nucleotides at the 3' end related to the mode of in vitro or in vivo transcription. If the T7 promoter is used for in vitro transcription of gRNA, these nucleotides may be any nucleotides present up to the 3' end of the template DNA. If the U6 promoter is used for in vivo transcription, these nucleotides may be the sequence UUUUUU. If alternative pol-III promoters are used, these nucleotides may be different numbers or uracil bases, or they may include alternative bases.

As a non-limiting example, in various embodiments, the proximal and tail domains taken together include the following sequences: AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU (SEQ ID NO:149), AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGGUGC (SEQ ID NO:150), AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGGAUC (SEQ ID NO:151), AAGGCUAGUCCGUUAUCAACUUGAAAAAGUG (SEQ ID NO:152), AAGGCUAGUCCGUUAUCA (SEQ ID NO:153), or AAGGCUAGUCCG (SEQ ID NO:154).

In some embodiments, the tail domain comprises a 3' UUUUUU sequence, for example, if the U6 promoter is used for transcription. In some embodiments, the tail domain comprises a 3' UUUU sequence, for example, if the H1 promoter is used for transcription. In some embodiments, the tail domain comprises a variable number of 3' U, depending on, for example, the stop codon of the pol-III promoter used. In some embodiments, the tail domain comprises a variable 3' sequence derived from a DNA template, for example, if the T7 promoter is used. In some embodiments, the tail domain comprises a variable 3' sequence derived from a DNA template, for example, if in vitro transcription is used to create an RNA molecule. In some embodiments, the tail domain comprises a variable 3' sequence derived from a DNA template, for example, if the pol-II promoter is used to carry out transcription.

In some embodiments, the gRNA has the following structure: 5' [targeting domain]-[first complementarity domain]-[binding domain]-[second complementarity domain]-[proximal domain]-[tail domain]-3', wherein the targeting domain comprises a core domain and, optionally, a secondary domain, and is 10-50 nucleotides in length; the first complementarity domain is 5-25 nucleotides in length and, in some embodiments, has at least 50, 60, 70, 80, 85, 90, 95, 98, or 99% homology to a reference first complementarity domain described herein; the binding domain is 1-5 nucleotides in length; the proximal domain is 5-20 nucleotides in length and, in some embodiments, has at least 50, 60, 70, 80, 85, 90, 95, 98 or 99% homology with the reference proximal domain described herein; and the tail domain is absent or has a nucleotide sequence of 1-50 nucleotides in length and, in some embodiments, has at least 50, 60, 70, 80, 85, 90, 95, 98 or 99% homology with the reference tail domain described herein.

I) Typical chimeric gRNAs

In some embodiments, the monomolecular or chimeric gRNA comprises, preferably from 5' to 3': a targeting domain, such as one comprising 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides (which are complementary to the target nucleic acid); a first complementarity domain; a binding domain; a second complementarity domain (which is complementary to the first complementarity domain); a proximal domain; and a tail domain, wherein (a) the proximal and tail domains taken together comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50 or 53 nucleotides; (b) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50 or 53 nucleotides from 3' to the last nucleotide of the second complementarity domain; or (c) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51 or 54 nucleotides from 3' to the last nucleotide of the second complementarity domain that are complementary to their corresponding nucleotides of the first complementarity domain.

In some embodiments, the sequences of (a), (b), or (c) have at least 60, 75, 80, 85, 90, 95, or 99% homology to the corresponding sequences of a naturally occurring gRNA or to an gRNA described herein. In some embodiments, the proximal and tail domains taken together comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides. In some embodiments, there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides from 3' to the last nucleotide of the second complementarity domain. In some embodiments, there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51 or 54 nucleotides from 3' to the last nucleotide of the second complementarity domain that are complementary to their corresponding nucleotides of the first complementarity domain. In some embodiments, the targeting domain comprises, has, or consists of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 consecutive nucleotides) that are complementary to the targeting domain, such as the targeting domain being 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides in length.

In some embodiments, a monomolecular or chimeric gRNA molecule (comprising a targeting domain, a first complementarity domain, a binding domain, a second complementarity domain, a proximal domain, and optionally a tail domain) comprises the following sequence, in which the targeting domain is depicted as 20 N but may be any sequence and is 16 to 26 nucleotides in length, and in which the gRNA sequence is followed by 6 U that serve as a stop codon for the U6 promoter, but which may be absent or in fewer numbers: NNNNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU (SEQ ID NO:155). In some embodiments, the monomolecular or chimeric gRNA molecule is an S. pyogenes gRNA molecule.

In some embodiments, a monomolecular or chimeric gRNA molecule (comprising a targeting domain, a first complementarity domain, a binding domain, a second complementarity domain, a proximal domain, and optionally a tail domain) comprises the following sequence, in which the targeting domain is depicted as 20 N but may be any sequence and is 16 to 26 nucleotides in length, and in which the gRNA sequence is followed by 6 U that serve as a stop codon for the U6 promoter, but which may be absent or in fewer numbers: NNNNNNNNNNNNNNNNNNNNNNGUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUUUU (SEQ ID NO:156). In some embodiments, the monomolecular or chimeric gRNA molecule is an S. aureus gRNA molecule. Sequences and structures of exemplary chimeric gRNAs are also shown in WO2015/161276, such as in FIGS. 10A-10B herein.

J) Typical modular gRNAs

In some embodiments, the modular gRNA comprises a first and a second strand. The first strand comprises, preferably, from 5' to 3'; a targeting domain, such as comprising 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides; a first complementarity domain. The second strand comprises, preferably, from 5' to 3': optionally a 5' extension domain; a second complementarity domain; a proximal domain; and a tail domain, wherein: (a) the proximal and tail domains taken together comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50 or 53 nucleotides; (b) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50 or 53 nucleotides from 3' to the last nucleotide of the second complementarity domain; or (c) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51 or 54 nucleotides from 3' to the last nucleotide of the second complementarity domain that are complementary to their corresponding nucleotides of the first complementarity domain.

In some embodiments, the sequences of (a), (b), or (c) have at least 60, 75, 80, 85, 90, 95, or 99% homology to the corresponding sequences of a naturally occurring gRNA or to an gRNA described herein. In some embodiments, the proximal and tail domains taken together comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides. In some embodiments, there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides from 3' to the last nucleotide of the second complementarity domain.

In some embodiments, there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51 or 54 nucleotides from 3' to the last nucleotide of the second complementarity domain that are complementary to their corresponding nucleotides of the first complementarity domain.

In some embodiments, the targeting domain comprises, has, or consists of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 consecutive nucleotides) that are complementary to the targeting domain, such as the targeting domain being 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides in length.

K) Methods for constructing gRNA

Methods for constructing gRNAs are described here, including methods for selecting, constructing, and validating targeting domains. Exemplary targeting domains are also presented here. The targeting domains discussed here can be incorporated into the gRNAs described here.

In some embodiments, a guide RNA (gRNA) specific for a target gene ( e.g., TRAC , TRBC1 , and/or TRBC2 in humans) is used for RNA-guided nucleases, such as Cas, to cause a DNA break at a target site or target position. Methods for constructing gRNAs and exemplary targeting domains may include those described in, for example, PCT International Publication No. WO2015/161276. Targeting domains may be introduced into gRNAs that are used to target a Cas9 nuclease to a target site or target position.

Methods for selecting and justifying target sequences, as well as untargeted analyses, are described, for example, in Mali et al., 2013 Science 339(6121): 823–826; Hsu et al. Nat Biotechnol, 31(9): 827–32; Fu et al., 2014 Nat Biotechnol, doi: 10,1038/nbt,2808. PubMed PMID: 24463574; Heigwer et al., 2014 Nat Methods 11(2):122–3. doi: 10,1038/nmeth,2812. PubMed PMID: 24481216; Bae et al., 2014 Bioinformatics PubMed PMID: 24463181; Xiao A et al., 2014 Bioinformatics PubMed PMID: 24389662.

In some embodiments, the software may be used to optimize the selection of gRNAs within user-defined target sequences, such as to minimize overall off-target activity across the genome. Off-target activity may be distinct from cleavage. For example, for each possible selection of gRNAs using S. pyogenes Cas9, the software may identify all potential off-target sequences (preceding either the NAG or NGG PAM) across the genome that contain up to a specified number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) mismatches. The cleavage efficiency at each off-target sequence may be predicted, such as using an experimentally derived weighting scheme. Each possible gRNA may then be ranked according to its overall predicted off-target cleavage; Leading gRNAs are those that are likely to have the most on-target and the least off-target cleavage. Other features, such as automated reagent design for gRNA vector construction, primer design for Surveyor target assay, and primer design for high-throughput detection and quantification of off-target cleavage via next-generation sequencing, may also be included in the tool. Potential gRNA molecules may be evaluated by methods known in the art or as described herein.

In some embodiments, gRNAs for use with S. pyogenes , S. aureus , and N. meningitidis Cas9s are identified using a DNA sequence search algorithm, such as custom gRNA design software based on the publicly available Cas-OFFinder tool (Bae et al. Bioinformatics. 2014; 30(10): 1473-1475). The custom gRNA design software scores guides after calculating their genome-wide off-target propensity. Typically, pairs ranked from a perfect match to 7 mismatches are considered for guides of length 17 to 24. In some aspects, once the computer has identified off-target sites, an aggregate score is calculated for each guide and tabulated using a web interface. In addition to identifying potential gRNA sites adjacent to PAM sequences, the program can also identify all PAM neighboring sequences that differ by 1, 2, 3, or more nucleotides from the selected gRNA sites. In some embodiments, genomic DNA sequences for each gene are obtained from the UCSC Genome Browser, and the sequences can be screened for the presence of repetitive elements using the publicly available RepeatMasker program. RepeatMasker screens input DNA sequences for the presence of repetitive elements and low-complexity regions. The output is a detailed annotation of the repeats present in a given target sequence.

Once identified, gRNAs can be ranked into ranks based on one or more of their distance to the target site, their orthogonality, and the presence of a 5' G (based on the identification of close matches in the human genome containing the corresponding PAM, e.g., in the case of S. pyogenes , the NGG PAM, in the case of S. aureus , the NNGRR (e.g., NNGRRT or NNGRRV) PAM, and in the case of N. meningtidis , the NNNNGATT or NNNNGCTT PAM). Orthogonality refers to the number of sequences in the human genome that contain a minimal number of mismatches with the target sequence. “High orthogonality” or “good orthogonality” may, for example, refer to 20-mer targeting domains that have no identical sequences in the human genome other than the intended target and no sequences that contain one or two mismatches in the target sequence. Targeting domains with good orthogonality are selected to minimize off-target DNA cleavage. It should be understood that this is a non-limiting example and that multiple strategies may be used to identify gRNAs for use with S. pyogenes , S. aureus , and N. meningitidis or other Cas9 enzymes.

In some embodiments, gRNAs for use with S. pyogenes Cas9 can be identified using the publicly available ZiFiT web server (Fu et al., Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol. 2014 Jan 26. doi: 10,1038/nbt,2808. PubMed PMID: 24463574, original references provided in Sander et al., 2007, NAR 35:W599-605; Sander et al., 2010, NAR 38: W462-8). In addition to identifying potential gRNA sites adjacent to PAM sequences, the program also identifies all PAM neighboring sequences that differ by 1, 2, 3, or more nucleotides from the selected gRNA sites. In some aspects, genomic DNA sequences for each gene can be retrieved from the UCSC Genome Browser, and the sequences can be screened for the presence of repetitive elements using the publicly available RepeatMasker program. RepeatMasker screens input DNA sequences for the presence of repetitive elements and low-complexity regions. The output is a detailed annotation of the repeats present in a given target sequence.

Once identified, the gRNAs for use with S. pyogenes Cas9 can be ranked into tiers, such as 5 tiers. In some embodiments, the targeting domains of the first tier of gRNA molecules are selected based on their distance to the target site, their orthogonality, and the presence of a 5' G (based on ZiFiT identification of close matches in the human genome containing the NGG PAM). In some embodiments, both 17-mer and 20-mer gRNAs are designed for targeting. In some aspects, gRNAs are also selected for a single-gRNA nuclease cleavage strategy and a dual-gRNA nickase strategy. Criteria for selecting gRNAs and determining which gRNAs can be used for which strategy can be based on several considerations. In some embodiments, gRNAs are identified for a single-gRNA nuclease cleavage strategy and for a dual-gRNA nickase strategy. In some embodiments, for gRNA selection, including determining which gRNAs can be used for the dual-nRNA nickase strategy, the gRNA pairs should be oriented toward the DNA such that the PAMs, facing outward and truncated with D10A Cas9 nickase, will yield 5' sticky ends. In some aspects, it may be expected that dual-nickase cleavage will result in deletion of all intervening sequence at a reasonable frequency. However, dual-nickase cleavage can also frequently result in insertion-deletion mutations in only one of the gRNAs. Potential pair members can be tested for how efficiently they remove the entire sequence compared to simply causing insertion-deletion mutations at the site of a single gRNA.

In some embodiments, the targeting domains of the first row of gRNA molecules may be selected based on (1) a reasonable distance to the target position, such as within the first 500 bp of the coding sequence downstream of the start codon, (2) a high level of orthogonality, and (3) the presence of a 5' G. In some embodiments, for the selection of the second row of gRNAs, the 5'G requirement may be waived, but the distance constraints are required and a high level of orthogonality is required. In some embodiments, for the selection of the third row, the same distance constraints and the 5'G requirement apply, but the requirement for good orthogonality is waived. In some embodiments, for the selection of the fourth row, the same distance constraints apply, but the requirement for good orthogonality and starting with a 5'G are waived. In some embodiments, the fifth row is chosen by omitting the requirement for good orthogonality and 5'G, and scanning longer sequences (e.g., the remaining coding sequences, e.g., an additional 500 bp upstream or downstream of the target site transcription). In some cases, no gRNAs are identified by the criteria of a particular row.

In some embodiments, gRNA is identified for single-gRNA nuclease cleavage as well as for dual-gRNA paired nickase strategy.

In some aspects, gRNAs for use with N. meningitidis and S. aureus Cas9s can be identified by manually scanning the genomic DNA sequence for the presence of PAM sequences. These gRNAs can be divided into two tiers. In some embodiments, for the first tier gRNAs, the targeting domains are selected within the first 500 bp of the coding sequence downstream of the start codon. In some embodiments, for the second tier gRNAs, the targeting domains are selected within the remaining coding sequence (downstream of the first 500 bp). In certain embodiments, no gRNAs are identified based on a particular tier criterion.

In some embodiments, another strategy for identifying guide RNAs (gRNAs) for use with S. pyogenes , S. aureus , and N. meningtidis Cas9s may employ a DNA sequence search algorithm. In some aspects, guide RNA design is performed using a custom guide RNA design software based on the publicly available Cas-OFFinder tool (Bae et al. Bioinformatics. 2014; 30(10): 1473-1475). The custom gRNA design software scores guides after calculating their genome-wide off-target propensity. Typically, pairs ranked from a perfect match to 7 mismatches are considered for guides of length 17 to 24. Once the computer has identified off-target sites, an aggregate score is calculated for each guide and tabulated using a web interface. In addition to identifying potential gRNA sites adjacent to PAM sequences, the program can also identify all PAM neighboring sequences that differ by 1, 2, 3, or more nucleotides from the selected gRNA sites. In some embodiments, genomic DNA sequences for each gene are obtained from the UCSC Genome Browser, and the sequences can be screened for the presence of repetitive elements using the publicly available RepeatMasker program. RepeatMasker screens input DNA sequences for the presence of repetitive elements and low-complexity regions. The output is a detailed annotation of the repeats present in a given target sequence.

In some embodiments, after identification, the gRNAs are ranked into ranks based on their distance to the target site or their orthogonality (based on the identification of close matches in the human genome containing the corresponding PAM, such as in the case of S. pyogenes , the NGG PAM, in the case of S. aureus , the NNGRR (e.g., NNGRRT or NNGRRV) PAM, and in the case of N. meningtidis, the NNNNGATT or NNNNGCTT PAM. In some aspects, a targeting domain with good orthogonality is selected to minimize off-target DNA cleavage.

As an example, for S. pyogenes and N. meningtidis targets, 17-mer or 20-mer gRNAs can be designed. As another example, for S. aureus targets, 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, and 24-mer gRNAs can be designed.

In some embodiments, gRNAs are identified for both single-gRNA nuclease cleavage and a dual-gRNA nickase strategy. In some embodiments, to select gRNAs, including determining which gRNAs can be used for a dual-gRNA nickase strategy, the gRNA pairs must be oriented on the DNA such that the PAMs facing outward and truncated with D10A Cas9 nickase will yield 5' sticky ends. In some aspects, dual-nickase cleavage can be expected to result in deletion of all intervening sequence at a reasonable frequency. However, dual-nickase cleavage can also frequently result in insertion-deletion mutations in only one of the gRNAs. Potential pair members can be tested for how efficiently they remove the entire sequence compared to simply causing insertion-deletion mutations at the site of a single gRNA.

To design strategies for genetic disruption, in some embodiments, targeting domains for set 1 gRNA molecules for S. pyogenes are selected based on their distance to the target site and their orthogonality (PAM is NGG). In some cases, targeting domains for set 1 gRNA molecules are selected based on (1) a reasonable distance to the target position, such as within the first 500 bp of the coding sequence downstream of the start codon and (2) a high level of orthogonality. In some aspects, a high level of orthogonality is not required for the selection of set 2 gRNAs. In some cases, set 3 gRNAs remove the requirement for good orthogonality and a longer sequence (e.g., the remaining coding sequence) can be scanned. In certain cases, no gRNAs are identified based on set-specific criteria.

To design strategies for genetic disruption, in some embodiments, the targeting domain for row 1 of the N. meningtidis gRNA molecules is selected within the first 500 bp of the coding sequence and has a high level of orthogonality. The targeting domain for row 2 of the N. meningtidis gRNA molecules is selected within the first 500 bp of the coding sequence and does not require high orthogonality. The targeting domain for row 3 of the N. meningtidis gRNA molecules is selected within the remainder of the coding sequence below 500 bp. It should be noted that the rows are non-inclusive (each gRNA is listed only once). In some cases, no gRNAs are identified based on the criteria of a particular row.

To design strategies for genetic disruption, in some embodiments, the targeting domain for set 1 of the S. aureus nRNA molecules is selected within the first 500 bp of the coding sequence, has a high level of orthogonality, and comprises an NNGRRT PAM. In some embodiments, the targeting domain for set 2 of the S. aureus nRNA molecules is selected within the first 500 bp of the coding sequence, does not require a level of orthogonality, and comprises an NNGRRT PAM. In some embodiments, the targeting domain for set 3 of the S. aureus nRNA molecules is selected within the remainder of the coding sequence downstream, and comprises an NNGRRT PAM. In some embodiments, the targeting domain for set 4 of the S. aureus nRNA molecules is selected within the first 500 bp of the coding sequence, and comprises an NNGRRV PAM. In some embodiments, the targeting domain for the set of 5 nRNA molecules for S. aureus is selected within the remainder of the coding sequence downstream and comprises the NNGRRV PAM. In certain cases, no nRNAs are identified based on the criteria of a particular set.

2) Cas9

Cas9 molecules from a variety of species can be used in the methods and compositions described herein. Although S. pyogenes, S. aureus, N. meningitidis , and S. thermophilus Cas9 molecules are the subject of much of the disclosure herein, Cas9 molecules derived from or based on Cas9 proteins of other species listed herein can also be used. In other words, although much of the disclosure herein utilizes S. pyogenes, S. aureus, N. meningitidis , and S. thermophilus Cas9 molecules, Cas9 molecules from other species can be substituted for them. Such species include: Acidov or ax avenae, Actinobacillus pleuropneumoniae, Actinobacillus succinogenes, Actinobacillus suis, Actinomyces sp., Cycliphilusdenitrificans, Aminomonas paucivorans, Bacillus cereus, Bacillus smithii, Bacillus thuringiensis, Bacteroides sp., Blastopirellula marina, Bradyrhizobium sp., Brevibacillus laterosp or us, Campylobacter coli, Campylobacter jejuni, Campylobacter lari, Candidatus puniceispirillum, Clostridium cellulolyticum, Clostridium perfringens, Corynebacterium accolens, Corynebacterium diphtheria, Corynebacterium matruchotii, Dinoroseobacter shibae, Eubacterium dolichum, Gammaproteobacterium, Gluconacetobacter diazotrophicus, Haemophilus parainfluenzae, Haemophilus sput or um, Helicobacter canadensis, Helicobacter cinaedi, Helicobacter mustelae, Ilyobacter polytropus, Kingella kingae, Lactobacillus crispatus, Listeria ivanovii, Listeria monocytogenes, Listeriaceae bacterium, Methylocystis sp., Methylosinus trichosp or ium, Mobiluncus mulieris, Neisseria bacillif or mis, Neisseria cinerea, Neisseria flavescens, Neisseria lactamica, Neisseria meningitidis, Neisseria sp., Neisseria wadsworthii, Nitrosomonas sp., Parvibaculum lavamentiv or ans, Pasteurella multocida, Phascolarctobacterium succinatutens, Ralstonia syzygii, Rhodopseudomonas palustris, Rhodovulum sp., Simonsiella muelleri, Sphingomonas sp., Sporolactobacillus vineae, Staphylococcus aureus, Staphylococcus lugdunensis, Streptococcus sp., Subdoligranulum sp., Tistrella mobilis, Treponema sp., or Verminephrobacter eiseniae. Examples of Cas9 molecules may include those described in, for example, WO2015/161276, WO2017/193107, WO2017/093969, US2016/272999, and US2015/056705.

A Cas9 molecule or a Cas9 polypeptide, as used herein, refers to a molecule or polypeptide that can interact with an gRNA molecule and, together with the gRNA molecule, is located or localized at a site that contains a target domain and a PAM sequence. A Cas9 molecule and a Cas9 polypeptide, as used herein, refers to naturally occurring Cas9 molecules and to engineered, altered, or modified Cas9 molecules or Cas9 polypeptides that differ, for example, by at least one amino acid residue, from a reference sequence, for example, the most similar naturally occurring Cas9 molecule.

Crystal structures have been determined for two different natural bacterial Cas9 molecules (Jinek et al., Science, 343(6176):1247997, 2014) and for S. pyogenes Cas9 with a guide RNA (e.g., a synthetic fusion of crRNA and tracrRNA) (Nishimasu et al., Cell, 156:935–949, 2014; and Anders et al., Nature, 2014, doi: 10.1038/nature13579).

The natural Cas9 molecule comprises two lobes: a recognition (REC) lobe and a nuclease (NUC) lobe; each of which further comprises domains described herein. A typical scheme for organizing the important Cas9 domains in a primary structure is described in WO2015/161276, for example, in FIGS. 8A-8B therein. The domain nomenclature and the numbering of amino acid residues encompassed by each domain used in this disclosure are as described in Nishimasu et al. The amino acid residue numbering is given with reference to Cas9 from S. pyogenes .

The REC domain contains an arginine-rich bridging helix (BH), the REC1 domain, and the REC2 domain. The REC domain has no structural similarity to any other known proteins, indicating that it is a Cas9-specific functional domain. The BH domain is a long α-helix and an arginine-rich region, and contains amino acids 60–93 of the S. pyogenes Cas9 sequence. The REC1 domain is essential for the recognition of repeat:antirepeat duplexes, such as gRNA or tracrRNA, and is therefore critical for Cas9 activity through target sequence recognition. The REC1 domain contains two REC1 motifs at amino acids 94–179 and 308–717 of the S. pyogenes Cas9 sequence. These two REC1 domains, although separated by the REC2 domain in the linear primary structure, assemble in a tertiary structure to form the REC1 domain. The REC2 domain or parts of it may also play a role in repeat:antirepeat duplex recognition. The REC2 domain contains amino acids 180-307 of the S. pyogenes Cas9 sequence.

The NUC lobe contains the RuvC domain (also referred to herein as the RuvC-like domain), the HNH domain (also referred to herein as the HNH-like domain), and the PAM-interacting (PI) domain. The RuvC domain has structural similarities to members of the retroviral integrase superfamily and cleaves one strand, e.g., the non-complementary strand, of a target nucleic acid molecule. The RuvC domain is assembled from three separated RuvC motifs (RuvC I, RuvCII, and RuvCIII, which are often collectively referred to as the RuvCI domain or N-terminal RuvC domain, RuvCII domain, and RuvCIII domain) at amino acids 1–59, 718–769, and 909–1098, respectively, of the S. pyogenes Cas9 sequence. Like the REC1 domain, the three RuvC motifs are linearly separated by other domains in the primary structure, but in the tertiary structure, the three RuvC motifs assemble to form the RuvC domain. The HNH domain has structural similarities to HNH endonucleases and cleaves one strand, for example, the complementary strand of the target nucleic acid molecule. The HNH domain lies between the RuvC II-III motifs and contains amino acids 775-908 of the S. pyogenes Cas9 sequence. The PI domain interacts with the PAM of the target nucleic acid molecule and contains amino acids 1099-1368 of the S. pyogenes Cas9 sequence.

A) RuvC-like domain and HNH-like domain

In some embodiments, the Cas9 molecule or Cas9 polypeptide comprises an HNH-like domain and a RuvC-like domain. In some embodiments, the cleavage activity is dependent on the RuvC-like domain and the HNH-like domain. The Cas9 molecule or Cas9 polypeptide, such as the eaCas9 molecule or eaCas9 polypeptide, may comprise one or more of the following domains: a RuvC-like domain and an HNH-like domain. In some embodiments, the Cas9 molecule or Cas9 polypeptide is an eaCas9 molecule or eaCas9 polypeptide, and the eaCas9 molecule or eaCas9 polypeptide comprises a RuvC-like domain, such as the RuvC-like domain described herein, and/or an HNH-like domain, such as the HNH-like domain described herein.

B) RuvC-like domains

In some embodiments, the RuvC-like domain cleaves a single strand, such as a non-complementary strand, of a target nucleic acid molecule. The Cas9 molecule or Cas9 polypeptide may include more than one RuvC-like domain (e.g., one, two, three or more RuvC-like domains). In some embodiments, the RuvC-like domain is at least 5, 6, 7, 8 amino acids in length, but not more than 20, 19, 18, 17, 16 or 15 amino acids in length. In some embodiments, the Cas9 molecule or Cas9 polypeptide comprises an N-terminal RuvC-like domain that is about 10-20 amino acids in length, such as about 15 amino acids in length.

C) N-terminal RuvC-like domains

Some natural Cas9 molecules contain more than one RuvC-like domain, where cleavage is dependent on the N-terminal RuvC-like domain. Therefore, a Cas9 molecule or Cas9 polypeptide may contain an N-terminal RuvC-like domain.

In a variant, the N-terminal RuvC-like domain is cleavage-competent.

In a variant, the N-terminal RuvC-like domain is cleavage-incompetent.

In some embodiments, the N-terminal RuvC-like domain differs from the sequence of the N-terminal RuvC-like domain described herein, such as in WO2015/161276, such as in FIGS. 3A-3B or FIGS. 7A-7B herein, by at least 1, but no more than 2, 3, 4, or 5 residues. In some embodiments, 1, 2, or all 3 of the highly conserved residues identified in WO2015/161276, such as in FIGS. 3A-3B or FIGS. 7A-7B herein, are present.

In some embodiments, the N-terminal RuvC-like domain differs from the sequence of the N-terminal RuvC-like domain described herein, such as in WO2015/161276, such as in FIGS. 3A-3B or FIGS. 7A-7B herein, by at least 1, but no more than 2, 3, 4, or 5 residues. In some embodiments, 1, 2, 3, or all 4 of the highly conserved residues identified in WO2015/161276, such as in FIGS. 4A-4B or FIGS. 7A-7B herein, are present.

D) Additional RuvC-like domains

In addition to the N-terminal RuvC-like domain, a Cas9 molecule or Cas9 polypeptide, such as an eaCas9 molecule or eaCas9 polypeptide, may comprise one or more additional RuvC-like domains. In some embodiments, a Cas9 molecule or Cas9 polypeptide may comprise two additional RuvC-like domains. Preferably, the additional RuvC-like domain is at least 5 amino acids in length and, for example, less than 15 amino acids in length, such as 5-10 amino acids in length, such as 8 amino acids in length.

E) HNH-like domains

In some embodiments, the HNH-like domain cleaves a single-stranded complementary domain, such as a complementary strand of a double-stranded nucleic acid molecule. In some embodiments, the HNH-like domain is at least 15, 20, 25 amino acids in length, but not more than 40, 35 or 30 amino acids in length, such as 20-35 amino acids in length, such as 25-30 amino acids in length. Exemplary HNH-like domains are described herein.

In some variants, the HNH-like domain is cleavage-competent.

In some variants, the HNH-like domain is cleavage-incompetent.

In some embodiments, the HNH-like domain differs from the sequence of the HNH-like domain described herein, such as in WO2015/161276, such as in FIGS. 5A-5C or FIGS. 7A-7B herein, by at least 1, but no more than 2, 3, 4, or 5 residues. In some embodiments, 1 or both of the highly conserved residues identified in WO2015/161276, such as in FIGS. 5A-5C or FIGS. 7A-7B herein, are present.

In some embodiments, the HNH-like domain differs from the sequence of the HNH-like domain described herein, such as in WO2015/161276, such as in FIGS. 5A-5C or FIGS. 7A-7B herein, by at least 1, but no more than 2, 3, 4, or 5 residues. In some embodiments, 1, 2, all 3 of the highly conserved residues identified in WO2015/161276, such as in FIGS. 6A-6B or FIGS. 7A-7B herein, are present.

F) Nuclease and helicase activity

In some embodiments, the Cas9 molecule or Cas9 polypeptide is capable of cleaving a target nucleic acid molecule. Typically, wild-type Cas9 molecules cleave both strands of a target nucleic acid molecule. Cas9 molecules and Cas9 polypeptides can be engineered to alter the nuclease cleavage (or other properties), such as to produce a Cas9 molecule or Cas9 polypeptide that is a nicase or lacks the ability to cleave a target nucleic acid. A Cas9 molecule or Cas9 polypeptide that is capable of cleaving a target nucleic acid molecule is referred to herein as an eaCas9 molecule or eaCas9 polypeptide.

In some embodiments, the eaCas9 molecule or eaCas9 polypeptide has one or more of the following activities: a nickase activity, e.g., the ability to cleave one strand, e.g., the non-complementary strand or the complementary strand, of a nucleic acid molecule; a double-stranded nuclease activity, i.e., the ability to cleave both strands of a double-stranded nucleic acid and create a double-strand break, which, in some embodiments, is the presence of two nickase activities; an endonuclease activity; an exonuclease activity; and a helicase activity, i.e., the ability to unwind the helical structure of a double-stranded nucleic acid.

In some embodiments, the enzymatically active or eaCas9 molecule or eaCas9 polypeptide cleaves both strands and causes a double-strand break. In some embodiments, the eaCas9 molecule cleaves only one strand, such as the strand to which the gRNA hybridizes or the strand complementary to the strand of the gRNA to which it hybridizes. In some embodiments, the eaCas9 molecule or eaCas9 polypeptide has cleavage activity associated with an HNH-like domain. In some embodiments, the eaCas9 molecule or eaCas9 polypeptide has cleavage activity associated with an N-terminal RuvC-like domain. In some embodiments, the eaCas9 molecule or eaCas9 polypeptide has cleavage activity associated with an HNH-like domain and cleavage activity associated with an N-terminal RuvC-like domain. In some embodiments, the eaCas9 molecule or eaCas9 polypeptide comprises an active or cleavage-competent HNH-like domain and an inactive or cleavage-incompetent N-terminal RuvC-like domain. In some embodiments, the eaCas9 molecule or eaCas9 polypeptide comprises an inactive or cleavage-incompetent HNH-like domain and an active or cleavage-competent N-terminal RuvC-like domain.

Some Cas9 molecules or Cas9 polypeptides have the ability to interact with a gRNA molecule, and in combination with the gRNA molecule, localize to the core target domain, but are unable to cleave the target nucleic acid or are unable to cleave at effective rates. Cas9 molecules that lack or substantially lack cleavage activity are referred to herein as an eiCas9 molecule or eiCas9 polypeptide. For example, an eiCas9 molecule or eiCas9 polypeptide may lack cleavage activity or have substantially less, such as less than 20, 10, 5, 1, or 0.1%, cleavage activity compared to a reference Cas9 molecule or eiCas9 polypeptide, as measured by the assay described herein.

G) Targeted Impact and PAM

A Cas9 molecule or Cas9 polypeptide is a polypeptide that can interact with a guide RNA (gRNA) molecule and, in combination with the gRNA molecule, localize to a site that contains the target domain and the PAM sequence.

In some embodiments, the ability of an eaCas9 molecule or eaCas9 polypeptide to interact with and cleave a target nucleic acid depends on a PAM sequence. A PAM sequence is a sequence in a target nucleic acid. In some embodiments, cleavage of a target nucleic acid occurs upstream of the PAM sequence. EaCas9 molecules from different bacterial species may recognize different sequence motifs (e.g., PAM sequences). In some embodiments, a S. pyogenes eaCas9 molecule recognizes a NGG, NAG, NGA sequence motif and directs cleavage of a target nucleic acid sequence 1-10, e.g., 3-5, base pairs upstream of that sequence. See, e.g., Mali et al., Science 2013; 339(6121): 823-826. In some embodiments, the S. thermophilus eaCas9 molecule recognizes an NGGNG and/or NNAGAAW (W=A or T) sequence motif and directs cleavage of a target nucleic acid sequence 1-10, e.g., 3-5, base pairs upstream of these sequences. See, e.g., Horvath et al. , Science 2010; 327(5962):167-170, and Deveau et al. , J Bacteriol 2008; 190(4): 1390-1400. In some embodiments, the S. mutans eaCas9 molecule recognizes an NGG and/or NAAR (R=A or G) sequence motif and directs cleavage of a core target nucleic acid sequence 1-10, e.g., 3-5 base pairs, upstream of this sequence. See, e.g., Deveau et al. , J Bacteriol 2008; 190(4): 1390-1400. In some embodiments, the S. aureus eaCas9 molecule recognizes a NNGRR (R=A or G) sequence motif and directs cleavage of a target nucleic acid sequence 1-10, e.g., 3-5, base pairs upstream of that sequence. In some embodiments, the S. aureus eaCas9 molecule recognizes a NNGRRT (R=A or G) sequence motif and directs cleavage of a target nucleic acid sequence 1-10, e.g., 3-5, base pairs upstream of that sequence. In some embodiments, the S. aureus eaCas9 molecule recognizes a NNGRRV (R=A or G) sequence motif and directs cleavage of a target nucleic acid sequence 1-10, e.g., 3-5, base pairs upstream of that sequence. In some embodiments, the N. meningitidis eaCas9 molecule recognizes a NNNNGATT or NNNGCTT sequence motif (R=A or G, V=A, G, or C and directs cleavage of a target nucleic acid sequence 1-10, e.g., 3-5, base pairs upstream of that sequence. See, e.g., Hou et al., PNAS Early Edition 2013, 1-6. The ability of a Cas9 molecule to recognize a PAM sequence can be determined, e.g., using the conversion assay described in Jinek et al. , Science 2012 337:816. In the above embodiments, N can be any nucleotide residue, e.g., any of A, G, C, or T.

As discussed here, Cas9 molecules can be engineered to alter the PAM specificity of the Cas9 molecule.

Typical natural Cas9 molecules are described in Chylinski et al., RNA Biology 2013 10:5, 727–737. Such Cas9 molecules include the bacterial family cluster 1–78 Cas9 molecules.

Typical natural Cas9 molecules include the bacterial family cluster 1 Cas9 molecule. Examples include the Cas9 molecule: S. pyogenes (e.g., strains SF370, MGAS10270, MGAS10750, MGAS2096, MGAS315, MGAS5005, MGAS6180, MGAS9429, NZ131, and SSI-1), S. thermophilus (e.g., strain LMD-9), S. pseudoporcinus (e.g., strain SPIN 20026), S. mutans (e.g., strain UA159, NN2025), S. macacae (e.g., strain NCTC11558), S. gallolyticus (e.g., strain UCN34, ATCC BAA-2069), S. equines (e.g., strain ATCC 9812, MGCS 124), S. dysdalactiae (e.g., strain GGS 124), S. bovis (e.g., strain ATCC 700338), S. anginosus (e.g., strain F0211), S. agalactiae (e.g., strain NEM316, A909), Listeria monocytogenes (e.g., strain F6854), Listeria innocua ( L. innocua , e.g., strain Clip11262), Enterococcus italicus (e.g., strain DSM 15952), or Enterococcus faecium (e.g., strain 1,231,408). Another exemplary Cas9 molecule is the Cas9 molecule of Neisseria meningitidis (Hou et al., PNAS Early Edition 2013, 1-6).

In some embodiments, a Cas9 molecule or Cas9 polypeptide, e.g., an eaCas9 molecule or eaCas9 polypeptide, comprises an amino acid sequence: having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology to; differing by no more than 2, 5, 10, 15, 20, 30, or 40% amino acid residues compared to; differing by at least 1, 2, 5, 10, or 20 amino acids, but no more than 100, 80, 70, 60, 50, 40, or 30 amino acids from; or identical to the sequence of any Cas9 molecule described herein or the sequence of a naturally occurring Cas9 molecule, such as a Cas9 molecule from a species listed herein (e.g., SEQ ID NOS:157-162) or described in Chylinski et al., RNA Biology 2013 10:5, 727-737; Hou et al., PNAS Early Edition 2013, 1-6. In some embodiments, the Cas9 molecule or Cas9 polypeptide has one or more of the following activities: a nickase activity; a double-strand cleavage activity (e.g., an endonuclease and/or exonuclease activity); a helicase activity; or the ability, in conjunction with a gRNA molecule, to localize to a target nucleic acid.

In some embodiments, the Cas9 molecule or Cas9 polypeptide comprises an amino acid sequence of the consensus sequence of WO2015/161276, such as in FIGS. 2A-2G herein, wherein “*” represents any amino acid found at the corresponding position of the amino acid sequence of the Cas9 molecule of S. pyogenes , S. thermophilus, S. mutans and L. innocua , and “-” represents any amino acid. In some embodiments, the Cas9 molecule or Cas9 polypeptide differs from the sequence of the consensus sequence of SEQ ID NOS: 157-162 or the consensus sequence described in WO2015/161276, such as in FIGS. 2A-2G herein, by at least 1, but not more than 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues. In some embodiments, the Cas9 molecule or Cas9 polypeptide comprises the amino acid sequence of SEQ ID NO: 162 or as described in WO2015/161276, such as in FIGS. 7A-7B herein, wherein “*” represents any amino acid found at the corresponding position in the amino acid sequence of the S. pyogenes or N. meningitidis Cas9 molecule, “-” represents any amino acid, and “-” represents any amino acid or the absence of an amino acid. In some embodiments, the Cas9 molecule or Cas9 polypeptide differs from the sequence of SEQ ID NO: 161 or 162 or as described in WO2015/161276, such as in FIGS. 7A-7B herein, by at least 1, but not more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues.

Sequence comparisons of multiple Cas9 molecules show that certain regions are conserved. These have been identified as: region 1 (residues 1–180, or in the case of region 1’, residues 120–180); region 2 (residues 360–480); region 3 (residues 660–720); region 4 (residues 817–900); and region 5 (residues 900–960).

In some embodiments, the Cas9 molecule or Cas9 polypeptide comprises regions 1-5, together with sufficient additional sequences of the Cas9 molecule to produce a biologically active molecule, e.g., a Cas9 molecule having at least one activity described herein. In some embodiments, each of regions 1-6, independently, has 50%, 60%, 70%, or 80% homology to the corresponding residues of a Cas9 molecule or Cas9 polypeptide described herein, e.g., as set forth in SEQ ID NOS:157-162, or a sequence described in WO2015/161276, e.g., from FIGS. 2A-2G or from FIGS. 7A-7B herein.

H) Engineered or modified Cas9 molecules and Cas9 polypeptides

The Cas9 molecules and Cas9 polypeptides described herein, e.g., naturally occurring Cas9 molecules, can have any number of properties, including: nickase activity, nuclease activity (e.g., endonuclease and/or exonuclease activity); helicase activity; the ability to operably associate with a gRNA molecule; and the ability to target (or localize to) a site on a nucleic acid (e.g., PAM recognition and specificity). In some embodiments, a Cas9 molecule or Cas9 polypeptide can include all or a subset of these properties. In exemplary embodiments, a Cas9 molecule or Cas9 polypeptide has the ability to interact with a gRNA molecule and, in combination with the gRNA molecule, localize to a site on a nucleic acid. Other activities, e.g., PAM specificity, cleavage activity, or helicase activity, can vary more widely among Cas9 molecules and Cas9 polypeptides.

Cas9 molecules include engineered Cas9 molecules and engineered Cas9 polypeptides (“engineered” in this context means that the Cas9 molecule or Cas9 polypeptide differs from reference sequences, and is not intended to be limited by process or origin). An engineered Cas9 molecule or Cas9 polypeptide may have altered enzymatic properties, such as an altered nuclease activity (relative to a natural or other reference Cas9 molecule) or an altered helicase activity. As discussed herein, an engineered Cas9 molecule or Cas9 polypeptide may have a nickase activity (as opposed to a double-stranded nuclease activity). In some embodiments, an engineered Cas9 molecule or Cas9 polypeptide may have an alteration that alters its size, such as a deletion of an amino acid sequence that reduces its size, for example, without significantly affecting one or more or any Cas9 activity. In some embodiments, an engineered Cas9 molecule or Cas9 polypeptide may comprise an alteration that affects PAM recognition. For example, an engineered Cas9 molecule may be altered to recognize a PAM sequence different from that recognized by the endogenous wild-type PI domain. In some embodiments, a Cas9 molecule or Cas9 polypeptide may differ in sequence from a native Cas9 molecule but have no significant change in one or more Cas9 activities.

Cas9 molecules or Cas9 polypeptides with desired properties can be obtained in a variety of ways, such as by altering parent, e.g., natural Cas9 molecules or Cas9 polypeptides, to produce an altered Cas9 molecule or Cas9 polypeptide having the desired property. For example, one or more mutations or changes can be introduced relative to a parent Cas9 molecule, e.g., a natural or engineered Cas9 molecule. Such mutations and changes include: substitutions (e.g., conservative substitutions or non-essential amino acid substitutions); insertions; or deletions. In some embodiments, a Cas9 molecule or Cas9 polypeptide can include one or more mutations or differences, such as at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, or 50 mutations, but less than 200, 100, or 80 mutations relative to a reference, e.g., parent Cas9 molecule.

In some embodiments, the mutation or mutations do not have a significant effect on Cas9 activity, such as the Cas9 activity described herein. In some embodiments, the mutation or mutations have a significant effect on Cas9 activity, such as the Cas9 activity described herein.

I) Cas9 molecules and Cas9 polypeptides, non-cleavage and with modified cleavage

In some embodiments, the Cas9 molecule or Cas9 polypeptide has a cleavage property that is different from naturally occurring Cas9 molecules, such as a cleavage property that is different from a naturally occurring Cas9 molecule that has the closest homology. For example, the Cas9 molecule or Cas9 polypeptide may differ from naturally occurring Cas9 molecules, such as a S. pyogenes Cas9 molecule, in the following: the ability to modulate, such as decrease or increase, the cleavage of a double-stranded nucleic acid (endonuclease and/or exonuclease activity), such as compared to a naturally occurring Cas9 molecule (e.g., a S. pyogenes Cas9 molecule); the ability to modulate, such as decrease or increase, the cleavage of a single strand of a nucleic acid, such as a non-complementary strand of a nucleic acid molecule or a complementary strand of a nucleic acid molecule (nickase activity), such as compared to a naturally occurring Cas9 molecule (e.g., a S. pyogenes Cas9 molecule); or the ability to cleave a nucleic acid molecule, such as a double-stranded or single-stranded nucleic acid molecule, which can be removed.

J) Modified cleavage of eaCas9 molecules and eaCas9 polypeptides

In some embodiments, the eaCas9 molecule or eaCas9 polypeptide has one or more of the following activities: a cleavage activity associated with an N-terminal RuvC-like domain; a cleavage activity associated with an HNH-like domain; a cleavage activity associated with an HNH-like domain, and a cleavage activity associated with an N-terminal RuvC-like domain.

In some embodiments, the eaCas9 molecule or eaCas9 polypeptide comprises an active or cleavage-competent HNH-like domain and an inactive or cleavage-incompetent N-terminal RuvC-like domain. An exemplary inactive or cleavage-incompetent N-terminal RuvC-like domain may have an aspartic acid mutation in the N-terminal RuvC-like domain, such as the aspartic acid at position 9 of the consensus sequence of SEQ ID NOS: 157-162 or the consensus sequence disclosed in WO2015/161276, such as in FIGS. 2A-2G herein, or the aspartic acid at position 10 of SEQ ID NO: 162, such as which may be substituted with alanine. In some embodiments, the eaCas9 molecule or eaCas9 polypeptide differs from the wild type in the N-terminal RuvC-like domain and does not cleave a target nucleic acid or cleaves with significantly less efficiency, such as less than 20, 10, 5, 1, or 1% of the cleavage activity of a reference Cas9 molecule, such as measured in an assay described herein. The reference Cas9 molecule may be a naturally occurring, unmodified Cas9 molecule, such as a naturally occurring Cas9 molecule such as a S. pyogenes or S. thermophilus Cas9 molecule. In some embodiments, the reference Cas9 molecule may be a naturally occurring Cas9 molecule having the closest sequence identity or homology.

In some embodiments, the eaCas9 molecule or eaCas9 polypeptide comprises an inactive or cleavage-incompetent HNH domain and an active or cleavage-competent N-terminal RuvC-like domain. Exemplary inactive or cleavage-incompetent HNH-like domains may have a mutation in one or more of: a histidine in the HNH-like domain, such as the histidine shown at position 856 of the consensus sequence of SEQ ID NOS:157-162 or the consensus sequence disclosed in WO2015/161276, such as in FIGS. 2A-2G herein, for example, it may be substituted with alanine; and one or more asparagines in the HNH-like domain, for example, the asparagine shown at position 870 of the consensus sequence SEQ ID NOS:157-162 or the consensus sequence disclosed in WO2015/161276, for example in FIGS. 2A-2G herein and/or at position 879 of the consensus sequence SEQ ID NOS:157-162 or the consensus sequence disclosed in WO2015/161276, for example in FIGS. 2A-2G herein, for example, which may be substituted with alanine. In some embodiments, eaCas9 differs from wild-type in the HNH-like domain and does not cleave a target nucleic acid or cleaves with significantly less efficiency, such as less than 20, 10, 5, 1, or 1% of the cleavage activity of a reference Cas9 molecule, such as measured in an assay described herein. The reference Cas9 molecule may be a naturally occurring, unmodified Cas9 molecule, such as a naturally occurring Cas9 molecule such as a S. pyogenes or S. thermophilus Cas9 molecule. In some embodiments, the reference Cas9 molecule may be a naturally occurring Cas9 molecule that has the closest sequence identity or homology.

In some embodiments, the eaCas9 molecule or eaCas9 polypeptide comprises an inactive or cleavage-incompetent HNH domain and an active or cleavage-competent N-terminal RuvC-like domain. Exemplary inactive or cleavage-incompetent HNH-like domains may have a mutation in one or more of: a histidine in the HNH-like domain, such as the histidine shown at position 856 of the consensus sequence of SEQ ID NOS:157-162 or the consensus sequence disclosed in WO2015/161276, such as in FIGS. 2A-2G herein, for example, it may be substituted with alanine; and one or more asparagines in the HNH-like domain, for example, the asparagine shown at position 870 of the consensus sequence SEQ ID NOS:157-162 or the consensus sequence disclosed in WO2015/161276, for example in FIGS. 2A-2G herein and/or at position 879 of the consensus sequence SEQ ID NOS:157-162 or the consensus sequence disclosed in WO2015/161276, for example in FIGS. 2A-2G herein, for example, which may be substituted with alanine. In some embodiments, eaCas9 differs from wild-type in the HNH-like domain and does not cleave a target nucleic acid or cleaves with significantly less efficiency, such as less than 20, 10, 5, 1, or 1% of the cleavage activity of a reference Cas9 molecule, such as measured in an assay described herein. The reference Cas9 molecule may be a naturally occurring, unmodified Cas9 molecule, such as a naturally occurring Cas9 molecule such as a S. pyogenes or S. thermophilus Cas9 molecule. In some embodiments, the reference Cas9 molecule may be a naturally occurring Cas9 molecule that has the closest sequence identity or homology.

K) Changes in the ability to cleave one or both strands of the target nucleic acid

In some embodiments, an exemplary Cas9 activity comprises one or more of PAM specificity, cleavage activity, and helicase activity. Mutations can be present, for example, in: one or more RuvC-like domains, for example, an N-terminal RuvC-like domain; an HNH-like domain; a region outside the RuvC-like domains and the HNH-like domain. In some embodiments, mutations are present in a RuvC-like domain, for example, an N-terminal RuvC-like. In some embodiments, mutations are present in an HNH-like domain. In some embodiments, mutations are present in both a RuvC-like domain, for example, an N-terminal RuvC-like domain, and an HNH-like domain.

Exemplary mutations that can be made in the RuvC domain or HNH domain with reference to the S. pyogenes sequence include: D10A, E762A, H840A, N854A, N863A and/or D986A.

In some embodiments, the Cas9 molecule or Cas9 polypeptide is an eiCas9 molecule or eiCas9 polypeptide that comprises one or more differences in the RuvC domain and/or the HNH domain compared to a reference Cas9 molecule, and the eiCas9 molecule or eiCas9 polypeptide does not cleave nucleic acid or cleaves with significantly less efficiency than the wild type, such as compared to the wild type in a cleavage assay, such as as described herein, cleaving less than 50, 25, 10, or 1% compared to the reference Cas9 molecule, as measured in the assay described herein.

Whether or not a particular sequence, such as a substitution, can affect one or more activities, such as targeting, cleavage activity, etc., can be assessed or predicted, for example, by assessing the conservation of the mutation. In some embodiments, a “non-essential” amino acid residue, as used in the context of a Cas9 molecule, is a residue that can be changed in a wild-type Cas9 molecule, such as a natural Cas9 molecule, such as an eaCas9 molecule, without abolishing, or more preferably without substantially changing, the Cas9 activity (e.g., cleavage activity), while changing an “essential” amino acid residue can cause a significant loss of activity (e.g., cleavage activity).

In some embodiments, the Cas9 molecule or Cas9 polypeptide has a cleavage property that is different from naturally occurring Cas9 molecules, such as a cleavage property that is different from a naturally occurring Cas9 molecule that has the closest homology. For example, the Cas9 molecule or Cas9 polypeptide may be different from naturally occurring Cas9 molecules, such as a S aureus , S pyogenes , or C jejuni Cas9 molecule, in the following: the ability to modulate, such as decrease or increase, double-strand break cleavage (endonuclease and/or exonuclease activity), such as compared to a naturally occurring Cas9 molecule (e.g., a S aureus , S pyogenes , or C jejuni Cas9 molecule) ; the ability to modulate, e.g. decrease or increase, the cleavage of one strand of a nucleic acid, e.g. a non-complementary strand of a nucleic acid molecule or a complementary strand of a nucleic acid molecule (nickase activity), e.g. compared to a natural Cas9 molecule (e.g. the Cas9 molecule of S aureus , S pyogenes or C jejuni ); or the ability to cleave a nucleic acid molecule, e.g. a double-stranded or single-stranded nucleic acid molecule, that can be excluded.

In some embodiments, the altered Cas9 molecule or Cas9 polypeptide is an eaCas9 molecule or eaCas9 polypeptide having one or more of the following activities: a cleavage activity associated with a RuvC domain; a cleavage activity associated with an HNH domain; a cleavage activity associated with an HNH domain; and a cleavage activity associated with a RuvC domain.

In some embodiments, the altered Cas9 molecule or Cas9 polypeptide is an eiCas9 molecule or eaCas9 polypeptide that does not cleave a nucleic acid molecule (either a double-stranded or single-stranded nucleic acid molecule) or cleaves a nucleic acid molecule with significantly less efficiency, such as less than 20, 10, 5, 1, or 0.1% of the cleaving activity of a reference Cas9 molecule, such as measured by an assay described herein. The reference Cas9 molecule can be a naturally occurring, unmodified Cas9 molecule, such as a naturally occurring Cas9 molecule such as a S. pyogenes , S. thermophilus, S. aureus, C. jejuni, or N. meningitidis Cas9 molecule. In some embodiments, the reference Cas9 molecule is a naturally occurring Cas9 molecule having the closest sequence identity or homology. In some embodiments, the eiCas9 molecule or eiCas9 polypeptide lacks significant RuvC domain-associated cleavage activity and HNH domain-associated cleavage activity.

In some embodiments, the altered Cas9 molecule or Cas9 polypeptide is an eaCas9 molecule or eaCas9 polypeptide comprising the fixed amino acid residues of S. pyogenes shown in the consensus sequence disclosed in WO2015/161276, such as in FIGS. 2A-2G herein, and having one or more amino acids that differ from the amino acid sequence of S. pyogenes (e.g., have a substitution) at one or more residues (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acid residues) in SEQ ID NO: 162 or a residue represented by “-” in the consensus sequence described in WO2015/161276, such as in FIGS. 2A-2G herein.

In some embodiments, an altered Cas9 molecule or Cas9 polypeptide, such as an eaCas9 molecule, may be a fusion of, for example, two or more different Cas9 molecules or Cas9 polypeptides, such as two or more natural Cas9 molecules from different species. For example, a fragment of a natural Cas9 molecule from one species may be fused to a fragment of a Cas9 molecule from a second species. As an example, a fragment of a Cas9 molecule from S. pyogenes containing an N-terminal RuvC-like domain may be fused to a fragment of a Cas9 molecule from a species other than S. pyogenes (e.g., S. thermophilus ) containing an HNH-like domain.

L) Cas9 Molecules with altered PAM recognition or without PAM recognition

Naturally occurring Cas9 molecules can recognize PAM sequences, e.g. recognition of PAM sequences is described here for S. pyogenes , S. thermophilus, S. mutans, S. aureus and N. meningitidis .

In some embodiments, the Cas9 molecule or Cas9 polypeptide has the same specificity for a PAM as a naturally occurring Cas9 molecule. In other embodiments, the Cas9 molecule or Cas9 polypeptide has a specificity for a PAM that is not related to a naturally occurring Cas9 molecule, or a specificity for a PAM that is not related to a naturally occurring Cas9 molecule to which it has the greatest sequence homology. For example, a naturally occurring Cas9 molecule can be altered, e.g., to alter PAM recognition, e.g., to alter the PAM sequence that the Cas9 molecule or Cas9 polypeptide recognizes to reduce target sites and/or improve specificity; or to eliminate the requirement for PAM recognition. In some embodiments, the Cas9 molecule can be altered, e.g., to increase the length of the PAM recognition sequence and/or improve the specificity of Cas9 to a high level of identity, e.g., to reduce target sites and improve specificity. In some embodiments, the length of the PAM recognition sequence is at least 4, 5, 6, 7, 8, 9, 10, or 15 amino acids in length.

Cas9 molecules or Cas9 polypeptides that recognize different PAM sequences and/or reduce off-target activity can be generated using targeted evolution. Exemplary methods and systems that can be used to targeted evolve Cas9 molecules are described, for example, in Svelte et al. Nature 2011, 472(7344): 499-503. Candidate Cas9 molecules can be evaluated, for example, by the methods described herein.

Alterations in the PI domain that mediates PAM recognition are discussed here.

M) Synthetic Cas9 molecules and Cas9 polypeptides with altered PI domains

Current genome editing techniques are limited by the diversity of target sequences that can be targeted by the PAM sequences recognized by the Cas9 molecule used. A synthetic Cas9 molecule (or Syn-Cas9 molecule) or synthetic Cas9 polypeptide (or Syn-Cas9 polypeptide), as the term is used herein, refers to a Cas9 molecule or Cas9 polypeptide that contains a Cas9 core domain from one bacterial species and a functionally altered PI domain, i.e., a PI domain different from that associated with the natural Cas9 core domain, such as from another bacterial species.

In some embodiments, the altered PI domain recognizes a PAM sequence that is different from the PAM sequence recognized by the naturally occurring Cas9 from which the Cas9 core domain is derived. In some embodiments, the altered PI domain recognizes the same PAM sequence recognized by the naturally occurring Cas9 from which the Cas9 core domain is derived, but with different affinity or specificity. The Syn-Cas9 molecule or Syn-Cas9 polypeptide can be, respectively, a Syn-eaCas9 molecule or a Syn-eaCas9 polypeptide or a Syn-eiCas9 molecule, a Syn-eiCas9 polypeptide.

A typical Syn-Cas9 molecule or Syn-Cas9 polypeptide comprises: a) a Cas9 core domain, e.g., a Cas9 core domain, e.g., a S. aureus, S. pyogenes , or C. jejuni Cas9 core domain; and b) an altered PI domain from the Cas9 sequence of species X.

In some embodiments, the RKR motif (PAM binding motif) of such an altered PI domain comprises: differences in 1, 2, or 3 amino acid residues; a difference in the amino acid sequence at the first, second, or third position; differences in the amino acid sequence at the first and second positions, the first and third positions, or the second and third positions; compared to the sequence of the RKR motif of a natural or endogenous PI domain associated with the Cas9 core domain.

In some embodiments, the Syn-Cas9 molecule or Syn-Cas9 polypeptide may also be size optimized, such that the Syn-Cas9 molecule or Syn-Cas9 polypeptide comprises one or more deletions, and optionally, one or more linkers located between amino acid residues adjacent to the deletions. In some embodiments, the Syn-Cas9 molecule or Syn-Cas9 polypeptide comprises a REC deletion.

N) Size-Optimized Cas9 Molecules and Cas9 Polypeptides

The engineered Cas9 molecules and engineered Cas9 polypeptides described herein include a Cas9 molecule or Cas9 polypeptide comprising a deletion that reduces the size of the molecule while maintaining desirable properties of Cas9, such as substantially natural conformation, Cas9 nuclease activity, and/or target recognition of nucleic acid molecules. Provided herein are Cas9 molecules or Cas9 polypeptides comprising one or more deletions and, optionally, one or more linkers, wherein the linker is located between amino acid residues that are adjacent to the deletion. Methods for identifying suitable deletions in a reference Cas9 molecule, methods for creating Cas9 molecules with a deletion and a linker, and methods for using such Cas9 molecules will be apparent from a review of this document.

A Cas9 molecule, such as a S. aureus , S. pyogenes , or C. jejuni Cas9 molecule having a deletion is smaller, such as having a reduced number of amino acids, than the corresponding naturally occurring Cas9 molecule. The smaller size of Cas9 molecules allows for increased flexibility in delivery methods and thereby increases utility for genome editing. A Cas9 molecule or Cas9 polypeptide may comprise one or more deletions that do not substantially affect or reduce the activity of the resulting Cas9 molecules or Cas9 polypeptides described herein. Activities retained in Cas9 molecules or Cas9 polypeptides comprising a deletion as described herein include one or more of the following: nickase activity, i.e., the ability to cleave one strand, such as the non-complementary strand or the complementary strand, of a nucleic acid molecule; double-stranded nuclease activity, i.e., the ability to cleave both strands of a double-stranded nucleic acid and create a double-strand break, which, in some embodiments, is the presence of two nickase activities; endo-nuclease activity; exonuclease activity; helicase activity, i.e., the ability to unwind the helical structure of a double-stranded nucleic acid; and nucleic acid molecule recognition activity, such as a target nucleic acid or gRNA.

The activity of the Cas9 molecules or Cas9 polypeptides described herein can be assessed using activity assays described herein or known.

O) Identifying regions suitable for deletion

Suitable regions of Cas9 molecules for deletion can be identified in a variety of ways. Naturally occurring orthologous Cas9 molecules from different bacterial species can be modeled into the crystal structure of S. pyogenes Cas9 (Nishimasu et al., Cell, 156:935–949, 2014) to examine the level of conversion of selected Cas9 orthologs with respect to the three-dimensional conformation of the protein. Less conserved or non-conserved regions that are spatially distant from regions involved in Cas9 activity, such as the interface with the target nucleic acid molecule and/or gRNA, represent regions or domains that are candidates for deletion without significantly affecting or reducing Cas9 activity.

R) REC-optimized Cas9 molecules and Cas9 polypeptides

A REC-optimized Cas9 molecule or REC-optimized Cas9 polypeptide, as used herein, refers to a Cas9 molecule or Cas9 polypeptide that comprises a deletion in one or both of the REC2 domains and the RE1 _CT domain (collectively, a REC deletion), wherein the deletion comprises at least 10% of the amino acid residues in the cognate domain. The REC-optimized Cas9 molecule or Cas9 polypeptide can be an eaCas9 molecule or eaCas9 polypeptide, or an eiCas9 molecule or eiCas9 polypeptide. An exemplary REC-optimized Cas9 molecule or REC-optimized Cas9 polypeptide comprises: a) a deletion selected from: i) a REC2 deletion; ii) a REC1 _CT deletion; or iii) a REC1 _SUB deletion.

Optionally, the linker is located between amino acid residues that surround the deletion. In some embodiments, the Cas9 molecule or Cas9 polypeptide includes only one deletion or only two deletions. The Cas9 molecule or Cas9 polypeptide can comprise a REC2 deletion and a REC1 _CT deletion. The Cas9 molecule or Cas9 polypeptide can comprise a REC2 deletion and a REC1 _SUB deletion.

In general, the deletion will comprise at least 10% of the amino acids in the cognate domain, for example, a REC2 deletion will include at least 10% of the amino acids in the REC2 domain. The deletion may comprise: at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% of the amino acid residues of its cognate domain; all of the amino acid residues of its cognate domain; an amino acid residue outside of its cognate domain; a plurality of amino acid residues outside of its cognate domain; an amino acid residue immediately N-terminal to its cognate domain; an amino acid residue immediately C-terminal to its cognate domain; an amino acid residue immediately N-terminal to its cognate and an amino acid residue immediately C-terminal to its cognate domain; a plurality of, for example, up to 5, 10, 15 or 20 amino acid residues N-terminal to its cognate domain; a plurality of, for example, up to 5, 10, 15 or 20 amino acid residues C-terminal to its cognate domain; a plurality of, for example, up to 5, 10, 15 or 20 amino acid residues N-terminal to its cognate domain; and a plurality of, for example, up to 5, 10, 15 or 20 amino acid residues C-terminal to its cognate domain.

In some embodiments, the deletion does not extend beyond: its cognate domain; the N-terminal amino acid residue of its cognate domain; or the C-terminal amino acid residue of its cognate domain.

The REC-optimized Cas9 molecule or REC-optimized Cas9 polypeptide may include a linker located between amino acid residues that surround the deletion. Suitable linkers for use between amino acid residues that surround the REC deletion in the REC-optimized Cas9 molecule are described herein.

In some embodiments, a REC-optimized Cas9 molecule or REC-optimized Cas9 polypeptide comprises an amino acid sequence, minus any REC deletions and an associated linker, that has at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% homology to the amino acid sequence of a naturally occurring Cas9, such as a S. aureus Cas9 molecule, a S. pyogenes Cas9 molecule, or a C. jejuni Cas9 molecule.

In some embodiments, a REC-optimized Cas9 molecule or REC-optimized Cas9 polypeptide comprises an amino acid sequence that, other than any REC deletions and an associated linker, differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 amino acid residues from the amino acid sequence of a naturally occurring Cas9, such as a S. aureus Cas9 molecule, a S. pyogenes Cas9 molecule, or a C. jejuni Cas9 molecule.

In some embodiments, the REC-optimized Cas9 molecule or REC-optimized Cas9 polypeptide comprises an amino acid sequence that, other than any REC deletion and associated linker, differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25% amino acid residues from the amino acid sequence of a naturally occurring Cas9, such as a S. aureus Cas9 molecule, a S. pyogenes Cas9 molecule, or a C. jejuni Cas9 molecule.

For sequence comparison, typically one sequence is a reference sequence to which the test sequences are compared. In applying a sequence comparison algorithm, the test and reference sequences are entered into the computer, subsequence coordinates are specified, if appropriate, and the program parameters of the sequence algorithm are specified. The program's "default" parameters may be used, or alternative parameters may be specified. The sequence comparison algorithm then calculates the percent sequence identity for the test sequences relative to the reference sequence, based on the program parameters. Methods for aligning sequences for comparison are well known. Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman, (1970) Adv. Appl. Math. 2:482c, the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443, or the similarity search method of Pearson and Lipman, (1988) Proc. Nat’l. Acad. Sci. USA 85:2444, computerized versions of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or manual alignment and visual inspection (see, e.g., Brent et al., (2003) Current Protocols in Molecular Biology).

Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity include the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., (1977) Nuc. Acids Res. 25:3389–3402; and Altschul et al., (1990) J. Mol. Biol. 215:403–410, respectively. The BLAST analysis program is publicly available through the National Center for Biotechnology Information.

The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (1988) Comput. Appl. Biosci. 4:11–17), which is included in the ALIGN program (version 2.0), using the PAM120 residue substitution weight table, a gap extension penalty of 12, and a gap penalty of 4. Alternatively, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:444-453), which is included in the GAP program in the GCG software package (available at www.gcg.com), using either the Blossom 62 matrix or the PAM250 matrix, and a gap opening penalty of 16, 14, 12, 10, 8, 6, or 4 and a gap extension penalty of 1, 2, 3, 4, 5, or 6.

Sequence information for typical REC deletions is provided for 83 naturally occurring Cas9 orthologs described in, for example, PCT International Publication Nos. WO2015/161276, WO2017/193107 and WO2017/093969.

Q) Nucleic acids encoding Cas9 molecules

Nucleic acids encoding Cas9 molecules or Cas9 polypeptides, such as an eaCas9 molecule or eaCas9 polypeptide, can be used in connection with any of the embodiments provided herein.

Exemplary nucleic acids encoding Cas9 molecules or Cas9 polypeptides are described in Cong et al., Science 2013, 399(6121):819-823; Wang et al., Cell 2013, 153(4):910-918; Mali et al., Science 2013, 399(6121):823-826; Jinek et al., Science 2012, 337(6096):816-821, and WO2015/161276, e.g., in FIG. 8 herein.

In some embodiments, the nucleic acid encoding the Cas9 molecule or Cas9 polypeptide may be a synthetic nucleic acid sequence. For example, the synthetic nucleic acid molecule may be chemically modified. In some embodiments, the Cas9 mRNA has one or more (e.g., all) of the following properties: it is capped, polyadenylated, substituted with 5-methylcytidine and/or pseudouridine.

Additionally or alternatively, the synthetic nucleic acid sequence may be codon optimized, such that at least one non-conventional codon or less common codon may be substituted for a common codon. For example, the synthetic nucleic acid may direct the synthesis of an optimized messenger mRNA, such as optimized for expression in a mammalian expression system, such as that described herein.

Additionally or alternatively, the nucleic acid encoding the Cas9 molecule or Cas9 polypeptide may contain a nuclear localization sequence (NLS). The NLS is known.

If any of the Cas9 sequences is fused to a peptide or polypeptide at the C-terminus, it is clear that the stop codon will be removed.

R) Other Cas molecules and Cas polypeptides

Various types of Cas molecules or Cas polypeptides can be used to practice the inventions disclosed herein. In some embodiments, Cas molecules of type II Cas systems can be used. In other embodiments, Cas molecules of other Cas systems can be used. For example, type I or type III Cas molecules can be used. Exemplary Cas molecules (and Cas systems) are described, for example, in Haft et al., PLoS Computational Biology 2005, 1(6): e60 and Makarova et al., Nature Review Microbiology 2011, 9:467-477, the contents of both references being incorporated herein by reference in their entireties. Exemplary Cas molecules (and Cas systems) are also shown in Table 6 .

Table 6 . Cas systems

Gene name ^‡ System type or subtype Name from Haft et al . ^§ Structure of the encoded protein (PDB access) ^¶ Encoded protein families (and superfamilies) ^#** Representatives cas1 • Type I • Type II • Type III cas1 3GOD, 3LFX and 2YZS COG1518 SERP2463, SPy1047 and ygbT cas2 • Type I • Type II • Type III cas2 2IVY, 2I8E and 3EXC COG1343 and COG3512 SERP2462, SPy1048, SPy1723 (N-terminal domain) and ygbF cas3 ' • Type I ^‡‡ cas3 ND COG1203 APE1232 and ygcB cas3 '' • Subtype I-A • Subtype I-B ND ND COG2254 APE1231 and BH0336 cas4 • Subtype I-A • Subtype I-B • Subtype I-C • Subtype I-D • Subtype II-B cas4 and csa1 ND COG1468 APE1239 and BH0340 cas5 • Subtype I-A • Subtype I-B • Subtype I-C • Subtype I-E cas5a , cas5d , cas5e , cas5h , cas5p, cas5t and cmx5 3KG4 COG1688 (RAMP) APE1234, BH0337, devS and ygcI cas6 • Subtype IA • Subtype IB • Subtype ID • Subtype III-A
• Subtype III-B cas6 and cmx6 3I4H COG1583 and COG5551 (RAMP) PF1131 and slr7014 cas6e • Subtype I-E cse3 1WJ9 (RAMP) ygcH cas6f • Subtype I-F csy4 2XLJ (RAMP) y1727 cas7 • Subtype I-A • Subtype I-B • Subtype I-C • Subtype I-E csa2 , csd2 , cse4 , csh2 , csp1 and cst2 ND COG1857 and COG3649 (RAMP) devR and ygcJ cas8a1 • Subtype IA ^‡‡ cmx1 , cst1 , csx8 , csx13 and CXXC-CXXC ND BH0338-like LA3191 ^§§ and PG2018 ^§§ cas8a2 • Subtype IA ^‡‡ csa4 and csx9 ND PH0918 AF0070, AF1873, MJ0385, PF0637, PH0918 and SSO1401 cas8b • Subtype IB ^‡‡ csh1 and TM1802 ND BH0338-like MTH1090 and TM1802 cas8c • Subtype IC ^‡‡ csd1 and csp2 ND BH0338-like BH0338 cas9 • Type II ^‡‡ csn1 and csx12 ND COG3513 FTN_0757 and SPy1046 cas10 • Type III ^‡‡ cmr2 , csm1 and csx11 ND COG1353 MTH326, Rv2823c ^§§ and TM1794 ^§§ cas10d • Subtype ID ^‡‡ csc3 ND COG1353 slr7011 csy1 • Subtype IF ^‡‡ csy1 ND y1724-like y1724 csy2 • Subtype I-F csy2 ND (RAMP) y1725 csy3 • Subtype I-F csy3 ND (RAMP) y1726 cse1 • IE subtype ^‡‡ cse1 ND YgcL-like ygcl cse2 • Subtype I-E cse2 2ZCA YgcK-like ygcK csc1 • Subtype I-D csc1 ND alr1563-like (RAMP) alr1563 csc2 • Subtype I-D csc1 and csc2 ND COG1337 (RAMP) slr7012 csa5 • Subtype I-A csa5 ND AF1870 AF1870, MJ0380, PF0643 and SSO1398 csn2 • Subtype II-A csn2 ND SPy1049-like SPy1049 csm2 • Subtype III-A ^‡‡ csm2 ND COG1421 MTH1081 and SERP2460 csm3 • Subtype III-A csc2 and csm3 ND COG1337 (RAMP) MTH1080 and SERP2459 csm4 • Subtype III-A csm4 ND COG1567 (RAMP) MTH1079 and SERP2458 csm5 • Subtype III-A csm5 ND COG1332 (RAMP) MTH1078 and SERP2457 csm6 • Subtype III-A APE2256 and csm6 2WTE COG1517 APE2256 and SSO1445 cmr1 • Subtype III-B cmr1 ND COG1367 (RAMP) PF1130 cmr3 • Subtype III-B cmr3 ND COG1769 (RAMP) PF1128 cmr4 • Subtype III-B cmr4 ND COG1336 (RAMP) PF1126 cmr5 • Subtype III-B ^‡‡ cmr5 2ZOP and 2OEB COG3337 MTH324 and PF1125 cmr6 • Subtype III-B cmr6 ND COG1604 (RAMP) PF1124 csb1 • Subtype I-U GSU0053 ND (RAMP) Balac_1306 and GSU0053 csb2 • Subtype IU ^§§ ND ND (RAMP) Balac_1305 and GSU0054 csb3 • Subtype I-U ND ND (RAMP) Balac_1303 ^§§ csx17 • Subtype I-U ND ND ND Btus_2683 csx14 • Subtype I-U ND ND ND GSU0052 csx10 • Subtype I-U csx10 ND (RAMP) Caur_2274 csx16 • Subtype III-U VVA1548 ND ND VVA1548 csaX • Subtype III-U csaX ND ND SSO1438 csx3 • Subtype III-U csx3 ND ND AF1864 csx1 • Subtype III-U csa3 , csx1 , csx2 , DXTHG, NE0113 and TIGR02710 1XMX and 2I71 COG1517 and COG4006 MJ1666, NE0113, PF1127 and TM1812 csx15 • Unknown ND ND TTE2665 TTE2665 csf1 • Type U csf1 ND ND AFE_1038 csf2 • Type U csf2 ND (RAMP) AFE_1039 csf3 • Type U csf3 ND (RAMP) AFE_1040 csf4 • Type U csf4 ND ND AFE_1037

3) Cpf1

In some embodiments, a guide RNA or gRNA facilitates the specific association of an RNA-guided nuclease, such as Cas9 or Cpf1, with a target sequence, such as a genomic or episomal sequence in a cell. In general, gRNAs can be unimolecular (comprising a single RNA molecule, and alternatively referred to as chimeric) or modular (comprising more than one, typically two, separate RNA molecules, such as crRNAs and tracrRNAs, which are typically linked to each other, such as by duplexing). gRNAs and portions thereof have been described in the literature, such as in Briner et al. (Molecular Cell 56(2), 333-339, October 23, 2014 (Briner), which is incorporated herein by reference), and in Cotta-Ramusino.

Guide RNAs, whether monomolecular or modular, typically include a targeting domain that is fully or partially complementary to the target and is typically 10-30 nucleotides in length, and in certain embodiments, is 16-24 nucleotides in length (e.g., 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides in length). In some aspects, the targeting domains are at or near the 5' end of the gRNA in the case of Cas9 gRNA, and at or near the 3' end in the case of Cpf1 gRNA. Although the foregoing description has focused on gRNAs for use with Cas9, it should be understood that other RNA-guided nucleases have been (or may be) discovered or developed that utilize gRNAs that differ in some way from those described herein. For example, Cpf1 (“CRISPR from Prevotella and Franciscella 1”) is a recently discovered RNA-guided nuclease that does not require tracrRNA to function (Zetsche et al., 2015, Cell 163, 759-771 October 22, 2015 (Zetsche I), incorporated herein by reference). gRNAs for use in the Cpf1 genome editing system typically include a targeting domain and a complementarity domain (alternatively called a “handle”). It should also be noted that in gRNAs for use with Cpf1, the targeting domain is typically present at or near the 3’ end, rather than at the 5’ end as described above for Cas9 gRNAs (the handle is near the 5’ end of Cpf1 gRNAs).

Although structural differences may exist between gRNAs from different prokaryotic species or between Cpf1 and Cas9 gRNAs, the operating principles of gRNAs are generally consistent. Because of their consistent operation, gRNAs may be defined broadly by their targeting domain sequences, and those skilled in the art will appreciate that a given targeting domain sequence may be introduced into any suitable gRNA, including a monomolecular or chimeric gRNA or a gRNA that includes one or more chemical modifications and/or sequence modifications (substitutions, additional nucleotides, truncations, etc.). Thus, in some aspects of this disclosure, gRNAs may be described solely in terms of their targeting domain sequences.

More generally, certain aspects of this disclosure relate to systems, methods, and compositions that can be used with a variety of RNA-guided nucleases. Unless otherwise specified, the term gRNA should be understood to encompass any suitable gRNA that can be used with any RNA-guided nuclease, and not just those gRNAs that are compatible with particular types of Cas9 or Cpf1. By way of illustration, the term gRNA can, in certain embodiments, include gRNAs for use with any RNA-guided nuclease that exists in a Class 2 CRISPR system, such as a Type II or Type V CRISPR system, or an RNA-guided nuclease derived from or adapted therefrom.

Certain exemplary modifications discussed in this section may be included at any position in the gRNA sequence including, but not limited to, near the 5' end (e.g., within 1-10, 1-5, or 1-2 nucleotides of the 5' end) and/or near the 3' end (e.g., within 1-10, 1-5, or 1-2 nucleotides of the 3' end). In some cases, the modifications are located in functional motifs such as the repeat-anti-repeat duplex of Cas9 gRNA, the stem-loop structure of Cas9 or Cpf1 gRNA, and/or a targeting domain of gRNA.

RNA-guided nucleases include, but are not limited to, naturally occurring class 2 CRISPR nucleases such as Cas9 and Cpf1, as well as other nucleases derived from or derived therefrom. Functionally, RNA-guided nucleases are defined as those nucleases that: (a) interact with (e.g., form a complex with) a gRNA; and (b) together with the gRNA bound thereto, and optionally cleave or modify a target region of DNA that includes (i) a sequence complementary to the targeting domain of the gRNA and, optionally, (ii) an additional sequence termed a “protospacer adjacent motif” or “PAM,” which is described in more detail below. As will be shown in the following examples, RNA-guided nucleases can be defined, broadly speaking, by their PAM specificity and cleavage activity, even though variations can exist between individual RNA-guided nucleases that have a common PAM specificity or cleavage activity. Those skilled in the art will appreciate that certain aspects of this disclosure relate to systems, methods, and compositions that can be used using any suitable RNA-guided nucleases that have a particular PAM specificity and/or cleavage activity. For this reason, unless otherwise specified, the term RNA-guided nuclease should be understood as a general term and not limited to any particular type (e.g., Cas9 to Cpf1), species (e.g., S. pyogenes to S. aureus ), or variation (e.g., full-length to truncated or cleaved; naturally occurring PAM specificity to engineered PAM specificity, etc.) of RNA-guided nuclease.

In addition to recognizing specific sequence orientations of PAMs and protospacers, RNA-guided nucleases in some variants can also recognize specific PAM sequences. S. aureus Cas9, for example, typically recognizes the PAM sequence NNGRRT or NNGRRV, where the N residues are immediately following the 3' region recognized by the gRNA targeting domain. S. pyogenes Cas9 typically recognizes NGG PAM sequences. And F. novicida Cpf1 typically recognizes the TTN PAM sequence.

The crystal structure of Acidaminococcus sp. Cpf1 in complex with crRNA and a double-stranded (ds) DNA target including the TTTN PAM sequence was solved by Yamano et al. (Cell. 2016 May 5; 165(4): 949–962 (Yamano), incorporated herein by reference). Cpf1, like Cas9, has two lobes: the REC (recognition) lobe and the NUC (nuclease) lobe. The REC lobe contains the REC1 and REC2 domains, which do not resemble any known protein structures. The NUC lobe, meanwhile, contains three RuvC domains (RuvC-I, -II, and -III) and a BH domain. However, unlike Cas9, the Cpf1 REC lobe lacks an HNH domain and includes other domains that also bear no resemblance to known protein structures: a structurally unique PI domain, three Wedge (WED) domains (WED-I, -II, and -III), and a nuclease (Nuc) domain.

Although Cas9 and Cpf1 are similar in structure and function, it should be clear that certain Cpf1 activities are mediated by structural domains that are not analogous to any Cas9 domains. For example, cleavage of the complementary strand of target DNA is likely mediated by the Nuc domain, which is sequenceally and spatially distinct from the HNH domain of Cas9. Additionally, the off-target portion of Cpf1 gRNA (the handle) adopts a pseudoknot structure rather than the stem-loop structure formed by the repeat:antirepeat duplex in Cas9 gRNA.

Nucleic acids encoding RNA-guided nucleases, such as Cas9, Cpf1, or their functional fragments, are presented here. Exemplary nucleic acids encoding RNA-guided nucleases have been described previously (see, e.g., Cong 2013; Wang 2013; Mali 2013; Jinek 2012).

3. Delivery of agents for genetic destruction

In some embodiments, targeted genetic disruption, e.g., DNA disruption, of endogenous genes encoding TCRs, such as TRAC and TRBC1 or TRBC2 in humans, is accomplished by delivering or introducing one or more agents capable of causing genetic disruption, e.g., Cas9 and/or gRNA components, into a cell using any number of known delivery methods or vehicles for introduction or transfer into cells, e.g., using lentiviral delivery vectors or any known methods or vehicles for delivering Cas9 molecules and gRNA. Exemplary methods are described in, e.g., Wang et al. (2012) J. Immunother. 35(9): 689-701; Cooper et al. (2003) Blood. 101:1637-1644; Verhoeyen et al. (2009) Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003) Blood. 102(2): 497-505. In some embodiments, nucleic acid sequences encoding one or more components of one or more agents capable of causing genetic disruption, such as DNA breakage, are introduced into cells, such as by any methods for introducing nucleic acids into a cell described herein or known. In some embodiments, a vector encoding components of one or more agents capable of causing genetic disruption, such as a CRISPR guide RNA and/or a Cas9 enzyme, can be delivered to a cell.

In some embodiments, one or more agents capable of causing genetic destruction, such as one or more agents that are Cas9/gRNA, are introduced into the cell as a ribonucleoprotein (RNP) complex. RNP complexes include a ribonucleotide sequence, such as an RNA or gRNA molecule, and a protein, such as a Cas9 protein or a variant thereof. For example, a Cas9 protein is delivered as an RNP complex that includes a Cas9 protein and a gRNA molecule for targeting a target sequence, such as by electroporation or another physical method. In some embodiments, the RNP is delivered into the cell by electroporation or other physical means, such as a gene gun, calcium phosphate transfection, cell condensation or compression. In some embodiments, the RNP can pass through the plasma membrane of the cell without the need for additional delivery agents (e.g., small molecule agents, lipids, etc.). In some embodiments, delivery of one or more agents capable of causing genetic disruption, such as CRISPR/Cas9, as an RNP has the advantage that the targeted disruption occurs transiently, such as in cells into which the RNP is introduced, without propagation of the agent to progeny cells. For example, delivery of an RNP minimizes the transmission of the agent to its progeny, thereby reducing the chance of off-target genetic disruption in the progeny. In such cases, the genetic disruption and transgene integration (discussed further in Section I.B) may be inherited by progeny cells, but without the agent itself, which may further cause off-target genetic disruption to be transmitted to progeny cells.

Agents and components capable of causing genetic destruction, such as a Cas9 molecule and a gRNA molecule, can be introduced into cells in a variety of forms using a variety of delivery methods and formulations, as shown in Tables 7 and 8 or the methods described in, for example, WO 2015/161276; US 2015/0056705, US 2016/0272999, US 2017/0211075; or US 2017/0016027. As described below, the delivery methods and formulations can be used to deliver template polynucleotides and/or other agents to cells in previous or subsequent steps of the methods described herein.

Table 7. Typical delivery methods

Elements Comments Cas9 molecules gRNA molecules DNA DNA In this variant, the Cas9 molecule and gRNA are read from DNA. In this variant, they are encoded into separate molecules. DNA In this variant, the Cas9 molecule and gRNA are read from DNA, here from one molecule. DNA RNA In this embodiment, the Cas9 molecule is read from DNA and gRNA is obtained as in vitro read or synthesized RNA. mRNA RNA In this embodiment, the Cas9 molecule is translated from in vitro transcribed mRNA, and gRNA is obtained as in vitro transcribed or synthesized RNA. mRNA DNA In this embodiment, the Cas9 molecule is translated from in vitro transcribed mRNA, and gRNA is transcribed from DNA. Protein DNA In this variant, the Cas9 molecule is produced as a protein and the gRNA is read from DNA. Protein RNA In this embodiment, the Cas9 molecule is produced as a protein and the gRNA is produced as either read or synthesized RNA.

Table 8. Comparison of typical delivery methods

Delivery Vector/Mode Delivery to non-dividing cells Duration of expression Genome integration Type of molecule delivered Physical (e.g. electroporation, gene gun, calcium phosphate transfection, cell condensation or compression) YES Temporary NO Nucleic acids and proteins Viral Retrovirus NO Stable YES RNA Lentivirus YES Stable YES/NO with modifications RNA Adenovirus YES Temporary NO DNA Adeno-associated virus (AAV) YES Stable NO DNA Vaccinia virus YES Very temporary NO DNA Herpes simplex virus YES Stable NO DNA Not viral Cationic liposomes YES Temporary Depends on what is being delivered Nucleic acids and proteins Polymer nanoparticles YES Temporary Depends on what is being delivered Nucleic acids and proteins Biological non-viral delivery systems weakened bacteria YES Temporary NO Nucleic acids Engineered bacteriophages YES Temporary NO Nucleic acids Mammalian virus-like particles YES Temporary NO Nucleic acids Biological liposomes: erythrocyte ghosts and exosomes YES Temporary NO Nucleic acids

In some embodiments, DNA encoding Cas9 molecules and/or gRNA molecules or RNP complexes containing a Cas9 molecule and/or gRNA molecules can be delivered to cells by known methods or as described herein. For example, Cas9-encoding and/or gRNA-encoding DNA can be delivered, for example, by vectors (e.g., viral or non-viral vectors), non-vector methods (e.g., using naked DNA or DNA complexes), or combinations thereof. In some embodiments, a polynucleotide containing the agents and/or their components is delivered by a vector (e.g., a viral vector/virus or plasmid). The vector can be any of those described herein.

In some aspects, a CRISPR enzyme ( e.g., Cas9 nuclease) in combination with (and optionally in complex with) a guide sequence is delivered to a cell. For example, one or more elements of a CRISPR system are derived from a type I, type II, or type III CRISPR system. For example, one or more elements of a CRISPR system are derived from a particular organism that contains an endogenous CRISPR system, such as Streptococcus pyogenes , Staphylococcus aureus , or Neisseria meningitides.

In some embodiments, a Cas9 nuclease ( e.g., that encoded by mRNA from Staphylococcus aureus or from Streptococcus pyogenes, e.g., pCW-Cas9, Addgene #50661, Wang et al. (2014) Science, 3:343-80-4; or nuclease or nickase lentiviral vectors available from Applied Biological Materials (ABM; Canada) under Cat. Nos. K002, K003, K005, or K006) and a guide RNA specific for a target gene ( e.g., TRAC , TRBC1 , and/or TRBC2 in humans) are introduced into the cells. In some embodiments, gRNA sequences that are or comprise a targeting domain of a targeting sequence for targeting a target site in a particular gene, such as the TRAC , TRBC1 , and/or TRBC2 genes, are designated or identified. The whole-genome gRNA database for CRISPR genome editing is publicly available and contains exemplary single guide RNA (sgRNA) sequences for targeting constitutive exons of genes in the human genome or mouse genome (see, e.g., genescript.com/gRNA-database.html; see also Sanjana et al. (2014) Nat. Methods, 11:783-4). In some aspects, the gRNA sequence is or comprises a sequence with minimal off-target binding to an off-target site or position.

In some embodiments, the polynucleotide comprising the agents and/or components thereof, or the RNP complex, is delivered by a non-vector method (e.g., using naked DNA or DNA complexes). For example, DNA or RNA or proteins or a combination thereof, such as ribonucleoprotein (RNP) complexes, can be delivered by, for example, organically modified silica or silicate (Ormosil), electroporation, transient cell concentration or compression (e.g., as described by Lee, et al. (2012) Nano Lett 12: 6322-27, Kollmannsperger et al. (2016) Nat Comm 7, 10372 doi:10,1038/ncomms10372), gene gun, sonoporation, magnetofection, lipid-mediated transfection, dendrimers, inorganic nanoparticles, calcium phosphates, or a combination thereof.

In some embodiments, electroporation delivery comprises mixing cells with Cas9 and/or gRNA-encoding DNA or RNP complex in a cartridge, chamber or cuvette, and applying one or more electrical pulses of a certain duration and amplitude. In some embodiments, electroporation delivery is performed using a system in which cells are mixed with Cas9 and/or gRNA-encoding DNA in a vessel connected to a device (e.g., a pump) that pumps the mixture into a cartridge, chamber or cuvette, where one or more electrical pulses of a certain duration and amplitude are applied, after which the cells are delivered to a second vessel.

In some embodiments, the delivery vehicle is a non-viral vector. In some embodiments, the non-viral vector is an inorganic nanoparticle. Exemplary inorganic nanoparticles include, for example, _magnetic nanoparticles (e.g., _Fe3MnO2 ) and silica. The outer surface of the nanoparticle can be conjugated to a positively charged polymer (e.g., polyethyleneimine, polylysine, polyserine) that allows attachment (e.g., conjugation or entrapment) of a payload. In some embodiments, the non-viral vector is an organic nanoparticle. Exemplary organic nanoparticles include, for example, SNALP liposomes that contain cationic fats together with neutral helper fats that are coated with polyethylene glycol (PEG) and protamine-gucleic acid complexes coated with fat. Exemplary fats and/or polymers are known and can be used in the present embodiments.

In some embodiments, the carrier has targeted modifications to enhance targeted cellular turnover of nanoparticles and liposomes, such as cell-specific antigens, monoclonal antibodies, single-chain antibodies, aptamers, polymers, sugars, and penetrating peptides (e.g., those described in US 2016/0272999). In some embodiments, the carrier utilizes fusogenic and endosome-destabilizing peptides/polymers. In some embodiments, the carrier undergoes acid-stimulated conformational changes (e.g., to enhance endosomal release of cargo). In some embodiments, a stimulus-cleavable polymer is used, such as for release into a cellular compartment. For example, cationic disulfide-based polymers that are cleaved in a reducing cellular environment can be used.

In some embodiments, the delivery vehicle is a biological, non-viral delivery vehicle. In some embodiments, the vehicle is an attenuated bacterium (e.g., a natural bacterium or an engineered bacterium that is invasive but attenuated to prevent pathogenesis and transgene expression (e.g., Listeria monocytogenes , certain strains of Salmonella , Bifidobacterium longum , and modified Escherichia coli ), a bacterium that has a nutrient and tissue-specific tropism for specific target cells, a bacterium that has modified surface proteins to alter target cell specificity). In some embodiments, the carrier is a genetically modified bacteriophage (e.g., phages engineered to have greater packaging capacity, less immunogenicity, contain mammalian plasmid maintenance sequences, and have targeting ligands introduced into them). In some embodiments, the carrier is a mammalian virus-like particle. For example, modified viral particles can be created (e.g., by purifying “empty” particles after ex vivo assembly of the virus with the desired cargo). The carrier can also be designed to introduce targeting ligands to alter the target tissue specificity. In some embodiments, the carrier is a biological liposome. For example, a biological liposome is a phospholipid-based particle derived from a human cell, such as a red blood cell ghost, which is a red blood cell broken into spherical structures derived from the subject (e.g., tissue targeting can be achieved by attaching different tissue- or cell-specific ligands) or secretory exosomes - subject-derived membrane-bound nanovesicles (30-100 nm) of endocytic origin (e.g., which can be derived from a variety of cell types and can therefore be taken up by the cells without the need for targeting ligands).

In some embodiments, RNA encoding Cas9 molecules and/or gRNA molecules can be delivered to cells, such as the target cells described herein, by known methods or as described herein. For example, Cas9-encoding and/or gRNA-encoding RNA can be delivered, such as by microinjection, electroporation, transient cell condensation or compression (e.g., as described in Lee, et al. (2012) Nano Lett 12: 6322-27), lipid-mediated transfection, peptide-mediated delivery, or a combination thereof.

In some embodiments, electroporation delivery comprises mixing cells with RNA encoding Cas9 molecules and/or gRNA molecules in a cartridge, chamber or cuvette and applying one or more electrical pulses of a certain duration and amplitude. In some embodiments, electroporation delivery is performed using a system in which cells are mixed with RNA encoding Cas9 molecules and/or gRNA molecules in a vessel connected to a device (e.g., a pump) that pumps the mixture into a cartridge, chamber or cuvette, where one or more electrical pulses of a certain duration and amplitude are applied, after which the cells are delivered to a second vessel.

In some embodiments, the Cas9 molecules can be delivered to cells by known methods or as described herein. For example, the Cas9 protein molecules can be delivered, for example, by microinjection, electroporation, transient cell condensation or compression (e.g., as described in Lee, et al. (2012) Nano Lett 12: 6322-27), lipid-mediated transfection, peptide-mediated delivery, or a combination thereof. The delivery can be accompanied by DNA encoding gRNA or gRNA.

In some embodiments, electroporation delivery includes mixing the cell with Cas9 molecules with or without gRNA molecules in a cartridge, chamber, or cuvette, using one or more electrical pulses of a certain duration and amplitude. In some embodiments, electroporation delivery is performed using a system in which the cells are mixed with Cas9 molecules with or without gRNA molecules in a vessel connected to a device (e.g., a pump) that pumps the mixture into a cartridge, chamber, or cuvette, where one or more electrical pulses of a certain duration and amplitude are applied, after which the cells are delivered to a second vessel.

In some embodiments, the polynucleotide containing the agents and/or their components is delivered by a combination of vector and non-vector methods. For example, a virosome comprises a liposome combined with an inactivated virus (e.g., HIV or influenza virus), which can provide more efficient gene transfer than either the viral or liposomal method alone.

In some embodiments, more than one agent or component thereof is delivered to a cell. For example, in some embodiments, agents capable of causing genetic disruption of two or more locations in the genome, TRAC, TRBC1 and/or TRBC2 loci, are delivered to a cell. In some embodiments, the agents and components thereof are delivered using a single method. For example, in some embodiments, agents that cause genetic disruption of TRAC, TRBC1 and/or TRBC2 loci are delivered as polynucleotides encoding components for genetic disruption. In some embodiments, one polynucleotide can encode agents that target TRAC, TRBC1 and/or TRBC2 loci. In some embodiments, two or more different nucleotides can encode agents that target TRAC, TRBC1 and/or TRBC2 loci. In some embodiments, the agents capable of causing genetic disruption may be delivered as ribonucleoprotein (RNP) complexes, and two or more different RNP complexes may be delivered together as a mixture or separately.

In some embodiments, one or more nucleic acid molecules other than one or more agents capable of causing genetic disruption and/or components thereof are delivered, such as a Cas9 component of a molecule and/or an gRNA component of a molecule, such as a template polynucleotide for HDR-directed integration (e.g., described in Section I.B. herein). In some embodiments, a nucleic acid molecule, such as a template polynucleotide, is delivered at the same time as one or more components of the Cas system. In some embodiments, the nucleic acid molecule is delivered before or after (e.g., less than about 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 9 hours, 12 hours, 1 day, 2 days, 3 days, 1 week, 2 weeks, or 4 weeks) the delivery of one or more components of the Cas system. In some embodiments, a nucleic acid molecule, such as a template polynucleotide, is delivered by means other than one or more components of the Cas system, such as a Cas9 component of the molecule and/or a gRNA component of the molecule. A nucleic acid molecule, such as a template polynucleotide, can be delivered by any of the delivery methods described herein. For example, a nucleic acid molecule, such as a template polynucleotide, can be delivered by a viral vector, such as a retrovirus or a lentivirus, and a Cas9 component of the molecule and/or a gRNA component of the molecule can be delivered by electroporation. In some embodiments, a nucleic acid molecule, such as a template polynucleotide, includes one or more transgenes, such as transgenes that encode a recombinant TCR, a recombinant CAR, and/or other gene products.

B. Directed integration via homology-directed repair (HDR)

In some embodiments provided herein, homology-directed repair (HDR) may be used to target the integration of a specific portion of a template polynucleotide containing a transgene, such as a nucleic acid sequence encoding a recombinant receptor, into a specific location in the genome, such as the TRAC, TRBC1 , and/or TRBC2 locus. In some embodiments, the presence of a genetic lesion (e.g., a DNA break such as those described in Section IA) and a template polynucleotide containing one or more homology arms (e.g., containing nucleic acid sequences homologous to sequences surrounding the genetic lesion) may induce or direct HDR, with the homologous sequences acting as a template for DNA repair. Based on the homology between the endogenous gene sequence surrounding the genetic disruption and the 5' and/or 3' homology arms included in the template polynucleotide, the cellular DNA repair machinery can utilize the template polynucleotide to repair the DNA break and resynthesize genetic information at the site of the genetic disruption, thereby effectively inserting or integrating transgene sequences in the template polynucleotide at or near the site of the genetic disruption. In some embodiments, the genetic disruption, such as the TRAC, TRBC1 and/or TRBC2 locus, can be created by any means to achieve the targeted genetic disruption described herein.

Also provided are polynucleotides, such as the template polynucleotides described herein. In some embodiments, the provided polynucleotides can be used in the methods described herein, such as those involving HDR, to target transgene sequences encoding a portion of a recombinant receptor, such as a recombinant TCR, at the endogenous TRAC, TRBC1 and/or TRBC2 locus.

In some embodiments, the template polynucleotide has or comprises a polynucleotide comprising a transgene (exogenous or heterologous nucleic acid sequences) encoding a recombinant receptor or a portion thereof (e.g., one or more chains, regions, or domains of a recombinant receptor), and homology sequences (e.g., homology arms) that are homologous to sequences at or near an endogenous genomic site, such as an endogenous TRAC, TRBC1 , and/or TRBC2 locus. In some aspects, the template polynucleotide is administered as a linear DNA fragment or is contained in a vector. In some aspects, the step of causing genetic disruption and the step of targeted integration (e.g., by administering the template polynucleotide) are performed simultaneously or sequentially.

1. Homology-directed repair (HDR)

In some embodiments, homology-directed repair (HDR) can be used to target the integration or insertion of one or more nucleic acid sequences, such as transgene sequences, into one or more target sites in the genome, such as the TRAC, TRBC1 , and/or TRBC2 locus. In some embodiments, nuclease-induced HDR can be used to alter a target sequence, integrate a transgene into a specific target location, and/or edit or repair a mutation in a specific target gene.

The alteration of the nucleic acid sequence at a target site can be carried out by HDR with exogenously obtained template polynucleotides (also referred to as a donor polynucleotide or template sequence). For example, a template polynucleotide provides an alteration of a target sequence, such as insertion of a transgene contained in a template polynucleotide. In some embodiments, a plasmid or vector can be used as a template for homologous recombination. In some embodiments, a linear DNA fragment can be used as a template for homologous recombination. In some embodiments, a single-stranded template polynucleotide can be used as a template for altering a target sequence by alternative homology-directed repair methods (e.g., single-strand annealing) between the target sequence and the template polynucleotide. The alteration of the target sequence caused by the template polynucleotide is dependent on cleavage by a nuclease, for example, a targeted nuclease such as CRISPR/Cas9. Nuclease cleavage may involve a double-strand break or two single-strand breaks.

In some embodiments, “recombination” refers to the process of exchanging genetic information between two polynucleotides. In some embodiments, “homologous recombination (HR)” refers to a specialized form of such exchange that occurs, for example, during the repair of double-strand breaks in cells via homology-directed repair mechanisms. These methods require nucleotide sequence homology, the use of a template polynucleotide to template the repair of target DNA (i.e., one that utilizes a double-strand break, such as a target site in an endogenous gene), and are variously known as “non-crossover gene conversion” or “short tract gene conversion” because they result in the transfer of genetic information from the template polynucleotide to the target. In some embodiments, such transfer may involve correction of a mismatch of heteroduplex DNA that forms between the broken target and the template polynucleotide, and/or "synthesis-dependent strand annealing" in which the template polynucleotide is used to resynthesize genetic information that becomes part of the target, and/or related methods. Such specialized GRs often produce a change in the sequence of the target molecule such that part or all of the sequence of the template polynucleotide is introduced into the target polynucleotide.

In some embodiments, a template polynucleotide, such as a polynucleotide containing a transgene, is integrated into the genome of a cell through homology-independent mechanisms. The methods include creating a double-strand break (DSB) in the genome of a cell and cleaving a template polynucleotide molecule using a nuclease such that the template polynucleotide integrates into the site of the DSB. In some embodiments, the template polynucleotide is integrated by homology-independent methods (e.g., NHEJ). When cleaved in vivo, template polynucleotides can be integrated into the genome of a cell by a targeted technique at the location of a DSB. The template polynucleotide can include one or more identical target sites for one or more nucleases used to create the DSB. Thus, the template polynucleotide can be cleaved by one or more identical nucleases used to cleave an endogenous gene into which integration is desired. In some embodiments, the template polynucleotide includes different nuclease target sites from the nuclease used to cause the DSB. As described herein, the genetic disruption of the target site or target position can be created by any mechanisms, such as ZFN, TALEN, CRISPR/Cas9 system or TtAgo nucleases.

In some embodiments, DNA repair mechanisms may be induced by a nuclease after (1) one double-strand break, (2) two single-strand breaks, (3) two double-strand breaks with a break occurring on each side of the target site, (4) one double-strand break and two single-strand breaks, where a double-strand break and two single-strand breaks occur on each side of the target site, (5) four single-strand breaks with a pair of single-strand breaks occurring on each side of the target site, or (6) one single-strand break. In some embodiments, a single-stranded template polynucleotide is used and the target site may be altered by alternative HDR.

The target site alteration induced by the template polynucleotide depends on cleavage by a nuclease molecule. The nuclease cleavage may contain a nick, a double-strand break, or two single-strand breaks, for example, on each DNA strand at the target site. After introducing breaks at the target site, resection occurs at the broken ends in single-stranded DNA overhangs.

In canonical HDR, a double-stranded template polynucleotide containing a homologous sequence is introduced into the target site, which is either directly introduced into the target site or used as a template for transgene insertion or target site sequence correction. Following resection of the break, repair can proceed via two different pathways, i.e., the double Holliday junction (or double-strand break repair, DSBR) model or the synthesis-dependent strand annealing (SDSA) pathway.

In the double Holliday junction model, the strand inserts two single-stranded sticky ends of the target site into a homologous sequence in the template polynucleotide, causing the formation of an intermediate product with two Holliday junctions. The junctions migrate as new DNA is synthesized from the ends of the inserted strand to fill the gap created by the resection. The end of the newly synthesized DNA is ligated to the resected end, and the junctions disintegrate to yield an insertion at the target site, such as a transgene insertion into the template polynucleotide. Crossover with the template polynucleotide may occur as the junctions disintegrate.

In the SDSA pathway, only the single-stranded sticky end is inserted into the template polynucleotide, and new DNA is synthesized from the end of the inserted strand to fill the gap created by the resection. The newly synthesized DNA is then annealed to the remaining single-stranded sticky end, new DNA is synthesized to fill the gap, and the strands are ligated to produce a modified DNA duplex.

In alternative HDR, a single-stranded template polynucleotide, such as a template polynucleotide, is introduced. A nick, single-strand break, or double-strand break at the target site to alter the desired target site is mediated by a nuclease molecule, and resection at the break occurs to open single-stranded sticky ends. Introduction of the template polynucleotide sequence to correct or alter the target DNA site is typically accomplished by SDSA, as described herein.

"Alternative HDR" or alternative homology-directed repair, in some embodiments, refers to a method of repairing DNA damage using a homologous nucleic acid (e.g., an endogenous homologous sequence, such as a sister chromatid, or exogenous nucleic acids, such as a template polynucleotide). Alternative HDR differs from canonical HDR in that the method uses pathways that differ from canonical HDR and can be inhibited by canonical HDR mediators, RAD51 and BRCA2. Alternative HDR also uses a single-stranded or gap-containing homologous nucleic acid to repair the gap. “Canonical HDR” or canonical homology-directed repair, in some embodiments, refers to a method of repairing DNA damage using a homologous nucleic acid (e.g., an endogenous homologous sequence, such as a sister chromatid, or an exogenous nucleic acid, such as a template nucleic acid). Canonical HDR typically operates when there is significant resection at a double-strand break, generating at least one single-stranded piece of DNA. In a normal cell, HDR typically involves a series of steps, such as break recognition, break stabilization, resection, single-stranded DNA stabilization, formation of a crossover intermediate DNA, resolution of the crossover intermediate, and ligation. The method requires RAD51 and BRCA2, and the homologous nucleic acid is typically double-stranded. Unless otherwise noted, the term “HDR” in some embodiments encompasses canonical HDR and alternative HDR.

In some embodiments, double-stranded cleavage is performed by a nuclease, such as a Cas9 molecule, having cleavage activity associated with an HNH-like domain and cleavage activity associated with a RuvC-like domain, such as the N-terminal RuvC-like domain, such as wild-type Cas9. Such embodiments require only one gRNA.

In some embodiments, the single-strand break or nick is produced by a nuclease molecule having nickase activity, such as Cas9 nickase. The DNA containing the nick can be a substrate for alternative HDR.

In some embodiments, the double-strand breaks or nicks are carried out by a nuclease, such as a Cas9 molecule having nickase activity, such as a cleavage activity associated with an HNH-like domain or a cleavage activity associated with an N-terminal RuvC-like domain. Such embodiments typically require two gRNAs, one to accommodate each single-strand break. In some embodiments, a Cas9 molecule having nickase activity cleaves the strand to which the gRNA hybridizes, but not the strand that is complementary to the strand to which the gRNA hybridizes. In some embodiments, a Cas9 molecule having nickase activity does not cleave the strand to which the gRNA hybridizes, but rather cleaves the strand that is complementary to the strand to which the gRNA hybridizes. In some embodiments, the nickase has HNH activity, for example, a Cas9 molecule having inactivated RuvC activity, for example, a Cas9 molecule having a mutation at D10, for example, a D10A mutation. D10A inactivates RuvC; therefore, the Cas9 nickase has (only) HNH activity and nicks the strand to which the gRNA hybridizes (e.g., the complementary strand that does not have the NGG PAM on it). In some embodiments, a Cas9 molecule having an H840, for example, H840A, mutation, can be used as a nickase. H840A inactivates HNH; therefore, the Cas9 nickase has (only) RuvC activity and nicks the non-complementary strand (e.g., the strand that has the NGG PAM and whose sequence is identical to the gRNA). In some embodiments, the Cas9 molecule is a nickase of the N-terminal RuvC-like domain, e.g., the Cas9 molecule contains a mutation at N863, e.g., N863A.

In some embodiments in which a nickase and two gRNAs are used to position two single-strand breaks, one break is on the + strand and one break is on the - strand of the target DNA. The PAM is exposed. The gRNAs can be selected such that the gRNAs are separated by about 0-50, 0-100, or 0-200 nucleotides. In some embodiments, there is no overlap between target sequences that are complementary to the targeting domains of the two gRNAs. In some embodiments, the gRNAs are non-overlapping and are separated by at least 50, 100, or 200 nucleotides. In some embodiments, the use of two gRNAs can improve specificity, for example, by reducing off-target binding (Ran et al., Cell 2013).

In some embodiments, a single strand break may be used to induce HDR, such as an alternative HDR. It is contemplated herein that a single strand break may be used to increase the ratio of HR to NHEJ at a given cleavage site, such as a target site. In some embodiments, a single strand break is formed on a DNA strand at a target site to which the targeting domain of said gRNA is complementary. In another embodiment, a single strand break is formed on a DNA strand at a target site other than the strand to which the targeting domain of said gRNA is complementary.

In some embodiments, other DNA repair pathways such as single-strand annealing (SSA), single-strand break repair (SSBR), mismatch repair (MMR), base excision repair (BER), nucleotide excision repair (NER), intrastrand cross-linking (ICL), through-lesion synthesis (TLS), and error-free post-replicative repair (PRR) may be used by the cell to repair a double-strand or single-strand break created by a nuclease.

2. Placement of genetic disruption (e.g. DNA strand break)

Targeted integration provides a transgene that integrates into a specific gene or locus in the genome. The transgene can be integrated at any location near one of at least one of the target sites or a site in the genome. In some embodiments, the transgene is integrated at or near one of at least one of the target sites, such as within 300, 250, 200, 150, 100, 50, 10, 5, 4, 3, 2, 1 or fewer base pairs before or after the cleavage site, such as within 100, 50, 10, 5, 4, 3, 2, 1 base pairs on either side of the target site, such as within 50, 10, 5, 4, 3, 2, 1 base pairs on either side of the target site. In some embodiments, the integrated sequence comprising the transgene does not include any sequence vectors (e.g., viral sequence vectors). In some embodiments, the integrated sequence includes a portion of the sequence vectors (e.g., viral sequence vectors).

The double-strand break or single-strand break in one of the strands must be close enough to the site for targeted integration so that the change occurs in a desired region, such as insertion of a transgene or correction of a mutation. In some embodiments, the distance is no more than 10, 25, 50, 100, 200, 300, 350, 400, or 500 nucleotides. In some embodiments, it is believed that the break must be close enough to the site for targeted integration so that the break occurs in a region that is susceptible to exonuclease-mediated excision during final resection. In some embodiments, the targeting domain is configured such that the cleavage agent, such as a double-strand or single-strand break, is located within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 350, 400 or 500 nucleotides of a region that is desired to be altered, such as a targeted insertion site. The break, such as a double-strand or single-strand break, can be located before or after the region that is desired to be altered, such as a targeted insertion site. In some embodiments, the break is located in a region that is desired to be altered, such as within a region defined by at least two mutant nucleotides. In some embodiments, the break is located directly adjacent to the area that you want to change, such as immediately before or after a site for targeted integration.

In some embodiments, the single-strand break is accompanied by an additional single-strand break located on a second gRNA molecule. For example, the targeting domains are configured such that a cleavage event, e.g., two single-strand breaks, are located within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 350, 400, or 500 nucleotides of the targeted integration site. In some embodiments, the first and second gRNA molecules are configured such that upon targeting of Cas9 nickase, the single-strand break will be accompanied by an additional single-strand break located on the second gRNA, sufficiently close to each other, to produce an alteration of the desired region. In some embodiments, the first and second gRNA molecules are configured such that the single-strand break located by said second gRNA is located within 10, 20, 30, 40, or 50 nucleotides of the break located by said first gRNA molecule, for example, if Cas9 is a nickase. In some embodiments, the two gRNA molecules are configured to position the breaks at the same position or within a few nucleotides of each other, on different strands, for example, substantially mimicking a double-strand break.

In some embodiments in which gRNA (monomolecular (or chimeric) or modular gRNA) and Cas9 nuclease cause a double-strand break for the purpose of inducing HDR-mediated transgene insertion or correction, the cleavage site is between 0-200 bp. (e.g., 0-175, 0-150, 0-125, 0-100, 0-75, 0-50, 0-25, 25-200, 25-175, 25-150, 25-125, 25-100, 25-75, 25-50, 50-200, 50-175, 50-150, 50-125, 50-100, 50-75, 75-200, 75-175, 75-150, 75-125, 75-100 bp) from the site of targeted integration. In some embodiments, the cleavage site is between 0-100 bp. (e.g. 0-75, 0-50, 0-25, 25-100, 25-75, 25-50, 50-100, 50-75 or 75-100 p.o.) from the site for targeted integration.

In some embodiments, HDR can be promoted through the use of nicks to create a break with sticky ends. In some embodiments, the single-stranded nature of the sticky ends may enhance the likelihood of the cell repairing the break via HDR, as opposed to, for example, NHEJ.

More specifically, in some embodiments, HDR is facilitated by selecting a first gRNA that directs a first nickase to a first target site and a second gRNA that directs a second nickase to a second target site that is on the opposite DNA strand from the first target site and away from the first nick. In some embodiments, the targeting domain of the gRNA molecule is configured to position the cleavage event far enough from a preselected nucleotide, such as a coding region nucleotide, such that the nucleotide is not altered. In some embodiments, the targeting domain of the gRNA molecule is configured to position the intron cleavage event far enough from an intron/exon boundary or a naturally occurring splicing signal to avoid altering the exonic sequence or unwanted splicing events. In some embodiments, the targeting domain of the gRNA molecule is configured to position an early exon so as to allow deletion or knockout of an endogenous gene and/or to allow in-frame integration of a transgene at or near at least one of the target sites.

In some embodiments, the double-strand break may be accompanied by an additional double-strand break located on a second gRNA molecule. In some embodiments, the double-strand break may be accompanied by two additional single-strand breaks located on a second gRNA molecule and a third gRNA molecule.

In some embodiments, two gRNAs, such as independent, monomolecular (or chimeric) or modular gRNAs, are configured to position a double-strand break on both sides of the site for targeted integration.

3. Matrix polynucleotides

A template polynucleotide having homology to sequences near one or more target sites in endogenous DNA can be used to alter the structure of a target DNA, such as targeted insertion of a transgene. In some embodiments, the template polynucleotide comprises homologous sequences (e.g., homology arms) located around a transgene, such as a nucleic acid sequence encoding a recombinant receptor, for targeted insertion. In some embodiments, the homologous sequences target the transgene to one or more TRAC , TRBC1 and/or TRBC2 loci. In some embodiments, the template polynucleotide includes additional sequences (coding or non-coding sequences) between the homology arms, such as regulatory sequences such as promoters and/or enhancers, splice donor and/or acceptor sites, an internal ribosome entry site (IRES), sequences encoding a ribosome skip element (e.g., 2A peptides), markers and/or SA sites, and/or one or more additional transgenes.

The sequences in question in the template polynucleotide may comprise one or more sequences encoding a functional polypeptide (e.g., cDNA), with or without a promoter.

In some embodiments, the transgene contained in the template polynucleotide comprises a sequence encoding a cell surface receptor (e.g., a recombinant receptor) or chain thereof, an antibody, an antigen, an enzyme, a growth factor, a nuclear receptor, a hormone, a lymphokine, a cytokine, a reporter, functional fragments or functional variants of any of the above, and a combination of the above. The transgene may encode one or more proteins used in cancer therapy, such as one or more chimeric antigen receptors (CARs) and/or a recombinant T cell receptor (TCR). In some embodiments, the transgene may encode any of the recombinant receptors as described in Section IV herein, or any chains, regions and/or domains thereof. In some embodiments, the transgene encodes a recombinant T cell receptor (TCR) or any chains, regions and/or domains thereof.

In certain embodiments, a polynucleotide, such as a template polynucleotide, comprises and/or includes a transgene encoding all or part of a recombinant receptor, such as a recombinant TCR or chain thereof. In particular embodiments, the transgene is directed to target sites that are within a gene, locus, or open reading frame that encodes an endogenous receptor, such as an endogenous gene encoding one or more regions, chains, or portions of a TCR.

In certain embodiments, the template polynucleotide comprises or contains a transgene, a portion of a transgene, and/or a nucleic acid encoding a recombinant receptor, which is a recombinant TCR or a chain thereof that comprises one or more variable domains and one or more constant domains. In certain embodiments, the recombinant TCR or a chain thereof comprises one or more constant domains that have complete, such as about 100% identity with all or part and/or a fragment of a constant domain of an endogenous TCR. In some embodiments, the transgene encodes a section or a portion of a constant domain, such as a portion or a fragment of a constant domain that is completely or partially identical to a constant domain of an endogenous TCR. In some embodiments, the transgene comprises nucleotides with a sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to all or a portion of the nucleic acid sequence set forth in SEQ ID NOS: 1, 2, or 3.

In some embodiments, the transgene comprises a sequence encoding a TCRα and/or TCRβ chain or a portion thereof that is codon optimized. In some embodiments, the transgene encodes a portion of a TCRα and/or TCRβ chain with less than 100% amino acid sequence identity to the corresponding portion of a natural or endogenous TCRα and/or TCRβ chain. In some embodiments, the encoded TCRα and/or TCRβ chain comprises an amino acid sequence with, about, or at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or more than 99% identity but less than 100% identity to the corresponding natural or endogenous TCRα and/or TCRβ chain. In particular embodiments, the transgene encodes a TCRα and/or TCRβ constant domain or a portion thereof with less than 100% amino acid sequence identity to the corresponding natural or endogenous TCRα and/or TCRβ constant domain. In some embodiments, the TCRα and/or TCRβ constant domain comprises an amino acid sequence with, about, or at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or more than 99% identity, but less than 100% identity to the corresponding natural or endogenous TCRα and/or TCRβ chain.

In certain embodiments, the transgene comprises one or more modifications to introduce one or more cysteine residues that are capable of forming one or more non-natural disulfide bridges between the TCRα chain and the TCRβ chain. In some embodiments, the transgene encodes a TCRα chain or a portion thereof comprising a TCRα constant domain comprising a cysteine at a position corresponding to position 48 as numbered in SEQ ID NO: 24. In some embodiments, the TCRα constant domain has an amino acid sequence as set forth in any of SEQ ID NOS: 19 or 24, or an amino acid sequence that has, has about, or has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% sequence identity thereto, comprising one or more cysteine residues capable of forming a non-naturally occurring disulfide bond with the TCRβ chain. In some embodiments, the transgene encodes a TCRβ chain or a portion thereof comprising a TCRβ constant domain comprising a cysteine at a position corresponding to position 57 as numbered in SEQ ID NO: 20. In some embodiments, the TCRβ constant domain has an amino acid sequence as set forth in any of SEQ ID NOS: 20, 21 or 25, or an amino acid sequence that has, has about, or has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% sequence identity thereto, comprising one or more cysteine residues capable of forming a non-naturally occurring disulfide bond with the TCRα chain.

In particular embodiments, the transgene encodes a TCRα and/or TCRβ chain and/or constant domains of a TCRα and/or TCRβ chain comprising one or more modifications to introduce one or more disulfide bonds. In some embodiments, the transgene encodes a TCRα and/or TCRβ chain and/or a TCRα and/or TCRβ with one or more modifications to remove or prevent a natural disulfide bond, such as between a TCRα encoded transgene and an endogenous TCRβ chain, or between a TCRβ encoded transgene and an endogenous TCRα chain. In some embodiments, one or more natural cysteines that form and/or are capable of forming a natural interchain disulfide bond are replaced with another residue, such as serine or alanine. In some embodiments, the TCRα and/or TCRβ chain and/or constant domains of the TCRα and/or TCRβ chain are modified to replace one or more non-cysteine residues with cysteine. In some embodiments, the one or more non-natural cysteine residues are capable of forming non-natural disulfide bonds, such as between recombinant TCRα and transgene-encoded TCRβ chains. In some embodiments, the cysteine is introduced at one or more of Thr48, Thr45, Tyr10, Thr45, and Ser15, with reference to the numbering of the TCRα constant domain set forth in SEQ ID NO: 24. In certain embodiments, the cysteines may be introduced at residues Ser57, Ser77, Ser17, Asp59, and Glu15 of the TCRβ chain, with reference to the numbering of the TCRβ chain set forth in SEQ ID NO: 20. Exemplary non-natural TCR disulfide bonds are described in Published International PCT Application Nos. WO2006/000830, WO2006/037960, and Kuball et al. (2007) Blood, 109:2331-2338. In some embodiments, the transgene encodes a portion of a TCRα chain and/or a TCRα constant domain containing one or more modifications to introduce one or more disulfide bonds.

In some embodiments, the transgene encodes all or a portion of the TCRα chain and/or the TCRα constant domain with one or more modifications to remove or prevent a natural disulfide bond, such as between the TCRα chain encoded by the transgene and an endogenous TCRβ chain. In some embodiments, one or more natural cysteines that form and/or are capable of forming a natural interchain disulfide bond are replaced with another residue, such as a serine or alanine. In some embodiments, a portion of the TCRα chain and/or the TCRα constant domain is modified to replace one or more non-cysteine residues with a cysteine. In some embodiments, the one or more non-natural cysteine residues are capable of forming non-natural disulfide bonds, such as with the TCRβ chain encoded by the transgene. In some embodiments, the transgene encodes all or a portion of the TCRβ chain and/or the TCRβ constant domain with one or more modifications to remove or prevent a natural disulfide bond, such as between the TCRβ chain encoded by the transgene and the endogenous TCRα chain. In some embodiments, one or more natural cysteines that form and/or are capable of forming a natural interchain disulfide bond are replaced with another residue, such as serine or alanine. In some embodiments, a portion of the TCRβ chain and/or the TCRβ constant domain is modified to replace one or more non-cysteine residues with a cysteine.

In some embodiments, one or more non-natural cysteine residues and are capable of forming non-natural disulfide bonds, such as with the TCRα chain encoded by the transgene. In some embodiments, one or more different template polynucleotides are used to target the integration of the transgene into one or more different target sites. To target integration into different target sites, one or more genetic disruptions (e.g., DNA breaks) are created at one or more target sites; and one or more different homologous sequences are used to target the integration of the transgene into the corresponding target site. In some embodiments, the transgene inserted into each site is the same or substantially the same. In some embodiments, the transgene inserted into each site is different. In some embodiments, two or more different transgenes encoding two or more different domains or chains of a protein are inserted into one or more target sites. In some embodiments, a transgene encoding a recombinant TCR or an antigen-binding fragment or chain thereof encodes one chain of a recombinant TCR and a second transgene encodes another chain of a recombinant TCR. In some embodiments, a transgene encoding a recombinant TCR or an antigen-binding fragment or chain thereof encodes an alpha (TCRα) chain of a recombinant TCR and a second transgene encodes a beta (TCRβ) chain of a recombinant TCR. In some embodiments, two or more transgenes encoding different domains of the recombinant receptors are directed to integrate at two or more target sites. For example, in some embodiments, a transgene encoding an alpha chain of a recombinant TCR is directed to integrate into the TRAC locus and a transgene encoding a beta chain of a recombinant TCR is directed to integrate into the TRBC1 and/or TRBC2 loci.

In some embodiments, two or more different template polynucleotides are used to target two or more transgenes for integration into two or more different endogenous gene loci. In some embodiments, the first template polynucleotide includes a transgene encoding a recombinant receptor. In some embodiments, the second template polynucleotide includes one or more second transgenes, such as one or more second transgenes encoding one or more different molecules, polypeptides, and/or factors. Any description or characterization of a template polynucleotide provided herein may also apply to one or more second template polynucleotides.

In some embodiments, the one or more second transgenes are targeted for integration at or near one of at least one target site in a TRAC gene. In some embodiments, the one or more second transgenes are targeted for integration at or near one of at least one target site in a TRBC1 or TRBC2 gene. In some embodiments, the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof is targeted for integration at or near one of at least one target site in a TRAC gene, a TRBC1 gene, or a TRBC2 gene, and the one or more second transgenes are targeted for integration at or near one or more target sites that are not targeted by the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof.

In some embodiments, the molecule, polypeptide, or factor encoded by one or more second transgenes is a molecule, polypeptide, factor, or agent that can provide a costimulatory signal to an immune cell, such as a T cell. In some embodiments, the molecule, polypeptide, factor, or agent encoded by the second transgene is an additional receptor, such as an additional recombinant receptor. In some embodiments, the additional receptor can provide a costimulatory signal and/or impulses or reverse an inhibitory signal.

In some embodiments, one or more second transgenes encode a molecule selected from a costimulatory ligand, a cytokine, a soluble single-stranded variable fragment (scFv), an immunomodulatory fusion protein, a chimeric switch receptor (CSR), or a coreceptor.

In some embodiments, the molecule, polypeptide, or factor encoded by one or more second transgenes is a costimulatory ligand. Exemplary costimulatory ligands include a tumor necrosis factor (TNF) ligand or an immunoglobulin (Ig) superfamily ligand. In some embodiments, exemplary TNF ligands include 4-1BBL, OX40L, CD70, LIGHT, and CD30L. In some embodiments, exemplary Ig superfamily ligands include CD80 and CD86. In some embodiments, the costimulatory ligand includes CD3, CD27, CD28, CD83, CD127, 4-1BB, PD-1, or PDIL. In some embodiments, the molecule, polypeptide or factor encoded by one or more second transgenes is a cytokine such as IL-2, IL-3, IL-6, IL-11, IL-12, IL7, IL-15, IL-21, granulocyte-monocyte colony-stimulating factor (GM-CSF), interferon alpha (IFN-α), interferon beta (IFN-β) or interferon gamma (IFN-γ) and erythropoietin. Exemplary costimulatory ligands and cytokines that may be encoded by one or more second transgenes include those described in, for example, WO 2008121420.

In some embodiments, the molecule, polypeptide or factor encoded by one or more second transgenes is a soluble single-stranded variable fragment (scFv), such as an scFv that binds a polypeptide that has an immunosuppressive effect or an immunostimulatory effect, such as CD47, PD-1, CTLA-4 and their ligands or CD28, OX-40, 4-1BB and their ligands. Exemplary scFvs that can be encoded by one or more second transgenes include those described in, for example, WO 2014134165.

In some embodiments, the molecule, polypeptide, or factor encoded by the one or more second transgenes is an immunomodulatory fusion protein or a chimeric switch receptor (CSR). In some embodiments, the encoded immunomodulatory fusion protein comprises (a) an extracellular component consisting of a binding domain that specifically binds a target, (b) an intracellular component consisting of an intracellular binding domain, and (c) a hydrophobic component connecting the extracellular and intracellular components. In some embodiments, the encoded immunomodulatory fusion protein comprises (a) an extracellular binding domain that specifically binds an antigen derived from CD200R, SIRPα, CD279 (PD-1), CD2, CD95 (Fas), CD152 (CTLA4), CD223 (LAG3), CD272 (BTLA), A2aR, KIR, TIM3, CD300, or LPA5; (b) an intracellular binding domain derived from CD3ε, CD3δ, CD3ζ, CD25, CD27, CD28, CD40, CD47, CD79A, CD79B, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD278 (ICOS), CD357 (GITR), XAPD11, DAP10, DAP12, FcRα, FcRβ, FcRγ, Fyn, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, ROR2, Ryk, Slp76, pTα, TCRα, TCRβ, TRFM, Zap70, PTCH2, or any combination thereof; and (c) a hydrophobic transmembrane domain derived from CD2, CD3ε, CD3δ, CD3ζ, CD25, CD27, CD28, CD40, CD79A, CD79B, CD80, CD86, CD95 (Fas), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD200R, CD223 (LAG3), CD270 (HVEM), CD272 (BTLA), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), CD279 (PD-1), CD300, CD357 (GITR), A2aR, DAP10, FcRα, FcRβ, FcRγ, Fyn, GAL9, KIR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPα, pTα, TCRα, TCRβ, TIM3, TRIM, LPA5, or Zap70. In some embodiments, the molecule, polypeptide, or factor encoded by the one or more second transgenes is a chimeric switch receptor (CSR), such as a CSR comprising a truncated extracellular domain of PD1 and the transmembrane and cytoplasmic signaling domains of CD28. Exemplary immunomodulatory fusion proteins or CSRs that can be encoded by one or more second transgenes include those described in, for example, WO2014134165, US2014/0219975, WO2013/019615, and Liu et al., Cancer Res. (2016) 76(6):1578-90.

In some embodiments, the molecule, polypeptide, or factor encoded by one or more second transgenes is a coreceptor. In some embodiments, exemplary coreceptors include CD4 or CD8.

In some embodiments, the one or more target sites are located at or adjacent to one or more of the TRAC , TRBC1 , and/or TRBC2 loci. In some embodiments, the first target site is at or adjacent to the coding sequence of the TRAC gene locus, and the second target site is at or adjacent to the coding sequence of the TRBC1 gene locus. In some embodiments, the first target site is at or adjacent to the coding sequence of the TRAC gene locus, and the second target site is at or adjacent to the coding sequence of the TRBC2 gene locus. In some embodiments, the first target site is at or adjacent to the coding sequence of the TRAC gene locus, and the second target site is at both the TRBC1 and TRBC2 loci, such as on a sequence that is conserved between TRBC1 and TRBC2 .

In some embodiments, one or more different DNA sites, such as the TRAC , TRBC1 , and/or TRBC2 loci, are targeted, and one or more transgenes are inserted into each site. In some embodiments, the transgene inserted into each site is the same or substantially the same. In some embodiments, the transgene inserted into each site is different. In some embodiments, the transgene is inserted into only one target site (e.g., the TRAC locus), and another target site is targeted for genome editing (e.g., knockout).

In some embodiments, any of the lengths and positions of the homology arms and related positions to the target sites, such as any described herein, may also apply to one or more second template polynucleotides.

In some embodiments, the HDR nuclease-induced insertion of a transgene (also referred to as an "exogenous sequence" or "transgene sequence") is used to express a transgene for a target insertion. The template polynucleotide sequence is typically not identical to the genomic sequence in which it is located. The template polynucleotide sequence may comprise a non-homologous sequence flanked by two regions of homology to allow for efficient insertion at the target location. Additionally, the template polynucleotide sequence may comprise a vector molecule comprising sequences that are non-homologous to the target region in cellular chromatin. The template polynucleotide sequence may comprise multiple, intermittent regions of homology to cellular chromatin. For example, to target insertion of sequences not normally present in the target region, the sequences may be present in the transgene and flanked by regions of homology to sequences in the target region.

In some aspects, the subject nucleic acid sequences, including coding and/or non-coding sequences and/or partial coding sequences that are inserted or integrated into a target location in the genome, may also be referred to as a "transgene", "transgene sequences", "exogenous nucleic acid sequences", "heterologous sequences" or "donor sequences". In some aspects, a transgene is a nucleic acid sequence that is exogenous or heterologous to endogenous genomic sequences, such as endogenous genomic sequences at a specific target locus or target location in the genome, of a T cell, such as a human T cell. In some aspects, a transgene is a sequence that is modified or altered compared to an endogenous genomic sequence at a target locus or target location of a T cell, such as a human T cell. In some aspects, a transgene is a nucleic acid sequence that is derived from or modified from a nucleic acid sequence from different genes, species, and/or origins. In some aspects, a transgene is a sequence that is derived from a sequence from another locus, such as another genomic region or another gene, of the same species.

Polynucleotides for insertion may also be referred to as “transgenes” or “exogenous sequences” or “donor” polynucleotides or molecules. The template polynucleotide may be DNA, single-stranded and/or double-stranded, and may be introduced into the cell in linear or circular form. The template polynucleotide may be RNA, single-stranded and/or double-stranded, and may be introduced as an RNA molecule (e.g., a portion of a viral RNA). See also U.S. Patent Publication Nos. 20100047805 and 20110207221. The template polynucleotide may also be introduced in DNA form, which may be introduced into the cell in circular or linear form. If introduced in linear form, the ends of the template polynucleotide may be protected (e.g., from exonucleolytic degradation) by known methods. For example, one or more dideoxynucleotide residues are added to the 3' end of the linear molecule and/or self-complementary oligonucleotides are ligated to one or both ends. See, for example, Chang et al. (1987) Proc. Natl. Acad. Sci. USA 84:4959-4963; Nehls et al. (1996) Science 272:886-889. Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, the addition of terminal amino groups and the use of modified internucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and O-methylribose or deoxyribose residues. If introduced in double-stranded form, the template polynucleotide may include one or more nuclease target sites, for example, nuclease target sites surrounding a transgene to be integrated into the genome of a cell. See, for example, U.S. Patent Publication No. 20130326645.

In some embodiments, the double-stranded template polynucleotide comprises sequences (also called a transgene) greater than 1 kb in length, such as between 2 and 200 kb, between 2 and 10 kb (or any intermediate value). The double-stranded template polynucleotide also comprises at least one nuclease target site, such as. In some embodiments, the template polynucleotide comprises at least 2 target sites, such as for a ZFN or TALEN pair. Typically, the nuclease target sites are outside the transgene sequences, such as 5' and/or 3' to the transgene sequences, for cleavage of the transgene. The nuclease cleavage sites can be for any nucleases. In some embodiments, the nuclease target sites contained in the double-stranded template polynucleotide are intended for the same nucleases that are used to cleave the endogenous target into which the cleaved template polynucleotide is integrated in a homology-independent manner.

In some embodiments, the nucleic acid template system is double-stranded. In some embodiments, the nucleic acid template system is single-stranded. In some embodiments, the nucleic acid template system comprises a single-stranded portion and a double-stranded portion.

In some embodiments, the template polynucleotide comprises a transgene, such as recombinant receptor-encoding nucleic acid sequences, flanked by homologous sequences (also called "homology arms") at the 5' and 3' ends, which allows DNA repair machinery, such as homologous recombination machinery, to be triggered to use the template polynucleotide as a repair template, effectively inserting the transgene into the target integration site in the genome. The homology arm should extend at least as far as the region in which the final resection can occur, such as to allow the excised single-stranded sticky end to find a complementary region in the template polynucleotide. The overall length should be limited by parameters such as plasmid size or virus packaging limits. In some embodiments, the homology arm does not extend into repetitive elements, such as ALU repeats or LINE repeats.

A typical homology arm length comprises at least or at least about, or is or is about 50, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900, 1000, 2000, 3000, 4000 or 5000 nucleotides. In some embodiments, the homology arm length is 50-100, 100-250, 250-500, 500-750, 750-1000, 1000-2000, 2000-3000, 3000-4000 or 4000-5000 nucleotides. exemplary homology arm lengths include less than or less than about, or are or are about 50, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900, 1000, 2000, 3000, 4000, or 5000 nucleotides. In some embodiments, the homology arm length is 50-100, 100-250, 250-500, 500-750, 750-1000, 1000-2000, 2000-3000, 3000-4000, or 4000-5000 nucleotides.

A target site (also known as a “target position,” “target DNA sequence,” or “target location”) in some embodiments refers to a site on a target DNA (e.g., a chromosome) that is modified by one or more agents capable of causing genetic disruption, such as a Cas9 molecule. For example, a target site can be modified by cleavage of DNA by a Cas9 molecule at the target site and by a modification directed by a template polynucleotide, such as targeted insertion of a transgene, at the target site. In some embodiments, a target site can be a site between two nucleotides, such as adjacent nucleotides, on DNA to which one or more nucleotides are added. The target site can comprise one or more nucleotides that are modified by the template polynucleotide. In some embodiments, the target site is within a target sequence (e.g., a sequence to which an gRNA is linked). In some embodiments, the target site is upstream or downstream of a target sequence (e.g., a sequence to which an gRNA is linked). In some aspects, a pair of single-strand breaks (e.g. nicks) may be created on each side of the target site.

In some embodiments, the template polynucleotide comprises about 500 to 1000, such as 600 to 900 or 700 to 800, base pairs of homology on each side of a target site on an endogenous gene. In some embodiments, the template polynucleotide comprises at least or less than or about 200, 300, 400, 500, 600, 700, 800, 900, or 1000 base pairs of homology to a 5' target site, a 3' target site, or both 5' and 3' target site, such as in a TRAC , TRBC1 , and/or TRBC2 gene, locus, or open reading frame (e.g., those described in Tables 1-3 herein).

In some embodiments, the template polynucleotide comprises about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, or 5000 base pairs of 3' homology to the target site. In some embodiments, the template polynucleotide comprises about 100 to 500, 200 to 400, or 250 to 350, base pairs of 3' homology to the transgene and/or target site. In some embodiments, the template polynucleotide comprises less than about 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, or 10 base pairs of homology to a 5' target site, such as in a TRAC , TRBC1 , and/or TRBC2 gene, locus, or open reading frame (e.g., those described in Tables 1-3 herein).

In some embodiments, the template polynucleotide comprises about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, or 5000 base pairs of 5' homology to the target site. In some embodiments, the template polynucleotide comprises about 100 to 500, 200 to 400, or 250 to 350, base pairs of 5' homology to the transgene and/or target site. In some embodiments, the template polynucleotide comprises less than about 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, or 10 base pairs of homology to a 3' target site, such as in a TRAC , TRBC1 , and/or TRBC2 gene, locus, or open reading frame (e.g., those described in Tables 1-3 herein).

In some embodiments, the template polynucleotide is a nucleic acid sequence that can be used in combination with one or more agents capable of causing genetic disruption to alter the structure of a target site. In some embodiments, the target site is modified to have some or all of the sequences of the template polynucleotide, typically near cleavage sites. In some embodiments, the template polynucleotide is single-stranded. In some embodiments, the template polynucleotide is double-stranded. In some embodiments, the template polynucleotide is DNA, such as double-stranded DNA. In some embodiments, the template polynucleotide is single-stranded DNA. In some embodiments, the template polynucleotide is encoded on the same vector backbone, such as an AAV genome, plasmid DNA such as Cas9, and gRNA. In some embodiments, the template polynucleotide is excised from the vector backbone in vivo, such as surrounded by gRNA recognition sequences. In some embodiments, the template polynucleotide is on a separate polynucleotide molecule, such as Cas9 and gRNA. In some embodiments, Cas9 and gRNA are administered in the form of a ribonucleoprotein (RNP) complex, and the template polynucleotide is administered as a polynucleotide molecule, such as in a vector.

In some embodiments, the template polynucleotide alters the structure of the target site, such as by inserting a transgene, participating in homology-directed repair, or by altering the sequence of the target site.

In some embodiments, the template polynucleotide includes a sequence that corresponds to a site on the target sequence that is cleaved by one or more agents capable of causing genetic destruction. In some embodiments, the template polynucleotide includes a sequence that corresponds to both a first site on the target sequence that is cleaved by a first agent capable of causing genetic destruction and a second site on the target sequence that is cleaved by a second agent capable of causing genetic destruction.

In some embodiments, the template polynucleotide comprises the following components: [5' homology arm]-[transgene]-[3' homology arm]. The homology arms are present for recombination into the chromosome, thereby inserting the transgene into the DNA at or near the cleavage site, such as the target sites. In some embodiments, the homology arms surround the most distal target sites.

In some embodiments, the 3' end of the 5' homology arm is at a position subsequent to the 5' end of the transgene. In some embodiments, the 5' homology arm can extend at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, or 5000 nucleotides 5' of the 5' end of the transgene.

In some embodiments, the 5' end of the 3' arm of homology is at a position subsequent to the 3' end of the transgene. In some embodiments, the 3' arm of homology can extend at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, or 5000 nucleotides 3' of the 3' end of the transgene.

In some embodiments, for targeted insertion, the homology arms, e.g., the 5' and 3' homology arms, may each contain about 1000 base pairs (bp) of sequence surrounding the most distal gRNAs (e.g., 1000 bp from the sequence on either side of the mutation).

In some embodiments, one or more second template polynucleotides comprising one or more second transgenes may be introduced. In some embodiments, the one or more second transgenes are targeted for integration at or near one of at least one target site via homology-directed repair (HDR).

In some embodiments, the one or more second template polynucleotides comprise a [second 5' homology arm]-[one or more second transgenes]-[second 3' homology arm] structure. The homology arms provide for recombination into chromosomes, thereby inserting the transgene into DNA at or near a cleavage site, such as a target site. In some embodiments, the homology arms surround the most distal cleavage sites. In some embodiments, the second 5' homology arm and the second 3' homology arm comprise nucleic acid sequences homologous to nucleic acid sequences surrounding at least one target site. In some embodiments, the second 5' homology arm comprises nucleic acid sequences that are homologous to nucleic acid sequences of the second 5' target site. In some embodiments, the second 3' homology arm comprises nucleic acid sequences that are homologous to nucleic acid sequences of the second 3' target site. In some embodiments, the second 5' homology arm and the second 3' homology arm independently have at least or at least about, or are or are about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 base pairs, or less or less than about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 base pairs. In some embodiments, the second 5' homology arm and the second 3' homology arm independently have between about 50 and 100, 100 and 250, 250 and 500, 500 and 750, 750 and 1000, 1000 and 2000 base pairs. In some embodiments, the second 5' homology arm and the second 3' homology arm independently have about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 base pairs.

In some embodiments, one or more second transgenes are targeted for integration at or near a target site in a TRAC gene (e.g., described in Table 1 herein). In some embodiments, one or more second transgenes are targeted for integration at or near a target site in a TRBC1 or TRBC2 gene (e.g., described in Tables 2-3 herein).

It is contemplated herein that one or both homology arms may be truncated to avoid inclusion of certain repetitive sequence elements, such as Alu repeats or LINE elements. For example, the 5' homology arm may be truncated to avoid repetitive sequence elements. In some embodiments, the 3' homology arm may be truncated to avoid repetitive sequence elements. In some embodiments, both the 5' and 3' homology arms may be truncated to avoid inclusion of certain repetitive sequence elements. It is contemplated herein that template polynucleotides for target insertion may be designed for use as a single-stranded oligonucleotide, such as a single-stranded oligodeoxynucleotide (ssODN). When using ssODNs, the 5' and 3' homology arms can vary up to about 200 base pairs (bp) in length, such as at least 25, 50, 75, 100, 125, 150, 175, or 200 bp in length. Longer homology arms are also contemplated for ssODNs as improvements in ongoing oligonucleotide synthesis. In some embodiments, the longer homology arm is produced by a method other than chemical synthesis, such as by denaturing a long double-stranded nucleic acid and purifying one of the strands, such as by affinity for a strand-specific sequence attached to a solid substrate.

In some embodiments, alternative HDR occurs more efficiently when the template polynucleotide extends homology 5' to the target site (i.e., in the 5' strand direction of the target site). Thus, in some embodiments, the template polynucleotide has a longer homology arm and a shorter homology arm, where the long homology arm can anneal 5' to the target site. In some embodiments, the arm that can anneal 5' to the target site is at least 25, 50, 75, 100, 125, 150, 175 or 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000 or 5000 nucleotides from the target site or the 5' or 3' ends of the transgene. In some embodiments, the arm that can anneal 5' to the target site is at least 10%, 20%, 30%, 40% or 50% longer than the arm that can anneal 3' to the target site. In some embodiments, the arm that can anneal 5' to the target site is at least 2x, 3x, 4x, or 5x longer than the arm that can anneal 3' to the target site. Depending on where the sDNA template can anneal to the intact strand or the target site strand, the homology arm that anneals 5' to the target site can be at the 5' end of the sDNA template or the 3' end of the sDNA template, respectively.

Also, in some embodiments, the template polynucleotide has a 5' homology arm, a transgene, and a 3' homology arm, such that the template polynucleotide has extended homology to the 5' of the target site. For example, the 5' homology arm and the 3' homology arm can be substantially the same length, but the transgene can extend the 5' of the target site rather than the 3' of the target site. In some embodiments, the homology arm extends at least 10%, 20%, 30%, 40%, 50%, 2x, 3x, 4x, or 5x to the 5' end of the target site rather than to the 3' end of the target site.

In some embodiments, alternative HDR is more efficient if the template polynucleotide is centered at the target site. Thus, in some embodiments, the template polynucleotide has two homology arms that are substantially the same size.

For example, the first homology arm of a template polynucleotide has a length that is within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the second homology arm of the template polynucleotide.

Also, in some embodiments, the template polynucleotide has a 5' homology arm, a transgene, and a 3' homology arm, such that the template polynucleotide extends substantially the same distance on either side of the target site. For example, the homology arms may be different lengths, but the transgene may be selected to compensate for this. For example, the transgene may be further 5' from the target site than the 3' of the target site, but the 5' homology arm of the target site is shorter than the 3' homology arm of the target site to compensate. The converse is also possible, such that the transgene may be further 3' from the target site than the 5' of the target site, but the 3' homology arm of the target site is shorter than the 5' homology arm of the target site to compensate.

In some embodiments, the template polynucleotide is a single-stranded nucleic acid. In another embodiment, the template polynucleotide is a double-stranded nucleic acid. In some embodiments, the template polynucleotide comprises a nucleotide sequence, such as one or more nucleotides, that will be added to or create a template for alteration in the target DNA. In some embodiments, the template polynucleotide comprises a nucleotide sequence that can be used to modify a target site. In some embodiments, the template polynucleotide comprises a nucleotide sequence, such as one or more nucleotides, that corresponds to a wild-type sequence of the target DNA, such as a target site.

The template polynucleotide may comprise a transgene. In some embodiments, the template polynucleotide comprises a 5' homology arm. In some embodiments, the nucleic acid template comprises a 3' homology arm.

In some embodiments, the template polynucleotide is double-stranded DNA. The length may be, for example, about 200-5000 base pairs, such as about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2500, 3000, 4000, or 5000 base pairs. The length can be, for example, at least 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2500, 3000, 4000 or 5000 base pairs. In some embodiments, the length is no more than 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2500, 3000, 4000 or 5000 base pairs. In some embodiments, the double-stranded template polynucleotide has a length of about 160 base pairs, such as about 200-4000, 300-3500, 400-3000, 500-2500, 600-2000, 700-1900, 800-1800, 900-1700, 1000-1600, 1100-1500, or 1200-1400 base pairs.

The template polynucleotide may be a linear single-stranded DNA. In some embodiments, the template polynucleotide has (i) a linear single-stranded DNA that can anneal with the nicked strand of the target DNA, (ii) a linear single-stranded DNA that can anneal with the intact strand of the target DNA, (iii) a linear single-stranded DNA that can anneal with the read strand of the target DNA, (iv) a linear single-stranded DNA that can anneal with the unread strand of the target DNA, or more than one of the above.

The length may be, for example, about 200-5000 base pairs, such as about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2500, 3000, 4000 or 5000 nucleotides. The length may be, for example, at least 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2500, 3000, 4000 or 5000 nucleotides. In some embodiments, the length is no more than 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2500, 3000, 4000, or 5000 nucleotides. In some embodiments, the single-stranded template polynucleotide has a length of about 160 nucleotides, such as about 200-4000, 300-3500, 400-3000, 500-2500, 600-2000, 700-1900, 800-1800, 900-1700, 1000-1600, 1100-1500, or 1200-1400 nucleotides.

In some embodiments, the template polynucleotide is a circular double-stranded DNA, such as a plasmid. In some embodiments, the template polynucleotide comprises about 500 to 1000 base pairs of homology on either side of the transgene and/or target site. In some embodiments, the template polynucleotide comprises about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 base pairs of homology to the 5' target site or transgene, the 3' target site or transgene, or both the 5' and 3' target site or transgene. In some embodiments, the template polynucleotide comprises at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 base pairs of homology to the 5' target site or transgene, the 3' target site or transgene, or both the 5' and 3' target site or transgene. In some embodiments, the template polynucleotide comprises no more than 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 base pairs of homology to the 5' target site or transgene, the 3' target site or transgene, or both the 5' and 3' target site or transgene.

In some embodiments, the length of any of the polynucleotides, such as the template polynucleotides, can be, for example, from about 200-10,000 nucleotides, such as from about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2500, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10,000 nucleotides, or a value between any of the above. In some embodiments, the length can be, for example, at least about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2500, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10000 nucleotides, or a value between any of the above. In some embodiments, the length is no more than about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2500, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000 nucleotides. In some embodiments, the length is from about 200-4000, 300-3500, 400-3000, 500-2500, 600-2000, 700-1900, 800-1800, 900-1700, 1000-1600, 1100-1500, or 1200-1400 nucleotides. In some embodiments, the polynucleotide is at least about 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4760, 5000, 5250, 5500, 5750, 6000, 7000, 7500, 8000, 9000 or 10000 nucleotides in length, or any value between any of the above. In some embodiments, the polynucleotide is between about 2500 and about 5000 nucleotides, between about 3500 and about 4500 nucleotides, or between about 3750 nucleotides and about 4250 nucleotides in length. In some embodiments, the polynucleotide is about 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4760, 5000, 5250, 5500, 5750, 6000, 7000, 7500, 8000, 9000, or 10000 nucleotides in length.

In some embodiments, the template polynucleotide comprises homology arms to target an endogenous TRAC locus (an exemplary nucleotide sequence for the human TRAC gene locus is provided in SEQ ID NO:1; NCBI Reference Sequence: NG_001332.3, TRAC or described in Table 1 herein). In some embodiments, the genetic disruption of the TRAC locus is introduced into an early coding region of the gene, including a sequence located immediately downstream of the transcription start site, within the first exon of the coding sequence, or within 500 bp of the transcription start site (e.g., less than 500, 450, 400, 350, 300, 250, 200, 150, 100, or 50 bp) or within 500 bp of the transcription start site. from the initiation codon (e.g., less than 500, 450, 400, 350, 300, 250, 200, 150, 100, or 50 bp). In some embodiments, the genetic disruption is introduced using any of the targeted nucleases and/or gRNAs described in Section IA herein. In some embodiments, the template polynucleotide comprises about 500 to 1000, such as 600 to 900 or 700 to 800, base pairs of homology on either side of the genetic disruption introduced by the targeted nuclease and/or gRNA. In some embodiments, the template polynucleotide comprises about 500, 600, 700, 800, 900, or 1000 base pairs of 5' homology arm sequences that are homologous to 500, 600, 700, 800, 900, or 1000 base pairs of 5' genetic disruption sequences (e.g., at the TRAC locus) of the transgene, and about 500, 600, 700, 800, 900, or 1000 base pairs of 3' homology arm sequences that are homologous to 500, 600, 700, 800, 900, or 1000 base pairs of 3' genetic disruption sequences (e.g., at the TRAC locus). In some embodiments, exemplary 5' and 3' homology arms for targeted integration at the TRAC locus are set forth in SEQ ID NOS: 124 and 125, respectively. In some embodiments, exemplary 5' and 3' homology arms for targeted integration at the TRAC locus are set forth in SEQ ID NOS: 227-233 and 234-240, respectively.

In some embodiments, the template polynucleotide comprises homology arms to target an endogenous TRBC1 or TRBC2 locus (an exemplary nucleotide sequence of the human TRBC1 gene locus is provided in SEQ ID NO:2; NCBI Reference Sequence: NG_001333,2, TRBC1, is described in Table 2 herein; an exemplary nucleotide sequence of the human TRBC2 gene locus is provided in SEQ ID NO:3; NCBI Reference Sequence: NG_001333,2, TRBC2 , is described in Table 3 herein). In some embodiments, the genetic disruption of the TRBC1 or TRBC2 locus is introduced at an early coding region of the gene, including a sequence located immediately downstream of the transcription start site, within the first exon of the coding sequence, or within 500 bp of the first exon of the coding sequence. transcription start site (e.g., less than 500, 450, 400, 350, 300, 250, 200, 150, 100, or 50 bp) or within 500 bp of the initiation codon (e.g., less than 500, 450, 400, 350, 300, 250, 200, 150, 100, or 50 bp). In some embodiments, the genetic disruption is introduced using any of the targeted nucleases and/or gRNAs described in Section IA herein. In some embodiments, the template polynucleotide comprises about 500 to 1000, such as 600 to 900 or 700 to 800, base pairs of homology on either side of the genetic disruption introduced by the targeted nuclease and/or gRNA. In some embodiments, the template polynucleotide comprises about 500, 600, 700, 800, 900, or 1000 base pairs of 5' homology arm sequences that are homologous to 500, 600, 700, 800, 900, or 1000 base pairs of 5' genetic disruption sequences (e.g., at the TRBC1 or TRBC2 locus), a transgene, and about 500, 600, 700, 800, 900, or 1000 base pairs of 3' homology arm sequences that are homologous to 500, 600, 700, 800, 900, or 1000 base pairs of 3' genetic disruption sequences (e.g., at the TRBC1 or TRBC2 locus).

In some embodiments, any lengths and positions of the homology arms and related positions to the target sites, such as those described herein, may also be used for one or more second template polynucleotides.

In some cases, the template polynucleotide comprises a promoter, such as a promoter that is exogenous and/or not present at or near the target locus. In some embodiments, the promoter directs expression only in a particular cell type (e.g., a T cell- or B cell-specific or NK cell-specific promoter). In some embodiments in which the functional polypeptide coding sequences do not comprise a promoter, expression of the integrated transgene is driven by transcription driven by an endogenous promoter or other control element in the region in question.

A transgene, including a transgene encoding a recombinant receptor or antigen binding, a portion or chain thereof, and/or one or more second transgenes, can be inserted such that expression is driven by an endogenous promoter at the integration site, namely, a promoter that drives expression of the endogenous gene into which the transgene is inserted (e.g., TRAC , TRBC1 , and/or TRBC2 ). For example, coding sequences in a transgene can be inserted without a promoter, but in-frame with the coding sequence of an endogenous target gene, such that expression of the integrated transgene is controlled by transcription of the endogenous promoter at the integration site. In some embodiments, a transgene encoding a recombinant TCR or an antigen binding fragment or chain thereof, and/or one or more second transgenes are independently operably linked to an endogenous promoter of the gene at the target site. In some embodiments, a ribosome skip element/self-cleavage element, such as a 2A element, is placed upstream of the transgene coding sequence such that the ribosome skip element/self-cleavage element is placed in-frame with the endogenous gene such that expression of the transgene encoding the recombinant or antigen-binding fragment or chain thereof and/or one or more second transgenes is operably linked to the endogenous TCRα promoter.

In some embodiments, the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof and/or one or more second transgenes independently comprises one or more multicistronic elements. In some embodiments, the one or more multicistronic elements are located upstream of the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof and/or one or more second transgenes. In some embodiments, the multicistronic elements are located between the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof and the one or more second transgenes. In some embodiments, the multicistronic elements are located between the nucleic acid sequence encoding TCRα or a portion thereof and the nucleic acid sequence encoding TCRβ or a portion thereof. In some embodiments, the ribosome skipping element comprises a sequence encoding a ribosome skipping element selected from T2A, P2A, E2A, or F2A or an internal ribosome entry site (IRES).

In some embodiments, the encoded TCRα chain and the TCRβ chain are separated by a linker or spacer region. In some embodiments, a linker sequence is included that links the TCRα and TCRβ chains to form a single polypeptide chain. In some embodiments, the linker is of sufficient length to span the distance between the C terminus of the α chain and the N terminus of the β chain, or vice versa, while also ensuring that the length of the linker is not so long as to block or reduce binding to the target peptide-MHC complex. In some embodiments, the linker can be any linker capable of forming a single polypeptide chain while maintaining specificity for TCR binding. In some embodiments, the linker can comprise from or about 10 to 45 amino acids, such as from 10 to 30 amino acids, or from 26 to 41 amino acid residues, such as 29, 30, 31, or 32 amino acids. In some embodiments, the linker has the formula -PGGG-(SGGGG)n-P-, where n is 5 or 6 and P is proline, G is glycine and S is serine (SEQ ID NO: 22). In some embodiments, the linker has the sequence GSADDAKKDAAKKDGKS (SEQ ID NO: 23). In some embodiments, a linker or spacer between the TCRα chain or a portion thereof and the TCRβ chain or a portion thereof that is recognized and/or cleavable by a protease. In certain embodiments, a linker or spacer between the TCRα chain or a portion thereof and the TCRβ chain or a portion thereof comprises a ribosome skip element or a self-cleavage element.

In some embodiments, the transgene is or includes a nucleotide sequence that is or includes a [TCRβ chain]-[linker]-[TCRα chain] structure. In particular embodiments, the transgene is or includes a nucleotide sequence that is or includes a [TCRβ chain]-[self-cleavage element]-[TCRα chain] structure. In certain embodiments, the transgene is or includes a nucleotide sequence that is or includes a [TCRβ chain]-[ribosome skip sequence]-[TCRα chain] structure. In some embodiments, the transgene is or includes a nucleotide sequence that is or includes a [TCRα chain]-[linker]-[TCRβ chain] structure. In particular embodiments, the transgene is or includes a nucleotide sequence that is or includes a [TCRα chain]-[self-cleavage element]-[TCRβ chain] structure. In certain embodiments, the transgene is or includes a nucleotide sequence that is or includes a [TCRα chain]-[ribosome skip sequence]-[TCRβ chain] structure. In some embodiments, the structures are an encoded polynucleotide strand or a single- or double-stranded polynucleotide, in a 5'-3' direction.

In some cases, a ribosome skip/self-cleavage element such as T2A can cause the ribosome to skip (ribosomal skip) the synthesis of a peptide bond at the C-terminus of the 2A element, resulting in separation between the end of the 2A sequence and the next peptide downstream (see, e.g., de Felipe, Genetic Vaccines and Ther. 2:13 (2004) and de Felipe et al. Traffic 5:616-626 (2004)). This allows control of the inserted transgene by transcription of an endogenous promoter at the integration site, e.g., the TRAC , TRBC1 and/or TRBC2 promoter. An exemplary ribosome skip element/self-cleavage element includes 2A sequences from pangolin virus (F2A, e.g., SEQ ID NO: 11), equine rhinitis virus A (E2A, e.g., SEQ ID NO: 10), Thosea asigna virus (T2A, e.g., SEQ ID NO: 6 or 7), and porcine teschovirus-1 (P2A, e.g., SEQ ID NO: 8 or 9) as described in U.S. Patent Publication No. 20070116690. In some embodiments, the template polynucleotide includes a P2A ribosome skip element (the sequence set forth in SEQ ID NO: 8 or 9) upstream of a transgene, e.g., a recombinant receptor encoding nucleic acids.

In some embodiments, the transgene may comprise a promoter and/or an enhancer, such as a constitutive promoter or an inducible or tissue-specific promoter. In some embodiments, the promoter is or comprises a constitutive promoter. Exemplary constitutive promoters include, for example, the simian virus 40 (SV40) early promoter, the cytomegalovirus (CMV) immediate early promoter, the human ubiquitin C (UBC) promoter, the human elongation factor 1α (EF1α) promoter, the mouse phosphoglycerate kinase 1 (PGK) promoter, and the chicken β-active coupled to the CMV early enhancer (CAGG) promoter. In some embodiments, the constitutive promoter is a synthetic or modified promoter. In some embodiments, the promoter is or comprises the MND promoter, a synthetic promoter that comprises the U3 region of a modified MoMuLV LTR with the myeloproliferative sarcoma virus enhancer (the sequence set forth in SEQ ID NO:18 or 126; see Challita et al. (1995) J. Virol. 69(2):748-755). In some embodiments, the promoter is a tissue-specific promoter. In another embodiment, the promoter is a viral promoter. In another embodiment, the promoter is a non-viral promoter. In some cases, the promoter is selected from the human elongation factor 1 alpha (EF1α) promoter (sequence as set forth in SEQ ID NO:4 or 5) or a modified form thereof (EF1α promoter with HTLV1 enhancer; sequence as set forth in SEQ ID NO:127) or the MND promoter (sequence as set forth in SEQ ID NO:18 or 126). In some embodiments, the transgene does not include a regulatory element, such as a promoter.

In some embodiments, a "tandem" cassette is integrated at a selected site. In some embodiments, one or more "tandem" cassettes encode one or more polypeptides or factors, each independently controlled by a regulatory element or all controlled as a multicistronic expression system. In some embodiments, for example, in which a polynucleotide comprises a first and second nucleic acid sequence, the coding sequences encoding each of the different polypeptide chains can be operably linked by a promoter, which can be the same or different. In some embodiments, a nucleic acid molecule can comprise a promoter that directs the expression of two or more different polypeptide chains. In some embodiments, such nucleic acid molecules can be multicistronic (bicistronic or tricistronic, see, for example, U.S. Patent No. 6,060,273). In some embodiments, the transcription units may be constructed as a bicistronic unit containing an IRES (internal ribosome entry site) that allows co-expression of gene products via mRNA from a single promoter. Alternatively, in some cases, a single promoter may direct the expression of RNA that contains, in a single open reading frame (ORF), two or three polypeptides separated from each other by sequences encoding a self-cleavage peptide ( e.g., the 2A sequence) or a protease recognition site ( e.g., furin), as described herein. The ORF thus encodes a single polypeptide that is processed into individual proteins either during (in the case of 2A) or after translation. In some embodiments, a "tandem cassette" includes a first cassette component containing a non-promoter sequence, followed by a transcriptional conversion sequence, and a second sequence encoding an autonomous expression cassette or a multicistronic expression sequence. In some embodiments, the tandem cassette encodes two or more different polypeptides or factors, such as two or more chains or domains of a recombinant receptor. In some embodiments, nucleic acid sequences encoding two or more chains or domains of a recombinant receptor are introduced as tandem expression cassettes or bi- or multicistronic cassettes, into a target DNA integration site.

The transgene may be inserted into an endogenous gene such that all, some, or none of the endogenous genes are expressed. In some embodiments, the transgene (e.g., with or without peptide coding sequences) is integrated into any endogenous locus. In some embodiments, the transgene is integrated into the TRAC , TRBC1 , and/or TRBC2 gene loci.

In some embodiments, the exogenous sequences may also include transcriptional or translational regulatory sequences, such as promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding 2A peptides and/or polyadenylation signals. Furthermore, the control elements of the genes in question may be operably linked to reporter genes to create chimeric genes (e.g., reporter expression cassettes). Additionally, splice acceptor sequences may be included. Exemplary known splice acceptor site sequences include, for example, CTGACCTCTTCTCTTCCTCCCACAG, (SEQ ID NO:119) (from the human HBB gene) and TTTCTCTCCACAG (SEQ ID NO:120) (from the human immunoglobulin gamma gene).

In a typical embodiment, the template polynucleotide includes arms of homology for targeting the TRAC locus, regulatory sequences, such as a promoter, and nucleic acid sequences encoding a recombinant receptor, such as a TCR. In a typical embodiment, an additional template polynucleotide is used that includes arms of homology for targeting the TRBC1 and/or TRBC2 loci, regulatory sequences, such as a promoter, and nucleic acid sequences encoding another factor.

In some embodiments, exemplary template polynucleotides comprise a transgene encoding a recombinant T cell receptor under the functional control of the human elongation factor 1 alpha (EF1α) promoter with the HTLV1 enhancer (the sequence presented in SEQ ID NO:127) or the MND promoter (the sequence presented in SEQ ID NO:126) or linked to nucleic acid sequences encoding the P2A ribosome skip element (the sequence presented in SEQ ID NO:8) to drive expression of the recombinant TCR from an endogenous target gene locus (e.g., TRAC ), a 5' arm of approximately 600 bp sequence homology (e.g., that presented in SEQ ID NO:124), a 3' arm of approximately 600 bp sequence homology, and a 5' arm of approximately 600 bp sequence homology. (e.g., as set forth in SEQ ID NO:125) that are homologous to sequences surrounding the target integration site in exon 1 of the human TCR α constant region ( TRAC ) gene. In some embodiments, the template polynucleotide further comprises other nucleic acid sequences, such as nucleic acid sequences encoding a marker, such as a surface marker or a selection marker. In some embodiments, the template polynucleotide further comprises a viral vector of sequences, such as an adeno-associated virus (AAV) vector of sequences.

The transgene contained on the template polynucleotide described herein can be isolated from plasmids, cells or other sources using known standard techniques such as PCR. The template polynucleotide for use can include different types of topology, including circular supercoiled, circular relaxed, linear and the like. Alternatively, they can be chemically synthesized using standard oligonucleotide synthesis techniques. In addition, the template polynucleotides can be methylated or unmethylated. The template polynucleotides can be in the form of bacterial or yeast artificial chromosomes (BAC or YAC).

The polynucleotide may be introduced into the cell as part of a vector molecule having additional sequences such as, for example, origins of replication, promoters and genes encoding antibiotic resistance. Moreover, template polynucleotides may be introduced as naked nucleic acids, as nucleic acids complexed with materials such as liposomes, nanoparticles or poloxamer, or may be delivered by viruses (e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus and integrase-defective lentivirus (IDLV)).

In other aspects, the template polynucleotide is delivered by viral and/or non-viral gene transfer methods. In some embodiments, the template polynucleotide is delivered to the cell via an adeno-associated virus (AAV). Any AAV can be used, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and combinations thereof. In some cases, the AAV comprises LTRs that are a heterologous serotype compared to the capsid serotype (e.g., AAV2 ITRs with AAV5, AAV6, or AAV8 capsids). The template polynucleotide can be delivered using the same gene transfer system that is used to deliver the nuclease (including on the same vector) or can be delivered using different delivery systems from the one that delivers the nuclease. In some embodiments, the template polynucleotide is delivered using a viral vector (e.g., AAV) and the nucleases are delivered in the form of mRNA. The cell can also be processed with one or more molecules that inhibit the binding of the viral vector to a cell surface receptor, as described herein, before, simultaneously with, and/or after delivery of the viral vector (e.g., carrying the nucleases and/or template polynucleotide).

In some embodiments, the template polynucleotide is contained in a viral vector and is at least about 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4760, 5000, 5250, 5500, 5750, 6000, 7000, 7500, 8000, 9000 or 10000 nucleotides in length, or any value between any of the above. In some embodiments, the polynucleotide is contained in a viral vector and is between about 2500 and about 5000 nucleotides, between about 3500 and about 4500 nucleotides, or between about 3750 nucleotides and about 4250 nucleotides in length. In some embodiments, the polynucleotide is contained in a viral vector and is about 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4760, 5000, 5250, 5500, 5750, 6000, 7000, 7500, 8000, 9000, or 10,000 nucleotides in length.

In some embodiments, the template polynucleotide is an adenoviral vector, such as an AAV vector, such as an ssDNA molecule of a length and sequence that allows it to be packaged into an AAV capsid. The vector may be, for example, less than 5 kb and may contain an ITR sequence that facilitates packaging into a capsid. The vector may be integration deficient. In some embodiments, the template polynucleotide contains about 150 to 1000 nucleotides of homology on each side of the transgene and/or target site. In some embodiments, the template polynucleotide comprises about 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides of the 5' target site or transgene, the 3' target site or transgene, or both the 5' and 3' target site or transgene. In some embodiments, the template polynucleotide comprises at least 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides of the 5' target site or transgene, the 3' target site or transgene, or both the 5' and 3' target site or transgene. In some embodiments, the template polynucleotide comprises no more than 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides of the 5' target site or transgene, the 3' target site or transgene, or both the 5' and 3' target site or transgene.

In some embodiments, the template polynucleotide is a lentiviral vector, such as IDLV (integration deficient lentivirus). In some embodiments, the template polynucleotide comprises about 500 to 1000 base pairs of homology on each side of the transgene and/or target site. In some embodiments, the template polynucleotide comprises about 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 base pairs of homology to the 5' target site or transgene, the 3' target site or transgene, or both the 5' and 3' target site or transgene. In some embodiments, the template polynucleotide comprises at least 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 base pairs of homology to the 5' target site or transgene, the 3' target site or transgene, or both the 5' and 3' target site or transgene. In some embodiments, the template polynucleotide comprises no more than 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 base pairs of homology to the 5' target site or transgene, the 3' target site or transgene, or both the 5' and 3' target site or transgene. In some embodiments, the template polynucleotide comprises one or more mutations, such as silent mutations, that prevent the template polynucleotide from recognizing and cleaving Cas9. The template polynucleotide can comprise, for example, at least 1, 2, 3, 4, 5, 10, 20 or 30 silent mutations relative to the corresponding sequence in the genome of the cell to be altered. In some embodiments, the template polynucleotide comprises no more than 2, 3, 4, 5, 10, 20, 30 or 50 silent mutations relative to the corresponding sequence in the genome of the cell to be altered. In some embodiments, the cDNA comprises one or more mutations, such as silent mutations, that prevent the template polynucleotide from recognizing and cleaving Cas9. The template polynucleotide may contain, for example, at least 1, 2, 3, 4, 5, 10, 20 or 30 silent mutations relative to the corresponding sequence in the genome of the cell being modified. In some embodiments, the template polynucleotide contains no more than 2, 3, 4, 5, 10, 20, 30 or 50 silent mutations relative to the corresponding sequence in the genome of the cell being modified.

The double-stranded template polynucleotides described herein may include one or more non-natural bases and/or backbones. In particular, insertion of a template polynucleotide with methylated cytosines may be accomplished using the methods described herein to achieve a state of transcriptional immobility in the region in question.

The template polynucleotide may contain any transgene of interest (exogenous sequence). Typical exogenous sequences include, but are not limited to, any polypeptide coding sequence (e.g., cDNA or fragments thereof), promoter sequences, enhancer sequences, epitope tags, marker genes, enzyme cleavage recognition sites, and various types of expression constructs. Marker genes include, but are not limited to, sequences encoding a protein that mediates antibiotic resistance (e.g., ampicillin resistance, neomycin resistance, G418 resistance, piromycin resistance), sequences encoding colored or fluorescent or luminescent proteins (e.g., green fluorescent protein, enhanced green fluorescent protein, red fluorescent protein, luciferase), and proteins that mediate enhanced cell growth and/or gene amplification (e.g., dihydrofolate reductase). Epitope tags include, for example, one or more copies of FLAG, His, myc, Tap, HA, or any detectable amino acid sequence.

In some embodiments, the transgene comprises a polynucleotide encoding any polypeptide whose expression in a cell is desired, including, but not limited to, antibodies, antigens, enzymes, receptors (cell surface or nuclear), hormones, lymphokines, cytokines, reporter polypeptides, growth factors, and functional fragments of any of the foregoing. In some embodiments, the exogenous sequence (transgene) comprises a polynucleotide encoding one or more recombinant receptors, such as functional non-TCR antigen receptors, chimeric antigen receptors (CARs), and T cell receptors (TCRs), such as transgenic TCRs, engineered TCRs, or recombinant TCRs, and components of any of the foregoing.

In some embodiments, the coding sequences can be, for example, cDNA. Exogenous sequences can also be a fragment of a transgene for linking to the endogenous gene sequence in question. For example, a transgene fragment containing a sequence at the 3' end of the gene in question can be used to correct, through insertion or substitution, a sequence encoding a mutation at the 3' end of the endogenous gene sequence. Also, the fragment can contain sequences similar to the 5' end of the endogenous gene for insertion/substitution of endogenous sequences for correction or modification of such endogenous sequence. Additionally, the fragment can encode a functional domain in question (catalytic, secretory or the like) for in situ binding to the endogenous gene sequence to produce a fusion protein.

In some embodiments, the transgene further encodes one or more markers. In some embodiments, the one or more markers are a transduction marker, a surrogate marker, and/or a selection marker.

In some embodiments, the marker is a transduction marker or a surrogate marker. A transduction marker or surrogate marker can be used to identify cells that have been administered with a polynucleotide, such as a polynucleotide encoding a recombinant receptor. In some embodiments, a transduction marker can indicate or confirm a modification of a cell. In some embodiments, a surrogate marker is a protein that is engineered to be coexpressed on the surface of a cell with a recombinant receptor, such as a TCR or CAR. In certain embodiments, such a surrogate marker is a surface protein that is modified to have little or no activity. In certain embodiments, the surrogate marker is encoded on the same polynucleotide that encodes the recombinant receptor. In some embodiments, a nucleic acid sequence encoding a recombinant receptor is operably linked to a nucleic acid sequence encoding a marker, optionally separated by an internal ribosome entry site (IRES) or a nucleic acid encoding a self-cleavage peptide or a peptide that causes ribosome skipping, such as a 2A sequence such as T2A, P2A, E2A or F2A. External marker genes, in some cases, can be used in combination with engineered cells to allow cell detection or selection, and, in some cases, also promote cell suicide.

Exemplary surrogate markers may include truncated forms of cell surface polypeptides, such as truncated forms that are non-functional and do not transduce or are incapable of transducing a signal or signal normally transduced by the full form of the cell surface polypeptide and/or do not or are incapable of internalization. Exemplary truncated cell surface polypeptides include truncated forms of growth factors or other receptors, such as truncated human epidermal growth factor receptor 2 (tHER2), truncated epidermal factor receptor (tEGFR, an exemplary tEGFR sequence is provided in SEQ ID NO:12 or 13), or prostate-specific membrane antigen (PSMA), or modified forms thereof. tEGFR may comprise an epitope recognized by the antibody cetuximab (Erbitux®) or another therapeutic anti-EGFR antibody or binding molecule, which can be used to identify or select cells that have been engineered with the tEGFR construct and the encoded exogenous protein, and/or to exclude or isolate cells that express the encoded exogenous protein. See U.S. Patent No. 8,802,374 and Liu et al., Nature Biotech. 2016 April; 34(4): 430-434). In some aspects, a marker, e.g., a surrogate marker, comprises all or a portion ( e.g., a truncated form) of CD34, NGFR, CD19, or a truncated CD19, e.g., a truncated non-human CD19 receptor or epidermal growth factor receptor ( e.g., tEGFR). In some embodiments, the marker comprises a fluorescent protein such as green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP) such as super-folded GFP (sfGFP), red fluorescent protein (RFP) such as tdTomato, mCherry, mStrawberry, AsRed2, DsRed or DsRed2, cyan fluorescent protein (CFP), blue-green fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP) and yellow fluorescent protein (YFP), and variants thereof, including species variants, monomeric variants and codon-optimized and/or enhanced variants of fluorescent proteins. In some embodiments, the marker is or comprises an enzyme such as luciferase, the lacZ gene from E. coli , alkaline phosphatase, secreted embryonic alkaline phosphatase (SEAP), chloramphenicol acetyltransferase (CAT). Exemplary light-emitting reporter genes include luciferase (luc), β-galactosidase, chloramphenicol acetyltransferase (CAT), β-glucuronidase (GUS), or variants thereof.

In some embodiments, the marker is a selection marker. In some embodiments, the selection marker is or comprises a polypeptide that confers resistance to exogenous agents or drugs. In some embodiments, the selection marker is an antibiotic resistance gene. In some embodiments, the selection marker is an antibiotic resistance gene that confers antibiotic resistance to mammalian cells. In some embodiments, the selection marker is or comprises a Pyromycin resistance gene, a Hygromycin resistance gene, a Blasticidin resistance gene, a Neomycin resistance gene, a Geneticin resistance gene, or a Zeocin resistance gene, or modified forms thereof.

In some embodiments, a nucleic acid encoding a marker is operably linked to a polynucleotide encoding a linker sequence, such as a cleavable linker sequence, such as T2A. For example, the marker and, optionally, the linker sequence can be as described in PCT Publication No. WO2014031687. For example, the marker can be a truncated EGFR (tEGFR) that is optionally linked to a linker sequence, such as a T2A cleavable linker sequence. An exemplary polypeptide for a truncated EGFR ( e.g., tEGFR) comprises an amino acid sequence as set forth in SEQ ID NO: 12 or 13 or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12 or 13.

In some embodiments, the marker is a molecule, such as a cell surface protein, that is not naturally found on a T cell or is not naturally found on the surface of a T cell or a portion thereof.

In some embodiments, the molecule is not a self molecule, for example, a non-self protein, i.e. one that is not recognized as “self” by the immune system of the host into which the cells are adoptively transferred.

In some embodiments, the marker does not have a therapeutic function and/or does not have an effect other than being used as a marker for genetic engineering, such as for selecting cells that have been successfully engineered. In other embodiments, the marker may be a therapeutic molecule or a molecule that otherwise has some desired effect, such as a ligand for a cell found in vivo, such as a costimulatory molecule or an immune checkpoint to improve and/or reduce the response of cells upon adoptive transfer and encounter with a ligand.

In some embodiments, such a transgene further comprises a T2A ribosome skip element and/or a sequence encoding a marker, such as a tEGFR sequence, for example downstream of a TCR or CAR, such as that shown in SEQ ID NO: 12 or 13, respectively, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12 or 13.

In some embodiments, the template polynucleotide encodes a recombinant receptor that serves to direct T cell function. Chimeric antigen receptors (CARs) are molecules engineered to direct immune cells to specific molecular targets expressed on the surfaces of cells. In their most basic form, they are receptors introduced into a cell that combine a specificity domain expressed on the outside of the cell with signaling pathways inside the cell such that when the specificity domain interacts with its target, the cells are activated. Other CARs are derived from T cell receptor (TCR) variants where a specificity domain, such as an scFv or some type of receptor, is fused to the signaling domain of the TCR. These constructs are then introduced into the T cell, allowing the T cell to become activated in the presence of a cell expressing the target antigen, which causes the activated T cell to attack the target cell in an MHC-independent manner (see Chicaybam et al (2011) Int Rev Immunol 30:294–311). Alternatively, CAR expression cassettes can be introduced into an immune cell for further engraftment such that the CAR cassette is under the control of a T cell specific promoter (e.g., the FOXP3 promoter, see Mantel et. al (2006) J. Immunol 176: 3593-3602).

As an exemplary example, the template polynucleotide is included as an adeno-associated virus (AAV) vector construct comprising a nucleic acid sequence encoding a recombinant TCR α and TCR β chain and under the control of a constitutive promoter flanked by homology arms of about 600 base pairs each from the 5' and 3' side of the nucleic acid sequence encoding the recombinant TCR for targeting exon 1 of the endogenous TRAC gene. An exemplary 5' homology arm for targeting TRAC comprises the sequence set forth in SEQ ID NO:124. An exemplary 3' homology arm for targeting TRAC comprises the sequence set forth in SEQ ID NO:125.

In constructing such expression cassettes following the ideas of this specification, techniques well known in the field of molecular biology are used (see, for example, Ausubel or Maniatis). Before using the expression cassette to create a transgenic animal, the responsiveness of the expression cassette to the stress challenge associated with the selected control elements can be tested by introducing the expression cassette into a suitable cell colony (e.g., primary cells, transformed cells, or immortalized cell colonies).

Directed insertion of non-coding nucleic acid sequences can also be performed. Sequences encoding antisense RNA, RNAi, shRNA and micro RNA (miRNA) can also be used for directed insertions. In further embodiments, the template polynucleotide can contain non-coding sequences that are specific target sites for additional nuclease designs. Additional nucleases can then be expressed in cells such that the original template polynucleotide is cleaved and modified by the insertion of another target template polynucleotide. In this manner, reiterative integrations of template polynucleotides can be obtained, allowing for the priming of traits at a particular target locus, such as the TRAC , TRBC1 and/or TRBC2 gene loci.

In some embodiments, the polynucleotide comprises the structure: [5′ homology arm]-[transgene sequence]-[3′ homology arm]. In some embodiments, the polynucleotide comprises the structure: [5′ homology arm]-[multicistronic element]-[transgene sequence]-[3′ homology arm]. In some embodiments, the polynucleotide comprises the structure: [5′ homology arm]-[promoter]-[transgene sequence]-[3′ homology arm].

4. Delivery of template polynucleotides

In some embodiments, a polynucleotide, such as a template polynucleotide, encoding a chimeric receptor is introduced into cells in the form of a nucleotide, such as a polynucleotide or a vector. In particular embodiments, the polynucleotide comprises a transgene that encodes a chimeric receptor or a portion thereof.

In some embodiments, the template polynucleotide is introduced into the cell for engineering, in addition to agents capable of causing targeted genetic destruction, such as nuclease and/or gRNA. In some embodiments, the template polynucleotides can be delivered before, simultaneously with, or after agents capable of causing targeted genetic destruction are introduced into the cell. In some embodiments, the template polynucleotides are delivered simultaneously with the agents. In some embodiments, the template polynucleotides are delivered before the agents, such as seconds to hours to days before the agents, including, but not limited to, 1-60 minutes (or any time in between) before the agents, 1-24 hours (or any time in between) before the agents, or more than 24 hours before the agents. In some embodiments, the template polynucleotides are delivered after the agents, from seconds to hours to days after the agents, including immediately after the agent is delivered, such as between or between about 30 seconds to 4 hours, such as about 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 6 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours or 4 hours after the agents are delivered and/or, preferably, within 4 hours after the agents are delivered. In some embodiments, the template polynucleotide is delivered more than 4 hours after the agents are delivered. In some embodiments, the template polynucleotides are delivered after the agents, such as, but not limited to, within 1 second to 60 minutes (or any time in between) after the agents, 1 to 4 hours (or any time in between) after the agents, or more than 4 hours after the agents.

In some embodiments, the template polynucleotides can be delivered using the same delivery systems as the agents capable of causing targeted genetic destruction, such as a nuclease and/or gRNA. In some embodiments, the template polynucleotides can be delivered using systems different from the delivery systems of the agents capable of causing targeted genetic destruction, such as a nuclease and/or gRNA. In some embodiments, the template polynucleotide is delivered simultaneously with the agents. In other embodiments, the template polynucleotide is delivered at a different time, before or after delivery of the agents. Any of the delivery methods described herein in Section IA3 (e.g., Tables 7 and 8 ) for delivering nucleic acids to agents capable of causing targeted genetic destruction, such as a nuclease and/or gRNA, can be used to deliver the template polynucleotide.

In some embodiments, one or more agents and the template polynucleotide are delivered in the same format or method. For example, in some embodiments, both one or more agents and the template polynucleotide are contained in a vector, such as a viral vector. In some embodiments, the template polynucleotide is encoded on the same vector backbone, such as an AAV genome, a plasmid DNA such as Cas9, and gRNA. In some aspects, one or more agents and the template polynucleotide are in different formats, such as a ribonucleic acid-protein (RNP) complex for a Cas9-gRNA agent and linear DNA for a template polynucleotide, but are delivered using the same method. In some aspects, one or more agents and the template polynucleotide are in different formats, for example, a ribonucleic acid-protein (RNP) complex for a Cas9-nRNA agent, and the template polynucleotide is contained in an AAV vector, and the RNP is delivered using a physical delivery method (e.g., electroporation) and the template polynucleotide is delivered through transduction of AAV viral preparations. In some aspects, the template polynucleotide is delivered immediately after, for example, within about 1, 2, 3, 4, 5, 10, 20, 30, 40, 50 or 60 minutes after delivery of one or more agents.

In some embodiments, the template polynucleotide is a linear or circular nucleic acid molecule, such as linear or circular DNA or linear RNA, and can be delivered using any of the methods described in Section IA3 herein (e.g., Tables 7 and 8 ) for delivering nucleic acid molecules into a cell.

In particular embodiments, a polynucleotide, such as a template polynucleotide, is introduced into cells in nucleotide form, such as in or within a non-viral vector. In some embodiments, the non-viral vector is or includes a polynucleotide, such as a DNA or RNA polynucleotide, that is suitable for transduction and/or transfection by any suitable and/or known non-viral gene delivery method, such as, but not limited to, microinjection, electroporation, transient cell condensation or compression (e.g., as described in Lee, et al. (2012) Nano Lett 12: 6322-27), lipid-mediated transfection, peptide-mediated delivery, such as cell-penetrating peptides, or a combination thereof. In some embodiments, the non-viral polynucleotide is delivered to the cell by a non-viral method described herein, such as the non-viral methods listed in Table 8 herein.

In some embodiments, the template polynucleotide sequence may be contained in a vector molecule comprising sequences that are not homologous to the region in question in genomic DNA. In some embodiments, the virus is a DNA virus (e.g., a dsDNA or ssDNA virus). In some embodiments, the virus is an RNA virus (e.g., a ssRNA or ssRNA virus). Exemplary viral vectors/viruses include, for example, retroviruses, lentiviruses, adenovirus, adeno-associated virus (AAV), vaccinia viruses, poxviruses, and herpes simplex viruses, or any of the viruses described herein.

In some embodiments, the template polynucleotide can be transferred into cells using recombinant infectious viral particles, such as, for example, vectors derived from simian virus 40 (SV40), adenoviruses, adeno-associated viruses (AAV). In some embodiments, the template polynucleotide is transferred into T cells using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors (see, e.g., Koste et al. (2014) Gene Therapy 2014 Apr 3. doi: 10,1038/gt,2014,25; Carlens et al. (2000) Exp Hematol 28(10): 1137-46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. 2011 November 29(11): 550-557) or HIV-1-derived lentiviral vectors.

In some embodiments, the retroviral vector has a long terminal repeat (LTR), such as a retroviral vector derived from Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), mouse embryonic stem cell virus (MESV), mouse stem cell virus (MSCV), or spleen necrosis virus (SFFV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, retroviruses include those derived from any avian or mammalian cell source. Retroviruses are typically amphotropic, meaning that they are capable of infecting host cells from multiple species, including humans. In one embodiment, the gene to be expressed replaces the retroviral gag, pol, and/or env sequences. A variety of illustrative retroviral systems have been described (e.g., U.S. Patent Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109).

In some embodiments, the template polynucleotides and nucleases can be on the same vector, such as an AAV vector (e.g., AAV6). In some embodiments, the template polynucleotides are delivered using an AAV vector, and the agents capable of causing targeted genetic destruction, such as a nuclease and/or gRNA, are delivered in another form, such as an mRNA encoding the nuclease and/or gRNA. In some embodiments, the template polynucleotides and nucleases are delivered using the same type of method, such as a viral vector, but on separate vectors. In some embodiments, the template polynucleotides are delivered in different delivery systems as the agents capable of causing genetic destruction, such as nucleases and/or gRNA. In some embodiments, the template polynucleotide is excised from the vector backbone in vivo, such as by surrounding it with gRNA recognition sequences. In some embodiments, the template polynucleotide is on a separate polynucleotide molecule, such as Cas9 and gRNA. In some embodiments, Cas9 and gRNA are administered in the form of a ribonucleoprotein (RNP) complex, and the template polynucleotide is administered as a polynucleotide molecule, such as in a vector or a linear nucleic acid molecule, such as linear DNA. Types or nucleic acids and vectors for delivery include any of those described in Section III herein.

C. Evaluation of engineered T cells and compositions

In some embodiments, the methods include evaluating T cells or T cell compositions engineered to express recombinant TCRs for specific properties. For example, the methods include evaluating T cells or T cell compositions for expressing recombinant TCRs on the cell surface and/or for recognizing a peptide in the context of an MHC molecule. For example, in any of the embodiments provided herein, functional assays can be performed on T cells or T cell compositions expressing exogenous recombinant TCRs engineered or produced using any of the methods described herein. In some embodiments, assays can also be performed to determine TCR functionality and TCR signaling activity.

In some embodiments, T cells or T cell compositions are assessed for expression of a recombinant TCR on the cell surface, such as the ability or capacity to express a functional TCR, such as TCRαβ, on the cell surface. In some embodiments, T cells or T cell compositions are assessed for the ability or capacity of the expressed TCR to recognize a peptide in the context of an MHC molecule, such as binding antigens or epitopes in the context of an MHC molecule. In some embodiments, the methods include assessing T cells or T cell compositions for T cell activity and/or functionality. In some embodiments, T cells or T cell compositions are assessed for expression of a marker for transduction or transgene delivery.

In some embodiments, the T cells or T cell compositions are assessed for expression of a recombinant TCR on the cell surface, such as the potential or ability to express a functional TCR, such as TCRαβ, on the cell surface. In some embodiments, assessing TCR surface expression comprises contacting the cells of each T cell composition with a binding reagent specific for the TCRα chain or the TCRβ chain and assessing binding of the reagent to the cells. In some embodiments, the binding reagent is an antibody. In some embodiments, the binding reagent is detectably labeled, optionally fluorescently labeled, directly or indirectly. In some embodiments, the binding reagent is a fluorescently labeled antibody, such as an antibody labeled directly or indirectly. In some embodiments, the binding reagent is an anti-pan-TCR Vβ antibody or is an anti-pan-TCR Vα antibody. In some embodiments, the binding reagent recognizes a specific chain family. In some embodiments, the binding reagent is an anti-TCR Vβ or anti-TCR Vα antibody that recognizes or binds to a specific family, such as an anti-TCR Vβ22 antibody or an anti-TCR Vβ2 antibody. In some embodiments, expression is detected using antibodies against one or more common portions, such as extracellular portions, of the TCR. For example, cell surface expression of the TCR can be detected using a pan-reactive anti-TCR antibody, such as a pan-reactive TCR Vβ antibody or a pan-reactive TCR Vα antibody. Pan-reactive antibodies can detect regions of the TCR regardless of their antigen or epitope binding specificity. In some embodiments, the cells are stained using a binding reagent, such as a labeled antibody that recognizes TCR expression on the cell surface, such as a labeled pan-reactive TCR Vα antibody or an antigen-binding fragment thereof, and detected using fluorescence microscopy, flow cytometry, or fluorescence-activated cell sorting (FACS). In some embodiments, T cells or T cell compositions that express TCR on the cell surface are identified and/or selected, such as those that are positive for staining using pan-reactive anti-TCR antibodies, such as a pan-reactive TCR Vβ antibody or a pan-reactive TCR Vα antibody.

In some embodiments, T cells or T cell compositions are assessed for the ability or capability of the expressed TCRs to recognize a peptide in the context of an MHC molecule, such as binding antigens or epitopes in the context of an MHC molecule. For example, in some embodiments, assessing T cells or T cell compositions for recognizing a peptide in the context of an MHC molecule includes: (1) contacting the cells or cells of a T cell composition with a target antigen comprising a peptide-MHC complex, and (2) determining the presence or absence of binding of the peptide-MHC complex to the cells and/or determining the presence or absence of activation of the T cell TCR-expressing cells upon interaction with the peptide-MHC complex.

In some embodiments, T cells or T cell compositions into which nucleic acid sequences encoding recombinant TCRs are introduced are tested by confirming that the recombinant TCRs bind to desired or known antigens, such as a TCR ligand (MHC-peptide complex). In some embodiments, cell binding to an antigen or epitope can be determined in a variety of ways. In some methods, a particular antigen, such as an MHC-peptide complex, can be detectably labeled so that binding to a receptor, such as a TCR, can be visualized. In some embodiments, the antigen can be soluble or expressed in a soluble form. In some embodiments, the TCR ligand can be a peptide-MHC tetramer, and in some cases, the peptide-MHC tetramer can be detectably labeled, such as labeled with a fluorescent label. The peptide-MHC tetramer can be labeled directly or indirectly. In some embodiments, the fluorescent label can be detected using flow cytometry or fluorescence-activated cell sorting (FACS) or fluorescence microscopy. In some embodiments, the methods include identifying one or more T cells or T cell compositions that recognize the peptide in the context of an MHC molecule, i.e., a peptide-MHC complex.

In some cases, binding of a TCR, such as a recombinant TCR, to a peptide epitope, such as in a complex with MHC, confers or triggers a functional property of the interaction. For example, a T cell expressing a TCR, such as a recombinant TCR, upon specific binding to an MHC-peptide complex, can induce a signal transduction pathway in the cell, causing the cell to express or secrete an effector molecule ( e.g., a cytokine), a reporter, or other detectable readout of the interaction, or cause T cell activation or a T cell response, such as T cell proliferation, cytokine production, a T cell cytotoxic response, or other responses. In some embodiments, a TCR, such as a recombinant TCR, can specifically bind to, and immunologically recognize, a peptide epitope, such that binding to the peptide epitope elicits an immune response.

Methods for testing a TCR for the ability to recognize a peptide epitope of a target polypeptide and antigen specificity are known. In some embodiments, T cells or T cell compositions produced by the present methods are contacted with a peptide-MHC complex, either in soluble form or through co-cultivation with peptide-activated antigen-presenting cells (e.g., T2 cells or other known antigen-presenting cells that match an MHC allele of a recombinant TCR). Exemplary antigens and MHC alleles of a recombinant TCR are described in Section III. In some embodiments, the methods include assessing properties, such as functional properties, of an exogenous recombinant TCR. In some embodiments, the method includes assessing T cell activation via an exogenous recombinant TCR, such as determining the presence or absence of T cell activation by TCR-expressing cells upon interaction with a peptide-MHC complex. In some embodiments, the detection of T cell activation by such methods includes the release of cytokines ( e.g., interferon-γ, granulocyte/monocyte colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF-α), or interleukin 2 (IL-2)). Additionally, TCR function can be assessed by measuring cellular cytotoxicity, as described in Zhao et al. , J. Immunol., 174:4415-4423 (2005).

In some embodiments, assessing T cell activation includes assessing the activity or expression of nucleic acid molecules encoding a reporter, such as a T cell activation reporter, assessing cytokine release, and/or assessing the functional activity of the T cell.

In some embodiments, one or more assays include one or more measuring devices, a type of result or assay, and/or data reading. In some embodiments, one or more assays are performed using fluorescently labeled reagents, such as antibodies directly or indirectly labeled with fluorophores, and are detected using a flow cytometry or fluorescence-activated cell sorting (FACS) instrument. For example, for flow cytometry or FACS, a plurality of different fluorophores can be detected that have different excitation and emission wavelength peaks. Thus, a plurality of fluorophore labels can be used to assess a plurality of properties, such as TCR expression, peptide recognition in the context of an MHC molecule, and/or T cell activation reporter expression, in a single experimental reaction. In some embodiments, one or more assays are performed in a high-throughput, multiplexed, and/or large-scale manner.

In some embodiments, the methods further include aspects of assessing T cell activation, such as assessing cytokine release and/or assessing the functional activity of the T cell, such as cytolytic activity and/or helper T cell activity. In some embodiments, the assessments can be performed on T cells or T cell compositions produced using the embodiments described herein.

In some embodiments, functional assays are performed on primary T cells, such as those isolated directly from a subject and/or isolated from a subject and frozen, such as primary CD4+ and/or CD8+ T cells that have been engineered using the embodiments provided herein.

In some embodiments, the methods include performing functional assays or determining the function of a TCR or a T cell. For example, functional assays for determining TCR activity or T cell activity include determining cytokine secretion, cytolytic activity, and/or helper T cell activity. For example, assessing T cell activation includes assessing cytokine secretion and/or assessing the functional activity of a T cell. In some embodiments, upon binding of a TCR to an antigen or epitope, the cytoplasmic domain or intracellular binding domain of the TCR activates at least one of the normal effector functions or responses of an immune cell, such as a T cell engineered to express a TCR. For example, in some contexts, a TCR causes a T cell function, such as cytolytic activity, and/or a helper T cell activity, such as secretion of cytokines or other factors. In some embodiments, the intracellular binding domain or domains comprise cytoplasmic sequences of a T-cell receptor (TCR), and in some aspects, also co-receptors that in a natural context act in concert with such a receptor to initiate signal transduction upon antigen receptor interaction, and/or any derivative or variant of such molecules, and/or any synthetic sequence that has the same functional capacity.

In some embodiments, T cells or T cell compositions comprising exogenous recombinant TCRs are assessed for immunological readout, such as using a T cell assay. In some embodiments, TCR-expressing cells can activate a CD8+ T cell response. In some embodiments, CD8+ T cell responses can be assessed by monitoring CTL reactivity using assays that include, but are not limited to, targeted cell lysis via ⁵¹ Cr release, targeted cell lysis assays using real-time imaging reagents, targeted cell lysis assays using an apoptosis detection reagent ( e.g., Caspase 3/7 reagent), or detection of interferon gamma release, such as an ELISA, intracellular cytokine staining, or ELISPOT. In some embodiments, TCR-expressing cells can activate a CD4+ T cell response. In some aspects, CD4+ T cell responses can be assessed by assays that measure proliferation, such as [ ^3H ]-thymidine incorporation into cellular DNA, and/or cytokine production, such as ELISA, intracellular cytokine staining, or ELISPOT. In some cases, the cytokine can include, for example, interleukin-2 (IL-2), interferon-gamma (IFN-gamma), interleukin-4 (IL-4), TNF-α, interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12 (IL-12), or TGF β. In some embodiments, recognition of or binding to a peptide epitope, such as an MHC class I or class II epitope, through the TCR can induce or activate a CD8+ T cell response and/or a CD4+ T cell response.

II. CELLS FOR GENETIC ENGINEERING

In some of the embodiments provided, the cells to be engineered are immune cells, such as T cells. Genetically engineered cells or populations of cells are provided, wherein one or more of the cells comprises a knockout of one or more endogenous TCR genes and recombinant receptor-encoding nucleic acids and/or another transgene that are integrated into one or more endogenous TCR genes. Populations or compositions of such cells, compositions comprising such cells and/or enriched in cells that are engineered using the methods provided are also provided.

In some embodiments, the cells to be engineered comprise one or more subsets of T cells or other cell types, such as populations of total T cells, CD4+ cells, CD8+ cells and subpopulations thereof, such as those defined by function, activation state, maturity, differentiation potential, expansion, recycling, localization and/or survival potential, antigen specificity, antigen receptor type, presence in a particular organ or compartment, marker or cytokine selection profile and/or degree of differentiation. With reference to the subject to be treated, the cells may be allogeneic and/or autologous. The methods include available methods. In some aspects, such as available techniques, the cells are pluripotent and/or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, the methods include isolating cells from a subject, preparing, processing, culturing, and/or engineering them as described herein, and re-administering them to the same patient, before or after cryopreservation.

T cell and/or CD4+ and/or CD8+ T cell subtypes and subpopulations include naive T (T _N ) cells, effector T cells ( _TEFF ), memory T cells and their subtypes such as stem memory T cells ( _TSCM ), central memory T cells ( _TCM ), effector memory T cells ( _TEM ) or terminally differentiated effector memory T cells, tumor infiltrating lymphocytes (TILs), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells such as T _H1 cells, T _H2 cells, T _H3 cells, T _H17 cells, T _H9 cells, T _H22 cells, follicular helper T cells, alpha/beta T cells, and delta/gamma T cells. In some embodiments, the cell is a regulatory T cell (Treg). In some embodiments, the cell further comprises recombinant FOXP3 or a variant thereof.

In some embodiments, the cells include one or more nucleic acids that have been genetically engineered and therefore express recombinant or genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous, i.e., not normally present in a cell or sample obtained from a cell, such as those obtained from another organism or cell that, for example, is not normally found in the engineered cell, and/or the organism from which such a cell is obtained. In some embodiments, the nucleic acids are non-naturally occurring, such as a nucleic acid not found in nature, including one that contains chimeric combinations of nucleic acids encoding different domains from a plurality of different cell types.

In some embodiments, obtaining engineered cells includes one or more culturing and/or preparation steps. The cells for engineering may be isolated from a sample, such as a biological sample, such as one obtained from or derived from a subject. In some embodiments, the subject from which the cells are isolated is one that has a disease or condition or is in need of cell therapy, or is being administered cell therapy. The subject in some embodiments is a human in need of a particular therapeutic intervention, such as adoptive cell therapy, for which the cells are isolated, processed, and/or engineered.

Therefore, cells in some embodiments are primary cells, such as primary human cells. Samples include tissue, fluid, and other samples taken directly from a subject, as well as samples obtained by one or more processing steps, such as separation, centrifugation, genetic engineering ( e.g., transduction with a viral vector), washing, and/or incubation. A biological sample may be a sample obtained directly from a biological source or a sample that has been processed. Biological samples include, but are not limited to, body fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat, tissue and organ samples, including processed samples obtained therefrom.

In some aspects, the sample from which the cells are obtained or isolated is blood or a blood-derived sample, or is or is a leukapheresis apheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMC), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, intestinal lymphoid tissue, mucosal lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testicles, ovaries, tonsils or other organ, and/or cells derived therefrom. Samples include, in the context of cell therapy, such as adoptive cell therapy, samples from autologous and allogeneic sources.

In some embodiments, the cells are obtained from cell colonies, such as T cell colonies. The cells in some embodiments are obtained from a xenogeneic source, such as a mouse, rat, non-human primate, or pig.

In some embodiments, isolating cells includes one or more steps of preparation and/or non-affinity cell separation. In some examples, cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, such as to remove unwanted components, enrich for desired components, lyse, or remove cells sensitive to specific reagents. In some examples, cells are separated based on one or more properties, such as density, adherence properties, size, sensitivity, and/or resistance to specific components.

In some examples, cells from the bloodstream of a subject are obtained, for example, by apheresis or leukapheresis. The samples, in some aspects, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells and/or platelets, and in some aspects, contain cells other than red blood cells and platelets.

In some embodiments, blood cells collected from a subject are washed, such as to remove the plasma fraction and to place the cells in a suitable buffer or medium for further processing. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the washing solutions do not contain calcium and/or magnesium and/or many or all divalent cations. In some aspects, the washing step is performed in a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In some aspects, the washing step is performed by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended in a plurality of biocompatible buffers after washing, such as, for example, Ca ⁺⁺ /Mg ^++- free PBS. In certain embodiments, components of the blood cell sample are removed and the cells are resuspended directly in the culture medium.

In some embodiments, the methods include density-based cell separation methods, such as obtaining white blood cells from peripheral blood by lysing red blood cells and centrifuging through a Percoll or Ficoll gradient.

In some embodiments, the methods of isolation include separating different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, such as surface proteins, intracellular markers, or nucleic acids. In some embodiments, any method of separation based on such markers can be used. In some embodiments, the separation is based on affinity or immunoaffinity. For example, isolation in some aspects includes separating cells and cell populations based on the expression of cells or the expression level of one or more markers, typically cell surface markers, such as by incubation with an antibody or binding partner that specifically binds to such markers, typically followed by washing steps and separating cells bound to the antibody or binding partner from cells that do not have binding to the antibody or binding partner.

Such separation steps may be based on positive selection, in which cells that bind the reagents are retained for further use, and/or negative selection, in which cells that do not bind to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection may be particularly useful if an antibody that specifically identifies a cell type in a heterogeneous population is not available, since such separation is best performed based on markers expressed by cells other than the desired population.

Separation does not necessarily result in 100% enrichment or removal of a particular population of cells or cells expressing a particular marker. For example, positive selection or enrichment of a particular cell type, such as those expressing a marker, refers to an increase in the number or proportion of such cells, but does not necessarily result in the complete absence of cells not expressing the marker. Likewise, negative selection, removal or depletion of a particular cell type, such as those expressing a marker, refers to a decrease in the number or proportion of such cells, but does not necessarily result in the complete removal of all such cells.

In some examples, multiple rounds of separation steps are performed, wherein a positively or negatively selected fraction from one step is subjected to another separation step, such as subsequent positive or negative selection. In some examples, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating the cells with multiple antibodies or binding partners, each specific for a marker targeted for negative selection. Similarly, multiple cell types can be positively selected simultaneously by incubating cells with multiple antibodies or binding partners expressed on different cell types.

For example, in some aspects, specific subsets of T cells, such as cells that are positive or express high levels of one or more surface markers, such as CD28 ⁺ , CD62L ⁺ , CCR7 ⁺ , CD27 ⁺ , CD127 ⁺ , CD4 ⁺ , CD8 ⁺ , CD45RA ⁺ , and/or CD45RO ⁺ T cells, are isolated by positive or negative selection methods.

For example, CD3 ⁺ , CD28 ⁺ T cells can be positively selected using anti-CD3/anti-CD28 conjugated magnetic beads ( e.g., DYNABEADS® M-450 CD3/CD28 T Cell Expander).

In some embodiments, selection is carried out by enrichment of a specific population of cells by positive selection or depletion of a specific population of cells by negative selection. In some embodiments, positive or negative selection is carried out by incubating cells with one or more antibodies or other binding agent that specifically binds to one or more surface markers expressed or expressed (marker ⁺ ) at a relatively high level (marker ^high ) of positively or negatively selected cells, respectively.

In some embodiments, T cells are separated from a PBMC sample by negative selection for markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD14. In some aspects, a CD4 ⁺ or CD8 ⁺ selection step is used to separate CD4 ⁺ helper and CD8 ⁺ cytotoxic T cells. Such CD4 ⁺ and CD8 ⁺ populations can be further sorted into subpopulations by positive or negative selection for markers expressed or expressed to a relatively high degree on one or more subpopulations of naive, memory, and/or effector T cells.

In some embodiments, the CD8 ⁺ cells are further enriched or depleted in naive, central memory cells, effector memory cells, and/or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the relevant subset. In some embodiments, enrichment in central memory T cells (T _CM ) is performed to improve efficacy, such as to improve long-term survival, expansion, and/or engraftment following administration, which in some aspects is particularly robust in such subsets. See Terakura et al. (2012) Blood,1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In some embodiments, combining T _CM -enriched CD8 ⁺ T cells and CD4 ⁺ T cells further improves efficacy.

In variants, memory T cells are present in both CD62L ⁺ and ^CD62L- subsets of CD8 ⁺ peripheral blood lymphocytes. PBMCs can be enriched or depleted in ^CD62L- CD8 ⁺ and/or CD62L ⁺ CD8 ⁺ fractions, for example, using anti-CD8 and anti-CD62L antibodies.

In some embodiments, enrichment of central T memory cells (T _CM ) is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and/or CD 127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and/or granzyme B. In some aspects, isolation of a CD8 ⁺ population enriched in T _CM cells is carried out by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment of cells expressing CD62L. In one aspect, enrichment of central T memory cells (T _CM ) cells is carried out starting from a negative fraction of cells selected based on CD4 expression, which is subjected to negative selection based on CD14 and CD45RA expression, and positive selection based on CD62L. Such changes are in some aspects performed simultaneously, and in other aspects, performed sequentially, in any order. In some aspects, the same CD4 expression-based selection step used to obtain a population or subpopulation of CD8 ⁺ cells is also used to create a population or subpopulation of CD4 ⁺ cells, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the methods, optionally after one or more additional positive or negative selection steps.

In a specific example, a PBMC sample or other white blood cell sample is subjected to CD4 ⁺ cell selection, where both negative and positive fractions are retained. The negative fraction is then subjected to negative selection based on CD14 and CD45RA or ROR1 expression, and positive selection based on central memory T cell marker characteristics such as CD62L or CCR7, where positive and negative selections are performed in any order.

CD4 ⁺ T helper cells are sorted into naive, central memory, and effector cells by identifying cell populations that have cell surface antigens. CD4 ⁺ lymphocytes can be obtained by standard methods. In some embodiments, the naive CD4 ⁺ T lymphocytes are ^CD45RO- , CD45RA ⁺ , CD62L ⁺ , CD4 ⁺ T cells. In some embodiments, the central memory CD4 ⁺ cells are CD62L ⁺ and CD45RO ⁺ . In some embodiments, the effector CD4 ⁺ cells are ^CD62L- and ^CD45RO- .

In one example, to enrich for CD4 ⁺ cells for negative selection, the monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as magnetic beads or paramagnetic beads, to allow separation of cells for positive and/or negative selection. For example, in some embodiments, cells and cell populations are separated or isolated using immunomagnetic (or affinity magnetic) separation techniques (reviewed in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo , p 17-25 Edited by: S. A. Brooks and U. Schumacher © Humana Press Inc., Totowa, NJ).

In some aspects, the sample or composition of the separated cells is incubated with a small magnetizable or magnetically susceptible material, such as magnetically susceptible particles or microparticles, such as paramagnetic ( e.g., such as Dynalbeads or MACS spheres). The magnetically susceptible material, such as a particle, is generally directly or indirectly attached to a binding partner, such as an antibody, which specifically binds to a molecule, such as a surface marker, present on a cell, cells, or cell population that is desired to be separated, such as that which is desired to be negatively or positively selected.

In some embodiments, the magnetic particle or sphere comprises a magnetically susceptible material associated with a specific binding member, such as an antibody or other binding partner. There are many well-known magnetically susceptible materials useful in magnetic separation methods. Suitable magnetic particles include those described in Molday, U.S. Patent No. 4,452,773, and in European Patent Specification EP 452,342 B, which are incorporated herein by reference. Colloidal sorted particles, such as those described in Owen, U.S. Patent No. 4,795,698, and Liberti et al. , U.S. Patent No. 5,200,084, are other examples.

Incubation is typically carried out under conditions where antibodies or binding partners or molecules, such as secondary antibodies or other reagents that specifically bind to such antibodies or binding partners that are attached to a magnetic particle or sphere, specifically bind to cell surface molecules if present on cells in the sample.

In some aspects, the sample is placed in a magnetic field, and cells having magnetically susceptible or magnetizable particles attached thereto will be attracted to the magnet and separated from unlabeled cells. For positive selection, cells that are attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained. In some aspects, a combination of positive and negative selection is performed during the same selection step, wherein positive and negative fractions are retained and further processed or further separated.

In certain embodiments, the magnetically susceptible particles are coated with primary antibodies or other binding partners, secondary antibodies, lectins, enzymes, or streptavidin. In certain embodiments, the magnetic particles are attached to the cells via coating with primary antibodies specific for one or more markers. In certain embodiments, the cells, rather than the beads, are labeled with the primary antibody or binding partner, and the cell type-specific secondary antibodies or other binding partner ( e.g., streptavidin)-coated magnetic particles are then added. In certain embodiments, streptavidin-coated magnetic particles are used in combination with biotinylated primary or secondary antibodies.

In some embodiments, the magnetically susceptible particles remain attached to the cells, which are then incubated, cultured, and/or engineered; in some aspects, the particles remain attached to the cells for administration to a patient. In some embodiments, the magnetizable or magnetically susceptible particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, for example, the use of competing unlabeled antibodies, magnetizable particles, or antibodies conjugated to cleavable linkers, etc. In some embodiments, the magnetizable particles are biodegradable.

In some embodiments, affinity-based selection is performed via magnetically activated cell sorting (MACS) (Miltenyi Biotec, Auburn, CA). Magnetically activated cell sorting (MACS) systems are capable of selecting cells with high purity that have magnetizable particles attached thereto. In certain embodiments, MACS operates in a mode where non-target and target species are subsequently eluted after application of an external magnetic field. That is, cells attached to magnetizable particles are kept in place until non-attached species are eluted. Then, after this first elution step is complete, species that are captured by the magnetic field and preserved from elution are released by any means so as to elute and recover them. In certain aspects, non-target cells are labeled and depleted from a heterogeneous cell population.

In certain embodiments, the isolation or separation is performed using a system, device, or apparatus that performs one or more of the isolation, cell preparation, separation, processing, culturing, and/or method formulation steps. In some aspects, the system is used to perform each of these steps in a closed or sterile environment, such as to minimize error, manual manipulation, and/or contamination. In one example, the system is the system described in PCT International Publication No. WO2009/072003 or US 20110003380 A1.

In some embodiments, the system or apparatus performs one or more, for example, all of the extraction, processing, design, and composition steps in an integrated or closed system and/or in an automated or programmable mode. In some aspects, the system or apparatus includes a computer and/or a computer program associated with the system or apparatus that allows a user to program, control, evaluate the result, and/or adjust various aspects of the processing, extraction, design, or composition steps.

In some aspects, the separation and/or other steps are performed using a CliniMACS system (Miltenyi Biotec), for example, for automated cell separation at clinical scale in a closed and sterile system. The components may include an integrated microcomputer, a magnetic separation unit, a peristaltic pump, and various check valves. The integrated computer in some aspects controls all components of the instrument and directs the system to perform repetitive procedures in a standardized sequence. The magnetic separation unit in some aspects includes a movable permanent magnet and a holder for a separation column. The peristaltic pump controls the flow rate through the tubing set and, together with the check valves, provides a controlled flow of buffer through the system and a continuous suspension of cells.

The CliniMACS system, in some aspects, uses antibody-conjugated magnetizable particles that are supplied in a sterile, non-pyrogenic solution. In some embodiments, after cells are labeled with magnetic particles, the cells are washed to remove excess particles. The cell preparation bag is then connected to a tubing set, which is then connected to a bag containing a buffer and a cell collection bag. The tubing set consists of pre-assembled sterile tubing that includes a pre-column and a separation column and is intended for single use only. Once the separation program is initiated, the system automatically applies a cell sample to the separation column. Labeled cells remain within the column, and unlabeled cells are removed by multiple wash steps. In some embodiments, cell populations for use in the methods described herein are not labeled and do not remain in the column. In some embodiments, cell populations for use in the methods described herein are labeled and remain in the column. In some embodiments, cell populations for use in the methods described herein are eluted from the column after removal of the magnetic field and collected in a cell collection bag.

In certain embodiments, the separation and/or other steps are performed using a CliniMACS Prodigy system (Miltenyi Biotec). The CliniMACS Prodigy system, in some aspects, is equipped with a cell processing unit that allows for automated washing and fractionation of cells by centrifugation. The CliniMACS Prodigy system may also include an integrated camera and image recognition software that determines the optimal cell fractionation endpoint by recognizing macroscopic layers of a source cell product. For example, peripheral blood may be automatically separated into red blood cell, white blood cell, and plasma layers. The CliniMACS Prodigy system may also include an integrated cell culture chamber that performs cell culture protocols such as, for example, cell differentiation and expansion, antigen loading, and long-term cell culture. Inlet ports may allow for sterile removal and replenishment of media, and cells may be monitored using an integrated microscope. See, for example, Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood, 1:72-82, and Wang et al. (2012) J Immunother.35(9):689-701.

In some embodiments, the cell population described herein is collected and enriched (or depleted) by flow cytometry, in which cells stained for a plurality of cell surface markers are introduced into a fluid flow. In some embodiments, the cell population described herein is collected and enriched (or depleted) by preparative (FACS) sorting. In certain embodiments, the cell population described herein is collected and enriched (or depleted) by using microelectromechanical systems (MEMS) chips in conjunction with a FACS-based detection system (see, e.g., WO 2010/033140, Cho et al. (2010) Lab Chip 10:1567-1573; and Godin et al. (2008) J Biophoton. 1(5):355-376). In both cases, cells can be labeled with a variety of markers, allowing the isolation of well-defined T cell subsets with high purity.

In some embodiments, antibodies or binding partners are labeled with one or more detectable markers to facilitate separation for positive and/or negative selection. For example, separation can be based on binding to fluorescently labeled antibodies. In some examples, cell separation based on binding of antibodies or other binding partners specific for one or more surface markers is performed in a fluid flow, such as fluorescence-activated cell sorting (FACS), including preparative (FACS) and/or microelectromechanical system (MEMS) chips, for example, in combination with a flow cytometric detection system. Such methods allow positive and negative selection based on multiple markers simultaneously.

In some embodiments, the methods of preparation include freezing steps, such as cryopreservation of cells, before or after isolation, incubation and/or engineering. In some embodiments, the freezing and subsequent thawing step removes granulocytes and, to some extent, monocytes from the cell population. In some embodiments, the cells are suspended in a freezing solution, such as after a washing step to remove plasma and platelets. In some aspects, any of the known freezing solutions and parameters can be used. One example includes using PBS containing 20% DMSO and 8% human serum albumin (HSA) or other suitable cell freezing medium. It is then diluted 1:1 with the medium such that the final concentration of DMSO and HSA is 10% and 4%, respectively. The cells are then frozen to -80 °C at a rate of 1 ° per minute and stored in the vapor phase in a storage tank in liquid nitrogen.

In some embodiments, the provided methods include growing, incubating, culturing, and/or genetic engineering steps. For example, in some embodiments, methods of incubating and/or constructing depleted cell populations and culture-initiating compositions are provided.

Thus, in some embodiments, the cell populations are incubated in a culture initiating composition. Incubation and/or construction can be carried out in a culture vessel, such as a block, chamber, cell, column, tube, tube set, valve, flask, culture dish, bag or other container for culturing or growing cells.

In some embodiments, the cells are incubated and/or cultured prior to or in combination with genetic engineering. Incubation steps may include culturing, growing, stimulating, activating, and/or expanding. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulating agent. Such conditions include conditions designed to induce proliferation, expansion, activation, and/or survival of cells in a population, to mimic exposure to an antigen, and/or to prime cells for genetic engineering, such as the introduction of nucleic acids encoding a recombinant receptor, such as a recombinant TCR.

The conditions may include one or more of a specific environment, temperature, oxygen content, carbon dioxide content, time, agents such as nutrients, amino acids, antibiotics, ions, and/or stimulatory factors such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and other agents designed to activate cells.

In some embodiments, the stimulatory conditions or agents comprise one or more agents, such as a ligand, that are capable of activating the intracellular signaling region of the TCR complex. In some aspects, the agent initiates the TCR/CD3 intracellular signaling cascade in a T cell. Such agents include antibodies, such as antibodies specific to the TCR, such as anti-CD3. In some embodiments, the stimulatory conditions comprise one or more agents, such as a ligand, that are capable of stimulating a costimulatory receptor, such as anti-CD28. In some embodiments, such agents and/or ligands can be associated with a solid support, such as beads, and/or one or more cytokines. Optionally, the method of propagation can further comprise the step of adding anti-CD3 and/or anti-CD28 antibodies to the culture medium (e.g., at a concentration of at least about 0.5 ng/mL). In some embodiments, the stimulatory agents comprise IL-2, IL-15, and/or IL-7. In some aspects, the IL-2 concentration is at least about 10 units/mL.

In some aspects, the incubation is carried out as described in U.S. Patent No. 6,040,177 to Riddell et al. , Klebanoff et al. (2012) J Immunother. 35(9):651-660, Terakura et al. (2012) Blood,1:72-82, and/or Wang et al. (2012) J Immunother. 35(9):689-701.

In some embodiments, the T cells are expanded by adding a culture-initiating composition of support cells, such as non-dividing peripheral blood mononuclear cells (PBMCs), ( e.g., such that the resulting cell population contains at least about 5, 10, 20, or 40 or more PBMC support cells for each T lymphocyte in the initial population being expanded); and incubating the culture ( e.g., for a time sufficient to expand the number of T cells). In some aspects, the non-dividing support cells can comprise gamma-irradiated PBMC support cells. In some embodiments, the PBMCs are irradiated with gamma rays in the range of about 3000 to 3600 rads to prevent cell division. In some aspects, the support cells are added to the culture prior to adding the T cell populations.

In some embodiments, the stimulating conditions include a temperature suitable for the growth of human T lymphocytes, such as at least about 25 degrees Celsius, generally at least about 30 degrees, and generally from about 37 degrees Celsius. Optionally, the incubation can further include adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as support cells. The LCL can be irradiated with gamma rays in the range of about 6,000 to 10,000 rads. The LCL support cells in some aspects are present in any suitable amount, such as a ratio of LCL support cells to parental T lymphocytes of at least about 10:1.

In some embodiments, antigen-specific T cells, such as antigen-specific CD4+ and/or CD8+ T cells, are produced by stimulating naive or antigen-specific T lymphocytes with an antigen. For example, colonies or clones of antigen-specific T cells can be generated for cytomegalovirus antigens by isolating T cells from infected subjects and stimulating the cells in vitro with the same antigen.

Various methods for introducing genetically engineered components, such as agents for causing genetic disruption and/or nucleic acids encoding recombinant receptors, such as CARs or TCRs, are known and can be used with the present methods and compositions. Exemplary methods include methods for transferring nucleic acids encoding polypeptides or receptors, including transfer via viral vectors, such as retroviral or lentiviral vectors, non-viral vectors, or transposons, such as the Sleeping Beauty transposon system. Methods of gene transfer can include transduction, electroporation, or another method that results in transfer of a gene into a cell, or any of the delivery methods described in Section IA herein. Other approaches and vectors for transferring nucleic acids encoding recombinant products are described, for example, in WO2014055668 and U.S. Patent No. 7,446,190.

In some embodiments, recombinant nucleic acids are transferred into T cells via electroporation (see, e.g., Chicaybam et al, (2013) PLoS ONE 8(3):e60298 and Van Tedeloo et al. (2000) Gene Therapy 7(16): 1431–1437). In some embodiments, recombinant nucleic acids are transferred into T cells via transposition (see, e.g., Manuri et al. (2010) Hum Gene Ther 21(4): 427–437; Sharma et al. (2013) Molec Ther Nucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506: 115–126). Other methods for introducing and expressing genetic material into immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York. NY), protoplast fusion, liposome-mediated cationic transfection; tungsten-enhanced microparticle ballistic transfection (Johnston, Nature, 346: 776–777 (1990)); and strontium phosphate–DNA coprecipitation (Brash et al., Mol. Cell Biol., 7: 2031–2034 (1987)).

In some embodiments, gene transfer is accomplished by first stimulating the cell, such as by combining it with a stimulus that elicits a response such as proliferation, survival, and/or activation, such as measured by expression of a cytokine or activation marker, following transduction of activated cells, and expanding in culture to quantities sufficient for clinical use.

In some contexts, it may be desirable to guard against the possibility that overexpression of a stimulatory factor (e.g., a lymphokine or cytokine) could potentially produce an undesirable result or reduce efficacy in a subject, such as a factor associated with toxicity in the subject. Thus, in some contexts, the engineered cells include gene segments that render the cells susceptible to negative selection in vivo , such as when administered in adoptive immunotherapy. For example, in some aspects, the cells are engineered such that they can be excluded as a result of an in vivo change in the condition of the patient to whom they are administered. A negatively selected phenotype can be obtained by inserting a gene that confers sensitivity to an administered agent, such as a compound. Negatively selected genes include the herpes simplex virus type I thymidine kinase (HSV-I TK) gene (Wigler et al., Cell 11:223, 1977), which confers sensitivity to ganciclovir; cellular hypoxanthine phosphoribosyltransferase (HPRT) gene, cellular adenine phosphoribosyltransferase (APRT) gene, bacterial cytosine deaminase (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).

In some embodiments, cells, such as T cells, can be engineered either during or after expansion. Such engineering to introduce a gene for a desired polypeptide or receptor can be accomplished with any suitable retroviral vector, for example. The genetically modified cell population can then be released from the original stimulus (a CD3/CD28 stimulus, for example) and then stimulated with a second type of stimulus (e.g., via a de novo introduced receptor). This second type of stimulus can include an antigenic stimulus in the form of a peptide/MHC molecule, a cognate ligand of the genetically introduced receptor (e.g., a natural CAR ligand), or any ligand (such as an antibody) that binds directly to the frame of the new receptor (e.g., via recognition of constant regions by the receptor). See, for example, Cheadle et al, “Chimeric antigen receptors for T-cell based therapy” Methods Mol Biol. 2012; 907:645-66 or Barrett et al., Chimeric Antigen Receptor Therapy for Cancer Annual Review of Medicine Vol. 65: 333-347 (2014).

Additional nucleic acids, such as genes for introduction, include those that improve the efficacy of therapy by promoting the viability and/or function of the transferred cells; genes that provide a gene marker for cell selection and/or evaluation, such as for the evaluation ofin vivosurvival or localization; genes to improve safety, such as making cells susceptible to negative selectionin vivoas described by Lupton S. D. et al.,Mol. and Cell Biol.,11:6 (1991); and Riddell et al., Human Gene Therapy3:319–338 (1992); see also publications PCT/US91/08442 and PCT/US94/05601 by Lupton et al., describing the use of bifunctional selectable fusion genes obtained by fusing a dominant positive selectable marker with a negative selectable marker. See,For example, Riddell et al., U.S. Patent No. 6,040,177, at columns 14-17.

As described herein, in some embodiments, the cells are incubated and/or cultured prior to or in combination with genetic engineering. Incubation steps may include culturing, growing, stimulating, activating, expanding, and/or freezing for preservation, such as cryopreservation.

III. NUCLEIC ACIDS, VECTORS AND DELIVERY

In some embodiments, one or more agents for genetic disruption and/or template polynucleotides, for example, template polynucleotides containing a transgene encoding a recombinant receptor or an antigen-binding fragment or chain thereof, or one or more second template polynucleotides, are introduced into cells in the form of nucleic acid, for example, in the form of polynucleotides and/or vectors. As described in Section I herein, the components for engineering can be delivered in various forms using various delivery methods, including polynucleotide encoding components. Also provided are one or more polynucleotides (e.g., nucleic acid molecules) encoding one or more components of one or more agents capable of causing genetic disruption and/or one or more template polynucleotides containing a transgene, and vectors for genetically engineered cells for targeted integration of the transgene.

In some embodiments, template polynucleotides are provided, such as template polynucleotides for targeting a transgene to a specific genomic target location, such as a TRAC, TRBC1 , and/or TRBC2 locus. In some embodiments, any of the template polynucleotides described in Section IB herein are provided. In some embodiments, a template polynucleotide comprises a transgene that includes nucleic acid sequences that encode a recombinant receptor or other polypeptides and/or factors, and homology arms for targeting integration. In some embodiments, the template polynucleotide may be contained in a vector.

In some embodiments, agents capable of causing genetic destruction can be encoded in one or more polynucleotides. In some embodiments, a component of the agents, such as Cas9 molecules and/or gRNA molecules, can be encoded in one or more polynucleotides and introduced into cells. In some embodiments, a polynucleotide encoding one or more components of the agents can be included in a vector.

In some embodiments, the vector may contain a sequence that encodes a Cas9 molecule and/or a gRNA molecule and/or template polynucleotides. The vector may also contain a sequence encoding a signal peptide ( e.g., for nuclear localization, nucleolar localization, mitochondrial localization), fused, for example, to a Cas9 molecule sequence. For example, the vector may contain a nuclear localization sequence (e.g., from SV40), fused to a sequence encoding a Cas9 molecule.

One or more regulatory/control elements, such as a promoter, enhancer, intron, polyadenylation signal, Kozak consensus sequence, internal ribosome entry site (IRES), 2A sequence, and splice acceptor or dongs, may be included in the vectors. In some embodiments, the promoter is selected from an RNA pol I, pol II, or pol III promoter. In some embodiments, the promoter is recognized by RNA polymerase II (e.g., CMV, SV40 early region, or adenovirus major late region promoter). In another embodiment, the promoter is recognized by RNA polymerase III (e.g., U6 or H1 promoter).

In another embodiment, the promoter is a regulated promoter (e.g., an inducible promoter). In some embodiments, the promoter is an inducible promoter or a repressible promoter. In some embodiments, the promoter comprises a Lac operator sequence, a tetracycline operator sequence, a galactose operator sequence, or a doxycycline operator sequence, or is an analog thereof, or is capable of binding to or being recognized by a Lac repressor or a tetracycline repressor, or an analog thereof.

In some embodiments, the promoter is or comprises a constitutive promoter. Exemplary constitutive promoters include, for example, the simian early virus 40 (SV40) promoter, the cytomegalovirus (CMV) middle early promoter, the human Ubiquitin C (UBC) promoter, the human elongation factor 1α (EF1α) promoter, the mouse phosphoglycerate kinase 1 (PGK) promoter, and the chicken β-Actin coupled to the CMV early enhancer (CAGG) promoter. In some embodiments, the constitutive promoter is a synthetic or modified promoter. In some embodiments, the promoter is or comprises the MND promoter, a synthetic promoter that contains the U3 region of a modified MoMuLV LTR with the myeloproliferative sarcoma virus enhancer (the sequence set forth in SEQ ID NO:18 or 126; see Challita et al. (1995) J. Virol. 69(2):748-755). In some embodiments, the promoter is a tissue-specific promoter. In another embodiment, the promoter is a viral promoter. In another embodiment, the promoter is a non-viral promoter. In some embodiments, exemplary promoters may include, but are not limited to, the human elongation factor 1 alpha (EF1α) promoter (the sequence shown in SEQ ID NO:4 or 5) or a modified form thereof (the EF1α promoter with the HTLV1 enhancer; the sequence shown in SEQ ID NO:127) or the MND promoter (the sequence shown in SEQ ID NO:18 or 126). In some embodiments, the polynucleotide and/or vector does not include a regulatory element, such as a promoter.

In some embodiments, the vector or delivery vehicle is a viral vector (e.g., for creating recombinant viruses). In some embodiments, the virus is a DNA virus (e.g., a dsDNA or ssDNA virus). In some embodiments, the virus is an RNA virus (e.g., a ssRNA virus). Exemplary viral vectors/viruses include, for example, retroviruses, lentiviruses, adenoviruses, adeno-associated virus (AAV), vaccinia viruses, poxviruses, and herpes simplex viruses, or any of the viruses described elsewhere herein.

In some embodiments, the virus infects dividing cells. In another embodiment, the virus infects non-dividing cells. In another embodiment, the virus infects both dividing and non-dividing cells. In another embodiment, the virus can integrate into the host genome. In another embodiment, the virus is engineered to have reduced immunity, such as in humans. In another embodiment, the virus is capable of replication. In another embodiment, the virus is replicatingly impaired, such as having one or more coding regions for genes required for additional rounds of virion replication and/or packaging replaced by other genes or deleted. In another embodiment, the virus causes transient expression of a Cas9 molecule and/or a gRNA molecule for the purpose of transiently causing genetic disruption. In another embodiment, the virus causes long-term, such as at least 1 week, 2 weeks, 1 month, 2 months, 3 months, 6 months, 9 months, 1 year, 2 years, or persistent expression of a Cas9 molecule and/or a gRNA molecule. The packaging capacity of the viruses can vary, for example, from at least about 4 tons to at least about 30 tons, such as at least about 5 tons, 10 tons, 15 tons, 20 tons, 25 tons, 30 tons, 35 tons, 40 tons, 45 tons, or 50 tons.

In some embodiments, the polynucleotide containing the agents and/or the template polynucleotide is delivered by a recombinant retrovirus. In another embodiment, the retrovirus (e.g., Moloney murine leukemia virus) contains a reverse transcriptase, e.g., that allows integration into the host genome. In some embodiments, the retrovirus is replicating. In another embodiment, the retrovirus is incapable of replication, e.g., has one or more coding regions for genes required for additional rounds of virion replication and packaging replaced by other genes or deleted.

In some embodiments, the polynucleotide containing the agents and/or the template polynucleotide is delivered by a recombinant lentivirus. For example, the lentivirus is incapable of replication, for example, lacks one or more genes required for viral replication. In some embodiments, the lentivirus is a lentivirus derived from HIV.

In some embodiments, the polynucleotide containing the agents and/or the template polynucleotide is delivered by a recombinant adenovirus. In another embodiment, the adenovirus is engineered to have reduced immunity in humans.

In some embodiments, the polynucleotide containing the agents and/or the template polynucleotide is delivered by a recombinant AAV. In some embodiments, the AAV can introduce its genome into the genome of a host cell, such as a target cell, as described herein. In another embodiment, the AAV is a self-complementary adeno-associated virus (scAAV), such as an scAAV that packages both strands that anneal together to form double-stranded DNA. AAV serotypes that can be used in the disclosed methods include AAV1, AAV2, modified AAV2 (e.g., modifications at Y444F, Y500F, Y730F, and/or S662V), AAV3, modified AAV3 (e.g., modifications at Y705F, Y731F, and/or T492V), AAV4, AAV5, AAV6, modified AAV6 (e.g., modifications at S663V and/or T492V), AAV7, AAV8, AAV 8.2, AAV9, AAV.rh10, modified AAV.rh10, AAV.rh32/33, modified AAV.rh32/33, AAV.rh43, modified AAV.rh43, AAV.rh64R1, modified AAV.rh64R1, and pseudotyped AAV such as AAV2/8, AAV2/5 and AAV2/6, which may also be used in the disclosed methods.

In some embodiments, the polynucleotide containing the agents and/or the template polynucleotide is delivered by a hybrid virus, such as a hybrid of one or more viruses described herein.

A packaging cell is used to produce virus particles that can infect a target cell. Such cells include the 293 cell, which can package adenovirus, and the ψ2 cell or PA317 cell, which can package retrovirus. A viral vector used in gene therapy is typically created by a producer cell line that packages the nucleic acid vector into a viral particle. The vector typically contains the minimal viral sequences required for packaging and subsequent integration into the host or target cell (if used), with other viral sequences replaced by an expression cassette encoding the protein to be expressed, such as Cas9. For example, an AAV vector used in gene therapy typically possesses only the inverted terminal repeat (ITR) sequence from the AAV genome, which is required for packaging and gene expression in the host or target cell. The missing viral functions are transferred in trans by the packaging cell line. At this point, the viral DNA is packaged into a colony of cells that contains a helper plasmid encoding other AAV genes, namely rep and cap, but lacks the ITR sequence. The colony of cells is also infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of the AAV genes from the helper plasmid. The helper plasmid is not packaged in significant quantities because it lacks the ITR sequences. Adenovirus contamination can be reduced, for example, by heat treatment, to which adenovirus is more sensitive than AAV.

In some embodiments, the viral vector has cell type recognition capability. For example, the viral vector can be pseudotyped with different/alternative viral envelope glycoproteins; engineered with a cell type-specific receptor (e.g., genetic modification of viral envelope glycoproteins to introduce targeting ligands such as a peptide ligand, single-stranded antibody, growth factor); and/or engineered to have a dual-specificity molecular bridge with one end recognizing a viral glycoprotein and the other end recognizing a target cell surface moiety (e.g., ligand-receptor, monoclonal antibody, avidin-biotin, and chemical conjugation).

In some embodiments, the viral vector achieves cell type-specific expression. For example, a tissue-specific promoter can be engineered to restrict expression of the transgene (Cas9 and gRNA) to only a specific target cell. Vector specificity can also be mediated by microRNA-dependent control of transgene expression. In some embodiments, the viral vector enhances the efficiency of fusion of the viral vector and the target cell membrane. For example, a fusion protein such as fusogenic hemagglutinin (HA) can be introduced to enhance viral uptake into cells. In some embodiments, the viral vector has nuclear localization capability. For example, a virus that requires cleavage of the nuclear membrane (during cell division) and therefore will not infect a non-dividing cell can be altered to introduce a nuclear localization peptide into the viral matrix protein, thereby allowing transduction of non-proliferating cells.

IV. RECOMBINANT RECEPTORS

In some embodiments, the transgene for targeted integration encodes a recombinant receptor or an antigen-binding fragment or chain thereof. In some embodiments, the recombinant receptor is a recombinant antigen receptor or a recombinant receptor that binds an antigen. In some embodiments, the recombinant receptor is a recombinant or engineered T cell receptor (TCR) that is different from the endogenous TCR encoded by a T cell. In some embodiments, the recombinant receptor is a chimeric antigen receptor (CAR) or a TCR-like CAR. In some embodiments, the transgene may encode a domain, region, or chain of the recombinant receptor, and one or more second transgenes may encode other domains, regions, or chains of the recombinant receptor. In some embodiments, the present polynucleotides, vectors, compositions, methods, finished articles and/or kits are used to engineer cells that express a recombinant TCR or an antigen-binding fragment thereof.

In some embodiments, the present recombinant receptors, such as TCRs or CARs, are capable of binding to or recognizing, such as specifically binding to or recognizing, an antigen that is associated with, is specific for and/or expressed on a cell or tissue of a disease, disorder or condition, such as a cancer or tumor. In some aspects, the antigen is in the form of a peptide, such as a peptide antigen or a peptide epitope. In some embodiments, the present TCRs bind to, such as specifically bind to, an antigen that is a peptide, in the context of a major histocompatibility (MHC) molecule.

The observation that a recombinant receptor binds to an antigen, such as a peptide antigen, or specifically binds to an antigen, such as a peptide antigen, does not necessarily mean that it binds to an antigen of any species. For example, in some embodiments, the characteristics of binding to an antigen, such as a peptide antigen in the context of MHC, such as the ability to specifically bind to it and / or compete for binding to it with a reference binding molecule or receptor, and / or bind with a specific affinity or compete to a certain extent, in some embodiments, relate to the ability with respect to a human antigen, and the recombinant receptor may not have this characteristic with respect to an antigen from another species, such as a mouse. In some aspects, the extent of binding of the recombinant receptor or antigen-binding fragment thereof to an unrelated antigen or protein, such as an unrelated peptide antigen, is less than about 10% of the binding of the recombinant receptor or antigen-binding fragment thereof to an antigen, such as a cognate antigen, as measured, for example, by radioimmunoassay (RIA), peptide titration assay, or receptor assay.

A. T cell receptors (TCR)

In some embodiments, the recombinant receptor introduced into the cell is a T cell receptor (TCR) or an antigen-binding fragment thereof.

In some embodiments, a “T cell receptor” or “TCR” is a molecule that contains α and β chains (also known as TCRα and TCRβ, respectively) or γ and δ chains (also known as TCRγ and TCRδ, respectively), or antigen-binding portions thereof, and that is capable of specifically binding to an antigen, such as a peptide antigen or a peptide epitope associated with an MHC molecule. In some embodiments, the TCR has an αβ form. Typically, TCRs that exist in αβ and γδ forms are generally structurally similar, but the T cells expressing them may have different anatomical locations or functions. A TCR may be found on the cell surface or in soluble form. In general, a TCR is found on the surface of a T cell (or T lymphocyte), where it is typically responsible for recognizing antigens associated with major histocompatibility complex (MHC) molecules.

Typically, specific binding of a recombinant receptor, such as a TCR, to a peptide epitope, such as in a complex with MHC, is driven by the presence of an antigen-binding site containing one or more complementarity determining regions (CDRs). In general, it is understood that specific binding does not mean that a particular peptide epitope, such as in a complex with MHC, is the only one to which the MHC peptide molecule can bind, since non-specific binding to other molecules can also occur. In some embodiments, the binding of the recombinant receptor to the peptide in the context of the MHC molecule occurs with a higher affinity than binding to such other molecules, such as another peptide in the context of the MHC molecule or a non-target (control) peptide in the context of the MHC molecule, such as at least about 2-fold, at least about 10-fold, at least about 20-fold, at least about 50-fold, or at least about 100-fold compared to the binding affinity to such other molecules.

In some embodiments, a recombinant receptor, such as a TCR, can be assessed for safety or off-target binding activity using any of a variety of known screening assays. In some embodiments, the generation of an immune response to a particular recombinant receptor, such as a TCR, can be measured in the presence of a cell known to express a target peptide epitope, such as a cell derived from normal tissues, allogeneic cell colonies that express one or more different MHC types, or other tissue or cell sources. In some embodiments, the cells or tissues include normal cells or tissues. In some embodiments, binding to cells can be tested in 2-dimensional cultures. In some embodiments, binding to cells can be tested in 3-dimensional cultures. In some embodiments, as a control, the tissues or cells can be those known to express a target epitope. The immune response can be assessed directly or indirectly, for example, through assessment of activation of immune cells such as T cells (e.g., cytotoxic activity), cytokine production (e.g., interferon gamma), or activation of a signaling cascade such as a reporter gene assay.

Unless otherwise specified, the term "TCR" shall be understood to encompass complete TCRs as well as antigen-binding portions or antigen-binding fragments thereof. In some embodiments, a TCR is a native or full-length TCR, such as a TCR comprising an alpha (TCRα) chain and a beta (TCRβ) chain. In some embodiments, a TCR is an antigen-binding portion that is smaller than a full-length TCR but that binds to a specific peptide bond in an MHC molecule, such as that which binds to an MHC-peptide complex. In some cases, an antigen-binding portion or fragment of a TCR may comprise only a portion of the structural domains of a full-length or native TCR, but is also capable of binding a peptide epitope, such as an MHC-peptide complex to which a full-length TCR binds. In some cases, the antigen-binding portion contains variable domains of the TCR, such as the variable α (V _α ) chain and the variable β (V _β ) chain of the TCR or antigen-binding fragments thereof, sufficient to form a binding site for a specific MHC-peptide complex.

In some embodiments, the variable domains of TCRs comprise complementarity determining regions (CDRs), which are typically the primary contributors to antigen recognition and peptide, MHC, and/or MHC-peptide complex binding and specificity. In some embodiments, a TCR CDR or combination thereof forms all or substantially all of the antigen-binding site of a given TCR molecule. The different CDRs within the variable region of a TCR chain are typically separated by framework regions (FRs), which typically exhibit less variability among TCR molecules than CDRs (see, e.g., Jores et al ., Proc. Nat'l Acad. Sci. USA 87 :9138, 1990; Chothia et al ., EMBO J. 7 :3745, 1988; see also Lefranc et al ., Dev. Comp. Immunol. 27:55 , 2003). In some embodiments, CDR3 is the major CDR responsible for antigen binding or specificity, or is the most important among the CDRs in a given variable region of the TCR for antigen recognition and/or for interaction with the processed peptide portion of the peptide-MHC complex. In some contexts, CDR1 of the α chain may interact with the N-terminal portion of certain antigenic peptides. In some contexts, CDR1 of the β chain may interact with the C-terminal portion of the peptide. In some contexts, CDR2 makes the largest contribution to or is the primary CDR responsible for interaction with or recognition of the MHC portion of the MHC-peptide complex. In some embodiments, the β chain variable region may contain an additional hypervariable region (CDR4 or HVR4), which is typically involved in superantigen binding but not antigen recognition (Kotb (1995) Clinical Microbiology Reviews, 8:411-426).

In some embodiments, the α-chain and/or β-chain of the TCR may also comprise a constant domain, a transmembrane domain, and/or a short cytoplasmic tail (see, e.g., Janeway et al ., Immunobiology: The Immune System in Health and Disease , 3 ^rd Ed., Current Biology Publications, p. 4:33, 1997). In some aspects, each chain (e.g., alpha or beta) of the TCR may have one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminal end. In some embodiments, the TCR, e.g., via the cytoplasmic tail, associates with invariant proteins of the CD3 complex involved in mediating signal transduction. In some cases, the structure allows the TCR to associate with other molecules, such as CD3 and its subunits. For example, TCRs containing constant domains with a transmembrane region can anchor the protein in the cell membrane and associate with invariant subunits of the CD3 signaling apparatus or complex. The intracellular tails of CD3 signaling subunits (e.g., CD3γ, CD3δ, CD3ε, and CD3ζ chains) contain one or more immunoreceptor tyrosine-based activation motifs or ITAMs and are generally involved in the signaling capacity of the TCR complex.

In some embodiments, different domains or regions of the TCR may be defined. In some cases, the precise locus of a domain or region may vary depending on the particular structural or homology modeling or other characteristics used to describe the particular domain. It is understood that reference to amino acids, including specific sequences, set forth as SEQ ID NOs used to describe the organization of a domain of a recombinant receptor, e.g., a TCR, are for illustrative purposes and are not intended to limit the scope of the embodiments shown. In some cases, a specific domain (e.g., variable or constant) may be several amino acids (e.g., one, two, three, or four) longer or shorter. In some aspects, TCR residues are known or can be identified according to the International Immunogenetics Information System (IMGT) numbering system (see, e.g., www.imgt.org; see also Lefranc et al. (2003) Developmental and Comparative Immunology, 2&;55-77; and The T Cell Factsbook 2nd Edition, Lefranc and LeFranc Academic Press 2001). Using this system, CDR1 sequences within the TCR Vα chains and/or Vβ chains correspond to amino acids present between residues Nos. 27-38, inclusive, CDR2 sequences within the TCR Vα chains and/or Vβ chains correspond to amino acids present between residues Nos. 56-65, inclusive, and CDR3 sequences within the TCR Vα chains and/or Vβ chains correspond to amino acids present between residues Nos. 105-117, inclusive.

In some embodiments, each of the α chain and β chain of the TCR comprises a constant domain. In some embodiments, the α chain constant domain (Cα) and the β chain constant domain (Cβ) are each a mammalian constant domain, such as a human or a mouse. In some embodiments, the constant domain is proximal to the cell membrane. For example, in some cases, the extracellular portion of the TCR formed by the two chains comprises two membrane-proximal constant domains and two membrane-distal variable domains, where each variable domain comprises a CDR.

In some embodiments, each of the Cα and Cβ domains is human. In some embodiments, Cα is encoded by the TRAC gene (IMGT nomenclature) or is a variant thereof. In some embodiments, Cα has or comprises an amino acid sequence as set forth in SEQ ID NO: 19, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 19. In some embodiments, Cα has or comprises an amino acid sequence as set forth in any of SEQ ID NO: 19. In some embodiments, Cα has or comprises an amino acid sequence, such as a mature polypeptide encoded by the nucleic acid sequence set forth in SEQ ID NO:1, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to an amino acid sequence, such as a mature polypeptide encoded by the nucleic acid sequence set forth in SEQ ID NO:1. In some embodiments, Cβ is encoded by the TRBC1 or TRBC2 genes (IMGT nomenclature) or is a variant thereof. In some embodiments, Cβ has or comprises an amino acid sequence as set forth in SEQ ID NO:20 or 21, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the sequence of SEQ ID NO:20 or 21. In some embodiments, Cβ has or comprises an amino acid sequence as set forth in SEQ ID NO:20 or 21. In some embodiments, Cβ has or comprises an amino acid sequence, e.g., a mature polypeptide encoded by the nucleic acid sequence set forth in SEQ ID NO:2 or 3, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to an amino acid sequence, such as a mature polypeptide encoded by the nucleic acid sequence set forth in SEQ ID NO:2 or 3.

In some embodiments, any of the present TCRs or antigen-binding fragments thereof may be a human/mouse chimeric TCR. In some cases, the TCR or antigen-binding fragment thereof has an α chain and/or β chain comprising a murine constant region. In some aspects, the Cα and/or Cβ regions are murine constant regions. In some embodiments, Cα is a mouse constant region that is or comprises the amino acid sequence set forth in SEQ ID NO: 14, 15, 121, or 122, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 14, 15, 121, or 122. In some embodiments, Cα is or comprises the amino acid sequence set forth in SEQ ID NO: 14, 15, 121, or 122. In some embodiments, Cβ is a mouse constant region that is or comprises the amino acid sequence set forth in SEQ ID NO: 16, 17, or 123, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 16, 17, or 123. In some embodiments, Cβ is or comprises the amino acid sequence set forth in SEQ ID NO: 16, 17, or 123.

In some of any such embodiments, the TCR or antigen-binding fragment thereof comprises one or more modifications in the α chain and/or β chain, such that, when the TCR or antigen-binding fragment thereof is expressed in a cell, the frequency of mismatch between the TCR α chain and β chain and the endogenous TCR α chain and β chain is reduced, the expression of the TCR α chain and β chain is increased, and/or the stability of the TCR α chain and β chain is increased. In some embodiments, the one or more modifications comprise a substitution, deletion, or insertion of one or more amino acids in the Cα region and/or the Cβ region. In some aspects, the one or more modifications comprise substitutions to introduce one or more cysteine residues that are capable of forming one or more non-native disulfide bridges between the α chain and the β chain.

In some of any such embodiments, the TCR or antigen-binding fragment thereof comprises a Cα region comprising a cysteine at a position corresponding to position 48 as numbered in SEQ ID NO: 24 and/or a Cβ region comprising a cysteine at a position corresponding to position 57 as numbered in SEQ ID NO: 20. In some embodiments, said Cα region comprises an amino acid sequence as set forth in any of SEQ ID NOS: 19 or 24, or an amino acid sequence that has at least 90% sequence identity thereto, comprising one or more cysteine residues capable of forming a non-native disulfide bond with the β chain; and/or said Cβ region comprises an amino acid sequence as set forth in any of SEQ ID NOS: 20, 21 or 25, or an amino acid sequence that has at least 90% sequence identity thereto, comprising one or more cysteine residues capable of forming a non-native disulfide bond with the α chain.

In some of any such variants, the TCR or its antigen-binding fragment is encoded by a nucleotide sequence that is codon-optimized.

In some of any such embodiments, the binding molecule or TCR or antigen-binding fragment thereof is isolated or purified or is recombinant. In some of any such embodiments, the binding molecule or TCR or antigen-binding fragment thereof is human.

In some embodiments, the TCR may be a heterodimer of two chains, α and β, that are linked, for example, by a disulfide bond or disulfide bonds. In some embodiments, the constant domain of the TCR may contain short linking sequences in which cysteine residues form a disulfide bond, thereby linking the two TCR chains. In some embodiments, the TCR may have an additional cysteine residue on each of the α and β chains, such that the TCR contains two disulfide bonds in the constant domains. In some embodiments, each of the constant and variable domains contains disulfide bonds formed by cysteine residues.

In some embodiments, the TCR may comprise an engineered disulfide bond or bonds. In some embodiments, native disulfide bonds are not present. In some embodiments, one or more native cysteines (e.g., in the constant domain of the α chain and β chain) that form native interchain disulfide bonds are replaced by another residue, such as serine or alanine. In some embodiments, the engineered disulfide bonds may be formed by mutation of non-cysteine residues on the alpha and β chains, such as in the constant domain of the α chain and β chain, to cysteine. In some embodiments, the presence of non-native cysteine residues (e.g., yielding one or more non-native disulfide bonds) in a recombinant TCR may favor the formation of the desired recombinant TCR in the cell into which it is introduced over the expression of mismatched TCR pairs containing the native TCR chain.

Exemplary non-native TCR disulfide bonds are described in Published International PCT Application No. WO2006/000830, WO2006037960, and Kuball et al. (2007) Blood, 109:2331-2338. In some embodiments, cysteines may be introduced at Thr48 of the Cα chain and Ser57 of the Cβ chain, at Thr45 of the Cα chain and Ser77 of the Cβ chain, at Tyr10 of the Cα chain and Ser17 of the Cβ chain, at Thr45 of the Cα chain and Asp59 of the Cβ chain, and/or at Ser15 of the Cα chain and Glu15 of the Cβ chain with reference to the numbering of the Cα set forth in SEQ ID NO:24 or the Cβ set forth in SEQ ID NO:20. In some embodiments, any of the disclosed cysteine mutations may be made at a corresponding position in another sequence, such as the mouse Cα and Cβ sequences disclosed herein. The term “corresponding” when referring to protein positions, such as when describing amino acid positions “corresponding” to amino acid positions in a disclosed sequence such as that set forth in the Sequence Listing, refers to amino acid positions identified by alignment with the disclosed sequence based on a structural sequence alignment or by applying a standard alignment algorithm such as the GAP algorithm. For example, corresponding residues may be determined by aligning a reference sequence with the Cα sequence disclosed in any of SEQ ID NO: 24, or the Cβ sequence disclosed in SEQ ID NO: 20, using the structural alignment methods disclosed herein. By aligning the sequences, corresponding residues may be identified, for example, using conserved and identical amino acid residues as guides.

Typical sequences (e.g., CDR, V _α and/or V _β , and constant region sequences) of the TCRs present are described here.

In some embodiments, the recombinant TCR or antigen-binding portion thereof (or another MHC-peptide binding molecule, such as a TCR-like antibody) is known to, or is likely to, or is likely to recognize a peptide epitope or a T cell epitope of a target polypeptide when presented by cells in the context of an MHC molecule, i.e., an MHC-peptide complex of the target polypeptide. In some embodiments, the recombinant TCR (or another MHC-peptide binding molecule, such as a TCR-like antibody) is known to, or is likely to exhibit specific binding for a T cell epitope of a target polypeptide, such as when displayed as an MHC-peptide complex. Methods for assessing binding or interaction of an MHC-peptide binding molecule ( e.g., a TCR or TCR-like antibody) are known, including any of the exemplary methods described herein.

In some embodiments, the MHC molecule is an MHC class I or an MHC class II molecule. In some embodiments, the MHC comprises a polymorphic peptide binding site or binding cavity that can, in some cases, form a complex with peptide epitopes of polypeptides, including peptide epitopes processed by the cellular machinery. In some cases, MHC molecules can be displayed or expressed on the cell surface, including a complex with a peptide, i.e., an MHC-peptide complex, to present an antigen in a conformation recognized by the TCR on T cells or other MHC-peptide binding molecules. In general, class I MHC molecules are heterodimers having a transmembrane α chain, in some cases with three α domains, and a non-covalently associated β2 microglobulin. In general, class II MHC molecules are composed of two transmembrane glycoproteins, α and β, both of which typically span a membrane. An MHC molecule may include an effective portion of an MHC that contains an antigen-binding site or sites for peptide binding and sequences necessary for recognition by a suitable binding molecule, such as a TCR. In some embodiments, class I MHC molecules deliver peptides originating in the cytosol to the cell surface, where the peptide:MHC complex is recognized by T cells, such as typically CD8 ⁺ T cells, but in some cases, CD4+ T cells. In some embodiments, class II MHC molecules deliver peptides originating in the vesicular system to the cell surface, where they are typically recognized by CD4 ⁺ T cells. Typically, MHC molecules are encoded by a group of linked loci that are collectively referred to as H-2 in mouse and human leukocyte antigen (HLA) in humans. In some aspects, human MHC may also be referred to as human leukocyte antigen (HLA).

In some embodiments, the peptide epitope or T cell epitope is a peptide that can be derived from, or based on a fragment of, a larger biological molecule, such as a polypeptide or protein, and that is capable of associating with or forming a complex with an MHC molecule. In some embodiments, the peptide is from about 8 to about 24 amino acids in length. In some embodiments, the peptide is from or about 9-22 amino acids in length for recognition in an MHC class II complex. In some embodiments, the peptide is from or about 8-13 amino acids in length for recognition in an MHC class I complex. In some embodiments, the MHC molecule and the peptide epitope or T cell epitope form a complex or associate through non-covalent interactions of the peptide in the binding cavity or cleft of the MHC molecule.

In some embodiments, an MHC-peptide complex is present or displayed on the surface of a cell. In some embodiments, the MHC-peptide complex can be specifically recognized by a TCR or an antigen-binding portion thereof or another MHC-peptide binding molecule. In some embodiments, a T cell epitope or peptide epitope is capable of eliciting an immune response in an animal through its MHC molecule binding characteristics. In some embodiments, upon recognition of a T cell epitope, such as an MHC-peptide complex, the TCR (or another MHC-peptide binding molecule) produces or triggers an activation signal in a T cell that elicits a T cell response, such as T cell proliferation, cytokine production, a T cell cytotoxic response, or another response.

In some embodiments, the TCR or other MHC peptide binding molecule recognizes or potentially recognizes a T cell epitope in the context of a class I MHC molecule. Class I MHC proteins are expressed in all nucleated cells of higher vertebrates. The class I MHC molecule is a heterodimer consisting of a 46-kDa heavy chain that is non-covalently linked to a 12-kDa light chain of β-2 microglobulin. Humans have several MHC alleles, such as, for example, HLA-A2, HLA-A1, HLA-A3, HLA-A24, HLA-A28, HLA-A31, HLA-A33, HLA-A34, HLA-B7, HLA-B45, and HLA-Cw8. The sequences of the MHC alleles are known and can be found, for example, in the IMGT/HLA database, available at www.ebi.ac.uk/ipd/imgt/hla. In some embodiments, the MHC class I allele is the HLA-A2 allele, which in some populations is expressed by approximately 50% of the population. In some embodiments, the HLA-A2 allele may be the HLA-A*0201, *0202, *0203, *0206, or *0207 gene product. In some cases, there may be differences in the frequency of the subtypes among different populations. For example, in some embodiments, greater than 95% of the HLA-A2 positive Caucasian population is HLA-A*0201, and in the Chinese population the frequency is described as approximately 23% HLA-A*0201, 45% HLA-A*0207, 8% HLA-A*0206, and 23% HLA-A*0203.

In some embodiments, MHC class I restricted peptides are 8-15 amino acids in length, such as 8-10 amino acids in length. In some embodiments, MHC class I molecules bind peptides derived from endogenous antigens, such as tumor proteins, viral or bacterial proteins produced in diseased or infected cells, which are processed in the cytoplasm of the cell via the cytosolic pathway. In some embodiments, MHC class I-peptide complexes displayed on the cell surface are typically recognized by TCRs expressed on CD8+ T cells, such as cytotoxic T cells. In some embodiments, MHC class I-peptide complexes can be recognized by TCRs expressed on CD4+ T cells, such as TCRs exhibiting CD8- or partially CD8-independent binding.

In some embodiments, the TCR or other MHC peptide binding molecule recognizes or potentially recognizes a T cell epitope in the context of a class II MHC molecule. Class II MHC proteins are expressed in a subset of nucleated cells in vertebrates, commonly referred to as antigen-presenting cells (APCs). Humans have multiple MHC class II alleles, such as, for example, DR1, DR3, DR4, DR7, DR52, DQ1, DQ2, DQ4, DQ8, and DP1. In some embodiments, the MHC class II allele is HLA-DRB1*0101, HLA-DRB*0301, HLA-DRB*0701, HLA-DRB*0401, HLA-DQB1*0201. The sequences of MHC alleles are known and can be found, for example, in the IMGT/HLA database available at www.ebi.ac.uk/ipd/imgt/hla.

In some embodiments, MHC class II restricted peptides are typically about 9-25 residues in length, such as 15-25 residues or 13-18 residues in length, and in some cases comprise a binding core region of about 9 amino acids or about 12 amino acids. In some embodiments, MHC class II molecules bind peptides derived from exogenous antigens that are internalized by phagocytosis or endocytosis and processed in the endosomal/lysosomal pathway. In some embodiments, MHC class II peptide complexes displayed on the surface of cells are typically recognized by CD4+ cells, such as helper T cells. In some embodiments, displayed MHC class II peptide complexes can be recognized by TCRs expressed on CD8+ T cells.

Typically, the peptide epitope or T cell epitope is a peptide portion of an antigen. In some embodiments, the antigen is known, and in some cases, the peptide epitope recognized by the TCR or its antigen-binding portion (or other MHC-peptide binding molecules) may also be known, such as known before the present method is performed.

In some embodiments, the antigen is a tumor-associated antigen, an antigen expressed in a particular cell type associated with an autoimmune or inflammatory disease, or an antigen derived from a viral pathogen or a bacterial pathogen. In some embodiments, the antigen is an antigen involved in a disease. In some embodiments, the disease may be caused by malignancy or cell transformation, such as cancer. In some embodiments, the antigen may be an intracellular protein antigen from a tumor or cancer cell, such as a tumor-associated antigen. In some cases, because the majority of cancer antigens are derived from intracellular proteins that can only be affected on the cell surface in the context of an MHC molecule, TCRs are ideal candidates for therapy because they are involved in recognizing this class of antigens. In some embodiments, the disease may be caused by contamination, such as a bacterial or viral infection. In some embodiments, the antigen is a virus-associated cancer antigen. In some cases, the recombinant TCR or its antigen-binding portion (and other MHC-peptide binding molecules) recognizes or potentially recognizes peptides derived from viral proteins that are naturally processed in infected cells and displayed by the MHC molecule on the cell surface. In some embodiments, the disease may be an autoimmune disease. Other targets include those listed in the HLA Factsbook (Marsh et al. (2000)) and others known.

In some embodiments, the antigen is one that is associated with a tumor or cancer. In some embodiments, a tumor or cancer antigen is one that can be found on a malignant cell, found within a malignant cell, or is a mediator of tumor cell growth. In some embodiments, a tumor or cancer antigen is one that is preferentially expressed or overexpressed by a tumor cell or cancer cell. A variety of tumor antigens have been identified and known, including MHC-restricted, T cell-specific tumor antigens (see, e.g., cancerimmunity.org/peptide/; Boon and Old (1997) Curr Opin Immunol, 9:681-3; Cheever et al. (2009) Clin Cancer Res, 15:5323-37). In some embodiments, tumor antigens include, but are not limited to, mutated peptides, differentiation antigens, and overexpressed antigens, all of which serve as targets for therapy.

In some embodiments, the tumor or cancer antigen is a lymphoma antigen ( e.g., non-Hodgkin's lymphoma or Hodgkin's lymphoma), a B-cell lymphoma cancer antigen, a leukemia antigen, a myeloma antigen ( i.e. , multiple myeloma or plasma cell myeloma), an acute lymphoblastic leukemia antigen, a chronic myeloid leukemia antigen, or an acute myelogenous leukemia antigen. In some embodiments, the cancer antigen is an antigen that is overexpressed or associated with cancer, which are adenocarcinomas, such as pancreatic, colon, breast, ovarian, lung, prostate, head and neck cancer, including multiple myelomas and B cell lymphomas. In some embodiments, the antigen is associated with a cancer, such as prostate cancer, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, skin cancer, liver cancer ( eg, hepatocellular adenocarcinoma), colon cancer, or bladder cancer.

In some embodiments, the antigen is a tumor antigen, which may be glioma-associated antigen, β-human chorionic gonadotropin, alpha-fetoprotein (AFP), B-cell maturation antigen (BCMA, BCM), B-cell activating factor receptor (BAFFR, BR3) and/or transmembrane activator and associate of CAML (TACI), Fc receptor-like 5 (FCRL5, FcRH5), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, Melanin-A/MART-1, WT-1, S-100, MBP, CD63, MUC1 ( e.g., MUC1-8), p53, Ras, cyclin B1, HER-2/neu, carcinoembryonic antigen (CEA), gp100, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A11, MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-C1, BAGE, GAGE-1, GAGE-2, pl5, tyrosinase, tyrosinase-related protein 1 (TRP-1), tyrosinase-related protein 2 (TRP-2), β-catenin, NY-ESO-1, LAGE-1a, PP1, MDM2, MDM4, EGVFvIII, Tax, SSX2, telomerase, TARP, pp65, CDK4, vimentin, S100, eIF-4A1, IFN-inducible p78 and melanotransferrin (p97), Uroplakin II, prostate specific antigen (PSA), human kallikrein (huK2), prostate specific membrane antigen (PSM) and prostatic acid phosphatase (PAP), neutrophil elastase, ephrin B2, BA-46, beta-catenin, Bcr-abl, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Caspase 8 or B-Raf antigen. Other tumor antigens may include any derived from FRα, CD24, CD44, CD133, CD 166, epCAM, CA-125, HE4, Oval, estrogen receptor, progesterone receptor, uPA, PAI-1, CD19, CD20, CD22, ROR1, mesothelin, CD33/IL3Ra, c-Met, PSMA, Glycolipid F77, GD-2, insulin growth factor (IGF)-I, IGF-II, and IGF-I receptor. Specific tumor-associated antigens or T cell epitopes are known (see, e.g., van der Bruggen et al. (2013) Cancer Immun, available at www.cancerimmunity.org/peptide/; Cheever et al. (2009) Clin Cancer Res, 15, 5323–37).

In some embodiments, the antigen is a viral antigen. A variety of viral antigenic targets have been identified and are known, including peptides derived from viral genomes in HIV, HTLV, and other viruses (see, e.g., Addo et al. (2007) PLoS ONE, 2, e321; Tsomides et al. (1994) J Exp Med, 180, 1283–93; Utz et al. (1996) J Virol, 70, 843–51). exemplary viral antigens include, but are not limited to, an antigen from hepatitis A, hepatitis B ( e.g., HBVc and surface antigens (HBVc, HBVs)), hepatitis C (HCV), Epstein-Barr virus ( e.g., EBVA), human papillomavirus (HPV; e.g., E6 and E7), human immunodeficiency virus type-1 (HIV1), Kaposi's sarcoma herpesvirus (KSHV), human papillomavirus (HPV), influenza virus, Lassa virus, HTLN-1, HIN-1, HIN-II, CMN, EBN, or HPN. In some embodiments, the target protein is a bacterial antigen or other pathogenic antigen, such as Mycobacterium tuberculosis (MT) antigens, trypanosoma antigens such as Tiypansoma cruzi (T. cruzi) surface antigen (TSA), or malaria antigens. specific viral antigens or epitopes or other pathogenic antigens or T cell epitopes are known (see, e.g., Addo et al. (2007) PLoS ONE, 2:e321; Anikeeva et al. (2009) Clin Immunol, 130:98–109).

In some embodiments, the antigen is an antigen derived from a virus associated with cancer, such as an oncogenic virus. For example, an oncogenic virus is one in which infection with certain viruses results in the development of various types of cancer, such as antigens of hepatitis A virus, hepatitis B virus ( e.g., HBV core and surface antigens (HBVc, HBVs)), hepatitis C virus (HCV), human papillomavirus (HPV), hepatitis B virus infections, Epstein-Barr virus (EBV), human herpes virus 8 (HHV-8), human T-cell leukemia virus-1 (HTLV-1), human T-cell leukemia virus-2 (HTLV-2), or cytomegalovirus (CMV).

In some embodiments, the viral antigen is an HPV antigen, which in some cases may lead to an increased risk of developing cervical cancer. In some embodiments, the antigen may be an HPV-16 antigen, an HPV-18 antigen, an HPV-31 antigen, an HPV-33 antigen, or an HPV-35 antigen. In some embodiments, the viral antigen is an HPV-16 antigen ( e.g., the E1, E2, E6, and/or E7 seroreactive regions of the HPV-16 proteins, see, e.g., U.S. Patent No. 6,531,127) or an HPV-18 antigen ( e.g., the L1 and/or L2 seroreactive regions of the HPV-18 proteins, such as those described in U.S. Patent No. 5,840,306). In some embodiments, the viral antigen is an HPV-16 antigen from the E6 and/or E7 proteins of HPV-16. In some embodiments, the TCR is a TCR directed against HPV-16 E6 or HPV-16 E7. In some embodiments, the TCR is a TCR described in, for example, WO 2015/184228, WO 2015/009604, and WO 2015/009606.

In some embodiments, the viral antigen is an HBV or HCV antigen, which, in some cases, may result in a greater risk of developing liver cancer than in HBV or HCV negative subjects. For example, in some embodiments, the heterologous antigen is an HBV antigen, such as hepatitis B core antigen or hepatitis B envelope antigen (US2012/0308580).

In some embodiments, the viral antigen is the EBV antigen, which, in some cases, may confer a greater risk of developing Burkitt's lymphoma, nasopharyngeal carcinoma, and Hodgkin's disease than EBV-negative subjects. For example, EBV is a human herpesvirus that, in some cases, has been associated with numerous human tumors of various tissue origins. Although primarily found as an asymptomatic infection, EBV-positive tumors may be characterized by active expression of viral gene products such as EBNA-1, LMP-1, and LMP-2A. In some embodiments, the heterologous antigen is an EBV antigen, which may include Epstein-Barr nuclear antigen (EBNA)-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA leader protein (EBNA-LP), latent membrane proteins LMP-1, LMP-2A and LMP-2B, EBV-EA, EBV-MA, or EBV-VCA.

In some embodiments, the viral antigen is an HTLV-1 or HTLV-2 antigen, which, in some cases, may result in a greater risk of developing T-cell leukemia than in HTLV-1 or HTLV-2 negative subjects. For example, in some embodiments, the heterologous antigen is an HTLV antigen such as TAX.

In some embodiments, the viral antigen is an HHV-8 antigen, which, in some cases, may result in a greater risk of developing Kaposi's sarcoma than in HHV-8 negative subjects. In some embodiments, the heterologous antigen is a CMV antigen, such as pp65 or pp64 (see U.S. Patent No. 8,361,473).

In some embodiments, the antigen is a self-antigen, such as a polypeptide antigen associated with an autoimmune disease or disorder. In some cases, the autoimmune disease or disorder may be multiple sclerosis (MS), rheumatoid arthritis (RA), Sjögren's syndrome, scleroderma, poliomyositis, dermatomyositis, systemic lupus erythematosus, juvenile rheumatoid arthritis, ankylosing spondylitis, myasthenia gravis (MG), bullous pemphigoid (antibodies to the basement membrane at the dermal-epidermal junction), pemphigus (antibodies to the protein complex of mucopolysaccharide or intracellular cement substance), glomerulonephritis (antibodies to the glomerular basement membrane), Goodpasture's syndrome, autoimmune hemolytic anemia (antibodies to red blood cells), Hashimoto's disease (antibodies to the thyroid gland), pernicious anemia (antibodies to intrinsic factor), idiopathic thrombocytopenic purpura (antiplatelet antibodies), Graves' disease, or Addison's disease (antithyroglobulin antibodies). In some embodiments, the autoantigen, such as an autoantigen associated with one or more of the above autoimmune diseases, can be a collagen such as type II collagen, mycobacterial heat shock protein, thyroglobulin, acetylcholine receptor (AcHR), myelin basic protein (MBP), or proteolipid protein (PLP). Specific autoimmune associated epitopes or antigens are known (see, e.g., Bulek et al. (2012) Nat Immunol, 13:283-9; Harkiolaki et al. (2009) Immunity, 30:348-57; Skowera et al. (2008) J Clin Invest, 1(18): 3390-402).

In some embodiments, the identity of the peptide epitope of the target antigen is known, which in some cases can be used to produce or create the TCR in question or in assessing the functional activity or property, including in connection with the presented methods. In some embodiments, the peptide epitopes can be determined or identified based on an HLA-restricted motif in the target antigen in question. In some embodiments, the peptides are identified using known computer prediction models. In some embodiments, for predicting MHC class I binding sites, such models include, but are not limited to, ProPred1 (Singh and Raghava (2001) Bioinformatics 17(12):1236-1237, and SYFPEITHI (see Schuler et al. (2007) Immunoinformatics Methods in Molecular Biology, 409(1): 75-93 2007). In some embodiments, the MHC-restricted epitope is HLA-A0201, which is expressed in approximately 39-46% of all Caucasians and therefore represents a suitable choice of MHC antigen for use in producing a TCR or other MHC peptide binding molecule. In some aspects, HLA-A*0201 binding motifs and cleavage sites for proteasomes and immune proteasomes are known using computer prediction models. For predicting sites MHC class I binding models, such as ProPred1 (described in more detail in Singh and Raghava, ProPred: prediction of HLA-DR binding sites. BIOINFORMATICS 17(12):1236-1237 2001), and SYFPEITHI (see Schuler et al . SYFPEITHI, Database for Searching and T-Cell Epitope Prediction. in Immunoinformatics Methods in Molecular Biology, vol 409(1): 75-93 2007) are provided. Screening methods and cells used in screening methods, such as T cells, that recognize an antigen or epitope, in the context of a major histocompatibility complex (MHC) molecule, are provided.

In some embodiments, the MHC comprises a binding site or binding cavity for a polymorphic peptide that may, in some cases, form a complex with peptide epitopes of polypeptides, including peptide epitopes processed by the cellular machinery. In some cases, MHC molecules may be expressed or displayed on the cell surface, including as a complex with a peptide, i.e., an MHC-peptide complex, to present the antigen in a structure recognized by the TCR on T cells or other MHC-binding peptides. In general, class I MHC molecules are heterodimers having a transmembrane α chain, in some cases with three α domains, and a non-covalently associated β2 microglobulin. In general, class II MHC molecules are composed of two transmembrane glycoproteins, α and β, both of which typically span the membrane. An MHC molecule may include an effective portion of an MHC that contains an epitope binding site or peptide binding sites and a sequence necessary for recognition of a suitable binding molecule, such as a TCR. In some embodiments, class I MHC molecules deliver peptides originating in the cytosol to the cell surface, where the peptide:MHC complex is recognized by T cells, such as primarily CD8 ⁺ T cells, but in some cases, CD4+ T cells. In some embodiments, class II MHC molecules deliver peptides originating in the vesicular system to the cell surface, where they are typically recognized by CD4 ⁺ T cells. In general, MHC molecules are encoded by a group of related loci that are collectively referred to as H-2 in mouse and human leukocyte antigen (HLA) in humans. In some aspects, human MHC may also be referred to as human leukocyte antigen (HLA).

In some embodiments, the peptide epitope or T cell epitope is a peptide that can be derived from or based on a fragment of a larger biological molecule, such as a polypeptide or protein, and that is capable of associating with or forming a complex with an MHC molecule. In some embodiments, the peptide is from about 8 to about 24 amino acids in length. In some embodiments, the peptide is from or about 9-22 amino acids in length for recognition in an MHC class II complex. In some embodiments, the peptide is from or about 8-13 amino acids in length for recognition in an MHC class I complex. In some embodiments, the MHC molecule and the peptide epitope or T cell epitope form a complex or associate through non-covalent interactions of the peptide in the binding cavity or cleft of the MHC molecule.

In some embodiments, the MHC-peptide complex is present or displayed on the surface of a cell. In some embodiments, the MHC-peptide complex can be specifically recognized by a TCR or an antigen-binding portion thereof or another MHC-peptide binding molecule. In some embodiments, the T cell epitope or peptide epitope is capable of eliciting an immune response in an animal through its MHC molecule binding characteristics. In some embodiments, upon recognition of a T cell epitope, such as an MHC-peptide complex, the TCR (or another MHC-peptide binding molecule) elicits or triggers an activation signal to the T cell that elicits a T cell response, such as T cell proliferation, cytokine production, a cytotoxic T cell response, or another response.

In some embodiments, the MHC peptide binding molecule is a TCR or an epitope-binding fragment thereof. In some embodiments, the MHC peptide binding molecule is a TCR-like CAR that comprises an antibody or an epitope-binding fragment thereof, such as a TCR-like antibody, such as engineered to bind to MHC peptide complexes. In some embodiments, such a binding molecule binds to a binding sequence, such as a T cell epitope comprising an amino acid sequence or an antigen of a target polypeptide. In some embodiments, the binding sequence of the target peptide or target polypeptide is known. In some embodiments, the MHC peptide binding molecule may be obtained from natural sources, or may be partially or wholly synthetic or recombinant.

In some embodiments, an MHC peptide binding molecule is a molecule or portion thereof that has the ability to bind, e.g., specifically bind, to a peptide epitope that is present or displayed in the context of an MHC molecule, i.e. , an MHC peptide complex, e.g., on a cell surface. In some embodiments, the binding molecule can include any naturally occurring, synthetic, semi-synthetic, or recombinantly produced molecule that can bind, e.g., specifically bind, to an MHC peptide complex. Exemplary MHC peptide binding molecules include T cell receptors or antibodies or antigen-binding portions thereof, including individual chains of immunoglobulin variable regions ( e.g., scTCR, scFv), which exhibit specific ability to bind to an MHC peptide complex.

In some embodiments, the TCR is a full-length TCR. In some embodiments, the TCR is an antigen-binding portion. In some embodiments, the TCR is a dimeric TCR (dTCR). In some embodiments, the TCR is a single-stranded TCR (sc-TCR). The TCR may be cell-bound or in a soluble form. In some embodiments, the TCR has a cell-bound form expressed on the surface of a cell.

In some embodiments, the dTCR comprises a first polypeptide, wherein a sequence corresponding to the present variable region of the TCR α chain sequence is fused to the N terminus of a sequence corresponding to the constant region of the TCR α chain extracellular sequence, and a second polypeptide, wherein a sequence corresponding to the present variable region of the TCR β chain sequence is fused to the N terminus of a sequence corresponding to the constant region of the TCR β extracellular sequence, wherein the first and second polypeptides are linked by a disulfide bond. In some embodiments, the linkage may correspond to a native interstrand disulfide bond present in native dimeric αβ TCRs. In some embodiments, interstrand disulfide bonds are not present in the native TCR. For example, in some embodiments, one or more cysteines may be introduced into the constant region of the dTCR extracellular sequences of a polypeptide pair. In some cases, both native and non-native disulfide bonds may be desirable. In some variants, the TCR contains a transmembrane sequence for membrane anchoring.

In some embodiments, the dTCR comprises a TCR-present α chain comprising a variable α domain, a constant α domain, and a first dimerization motif attached to the C-terminus of the constant α domain, and a TCR-present β chain comprising a variable β domain, a constant β domain, and a first dimerization motif attached to the C-terminus of the constant β domain, wherein the first and second dimerization motifs can interact to form a covalent bond between amino acids in the first dimerization motif and amino acids in the second dimerization motif linking the TCR α chain and the TCR β chain together.

In some embodiments, the TCR is an scTCR, which is a single amino acid strand comprising an α chain and a β chain that are capable of binding to MHC-peptide complexes. Generally, an scTCR can be obtained using known methods, see, for example, International PCT Publication Nos. WO 96/13593, WO 96/18105, WO99/18129, WO 04/033685, WO2006/037960, WO2011/044186; U.S. Patent No. 7,569,664; and Schlueter, C. J. et al. J. Mol. Biol. 256, 859 (1996).

In some embodiments, the scTCR comprises a first segment formed by an amino acid sequence corresponding to the sequence of the present variable region of the TCR α chain, a second segment formed by an amino acid sequence corresponding to the sequence of the present variable region of the TCR β chain, fused to the N terminus of an amino acid sequence corresponding to the constant domain of the extracellular sequence of the TCR β chain, and a linker sequence linking the C terminus of the first segment to the N terminus of the second segment.

In some embodiments, the scTCR comprises a first segment formed by an amino acid sequence corresponding to the sequence of the present variable region of the TCR β chain, a second segment formed by an amino acid sequence corresponding to the sequence of the present variable region of the TCR α chain, fused to the N terminus of an amino acid sequence corresponding to the constant domain of the extracellular sequence of the TCR α chain, and a linker sequence linking the C terminus of the first segment to the N terminus of the second segment.

In some embodiments, the scTCR comprises a first segment formed by the present variable region of the α chain sequence fused to the N terminus of the constant domain of the extracellular sequence of the α chain, and a second segment formed by the variable region of the β chain sequence fused to the N terminus of the extracellular constant and transmembrane sequence of the β chain, and, optionally, a linker sequence linking the C terminus of the first segment to the N terminus of the second segment.

In some embodiments, the scTCR comprises a first segment formed by the present variable region of the TCR β chain sequence fused to the N terminus of the constant domain of the extracellular sequence of the β chain, and a second segment formed by the variable region of the α chain sequence fused to the N terminus of the extracellular constant and transmembrane sequence of the α chain, and, optionally, a linker sequence linking the C terminus of the first segment to the N terminus of the second segment.

In some embodiments, for the scTCR to bind the MHC-peptide complex, the α and β chains must be paired such that their variable sequence regions are oriented for such binding. Various methods for facilitating pairing of the α and β in the scTCR are known. In some embodiments, a linker sequence is included that links the α and β chains to form a single polypeptide strand. In some embodiments, the linker must be of sufficient length to span the distance between the C terminus of the α chain and the N terminus of the β chain, or vice versa, and to ensure that the length of the linker is not so long that it blocks or reduces binding of the scTCR to the target peptide-MHC complex.

In some embodiments, the scTCR linker that links the first and second segments of the TCR may be any linker capable of forming a single polypeptide chain while maintaining TCR binding specificity. In some embodiments, the linker sequence may, for example, have the formula -P-AA-P-, where P is proline and AA represents amino acid sequences, where the amino acids are glycine and serine. In some embodiments, the first and second segments are paired such that the variable region of their sequences are oriented for such binding. Therefore, in some cases, the linker is of sufficient length to span the distance between the C terminus of the first segment and the N terminus of the second segment, or vice versa, but not so long that it blocks or reduces the binding of the scTCR to the target ligand. In some embodiments, the linker may comprise from or about 10-45 amino acids, such as 10-30 amino acids, or 26-41 amino acid residues, such as 29, 30, 31 or 32 amino acids. In some embodiments, the linker has the formula -PGGG-(SGGGG) _n -P-, where n is 5 or 6 and P is proline, G is glycine and S is serine (SEQ ID NO: 22). In some embodiments, the linker has the sequence GSADDAKKDAAKKDGKS (SEQ ID NO: 23).

In some embodiments, the scTCR comprises a disulfide bond between residues of a single amino acid strand, which in some cases can promote pairing stability between the α and β regions of the single-stranded molecule (see, e.g., U.S. Patent No. 7,569,664). In some embodiments, the scTCR comprises a covalent disulfide bond linking a residue of an immunoglobulin region of the α chain constant domain to a residue of an immunoglobulin region of the β chain constant domain of the single-stranded molecule. In some embodiments, the disulfide bond corresponds to a native disulfide bond present in a native dTCR. In some embodiments, a disulfide bond is not present in a native TCR. In some embodiments, the disulfide bond is an introduced non-native disulfide bond, such as by introducing one or more cysteines into the constant region of the extracellular sequence of the first and second chain regions of the scTCR polypeptide. Typical cysteine mutations include any described here. In some cases, both native and non-native disulfide bonds may be present.

In some embodiments, the scTCR is a non-disulfide-linked truncated TCR in which heterologous leucine zippers fused to their C-termini facilitate chain association (see, e.g., PCT International Publication No. WO99/60120). In some embodiments, the scTCR comprises a TCRα variable domain covalently linked to a TCRβ variable domain via a peptide linker (see, e.g., PCT International Publication No. WO99/18129).

In some embodiments, any of the present TCRs, including a dTCR or scTCR, can be associated with signaling domains that provide an active TCR on the surface of a T cell. In some embodiments, the TCR is expressed on the surface of a cell. In some embodiments, the TCR does not comprise a sequence corresponding to a transmembrane sequence. In some embodiments, the transmembrane domain is positively charged. In some embodiments, the transmembrane domain can be a Cα or Cβ transmembrane domain. In some embodiments, the transmembrane domain can be from a non-TCR, such as a transmembrane region from CD3z, CD28, or B7.1. In some embodiments, the TCR does not comprise a sequence corresponding to cytoplasmic sequences. In some embodiments, the TCR comprises a CD3z signaling domain. In some embodiments, the TCR is capable of forming a TCR complex with CD3.

In some embodiments, the TCR is a soluble TCR. In some embodiments, the soluble TCR has a structure as described in WO99/60120 or WO 03/020763. In some embodiments, the TCR does not contain a sequence corresponding to a transmembrane sequence, for example, to allow the membrane to anchor in the cell in which it is expressed. In some embodiments, the TCR does not contain a sequence corresponding to cytoplasmic sequences.

In some embodiments, a recombinant receptor, e.g., a TCR or antigen-binding fragment thereof, is or has been modified compared to a known recombinant receptor. In certain embodiments, recombinant receptors, e.g., TCR or antigen-binding fragments thereof, include one or more amino acid variations, e.g., substitutions, deletions, insertions, and/or mutations compared to the sequence of a recombinant receptor, e.g., a TCR, described herein or known. Exemplary variants include those designed to improve the binding affinity and/or other biological properties of the binding molecule. Amino acid sequence variants of the binding molecule can be obtained by introducing suitable modifications into the nucleotide sequence encoding the binding molecule or by peptide synthesis. Such modifications include, for example, deletions from and/or insertions into and/or substitutions of residues in the amino acid sequences of the binding molecule. Any combination of deletions, insertions, and substitutions can be performed to obtain the final construct, provided that the final construct has the desired characteristics, such as antigen binding.

In some embodiments, targeted evolution methods are used to generate TCRs with altered properties, such as higher affinity for a particular peptide in the context of an MHC molecule. In some embodiments, targeted evolution is achieved by display methods, including, but not limited to, yeast display (Holler et al. (2003) Nat Immunol, 4, 55-62; Holler et al. (2000) Proc Natl Acad Sci U S A, 97, 5387-92), phage display (Li et al. (2005) Nat Biotechnol, 23, 349-54), or T cell display (Chervin et al. (2008) J Immunol Methods, 339, 175-84). In some embodiments, display approaches involve engineering or modifying known, parental, or reference TCRs. For example, in some cases, a reference TCR, such as any of those provided herein, can be used as a template to generate mutagenized TCRs in which one or more CDR residues are mutated, and mutants with a desired altered property, such as higher affinity for a peptide epitope in the context of an MHC molecule, are selected.

In certain embodiments, recombinant receptors, e.g., TCRs, or antigen-binding fragments thereof, comprise one or more amino acid substitutions, e.g., compared to a recombinant receptor, e.g., TCR, sequence compared to a sequence of a natural receptor, e.g., a human receptor. The sites of interest for substitution mutagenesis include CDRs, FRs, and/or constant regions. The amino acid substitutions can be introduced into the binding molecule of interest and the products screened for desired activity, e.g., preserved/improved antigen affinity or avidity, reduced immunogenicity, improved half-life, CD8-independent binding or activity, surface expression, facilitation of TCR chain pairing, and/or other improved properties or functions.

In some embodiments, one or more residues within a CDR of a recombinant receptor, such as a TCR, are substituted. In some embodiments, the substitution is made to fold a sequence or position in a sequence to a germline sequence, such as a binding molecule sequence found in a germline (e.g., a human germline), such as to reduce the likelihood of immunogenicity, such as when administered to a human.

In certain embodiments, substitutions, insertions, or deletions may be made within one or more CDRs to the extent that such changes do not substantially reduce the ability of the recombinant receptor, e.g., a TCR or antigen-binding fragment thereof, to bind antigen. For example, conservative changes (e.g., the conservative substitutions provided herein) that do not substantially reduce binding affinity may be made in the CDRs. Such changes may, for example, be outside of the antigen-contacting residues in the CDRs. In certain embodiments of the variable sequences provided herein, each CDR is either unaltered or contains no more than one, two, or three amino acid substitutions.

Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from a single residue to polypeptides containing hundreds or more residues, as well as intrasequence insertions of single or multiple amino acid residues.

In some aspects, the TCR or antigen-binding fragment thereof may comprise one or more modifications in the α chain and/or β chain such that when the TCR or antigen-binding fragment thereof is expressed in a cell, the frequency of mismatch between the α chain and β chain of the TCR and the α chain and β chain of the endogenous TCR is reduced, expression of the α chain and β chain of the TCR is increased, and/or the stability of the α chain and β chain of the TCR is increased.

In some embodiments, the TCR comprises one or more non-native residues for introducing a covalent disulfide bond linking a residue of the immunoglobulin region of the α chain constant domain to a residue of the immunoglobulin region of the β chain constant domain. In some embodiments, one or more cysteines may be introduced into the constant region of the extracellular sequences of the first and second segments of the TCR polypeptide. Exemplary non-limiting modifications to the TCR for introducing non-native cysteine residues are described herein (see also International PCT Nos. WO2006/000830 and WO2006037960). In some cases, both native and non-native disulfide bonds may be desirable. In some embodiments, the TCR or antigen-binding fragment thereof is modified such that interstrand disulfide bonds are not present in the native TCR.

In some embodiments, the transmembrane domain of the TCR constant region may be modified to contain a greater number of hydrophobic residues (see, e.g., Haga-Friedman et al. (2012) Journal of Immunology, 188:5538-5546). In some embodiments, the transmembrane region of the TCR α chain comprises one or more mutations corresponding to S116L, G119V, or F120L, with reference to the Cα numbering set forth in SEQ ID NO:24.

In some embodiments, the TCR or antigen-binding fragment thereof is encoded by a nucleotide sequence that is or has been codon-optimized. Exemplary codon-optimized embodiments are described elsewhere herein.

B. Chimeric antigen receptors (CAR)

In some embodiments, the recombinant receptor that is introduced into the cell is a chimeric antigen receptor (CAR) or an antigen-binding fragment thereof. In some embodiments, engineered cells, such as T cells, are provided that express a CAR with specificity for a particular antigen (or marker or ligand), such as an antigen expressed on the surface of a particular cell type. In some embodiments, the antigen is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen is selectively expressed or overexpressed on a cell of a disease or condition, such as a tumor or pathogenic cell, compared to normal or non-targeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and/or expressed on engineered cells.

In particular embodiments, a recombinant receptor, such as a chimeric receptor, comprises an intracellular signaling region that includes a cytoplasmic signaling domain (also interchangeably referred to as an intracellular binding domain), such as a cytoplasmic (intracellular) region capable of eliciting a primary activation signal in a T cell, such as a cytoplasmic signaling domain of a T cell receptor (TCR) component (e.g., a cytoplasmic signaling domain of the zeta chain of the CD3 zeta (CD3ζ) chain or a functional variant or signaling portion thereof) and/or that comprises an immunoreceptor tyrosine-based activation motif (ITAM).

In some embodiments, the chimeric receptor further comprises an extracellular ligand-binding domain that specifically binds to a ligand (e.g., an antigen) of the antigen. In some embodiments, the chimeric receptor is a CAR that comprises an extracellular antigen-recognition domain that specifically binds to an antigen. In some embodiments, the ligand, such as an antigen, is a protein expressed on the surface of cells. In some embodiments, the CAR is a TCR-like CAR and the antigen is a processed peptide antigen, such as a peptide antigen of an intracellular protein that, like the TCR, is recognized on the surface of a cell in the context of a major histocompatibility complex (MHC) molecule.

Exemplary antigen receptors, including CARs, and methods for constructing and introducing such receptors into cells include those described, for example, in International Patent Application Publication Nos. WO200014257, WO2013126726, WO2012/129514, WO2014031687, WO2013/166321, WO2013/071154, WO2013/123061, U.S. Patent Application Publication Nos. US2002131960, US2013287748, US20130149337, U.S. Patent Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European Patent Application No. EP2537416, and/or those described in Sadelain et al., Cancer Discov. 2013 April; 3(4): 388-398; Davila et al. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 March 18(2): 160-75. In some aspects, antigen receptors include CARs described in U.S. Patent No.: 7,446,190 and those described in International Patent Application Publication No.: WO/2014055668 A1. Examples of CARs include those described in any of the above publications, such as WO2014031687, US 8,339,645, US 7,446,179, US 2013/0149337, US Patent No.: 7,446,190, US Patent No.: 8,389,282, Kochenderfer et al., 2013, Nature Reviews Clinical Oncology, 10, 267-276 (2013); Wang et al. (2012) J. Immunother. 35(9): 689-701; and Brentjens et al., Sci Transl Med. 2013 5(177). See also WO2014031687, US 8,339,645, US 7,446,179, US 2013/0149337, US Patent No.: 7,446,190, and US Patent No.: 8,389,282.

In some embodiments, the CAR is designed to be specific for a particular antigen (or marker or ligand), such as an antigen expressed on a particular cell type that is the target of adoptive therapy, such as a cancer marker, and/or an antigen designed to elicit a debilitating response, such as an antigen expressed on a normal or non-disease cell type. Thus, the CAR typically includes in its extracellular portion one or more antigen-binding molecules, such as one or more antigen-binding fragments, domains, or portions, or one or more variable domains of an antibody, and/or antibody molecules. In some embodiments, the CAR includes an antigen-binding portion or portions of an antibody molecule, such as a single-stranded antibody fragment (scFv) derived from the variable heavy (V _H ) and variable light (V _L ) chains of a monoclonal antibody (mAb).

In some embodiments, the antibody or antigen-binding portion thereof is expressed on cells as part of a recombinant receptor, such as an antigen receptor. Antigen receptors include functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs). In general, a CAR comprising an antibody or antigen-binding fragment that exhibits TCR-like specificity directed against peptide-MHC complexes may also be referred to as a TCR-like CAR. In some embodiments, the extracellular antigen-binding domain specific for the MHC-peptide complex of the TCR-like CAR is linked to one or more intracellular signaling components, in some aspects, via linkers and/or transmembrane domains. In some embodiments, such molecules may typically mimic or approximate a signal through a natural antigen receptor, such as a TCR, and optionally a signal through such a receptor in combination with a costimulatory receptor.

In some embodiments, a recombinant receptor, such as a chimeric receptor (e.g., a CAR), comprises a ligand binding domain that binds, e.g., specifically binds, an antigen (or ligand). Antigens to which chimeric receptors are directed include those expressed in the context of a disease, condition, or cell type to which adoptive cell therapy is directed. Diseases and conditions include proliferative, neoplastic, and malignant diseases and disorders, including cancer and tumors, including hematological malignancies, cancers of the immune system such as lymphomas, leukemias, and/or myelomas such as B, T, and myeloid leukemias, lymphomas, and myelomas.

In some embodiments, the antigen (or ligand) is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen (or ligand) is selectively expressed or overexpressed on cells of a disease or condition, such as tumor or pathogenic cells, compared to normal or non-target cells or tissues. In other embodiments, the antigen is expressed on normal cells and/or expressed on engineered cells.

In some embodiments, a CAR comprises an antibody or antigen-binding fragment ( e.g., an scFv) that specifically recognizes an antigen, such as a native antigen, expressed on the surface of a cell.

In some embodiments, the antigen (or ligand) is a tumor antigen or cancer marker. In some embodiments, the antigen (or ligand) is or includes αvβ6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), cancer testicular antigen, cancer testicular antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), cyclin, cyclin A2, C-C motif chemokine ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), epidermal growth factor receptor type III mutation (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin A2 receptor (EPHa2), estrogen receptor, Fc receptor-like 5 (FCRL5; also known as Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), folate binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp100), glypican-3 (GPC3), G protein-coupled receptor class C group 5 member D (GPRC5D), Her2/neu (erb-B2 receptor tyrosine kinase), Her3 (erb-B3), Her4 (erb-B4), erbB dimers, human high molecular weight melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, human leukocyte antigen A1 (HLA-A1), human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha (IL-22Rα), IL-13 receptor alpha 2 (IL-13Rα2), receptor with an integrated kinase domain (kdr), kappa light chain, L1 cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM leucine-rich repeat-containing family member 8 A (LRRC8A), Lewis Y, melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer cell group 2 member D ligands (NKG2D), melan A (MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen, predominantly expressed melanoma antigen (PRAME), progesterone receptor, prostate-specific antigen, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), orphan receptor tyrosine kinase type 1 (ROR1), survivin, trophoblast glycoprotein (TPBG, also known as 5T4), tumor-associated glycoprotein 72 (TAG72), tyrosinase-related protein 1 (TRP1, also known as TYRP1 or gp75), tyrosinase-related protein 2 (TRP2, also known as dopachrome tautomerase, dopachrome delta isomerase, or DCT), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms tumor 1 (WT-1), a pathogen-specific or pathogen-expressed antigen, or an antigen associated with a universal tag, and/or biotinylated molecules, and/or molecules expressed by HIV, HCV, HBV, or other pathogens. Antigens to which the receptors are targeted, in some embodiments, include antigens associated with B cell malignancy, such as any of a variety of known B cell markers. In some embodiments, the antigen is or includes CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Ig kappa, Ig lambda, CD79a, CD79b, or CD30.

In some embodiments, the antigen is or includes a pathogen-specific or pathogen-expressed antigen. In some embodiments, the antigen is a viral antigen (such as a viral antigen from HIV, HCV, HBV, etc.), bacterial antigens, and/or parasite antigens.

The term “antibody” is used herein in the broadest sense and includes polyclonal and monoclonal antibodies, including native antibodies and functional (antigen-binding) antibody fragments, including fragment antigen-binding (Fab) fragments, F(ab') ₂ fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, variable heavy chain (V _H ) regions capable of specifically binding antigen, single-chain antibody fragments, including single-chain variable fragments (scFv), and single-domain antibody fragments ( e.g., sdAb, sdFv, nanobodies). The term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies and heteroconjugate antibodies, multispecific, such as bispecific antibodies, diabodies, triabodies and tetrabodies, tandem dia-scFv, tandem tri-scFv. Unless otherwise specified, the term "antibody" shall be understood to encompass functional fragments of an antibody. The term also encompasses native or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA and IgD.

In some embodiments, the antigen-binding proteins, antibodies and antigen-binding fragments thereof specifically recognize the antigen of a full-length antibody. In some embodiments, the heavy and light chains of the antibody may be full-length or may be an antigen-binding portion (a Fab, F(ab')2, Fv or a single-stranded Fv fragment (scFv)). In other embodiments, the constant region of the heavy chain of the antibody is selected from, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE, in particular selected from, for example, IgG1, IgG2, IgG3, and IgG4, more particularly, IgG1 ( e.g., human IgG1). In another embodiment, the constant region of the light chain of the antibody is selected from, for example, kappa or lambda, in particular kappa.

The disclosed antibodies include antibody fragments. An “antibody fragment” refers to a molecule other than a native antibody that comprises a portion of a native antibody that binds an antigen to which the native antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ ; diabodies; linear antibodies; heavy chain variable regions (V _H ), single-chain antibody molecules such as scFv and single-domain V _H single antibodies; and multispecific antibodies formed from antibody fragments. In particular embodiments, the antibodies are single-chain antibody fragments comprising a heavy chain variable region and/or a light chain variable region, such as scFv.

The term “variable region” or “variable domain” refers to a domain of the heavy or light chain of an antibody that is involved in binding of the antibody to an antigen. The variable domains of the heavy chain and light chain (V _H and V _L , respectively) of a natural antibody generally have similar structures, with each domain containing four conserved framework regions (FRs) and three CDRs. (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W H Freeman and Co., page 91 (2007)). A single V _H or V _L domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a particular antigen can be isolated by using the V _H or V _L domain from an antibody that binds the antigen to screen for the release of complementary V _L or V _H domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880–887 (1993); Clarkson et al., Nature 352:624-628 (1991).

Single domain antibodies include antibody fragments comprising all or part of a variable domain of a heavy chain or all or part of a variable domain of a light chain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody. In some embodiments, a CAR comprises a heavy chain domain of an antibody that specifically binds an antigen, such as a cancer marker or a cell surface antigen of a target cell or disease, such as a tumor cell or a cancer cell, such as the target antigens described herein or known.

Antibody fragments can be produced by a variety of methods, including, but not limited to, proteolytic cleavage of a native antibody, as well as production by recombinant host cells. In some embodiments, the antibodies are recombinantly produced fragments, such as fragments that contain arrangements that do not exist in nature, such as with two or more regions or chains of the antibody connected by synthetic linkers, such as peptide linkers, and/or that may not be produced by enzymatic cleavage of a naturally occurring native antibody. In some embodiments, the antibody fragments are scFvs.

A "humanized" antibody is an antibody in which all or substantially all of the amino acid residues of the CDRs are derived from non-human CDRs and all or substantially all of the amino acid residues of the FRs are derived from human FRs. A humanized antibody may optionally include at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of a non-human antibody refers to a variant of a non-human antibody that has been humanized, typically to reduce immunogenicity in humans while maintaining the specificity and affinity of the original non-human antibody. In some embodiments, some of the FR residues in the humanized antibody are replaced with corresponding residues from a non-human antibody ( e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve the specificity or affinity of the antibody.

Thus, in some embodiments, a chimeric antigen receptor, including a TCR-like CAR, comprises an extracellular portion comprising an antibody or an antibody fragment. In some embodiments, the antibody or fragment comprises an scFv. In some aspects, a chimeric antigen receptor comprises an extracellular portion comprising an antibody or fragment, and an intracellular signaling region. In some embodiments, the intracellular signaling region comprises an intracellular binding domain. In some embodiments, the intracellular binding domain is or comprises a primary signaling domain, a signaling domain that is capable of causing a primary activation signal in a T cell, a component of a T cell receptor (TCR) signaling domain, and/or a signaling domain comprising an immunoreceptor tyrosine activation motif (ITAM).

In some embodiments, a recombinant receptor, such as a CAR, such as an antibody portion thereof, further comprises a spacer, which can be or include at least a portion of an immunoglobulin constant region or a variant or modified version thereof, such as a hinge region, for example, an IgG4 hinge region, and/or a _CH1 / _CL and/or an Fc region. In some embodiments, the recombinant receptor further comprises a spacer and/or a hinge region. In some embodiments, the constant region or portion is a human IgG, such as IgG4 or IgG1. In some aspects, a portion of the constant region serves as a spacer region between an antigen recognition component, for example, an scFv, and a transmembrane domain. The spacer can have a length that provides increased responsiveness of the cell upon antigen binding, compared to the absence of the spacer.

In some examples, the spacer is about 12 amino acids in length or no more than 12 amino acids in length. Exemplary spacers include those that are at least about 10 to 229 amino acids, about 10 to 200 amino acids, about 10 to 175 amino acids, about 10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100 amino acids, about 10 to 75 amino acids, about 10 to 50 amino acids, about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20 amino acids, or about 10 to 15 amino acids, and including any integer between the extremes of any of the listed ranges. In some embodiments, the spacer region is about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less. In some embodiments, the spacer is less than 250 amino acids in length, less than 200 amino acids in length, less than 150 amino acids in length, less than 100 amino acids in length, less than 75 amino acids in length, less than 50 amino acids in length, less than 25 amino acids in length, less than 20 amino acids in length, less than 15 amino acids in length, less than 12 amino acids in length, or less than 10 amino acids in length. In some embodiments, the spacer is from or about 10 to 250 amino acids in length, 10 to 150 amino acids in length, 10 to 100 amino acids in length, 10 to 50 amino acids in length, 10 to 25 amino acids in length, 10 to 15 amino acids in length, 15 to 250 amino acids in length, 15 to 150 amino acids in length, 15 to 100 amino acids in length, 15 to 50 amino acids in length, 15 to 25 amino acids in length, 25 to 250 amino acids in length, 25 to 100 amino acids in length, 25 to 50 amino acids in length, 50 to 250 amino acids in length, 50 to 150 amino acids in length, 50 to 100 amino acids in length, 100 to 250 amino acids in length, 100 to 150 amino acids in length or 150 to 250 amino acids in length.

Exemplary spacers include an IgG4 hinge alone, an IgG4 hinge linked to the CH2 and CH3 domains, or an IgG4 hinge linked to the CH3 domain. Exemplary spacers include, but are not limited to, those described in Hudecek et al . (2013) Clin. Cancer Res ., 19:3153 or International Patent Application Publication No. WO2014031687. In some embodiments, the spacer has the sequence set forth in SEQ ID NO: 131 and is encoded by the sequence set forth in SEQ ID NO: 132. In some embodiments, the spacer has the sequence set forth in SEQ ID NO: 133. In some embodiments, the spacer has the sequence set forth in SEQ ID NO: 134.

In some embodiments, the constant region or portion is taken from IgD. In some embodiments, the spacer has a sequence as set forth in SEQ ID NO: 135. In some embodiments, the spacer has an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS: 131, 133, 134 and 135.

The antigen recognition domain is generally associated with one or more intracellular signaling components, such as signaling components that mimic activation through an antigen receptor complex, such as the TCR complex, in the case of a CAR, and/or a signal through another cell surface receptor. Thus, in some embodiments, the antigen binding component (e.g., an antibody) is associated with one or more transmembrane and intracellular signaling regions. In some embodiments, the transmembrane domain is fused to an extracellular domain. In one embodiment, a transmembrane domain is used that is naturally associated with one or more domains in a receptor, such as a CAR. In some cases, the transmembrane domain is selected or modified by amino acid substitution so as to avoid association of such domains with transmembrane domains of the same or other surface membrane proteins to minimize interactions with other members of the receptor complex.

The transmembrane domain in some embodiments is obtained from either a natural or synthetic source. If the source is natural, the domain in some aspects is obtained from any membrane-associated or transmembrane protein. The transmembrane region includes one that is obtained from (i.e., comprises at least the transmembrane regions of) the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. Alternatively, the transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues, such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan, and valine is found at each end of the synthetic transmembrane domain. In some embodiments, the linkage occurs through linkers, spacers, and/or transmembrane domains.

The intracellular signaling region includes one that mimics or closely matches the signal through a natural antigen receptor, the signal through such a receptor in combination with a costimulatory receptor, and/or the signal through a costimulatory receptor alone. In some embodiments, a short oligo- or polypeptide linker is present, such as a linker of 2 to 10 amino acids in length, such as one comprising glycines and serines, such as a glycine-serine doublet, and forms a link between the transmembrane domain and the cytoplasmic signaling domain of the CAR.

A receptor, such as a CAR, typically includes at least one intracellular signaling component or components. In some embodiments, the receptor includes an intracellular component of a TCR complex, such as a TCR CD3 chain that mediates T cell activation and cytotoxicity, such as a CD3 zeta chain. Thus, in some aspects, a ROR1-binding antibody is linked to one or more cell signaling molecules. In some embodiments, a cell signaling molecule includes a CD3 transmembrane domain, CD3 intracellular binding domains, and/or other CD transmembrane domains. In some embodiments, a receptor, such as a CAR, further includes a portion of one or more additional molecules, such as Fc receptor γ, CD8, CD4, CD25, or CD16. For example, in some aspects, a CAR includes a chimeric molecule between CD3 zeta (CD3-ζ) or Fc receptor γ and CD8, CD4, CD25, or CD16.

In some embodiments, when a CAR is cross-linked, the cytoplasmic domain or intracellular signaling region of the CAR activates at least one of the normal effector functions or responses of an immune cell, such as a T cell, engineered to express the CAR. For example, in some contexts, the CAR induces T cell function, such as cytolytic activity, or T helper activity, such as secretion of cytokines or other factors. In some embodiments, a truncated portion of the intracellular signaling region of an antigen receptor component or costimulatory molecule is used in place of a native immunostimulatory chain, such as if it transmits a signal for an effector function. In some embodiments, intracellular signaling regions, such as those comprising an intracellular domain or domains, comprise cytoplasmic sequences of a T cell receptor (TCR), and in some aspects also coreceptors that in a natural context act in concert with such a receptor to initiate signal transduction upon binding to an antigen receptor, and/or any derivative or variant of such molecules, and/or any synthetic sequence that has such functionality.

In the context of a natural TCR, full activation typically requires not only signaling through the TCR, but also a costimulatory signal. Thus, in some embodiments, to facilitate full activation, a component for generating a secondary or costimulatory signal is also included in the CAR. In other embodiments, the CAR does not include a component for generating a costimulatory signal. In some aspects, an additional CAR is expressed in the same cell and provides a component for generating a secondary or costimulatory signal.

T cell activation has been described in some aspects as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to produce a secondary or costimulatory signal (secondary cytoplasmic signaling sequences). In some aspects, a CAR includes one or both of these signaling components.

In some aspects, the CAR comprises a primary cytoplasmic signaling sequence that regulates primary activation of the TCR complex. Primary cytoplasmic signaling sequences that act in a stimulatory manner may comprise signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAM-containing primary cytoplasmic signaling sequences include those derived from the TCR or CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, FcR gamma, or FcR beta. In some embodiments, the cytoplasmic signaling molecules in the CAR comprise a cytoplasmic signaling domain, a portion thereof, or a sequence derived from CD3 zeta.

In some cases, CARs are referred to as first, second, and/or third generation CARs. In some aspects, first generation CARs are those that provide only a CD3 chain-evoked signal upon antigen binding; in some aspects, second generation CARs are those that provide such a signal and a costimulatory signal, such as including an intracellular binding domain from a costimulatory receptor such as CD28 or CD137; in some aspects, third generation CARs are in some aspects those that include multiple costimulatory domains of different costimulatory receptors.

In some embodiments, the chimeric antigen receptor comprises an extracellular portion comprising an antibody or fragment as described herein. In some aspects, the chimeric antigen receptor comprises an extracellular portion comprising an antibody or fragment as described herein and an intracellular binding domain. In some embodiments, the antibody or fragment comprises an scFv or a single domain V _H antibody and the intracellular domain comprises an ITAM. In some aspects, the intracellular binding domain comprises the zeta chain signaling domain of the CD3 zeta (CD3ζ) chain. In some embodiments, the chimeric antigen receptor comprises a transmembrane domain located between the extracellular domain and the intracellular signaling region.

In some aspects, the transmembrane domain comprises a transmembrane portion of CD28. The extracellular domain and the transmembrane may be linked directly or indirectly. In some embodiments, the extracellular domain and the transmembrane are linked by a spacer, such as any described herein. In some embodiments, the chimeric antigen receptor comprises an intracellular domain of a T cell costimulatory molecule, such as between the transmembrane domain and the intracellular binding domain. In some aspects, the T cell costimulatory molecule is CD28 or 4-1BB.

In some embodiments, the CAR comprises an antibody, such as an antibody fragment, a transmembrane domain that is or comprises a transmembrane portion of CD28 or a functional variant thereof, and an intracellular binding domain that comprises a CD28 signal portion or a functional variant thereof, and a CD3 zeta signal portion or a functional variant thereof. In some embodiments, the CAR comprises an antibody, such as an antibody fragment, a transmembrane domain that is or comprises a transmembrane portion of CD28 or a functional variant thereof, and an intracellular binding domain that comprises a 4-1BB signal portion or a functional variant thereof, and a CD3 zeta signal portion or a functional variant thereof. In some such embodiments, the receptor further comprises a spacer that comprises a portion of an Ig molecule, such as a human Ig molecule, such as an Ig hinge, such as an IgG4 hinge, such as a hinge only spacer.

In some embodiments, the transmembrane domain of a receptor, e.g., a CAR, is a transmembrane domain of human CD28 or a variant thereof, e.g., the 27-amino acid transmembrane domain of human CD28 (Accession No.: P10747.1), or a transmembrane domain that comprises the amino acid sequence set forth in SEQ ID NO: 136, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 136; in some embodiments, the transmembrane domain comprising the recombinant receptor portion comprises the amino acid sequence set forth in SEQ ID NO: 137 or an amino acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

In some embodiments, the chimeric antigen receptor comprises an intracellular domain of a T cell costimulatory molecule. In some aspects, the T cell costimulatory molecule is CD28 or 4-1BB.

In some embodiments, the intracellular signaling region comprises the intracellular costimulatory signaling domain of human CD28 or a functional variant or portion thereof, such as the 41 amino acid domain thereof and/or the domain with the LL to GG substitution at positions 186-187 of the native CD28 protein. In some embodiments, the intracellular binding domain may comprise the amino acid sequence set forth in SEQ ID NO: 138 or 139, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 138 or 139. In some embodiments, the intracellular region comprises the 4-1BB intracellular costimulatory signaling domain or a functional variant or portion thereof, such as the 42-amino acid cytoplasmic domain of human 4-1BB (Accession No. Q07011.1) or a functional variant or portion thereof, such as the amino acid sequence set forth in SEQ ID NO: 140, or the sequence amino acids that exhibit at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 140.

In some embodiments, the intracellular signaling region comprises a human CD3 chain, optionally a CD3 zeta stimulatory signaling domain or a functional variant thereof, such as the 112 AA cytoplasmic domain of human CD3ζ isoform 3 (Accession No.: P20963.2) or the CD3 zeta signaling domain described in U.S. Patent No.: 7,446,190 or U.S. Patent No. 8,911,993. In some embodiments, the intracellular signal region comprises an amino acid sequence as set forth in SEQ ID NO: 129, 130, or 141, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 129, 130, or 141.

In some aspects, the spacer comprises only an IgG hinge region, such as only an IgG4 or IgG1 hinge, such as only the hinge spacer shown in SEQ ID NO:131. In other embodiments, the spacer is an Ig hinge, such as an IgG4 hinge linked to the _CH2 and/or _CH3 domains. In some embodiments, the spacer is an Ig hinge, such as an IgG4 hinge linked to the _CH2 and _CH3 domains, such as shown in SEQ ID NO:133. In some embodiments, the spacer is an Ig hinge, such as an IgG4 hinge linked to only the _CH3 domain, such as shown in SEQ ID NO:134. In some embodiments, the spacer is or comprises a glycine-serine rich sequence or other flexible linker, such as known flexible linkers.

In some embodiments, the CAR comprises an anti-HPV 16 E6 or E7 antibody or fragment, including sdAbs (e.g., comprising only a V _H region) and an scFv, a spacer such as any of the Ig hinge spacers, a CD28 transmembrane domain, a CD28 intracellular binding domain, and a CD3 zeta signaling domain. In some embodiments, the CAR comprises an HPV 16 antibody or fragment, including sdAbs and scFvs, a spacer such as any of the Ig hinge spacers, a CD28 transmembrane domain, a CD28 intracellular binding domain, and a CD3 zeta signaling domain. In some embodiments, such CAR constructs further comprise a T2A ribosome skipping element and/or a tEGFR sequence, for example, downstream of the CAR.

In some embodiments, the CAR or antigen-binding fragment thereof is encoded by a nucleotide sequence that is or has been codon-optimized. Exemplary codon-optimized embodiments are described elsewhere herein.

C. TCR-like CARs

In some embodiments, the antibody or antigen-binding portion thereof is expressed on cells as part of a recombinant receptor, such as an antigen receptor. Antigen receptors include functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs). In general, a CAR comprising an antibody or antigen-binding fragment that exhibits TCR-like specificity directed against a peptide in the context of an MHC molecule may also be referred to as a TCR-like CAR.

In some embodiments, a CAR comprises a TCR-like antibody, such as an antibody or antigen-binding fragment ( e.g., scFv) that specifically recognizes an intracellular antigen, such as a tumor-associated antigen, presented on the cell surface as an MHC-peptide complex. In some embodiments, an antibody or antigen-binding portion thereof that recognizes an MHC-peptide complex can be expressed on cells as part of a recombinant receptor, such as an antigen receptor. Antigen receptors include functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs). In general, a CAR comprising an antibody or antigen-binding fragment that exhibits TCR-like specificity directed against peptide-MHC complexes may also be referred to as a TCR-like CAR.

Reference to a “major histocompatibility complex” (MHC) refers to a protein, usually a glycoprotein, that contains a polymorphic peptide binding site or binding cavity that may, in some cases, form a complex with peptide antigens of polypeptides, including peptide antigens processed by the cellular machinery. In some cases, MHC molecules may be produced or expressed on the cell surface, for example, as a complex with a peptide, i.e., an MHC-peptide complex, to present the antigen in a conformation recognized by an antigen receptor on T cells, such as a TCR or a TCR-like antibody. In general, MHC class I molecules are heterodimers having a transmembrane α chain, in some cases with three α domains, and a non-covalently associated β2 microglobulin. In general, class II MHC molecules consist of two transmembrane glycoproteins, α and β, both of which typically span a membrane. An MHC molecule may include an effective MHC portion that contains the antigen-binding site or sites for peptide binding and sequences necessary for recognition by an appropriate antigen receptor. In some embodiments, class I MHC molecules deliver peptides nascent in the cytosol to the cell surface, where the MHC-peptide complex is recognized by T cells, such as primarily CD8 ⁺ T cells but in some cases CD4+ T cells. In some embodiments, class II MHC molecules deliver peptides nascent in the vesicular system to the cell surface, where they are typically recognized by CD4 ⁺ T cells. In general, MHC molecules are encoded by a group of linked loci, collectively referred to as H-2, in mouse and human leukocyte antigen (HLA) in humans. Therefore, human MHC can also be called human leukocyte antigen (HLA).

The term "MHC-peptide complex" or "peptide-MHC complex" or variations thereof refers to a complex or association of a peptide antigen and an MHC molecule, such as, typically, non-covalent interactions of the peptide in a binding cavity or cleft of the MHC molecule. In some embodiments, the MHC-peptide complex is present or displayed on the surface of a cell. In some embodiments, the MHC-peptide complex can be specifically recognized by an antigen receptor, such as a TCR, a TCR-like CAR, or antigen-binding portions thereof.

In some embodiments, a peptide, such as a peptide antigen or epitope, of a polypeptide can be associated with an MHC molecule, such as for recognition by an antigen receptor. In general, the peptide is derived from or based on a fragment of a longer biological molecule, such as a polypeptide or protein. In some embodiments, the peptide is typically about 8 to about 24 amino acids in length. In some embodiments, the peptide is from or about 9 to 22 amino acids in length for recognition in an MHC class II complex. In some embodiments, the peptide is from or about 8 to 13 amino acids in length for recognition in an MHC class I complex. In some embodiments, upon recognition of the peptide in the context of an MHC molecule, such as an MHC-peptide complex, an antigen receptor, such as a TCR or a TCR-like CAR, elicits or triggers an activation signal in a T cell that elicits a T cell response, such as T cell proliferation, cytokine production, a cytotoxic T cell response, or another response.

In some embodiments, the TCR-like antibody or antigen-binding portion thereof is known or can be produced by known methods (see, e.g., U.S. Published Application Nos. US2002/0150914; US2003/0223994; US2004/0191260; US2006/0034850; US2007/00992530; US20090226474; US20090304679; and PCT International Publication No. WO 03/068201).

In some embodiments, an antibody or antigen-binding portion thereof that specifically binds to an MHC-peptide complex can be produced by immunizing a host with an effective amount of an immunogen comprising a particular MHC-peptide complex. In some cases, the peptide of the MHC-peptide complex is an epitope of an antigen capable of binding to MHC, such as a tumor antigen, for example, a universal tumor antigen, a myeloma antigen, or another antigen as described herein. In some embodiments, an effective amount of the immunogen is then administered to the host to elicit an immune response, wherein the immunogen maintains its three-dimensional shape for a period sufficient to elicit an immune response against the three-dimensional presentation of the peptide in the binding cavity of the MHC molecule. Serum collected from the host is then analyzed to determine whether antibodies have been produced that recognize the three-dimensional presentation of the peptide in the binding cavity of the MHC molecule. In some embodiments, the antibodies produced can be analyzed to assess that the antibody can distinguish between an MHC-peptide complex and an MHC molecule alone, the peptide in question alone, and a complex of MHC and an irrelevant peptide. The desired antibodies can then be isolated.

In some embodiments, an antibody or antigen-binding portion thereof that specifically binds to an MHC-peptide complex can be produced using antigen display library methods, such as phage-antibody libraries. In some embodiments, phage display libraries of mutant Fabs, scFvs, or other forms of antibodies can be generated, e.g., in which library members are mutated at one or more CDR or CDR residues. See, e.g., U.S. Published Application Nos. US20020150914, US2014/0294841; and Cohen CJ et al . (2003) J Mol. Recogn . 16:324-332.

The present embodiments include recombinant receptors, such as those that include antibodies, such as TCR-like antibodies. In some embodiments, antigen receptors and other chimeric receptors specifically bind to a region or epitope of an antigen, such as TCR-like antibodies. Antigen receptors are non-TCR antigen receptors, such as chimeric antigen receptors (CARs). Also provided are cells expressing CARs and their use in adoptive cell therapy, such as the treatment of diseases and disorders associated with antigen and/or epitope expression.

Thus, provided herein are TCR-like CARs that comprise a non-TCR molecule that exhibits specificity for a T cell receptor, such as a T cell epitope or a peptide epitope, when displayed or present in the context of an MHC molecule. In some embodiments, a TCR-like CAR can comprise an antibody or an antigen-binding portion thereof, for example, a TCR-like antibody as described herein. In some embodiments, the antibody or antibody-binding portion thereof is responsive to its specific peptide epitope in the context of an MHC molecule, wherein the antibody or antibody fragment can distinguish a specific peptide in the context of an MHC molecule from only an MHC molecule, only a specific peptide, and, in some cases, an irrelevant peptide in the context of an MHC molecule. In some embodiments, the antibody or antigen-binding portion thereof can exhibit a binding affinity greater than the T cell receptor.

In some aspects, the transgene may include nucleic acids encoding one or more CARs, such as a first CAR that contains signaling domains for eliciting a primary signal, and a second CAR that binds to a second antigen and contains a component for creating a costimulatory signal. For example, the first CAR may be an activating CAR, and the second CAR may be a costimulatory CAR. In some aspects, both CARs must be ligated in order to elicit a specific effector function in a cell, which can impart specificity and selectivity to a target cell type.

In some embodiments, the activating domain is included in one CAR, the costimulatory component is another chimeric receptor that recognizes a different antigen. In some embodiments, the CARs include activating or stimulatory CARs and costimulatory receptors, both of which are expressed on the same cell (see WO2014/055668). In some aspects, the receptor comprising the HPV 16 E6 or E7 antibody is a stimulatory or activating CAR; in other aspects, it is a costimulatory receptor. In some embodiments, the transgene further encodes an inhibitory CAR (iCAR, see Fedorov et al., Sci. Transl. Medicine , 5(215) (December, 2013), such as an inhibitory receptor that recognizes a peptide epitope other than HPV 16 E6 or HPV16 E7, whereby the activating signal delivered via the HPV 16-targeted CAR is attenuated or inhibited by binding of the inhibitory CAR to its ligand, e.g., to reduce off-target activity.

In some embodiments, the transgene may include nucleic acids encoding a recombinant receptor, which may further encode an additional receptor, such as a receptor capable of delivering a costimulatory or pro-survival signal, such as a costimulatory receptor (see WO2014/055668) and/or blocking or altering the output of an inhibitory signal, such as a signal normally delivered through an immune checkpoint or other immune inhibitory molecules, such as those expressed in the tumor microenvironment, for example, to promote increased efficacy of such engineered cells. See, for example, Tang et al., Am J Transl Res. 2015; 7(3): 460-473. In some embodiments, the cell may further comprise one or more exogenous or recombinant or engineered components, such as one or more exogenous factors and/or costimulatory ligands that are expressed on and in or secreted by the cells and may facilitate function, such as in the microenvironment. Examples of such ligands and components include, for example, TNFα and/or Ig family receptors or ligands, such as 4-1BBL, CD40, CD40L, CD80, CD86, cytokines, chemokines, and/or antibodies or other molecules, such as scFv. See, for example, Patent Application Publication Nos. WO2008121420 A1, WO2014134165 A1, US20140219975 A1.

D. Chimeric autoantibody receptor (CAAR)

In some embodiments, the chimeric receptor is a chimeric autoantibody receptor (CAAR). In some embodiments, the CAAR binds, such as specifically binds to or recognizes, an autoantibody. In some embodiments, a cell expressing a CAAR, such as a T cell engineered to express a CAAR, can be used to bind to and kill cells expressing an autoantibody, but not cells expressing a normal antibody. In some embodiments, CAAR-expressing cells can be used to treat an autoimmune disease associated with the expression of autoantigens, such as autoimmune diseases. In some embodiments, CAAR-expressing cells can target B cells that ultimately produce autoantibodies and display autoantibodies on their cell surfaces, and mark these B cells as disease-specific targets for therapeutic intervention. In some embodiments, CAAR-expressing cells can be used to effectively target and kill pathogenic B cells in autoimmune diseases by targeting disease-causing B cells using an antigen-specific chimeric autoantibody receptor. In some embodiments, the chimeric receptor is a CAAR, such as any of those described in U.S. Patent Application Publication No. US 2017/0051035.

In some embodiments, the CAAR comprises an autoantibody binding domain, a transmembrane domain, and one or more intracellular signaling regions or domains (also interchangeably referred to as a cytoplasmic signaling domain or region). In some embodiments, the intracellular signaling region comprises an intracellular binding domain. In some embodiments, the intracellular binding domain is or comprises a primary signaling region, a signaling domain that is capable of stimulating and/or inducing a primary activation signal in a T cell, a signaling domain of a T cell receptor (TCR) component (e.g., an intracellular binding domain or region of the CD3 zeta (CD3ζ) chain or a functional variant or signaling portion thereof), and/or a signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM).

In some embodiments, the autoantibody binding domain comprises an autoantigen or a fragment thereof. The choice of autoantigen may depend on the type of autoantibody being targeted. For example, the autoantigen may be selected because it recognizes an autoantibody on a target cell, such as a B cell, associated with a particular disease state, such as an autoimmune disease, such as an autoantibody-mediated autoimmune disease. In some embodiments, the autoimmune disease includes pemphigus vulgaris (PV). Exemplary autoantigens include desmoglein 1 (Dsg1) and Dsg3.

V. COMPOSITIONS AND STRUCTURES

Also provided are populations of engineered cells, compositions comprising such cells and/or enriched in such cells. The compositions include pharmaceutical compositions and formulations for administration, for example, for adoptive cell therapy. Also provided are therapeutic methods for administering cells and compositions to subjects, such as patients.

In some embodiments, the provided population of cells and/or composition comprising the engineered cells comprises a population of cells that exhibits a better, more uniform, homogeneous and/or stable expression and/or antigen binding of the recombinant receptor, such as exhibiting a reduced coefficient of variation, compared to the expression and/or antigen binding of cell populations and/or compositions created using conventional methods. In some embodiments, the population of cells and/or compositions exhibit at least a coefficient of variation of transgene expression and/or antigen binding of the recombinant receptor that is 100%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% lower than a corresponding population created using conventional methods, such as random integration of the transgene. The coefficient of variation is defined as the standard deviation of the expression of a subject nucleic acid (e.g., a transgene encoding a recombinant receptor) in a population of cells, such as CD4+ and/or CD8+ T cells, divided by the mean expression of the corresponding subject nucleic acid in the corresponding population of cells. In some embodiments, the population of cells and/or compositions exhibit a coefficient of variation that is lower than 0.70, 0.65, 0.60, 0.55, 0.50, 0.45, 0.40, 0.35, or 0.30 or less, when measured in populations of CD4+ and/or CD8+ T cells that are engineered using the methods provided herein.

In some embodiments, a population of cells and/or compositions are provided that include cells that have a targeted knock-in of a recombinant receptor-encoding transgene at one or more endogenous TCR gene loci, thereby having a knock-out of one or more endogenous TCR gene loci, such as a knock-out of a targeted gene for integration, such as TRAC, TRBC1 , and/or TRBC2 . In some embodiments, all or substantially all of the cells in the population of cells into which the recombinant receptor-encoding transgene is integrated also have a knock-out of one or more endogenous TCR gene loci. In some embodiments, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the cells in the population of cells and/or compositions that express the recombinant receptor comprise a knockout of one or more endogenous TCR gene loci, such as TRAC, TRBC1 and/or TRBC2 . Thus, in the present population of cells and/or compositions, all or substantially all of the engineered cells that express the recombinant receptor also comprise a knockout of the endogenous TCR, due to the targeted knockin of the transgene at the endogenous TCR gene loci.

In some embodiments, a population of cells and/or compositions are provided that comprise a plurality of engineered immune cells comprising a recombinant receptor or antigen-binding fragment thereof encoded by a transgene and a genetic disruption of at least one target site in a T cell receptor constant alpha (TRAC) gene and/or a T cell receptor constant beta (TRBC) gene, wherein at least or more than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the cells in the composition comprise a genetic disruption at a target position in a T cell receptor constant alpha (TRAC) gene and/or a T cell receptor constant beta (TRBC) gene; and a transgene encoding a recombinant TCR or an antigen-binding fragment or chain thereof directed to or near a target position via homology-directed repair (HDR).

In some embodiments, expression and/or antigen binding by the recombinant receptor can be assessed using any of the reagents and/or assays described herein, such as in Section I.C. In some embodiments, expression is measured using a binding molecule that recognizes and/or specifically binds to the recombinant receptor or a portion thereof. For example, in some embodiments, expression of the recombinant receptor encoded by the transgene is assessed using an anti-TCR Vβ 22 antibody, such as by flow cytometry. In some embodiments, antigen binding of the recombinant receptor, which is the TCR, can be assessed using an antigen that is isolated or purified or is recombinant, cells expressing a particular antigen, and/or using a TCR ligand (MHC-peptide complex).

In some embodiments, compositions are provided comprising cells, wherein cells expressing a recombinant receptor and/or comprising a knockout of one or more genes encoding an endogenous TCR constitute at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of all cells in the composition or of a particular cell type, such as T cells or CD8+ or CD4+ cells.

Also provided are compositions comprising cells for administration, including pharmaceutical compositions and formulations, such as unit dosage form compositions comprising a number of cells for administration in a given dose or a portion thereof. Pharmaceutical compositions and formulations typically include one or more optional pharmaceutically acceptable carriers or excipients. In some embodiments, the composition includes at least one additional therapeutic agent.

The term "pharmaceutical formulation" refers to a preparation that is in a form that permits effective biological activity of the active ingredient contained therein and that does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered.

"Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation other than the active ingredient that is nontoxic to the subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

In some aspects, the choice of carrier is determined in part by the particular cell and/or route of administration. Accordingly, there are a variety of suitable formulations. For example, the pharmaceutical composition may contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of from about 0.0001% to about 2% by weight of the total composition. Carriers are described, for example, in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants such as polyethylene glycol (PEG).

Buffering agents are, in some aspects, included in the compositions. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of from about 0.001% to about 4% by weight of the total composition. Methods for the preparation of pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams &Wilkins; 21st ed. (May 1, 2005).

The formulations may include aqueous solutions. The formulation or composition may also contain more than one active ingredient useful for a particular indication, disease or condition treated by the cells, preferably those whose activity is complementary to the cells, wherein the respective activities do not adversely affect each other. Such active ingredients are typically present in combination in amounts effective for the intended purpose. Thus, in some embodiments, the pharmaceutical composition further comprises other pharmaceutically active agents or drugs, such as chemotherapeutic agents, for example, asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitablastaxel, rituximab, vinblastine and/or vincristine.

The pharmaceutical composition in some embodiments comprises cells in amounts effective to treat or prevent a disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic effectiveness in some embodiments is monitored by periodic assessment of treated subjects. The desired dose can be delivered by a single bolus administration of cells, multiple bolus administration of cells, or continuous infusion of cells.

The cells and compositions can be administered using standard administration techniques, formulations and/or devices. Administration of the cells can be autologous or heterologous. In some aspects, the cells are isolated from a subject, engineered and administered to the same subject. In other aspects, they are isolated from one subject, engineered and administered to another subject. For example, immunoresponsive cells or progenitors can be obtained from one subject and administered to the same subject or to another compatible subject. Immunoreactive cells obtained from peripheral blood or their progeny (e.g., obtained in vivo, ex vivo or in vitro) can be administered by local injection, including catheter administration, systemic injection, local injection, intravenous injection or parenteral administration. When administering a therapeutic composition (e.g., a pharmaceutical composition comprising a genetically modified immunoresponsive cell), it will initially be formulated as a unit dosage form for injection (solution, suspension, emulsion).

The formulations include formulations for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell populations are administered parenterally. As used herein, the term "parenteral" includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the cells are administered to a subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

The compositions in some embodiments are presented as sterile liquid preparations, for example, isotonic aqueous solutions, suspensions, emulsions, dispersions or viscous compositions, which in some aspects can be buffered to a selected pH. Liquid preparations are generally easier to prepare than gels, other viscous compositions and solid compositions. In addition, liquid compositions are somewhat more convenient to administer, especially by injection. On the other hand, viscous compositions can be formulated with an appropriate range of viscosity to provide longer periods of contact with specific tissues. Liquid or viscous compositions can contain carriers, which can be a solvent or dispersion medium containing, for example, water, saline, phosphate-buffered saline, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.

Sterile injectable solutions can be prepared by incorporating the cells in a vehicle, for example, in admixture with a suitable carrier, diluent or excipient such as sterile water, saline, glucose, dextrose or the like. The compositions may contain auxiliary substances such as wetting, dispersing or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity-enhancing agents, preservatives, flavoring and/or coloring agents, depending on the route of administration and the desired preparation. In some cases, standard texts may be consulted for the preparation of suitable preparations.

Various additives that enhance the stability and sterility of the composition may be added, including antimicrobial preservatives, antioxidants, chelating agents and buffers. Prevention of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol and sorbic acid. Prolonged absorption of the injectable pharmaceutical form can be achieved by using agents that delay absorption, such as aluminum monostearate and gelatin.

Formulations used for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through sterile filter membranes.

VI. METHODS OF ADMINISTRATION AND USE IN ADOPTIVE CELL THERAPY

Provided are methods for administering cells, populations and compositions and using such cells, populations and compositions to prevent diseases, conditions and disorders, including cancer. In some embodiments, the cells, populations and compositions are administered to a subject or patient having a particular disease or condition to be treated, such as by adoptive cell therapy, such as adoptive T cell therapy. In some embodiments, cells and compositions prepared with the provided methods, such as engineered compositions and compositions at the end of the manufacturing cycle after incubation and/or other processing steps, are administered to a subject, such as a subject having or at risk for a disease or condition. In some aspects, the methods thus treat, such as alleviate one or more symptoms of a disease or condition, such as by reducing tumor burden in a cancer expressing an antigen recognized by an engineered T cell.

As used herein, the term "subject" is a mammal, such as a human or other animal, and is typically a human. In some embodiments, the subject, such as a patient, to whom the cells, cell populations, or compositions are administered is a mammal, typically a primate, such as a human. In some embodiments, the primate is a monkey or ape. The subject may be a male or female, and may be of any suitable age, including infants, children, adolescents, adults, and the elderly. In some embodiments, the subject is a non-primate mammal, such as a rodent.

As used herein, the term "treatment" (and its grammatical variations such as "treat" or "treatment") refers to the alleviation or reduction, in whole or in part, of a disease or condition or disorder, or of a symptom, adverse effect, or outcome or phenotype associated therewith. Desirable effects of treatment include, but are not limited to, prevention of occurrence or recurrence of the disease, relief of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. The terms do not imply a complete cure of the disease or a complete elimination of any symptom or an effect on all symptoms or outcomes.

as used herein, the term "arrest of disease progression" means postponing, containing, slowing, inhibiting, stabilizing, suppressing, and/or delaying the progression of a disease (e.g., cancer). This delay may be of varying duration, depending on the history of the disease and/or the individual being treated. A sufficient or significant delay may, in effect, involve prevention in the sense that the individual does not develop the disease. For example, advanced cancer, such as the development of metastases, may be delayed.

"Prevention" as used herein includes preventing the occurrence or recurrence of a disease in a subject who may be predisposed to the disease but who has not yet been diagnosed with the disease. In some embodiments, the cells and compositions provided are used to delay the development of a disease or to slow the progression of a disease.

As used herein, the term "suppression" of a function or activity means a decrease in the function or activity relative to other such conditions, excluding the condition or parameter of interest, or alternatively, relative to another condition. For example, tumor growth suppressor cells reduce the rate of tumor growth relative to the rate of tumor growth in the absence of the cells.

An "effective amount" of a pharmaceutical composition, cell, or formulation in the context of administration refers to an effective amount in doses/amounts and for periods of time necessary to achieve a desired result, such as a therapeutic or prophylactic result.

A "therapeutically effective amount" of a pharmaceutical composition or cells refers to an amount effective at dosages and for periods of time necessary to achieve a desired therapeutic result, such as treatment of a disease, condition or disorder and/or a pharmacokinetic or pharmacodynamic effect of treatment. A therapeutically effective amount may vary depending on factors such as the disease state, age, sex and weight of the subject, and the population of cells administered. In some embodiments, the methods provided include administering cells and/or compositions in effective amounts, such as therapeutically effective amounts.

"Prophylactically effective amount" refers to an amount effective at dosages and for periods of time necessary to achieve the desired prophylactic result. Typically, but not necessarily, because the prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount. In the context of a smaller tumor burden, the prophylactically effective amount will in some cases be higher than the therapeutically effective amount.

Methods of administering cells for adoptive cell therapy are known and can be used in connection with the present methods and compositions. For example, methods of adoptive T cell therapy are described, for example, in U.S. Patent Application Publication No. 2003/0170238 to Gruenberg et al; U.S. Patent No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, for example, Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338.

In some embodiments, cell therapy, such as adoptive T cell therapy, is performed by autologous transfer, in which cells are isolated and/or otherwise obtained from a subject to be treated with cell therapy or from a sample obtained from such a subject. Thus, in some aspects, cells are obtained from a subject, such as a patient, in need of treatment, and the cells, after isolation and processing, are administered to the same subject.

In some embodiments, the cell therapy, such as adoptive T cell therapy, is performed by allogeneic transfer, in which cells are isolated and/or otherwise obtained from a subject other than the subject to receive or who will ultimately receive the cell therapy, such as a first subject. In such embodiments, the cells are then administered to another subject, such as a second subject of the same species. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.

The cells may be administered by any suitable means. Dosing and administration may depend in part on whether the administration is short-term or continuous. Different dosing regimens include, but are not limited to, single or multiple administrations at different time points, bolus administration, and pulse infusion.

In some embodiments, the subject is treated with a therapeutic agent targeting a disease or condition, such as a tumor, prior to administration of the cell or composition comprising cells. In some aspects, the subject is difficult to treat or refractory to another therapeutic agent. In some embodiments, the subject has a difficult to treat or recurrent disease, such as after treatment with another therapeutic intervention, including chemotherapy, radiation, and/or hematopoietic stem cell transplant (HSCT), such as allogeneic HSCT. In some embodiments, the administration effectively treats the subject despite the fact that the subject has become refractory to another therapy.

In some embodiments, the subject is sensitive to another therapeutic agent, and treatment with the therapeutic agent reduces the severity of the disease. In some aspects, the subject is initially sensitive to the therapeutic agent, but over time, the subject experiences a relapse of the disease or condition. In some embodiments, the subject does not relapse. In some such embodiments, the subject is determined to be at risk for relapse, such as at high risk for relapse, and therefore the cells are administered prophylactically, such as to reduce the likelihood of or prevent relapse.

In some aspects, the subject has not previously received treatment with another therapeutic agent.

The disease or condition treated in some aspects can be any in which the expression of an antigen associated with, specific for and/or expressed on a cell or tissue of a disease, disorder or condition, and/or involved in the etiology of the disease, condition or disorder, e.g., causes, exacerbates or otherwise participates in such a disease, condition or disorder. Exemplary diseases and conditions can include diseases or conditions associated with malignancy or cell transformation (e.g., cancer), an autoimmune or inflammatory disease, or an infectious disease, e.g., caused by a bacterial, viral or other pathogen. Exemplary antigens, which include antigens associated with various diseases and conditions that can be treated, are described herein. In particular embodiments, an immunomodulatory polypeptide and/or a recombinant receptor, e.g., a chimeric antigen receptor or TCR, specifically binds to an antigen associated with a disease or condition. In some embodiments, the subject has a disease, disorder, or condition, optionally a cancer, tumor, autoimmune disease, disorder, or condition, or infectious disease.

In some embodiments, the disease, disorder, or condition includes tumors associated with various types of cancer. In some embodiments, the cancer can be any cancer localized in the subject's body, such as, but not limited to, cancers located in the head and neck, breast, liver, colon, ovaries, prostate, pancreas, brain, cervix, bone, skin, eye, bladder, stomach, esophagus, peritoneum, or lung. For example, the anti-cancer agent can be used to treat colon cancer, cervical cancer, central nervous system cancer, breast cancer, bladder cancer, anal carcinoma, head and neck cancer, ovarian cancer, endometrial cancer, small cell lung cancer, non-small cell lung carcinoma, neuroendocrine cancer, soft tissue carcinoma, penile cancer, prostate cancer, pancreatic cancer, stomach cancer, gallbladder cancer, or esophageal cancer. In some cases, the cancer can be a blood cancer. In some embodiments, the disease, disorder, or condition is a tumor, such as a solid tumor, lymphoma, leukemia, a blood tumor, a metastatic tumor, or another type of cancer or tumor. In some embodiments, the disease, disorder, or condition is selected from colon cancer, lung cancer, liver cancer, breast cancer, prostate cancer, ovarian cancer, skin cancer, melanoma, bone cancer, brain cancer, ovarian cancer, epithelial cancer, renal cell carcinoma, pancreatic adenocarcinoma, cervical carcinoma, colorectal cancer, glioblastoma, neuroblastoma, Ewing's sarcoma, medulloblastoma, osteosarcoma, synovial sarcoma, and/or mesothelioma.

Diseases, conditions, and disorders include tumors, including solid tumors, hematologic malignancies, and melanomas, and including localized and metastatic tumors, infectious diseases such as infection with a virus or other pathogen such as HIV, HCV, HBV, CMV, HPV, and parasitic diseases, autoimmune diseases, and inflammatory diseases. In some embodiments, the disease, disorder, or condition is a tumor, cancer, malignancy, neoplasm, or other proliferative disease or disorder. Such diseases include, but are not limited to, leukemia, lymphoma such as acute myeloid (or myelogenous) leukemia (AML), chronic myeloid (or myelogenous) leukemia (CML), acute lymphocytic (or lymphoblastic) leukemia (ALL), chronic lymphocytic leukemia (CML), hairy cell leukemia (HCL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt lymphoma, Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), anaplastic large cell lymphoma (ALCL), follicular lymphoma, intractable follicular lymphoma, diffuse large B-cell lymphoma (DLBCL), and multiple myeloma (MM), a B-cell malignancy selected from acute lymphoblastic leukemia (ALL), adult ALL, chronic lymphoblastic leukemia (CML), non-Hodgkin lymphoma (NHL) and diffuse large B-cell lymphoma (DLBCL).

In some embodiments, the disease or condition is an infectious disease or condition such as, but not limited to, viral, retroviral, bacterial and protozoal infections, immunodeficiency, cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus, BK polyomavirus. In some embodiments, the disease or condition is an autoimmune or inflammatory disease or condition such as arthritis, for example, rheumatoid arthritis (RA), type I diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease, psoriasis, scleroderma, autoimmune thyroid disease, Graves' disease, Crohn's disease, multiple sclerosis, asthma and/or a transplant-related disease or condition.

In some embodiments, the antigen associated with the disease or disorder is a tumor antigen, which may be glioma-associated antigen, β-human chorionic gonadotropin, alpha-fetoprotein (AFP), B-cell maturation antigen (BCMA, BCM), B-cell activating factor receptor (BAFFR, BR3) and/or transmembrane activator and CAML interactor (TACI), Fc receptor-like 5 (FCRL5, FcRH5), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, Melanin-A/MART-1, WT-1, S-100, MBP, CD63, MUC1 ( e.g., MUC1-8), p53, Ras, cyclin B1, HER-2/neu, carcinoembryonic antigen (CEA), gp100, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A11, MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-C1, BAGE, GAGE-1, GAGE-2, pl5, tyrosinase ( e.g. tyrosinase-related protein 1 (TRP-1) or tyrosinase-related protein 2 (TRP-2)), β-catenin, NY-ESO-1, LAGE-1a, PP1, MDM2, MDM4, EGVFvIII, Tax, SSX2, telomerase, TARP, pp65, CDK4, vimentin, S100, eIF-4A1, IFN-inducible p78 and melanotransferrin (p97), uroplakin II, prostate-specific antigen (PSA), human kallikrein (huK2), prostate-specific membrane antigen (PSM) and prostatic acid phosphatase (PAP), neutrophil elastase, ephrin B2, BA-46, beta-catenin, Bcr-abl, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Caspase 8 or B-Raf antigen. Other tumor antigens may include any of FRα-derived, CD24, CD44, CD133, CD 166, epCAM, CA-125, HE4, Oval, estrogen receptor, progesterone receptor, uPA, PAI-1, CD19, CD20, CD22, ROR1, CD33/IL3Ra, c-Met, PSMA, Glycolipid F77, GD-2, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin. Specific tumor-associated antigens or T cell epitopes are known (see, e.g., van der Bruggen et al. (2013) Cancer Immun, available at www.cancerimmunity.org/peptide/; Cheever et al. (2009) Clin Cancer Res, 15, 5323–37).

In some embodiments, the antigen associated with the disease or disorder is a viral antigen. A variety of viral antigenic targets have been identified and known, including peptides derived from the viral genomes of HIV, HTLV, and other viruses (see, e.g., Addo et al. (2007) PLoS ONE, 2, e321; Tsomides et al. (1994) J Exp Med, 180, 1283-93; Utz et al. (1996) J Virol, 70, 843-51). Typical viral antigens include, but are not limited to, antigen from hepatitis A, hepatitis B ( e.g., HBV core or surface antigens (HBVc, HBVs)), hepatitis C (HCV), Epstein-Barr virus ( e.g., EBVA), human papillomavirus (HPV; e.g., E6 and E7), human immunodeficiency virus type-1 (HIV1), Kaposi's sarcoma herpesvirus (KSHV), human papillomavirus (HPV), influenza virus, Lassa virus, HTLN-1, HIN-1, HIN-II, CMN, EBN, or HPN. In some embodiments, the target protein is a bacterial antigen or other pathogenic antigen, such as Mycobacterium tuberculosis (MT) antigens, trypanosomes such as Tiypansoma cruzi (T. cruzi), antigens such as T cell surface antigen (TSA), or malarial antigens. The specific viral antigen or epitopes or other pathogenic antigens or T cell epitopes are known (see, e.g., Addo et al. (2007) PLoS ONE, 2:e321; Anikeeva et al. (2009) Clin Immunol, 130:98-109).

In some embodiments, the antigen associated with a disease or disorder is an antigen derived from a virus associated with cancer, such as an oncogenic virus. For example, an oncogenic virus is one in which infection with certain viruses is known to lead to the development of various types of cancer, such as antigens of hepatitis A virus, hepatitis B virus (e.g., HBVc and surface antigens (HBVc, HBVs)), hepatitis C virus (HCV), human papillomavirus (HPV), hepatitis B virus infections, Epstein-Barr virus (EBV), human herpesvirus 8 (HHV-8), human T-cell leukemia virus-1 (HTLV-1), human T-cell leukemia virus-2 (HTLV-2), or cytomegalovirus (CMV), or any antigens targeted by the recombinant receptors described herein, for example, in Section IV.

In some embodiments, the antigen associated with the disease, disorder, or condition is selected from αvβ6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), cancer testicular antigen, cancer testicular antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), cyclin, cyclin A2, chemokine ligand 1 C-C motif 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), epidermal growth factor receptor type III (EGFR vIII) mutation, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin A2 receptor (EPHa2), estrogen receptor, Fc receptor-like 5 (FCRL5; also known as Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), folate binding protein (FBP), alpha folate receptor, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp100), glypican-3 (GPC3), group C G protein-coupled receptor 5 member D (GPRC5D), Her2/neu (erb-B2 receptor tyrosine kinase), Her3 (erb-B3), Her4 (erb-B4), erbB dimers, human high molecular weight melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, human leukocyte antigen A1 (HLA-A1), human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha (IL-22Rα), IL-13 receptor alpha 2 (IL-13Rα2), receptor kinase insertion domain (kdr), kappa light chain, L1 cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM, leucine-rich repeat-containing family 8 member A (LRRC8A), Lewis Y, melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer cell group 2 member D ligands (NKG2D), melan A (MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen, predominantly expressed melanoma antigen (PRAME), progesterone receptor, prostate-specific antigen, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), survivin, trophoblast-derived glycoprotein (TPBG, also known as 5T4), tumor-associated glycoprotein 72 (TAG72), tyrosinase-related protein 1 (TRP1, also known as TYRP1 or gp75), tyrosinase-related protein 2 (TRP2, also known as dopachrome tautomerase, dopachrome delta isomerase, or DCT), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms tumor 1 (WT-1), a pathogen-specific or pathogen-expressed antigen or an antigen associated with a universal tag, and/or biotinylated molecules, and/or molecules expressed by HIV, HCV, HBV, or other pathogens. Antigens to which the receptors are targeted, in some embodiments, include antigens associated with B cell malignancy, such as any of a variety of known B cell markers. In some embodiments, the antigen is or includes CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Ig kappa, Ig lambda, CD79a, CD79b, or CD30. In some embodiments, the antigen is or includes a pathogen-specific or pathogen-expressed antigen. In some embodiments, the antigen is a viral antigen (such as a viral antigen from HIV, HCV, HBV, etc.), bacterial antigens, and/or parasitic antigens.

In some embodiments, the cells are administered at a desired dose, which, in some aspects, includes a desired dose or number of cells or cell types, and/or a desired ratio of cell types. Thus, the cell dose in some embodiments is based on the total number of cells (or number per kg of body weight) and a desired ratio of populations or subtypes, such as the ratio of CD4+ to CD8+. In some embodiments, the cell dose is based on the desired total number (or number per kg of body weight) of cells in individual populations or individual cell types. In some embodiments, the dose is based on a combination of such characteristics as the desired total number of cells, the desired ratio, and the desired total number of cells in individual populations.

In some embodiments, populations or subtypes of cells, such as CD8 ⁺ and CD4 ⁺ T cells, are administered within an acceptable difference of a desired dose of total cells, such as a desired dose of T cells. In some aspects, the desired dose is a desired number of cells or a desired number of cells per unit body weight of the subject to which the cells are administered, such as cells/kg. In some aspects, the desired dose exceeds a minimum number of cells or a minimum number of cells per unit body weight. In some aspects, among the total number of cells administered in the desired dose, individual populations or subtypes are present with a close to the desired output ratio (such as the ratio of CD4 ⁺ to CD8 ⁺ ), such as within a certain acceptable difference or error of such ratio.

In some embodiments, the cells are administered within an acceptable difference between a desired dose of one or more individual cell populations or subtypes, such as a desired dose of CD4+ cells and/or a desired dose of CD8+ cells. In some aspects, the desired dose is a desired number of cells of a subtype or population, or a desired number of such cells per unit body weight of the subject to which the cells are administered, such as cells/kg. In some aspects, the desired dose exceeds a minimum number of cells of a population or subtype, or a minimum number of cells of a population or subtype per unit body weight.

Thus, in some embodiments, the dose is based on a desired fixed dose of total cells and a desired ratio and/or based on a desired fixed dose of one or more, for example, each of the individual subtypes or subpopulations. Thus, in some embodiments, the dose is based on a desired fixed or minimum dose of T cells and a desired ratio of CD4 ⁺ to CD8 ⁺ cells, and/or based on a desired fixed or minimum dose of CD4 ⁺ and/or CD8 ⁺ cells.

In certain embodiments, the cells or individual subpopulations of cell subtypes are administered to the subject in a range of from about one million to about 100 billion cells and/or such number of cells per kilogram of body weight as, for example, from 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing), for example, from about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of above values), and, in some cases, from about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells), or any value in between, and/or per kilogram of body weight. Doses may vary depending on the indications specific to the disease or disorder and/or the patient and/or other treatment.

In some embodiments, for example, if the subject is a human, the dose comprises less than about 5×10 ⁸ total recombinant receptor (e.g., CAR) expressing cells, T cells, or peripheral blood mononuclear cells (PBMCs), such as in the range of from about 1×10 ⁶ to 5×10 ⁸ such cells, such as 2×10 ⁶ , 5×10 ⁶ , 1×10 ⁷ , 5×10 ⁷ , 1×10 ⁸ or 5×10 ⁸ total such cells, or a range between any two of the foregoing.

In some embodiments, the dose of genetically engineered cells comprises from or about 1×10 ⁵ to 5×10 ⁸ total CAR-expressing T cells, 1×10 ⁵ to 2.5×10 ⁸ total CAR-expressing T cells, 1×10 ⁵ to 1×10 ⁸ total CAR-expressing T cells, 1×10 ⁵ to 5×10 ⁷ total CAR-expressing T cells, 1×10 ⁵ to 2.5×10 ⁷ total CAR-expressing T cells, 1×10 ⁵ to 1×10 ⁷ total CAR-expressing T cells, 1×10 ⁵ to 5×10 ⁶ total CAR-expressing T cells, 1×10 ⁵ to 2.5×10 ⁶ total CAR-expressing T cells, 1×10 ⁵ to 1×10 ⁶ total CAR-expressing T cells, 1×10 ⁶ to 5×10 ⁸ total CAR-expressing T cells, 1×10 ⁶ to 2.5×10 ⁸ total CAR-expressing T cells, 1×10 ⁶ to 1×10 ⁸ total CAR-expressing T cells, 1×10 ⁶ to 5×10 ⁷ total CAR-expressing T cells, 1×10 ⁶ to 2.5×10 ⁷ total CAR-expressing T cells, 1×10 ⁶ to 1×10 ⁷ total CAR-expressing T cells, 1×10 ⁶ to 5×10 ⁶ total CAR-expressing T cells, 1×10 ⁶ to 2.5×10 ⁶ total CAR-expressing T cells, 2.5×10 ⁶ to 5×10 ⁸ total CAR-expressing T cells, 2.5×10 ⁶ to 2.5×10 ⁸ total CAR-expressing T cells, 2.5×10 ⁶ to 1×10 ⁸ total CAR-expressing T cells, 2.5×10 ⁶ to 5×10 ⁷ total CAR-expressing T cells, 2.5×10 ⁶ to 2.5×10 ⁷ total CAR-expressing T cells, 2.5×10 ⁶ to 1×10 ⁷ total CAR-expressing T cells, 2.5×10 ⁶ to 5×10 ⁶ total CAR-expressing T cells, 5×10 ⁶ to 5×10 ⁸ total CAR-expressing T cells, 5×10 ⁶ to 2.5×10 ⁸ total CAR-expressing T cells, 5×10 ⁶ to 1×10 ⁸ total CAR-expressing T cells, 5×10 ⁶ to 5×10 ⁷ total CAR-expressing T cells, 5×10 ⁶ to 2.5×10 ⁷ total CAR-expressing T cells, 5×10 ⁶ to 1×10 ⁷ total CAR-expressing T cells, 1×10 ⁷ to 5×10 ⁸ total CAR-expressing T cells, 1×10 ⁷ to 2.5×10 ⁸ total CAR-expressing T cells, 1×10 ⁷ to 1×10 ⁸ total CAR-expressing T cells, 1×10 ⁷ to 5×10 ⁷ total CAR-expressing T cells, 1×10 ⁷ to 2.5×10 ⁷ total CAR-expressing T cells, 2.5×10 ⁷ to 5×10 ⁸ total CAR-expressing T cells, 2.5×10 ⁷ to 2.5×10 ⁸ total CAR-expressing T cells, 2.5×10 ⁷ to 1×10 ⁸ total CAR-expressing T cells, 2.5×10 ⁷ to 5×10 ⁷ total CAR-expressing T cells, 5×10 ⁷ to 5×10 ⁸ total CAR-expressing T cells, 5×10 ⁷ to 2.5×10 ⁸ total CAR-expressing T cells, 5×10 ⁷ to 1×10 ⁸ total CAR-expressing T cells, 1×10 ⁸ to 5×10 ⁸ total CAR-expressing T cells, 1×10 ⁸ to 2.5×10 ⁸ total CAR-expressing T cells, or 2.5×10 ⁸ to 5×10 ⁸ total CAR-expressing T cells.

In some embodiments, the dose of genetically engineered cells comprises at least or at least about or is or is about 1×10 ⁵ CAR-expressing cells, at least or at least about or is or is about 2.5×10 ⁵ CAR-expressing cells, at least or at least about or is or is about 5×10 ⁵ CAR-expressing cells, at least or at least about or is or is about 1×10 ⁶ CAR-expressing cells, at least or at least about or is or is about 2.5×10 ⁶ CAR-expressing cells, at least or at least about or is or is about 5×10 ⁶ CAR-expressing cells, at least or at least about or is or is about 1×10 ⁷ CAR-expressing cells, at least or at least about or equal to, or equal to, about 2.5×10 ⁷ CAR-expressing cells, at least or at least about or equal to, or equal to, about 5×10 ⁷ CAR-expressing cells, at least or at least about or equal to, or equal to, about 1×10 ⁸ CAR-expressing cells, at least or at least about or equal to, or equal to, about 2.5×10 ⁸ CAR-expressing cells, or at least or at least about or equal to, or equal to, about 5×10 ⁸ CAR-expressing cells.

In some embodiments, the cellular therapy comprises administering a dose comprising a cell quantity of from or about 1×10 ⁵ to 5×10 ⁸ total recombinant receptor-expressing cells, total T cells, or total peripheral blood mononuclear cells (PBMC), from or about 5×10 ⁵ to 1×10 ⁷ total recombinant receptor-expressing cells, total T cells, or total peripheral blood mononuclear cells (PBMC), or from or about 1×10 ⁶ to 1×10 ⁷ total recombinant receptor-expressing cells, total T cells, or total peripheral blood mononuclear cells (PBMC), all inclusive. In some embodiments, the cell therapy comprises administering a dose of cells comprising a cell count of at least or at least about 1×10 ⁵ total recombinant receptor expressing cells, total T cells, or total peripheral blood mononuclear cells (PBMCs), such as at least or at least 1×10 ⁶ , at least or at least about 1×10 ⁷ , at least or at least about 1×10 ⁸ such cells. In some embodiments, the count is in reference to the total number of CD3+ or CD8+, in some cases also recombinant receptor expressing (e.g., CAR+) cells. In some embodiments, the cellular therapy comprises administering a dose comprising a cell quantity of from or about 1×10 ⁵ to 5×10 ⁸ CD3+ or CD8+ total T cells or CD3+ or CD8+ recombinant receptor-expressing cells, from or about 5×10 ⁵ to 1×10 ⁷ CD3+ or CD8+ total T cells or CD3+ or CD8+ recombinant receptor-expressing cells, or from or about 1×10 ⁶ to 1×10 ⁷ CD3+ or CD8+ total T cells or CD3+ or CD8+ recombinant receptor-expressing cells, all inclusive. In some embodiments, the cellular therapy comprises administering a dose comprising a cell quantity of from or about 1×10 ⁵ to 5×10 ⁸ total CD3+/CAR+ or CD8+/CAR+ cells, from or about 5×10 ⁵ to 1×10 ⁷ total CD3+/CAR+ or CD8+/CAR+ cells, or from or about 1×10 ⁶ to 1×10 ⁷ total CD3+/CAR+ or CD8+/CAR+ cells, all inclusive.

In some embodiments, the T cells of the dose comprise CD4+ T cells, CD8+ T cells, or CD4+ and CD8+ T cells.

In some embodiments, for example, if the subject is a human, the CD8+ T cells of the dose, including a dose comprising CD4+ and CD8+ T cells, comprise from about 1×10 ⁶ to 5×10 ⁸ total recombinant receptor (e.g., CAR) expressing CD8+ cells, such as in the range of from about 5×10 ⁶ to 1×10 ⁸ such cells, such as 1×10 ⁷ , 2.5×10 ⁷ , 5×10 ⁷ , 7.5×10 ⁷ , 1×10 ⁸ or 5×10 ⁸ total such cells, or a range between any two of the above values. In some embodiments, a plurality of doses are administered to the patient, and each of the doses and the total dose can be within any of the above values. In some embodiments, the cell dosage comprises administering from or about 1×10 ⁷ to 0.75×10 ⁸ total recombinant CD8+ receptor-expressing T cells, from 1×10 ⁷ to 2.5×10 ⁷ total recombinant CD8+ receptor-expressing T cells, from or about 1×10 ⁷ to 0.75×10 ⁸ total recombinant CD8+ receptor-expressing T cells, all inclusive. In some embodiments, the cell dosage comprises administering about 1×10 ⁷ , 2.5×10 ⁷ , 5×10 ⁷ , 7.5×10 ⁷ , 1×10 ⁸ , or 5×10 ⁸ total recombinant CD8+ receptor-expressing T cells.

In some embodiments, a dose of cells, such as those expressing a recombinant T cell receptor, is administered to a subject as a single dose or is administered only once over a period of two weeks, one month, three months, six months, 1 year, or more.

In some embodiments, for example, if the subject is a human, the dose comprises less than about 1×10 ⁸ total recombinant receptor (e.g., recombinant TCR) expressing cells, T cells, or peripheral blood mononuclear cells (PBMC), for example, in the range of from about 1×10 ⁶ to 1×10 ⁸ such cells, for example, 2×10 ⁶ , 5×10 ⁶ , 1×10 ⁷ , 5×10 ⁷ , or 1×10 ⁸ or total such cells, or a range between any two of the above values.

In some embodiments, the total cell dose and/or the dose of individual subpopulations of cells is within the range of about 10 ⁴ to about 10 ⁹ cells/kilogram (kg) body weight, such as 10 ⁵ to 10 ⁶ cells/kg body weight, such as about 1×10 ⁵ cells/kg, 1.5×10 ⁵ cells/kg, 2×10 ⁵ cells/kg, or 1×10 ⁶ cells/kg body weight. For example, in some embodiments, the cells are administered within a defined error range of between about 10 ⁴ and about 10 ⁹ T cells/kilogram (kg) body weight, such as between 10 ⁵ and 10 ⁶ T cells/kg body weight, such as between about 1×10 ⁵ T cells/kg, 1.5×10 ⁵ T cells/kg, 2×10 ⁵ T cells/kg, or 1×10 ⁶ T cells/kg body weight.

In some embodiments, the cells are administered within a defined error range between about 10 ⁴ and about 10 ⁹ CD4 ⁺ and/or CD8 ⁺ cells/kilogram (kg) of body weight, such as between 10 ⁵ and 10 ⁶ CD4 ⁺ and/or CD8 ⁺ cells/kg of body weight, such as about 1×10 ⁵ CD4 ⁺ and/or CD8 ⁺ cells/kg, 1.5×10 ⁵ CD4 ⁺ and/or CD8 ⁺ cells/kg, 2×10 ⁵ CD4 ⁺ and/or CD8 ⁺ cells/kg, or 1×10 ⁶ CD4 ⁺ and/or CD8 ⁺ cells/kg of body weight.

In some embodiments, the cells are administered within a defined error range of greater than and/or at least about 1×10 ⁶ , about 2.5×10 ⁶ , about 5×10 ⁶ , about 7.5×10 ⁶ , or about 9×10 ⁶ CD4 ⁺ cells and/or at least about 1×10 ⁶ , about 2.5×10 ⁶ , about 5×10 ⁶ , about 7.5×10 ⁶ , or about 9×10 ⁶ CD8+ cells and/or at least about 1×10 ⁶ , about 2.5×10 ⁶ , about 5×10 ⁶ , about 7.5×10 ⁶ , or about 9×10 ⁶ T cells. In some embodiments, the cells are administered within a defined error range of between about 10 ⁸ and 10 ¹² or between about 10 ¹⁰ and 10 ¹¹ T cells, between about 10 ⁸ and 10 ¹² or between about 10 ¹⁰ and 10 ¹¹ CD4 ⁺ cells, and/or between about 10 ⁸ and 10 ¹² or between about 10 ¹⁰ and 10 ¹¹ CD8 ⁺ cells.

In some embodiments, the cells are administered within an acceptable range of a desired output ratio of a plurality of cell populations or subtypes, such as CD4+ and CD8+ cells or subtypes. In some aspects, the desired ratio may be a specific ratio or may be a range of ratios. For example, in some embodiments, the desired ratio (e.g., ratio of CD4 ⁺ to CD8 ⁺ cells) is between about 1:5 and about 5:1 (or greater than about 1:5 and less than about 5:1) or between about 1:3 and about 3:1 (or greater than about 1:3 and less than about 3:1), such as between about 2:1 and about 1:5 (or greater than about 1:5 and less than about 2:1, such as between about 5:1, 4.5:1, 4:1, 3.5:1, 3:1, 2.5:1, 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1:1, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9: 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 or 1:5. In some aspects, the allowable difference is within about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50% of the desired ratio, including any value in between these ranges.

For the prevention or treatment of a disease, the appropriate dose may depend on the type of disease to be treated, the type of cells or recombinant receptors, the severity and course of the disease, whether the cells are administered for prophylactic or therapeutic purposes, previous therapy, the subject's medical history and response to the cells, and the discretion of the treating physician. Compositions and cells in some embodiments are suitably administered to a subject at one time or over a series of treatment courses.

In some aspects, the dose amount is determined based on one or more criteria such as the subject's response to prior treatment, such as chemotherapy, the severity of the disease in the subject, such as tumor burden, volume, size or extent, prevalence or type of metastases, stage and/or the likelihood or frequency of toxic outcomes in the subject, such as CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, and/or the host immune response against the administered cells and/or recombinant receptors.

In some aspects, the dose size is determined by the severity of the disease or condition of the subject. For example, in some aspects, the number of cells administered in a dose is determined based on the tumor mass that is present in the subject immediately prior to the start of the cell dose. In some embodiments, the size of the first and/or subsequent dose is inversely correlated with the severity of the disease. In some aspects, such as in the context of a greater disease severity, a small number of cells are administered to the subject. In other embodiments, such as in the context of a lesser disease severity, a larger number of cells are administered to the subject.

The cells may be administered by any suitable means, such as bolus infusion, injection, such as intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectural injection, subconjunctival injection, sub-Tenon injection, retrobulbar injection, peribulbar injection, or posterior periscleral delivery. In some embodiments, they are administered parenterally, intrapulmonarily, and intranasally, and, if desired for local treatment, intralesional. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, the dose is administered by a single bolus of cells. In some embodiments, it is administered by multiple bolus administration of cells, such as over a period of no more than 3 days, or by continuous infusion of cells.

In some embodiments, the cells are administered as part of a combination therapy, such as simultaneously or sequentially with, in any order, another therapeutic intervention, such as an antibody or engineered cells or a receptor or agent, such as a cytotoxic or therapeutic. The cells in some embodiments are administered together with one or more additional therapeutic agents or in combination with another therapeutic intervention simultaneously or sequentially, in any order. In some contexts, the cells are administered together with another therapy sufficiently close in time that the cell population enhances the effect of the one or more additional therapeutic agents, or vice versa. In some embodiments, the cells are administered before one or more additional therapeutic agents. In some embodiments, the cells are administered after one or more additional therapeutic agents. In some embodiments, the one or more additional agents include a cytokine, such as IL-2, for example, to enhance persistence. In some embodiments, the methods include administering a chemotherapeutic agent.

After administration of the cells, the biological activity of the engineered cell populations is, in some embodiments, measured, for example, by any of a variety of known methods. Evaluation parameters include specific binding of the engineered or natural T cell or other immune cell to an antigen, in vivo, for example, by imaging, or ex vivo , for example, by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured by any suitable known methods, such as cytotoxicity assays, using any suitable known methods, such as the cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004). In certain embodiments, the biological activity of the cells is measured by analyzing the expression and/or secretion of one or more cytokines, such as CD 107a, IFNγ, IL-2, and TNF. In some aspects, the biological activity is measured by assessing clinical outcomes, such as a decrease in tumor burden or burden.

In certain embodiments, the engineered cells are further modified in any way so as to enhance therapeutic or prophylactic efficacy. For example, an engineered CAR or TCR expressed by the population can be conjugated either directly or indirectly via a linker to a targeting moiety. The practice of conjugating compounds, such as CARs or TCRs, to targeting moieties is known. See, for example, Wadwa et al., J. Drug Targeting 3: 1 1 1 (1995), and U.S. Patent 5,087,616.

VII. KITS AND FINISHED PRODUCTS

Also provided are articles of manufacture, systems, apparatuses, and kits useful in practicing the present embodiments. In some embodiments, the present articles of manufacture and kits comprise one or more components of one or more agents capable of genetic disruption and/or template polynucleotides, for example, template polynucleotides comprising a transgene encoding a recombinant receptor or an antigen-binding fragment or chain thereof, or one or more second template polynucleotides. In some embodiments, the articles of manufacture or kits can be used in methods of engineering T cells to express a recombinant receptor and/or other polypeptides, and assessing the resulting cells and/or cell population in accordance with the present methods. In some embodiments, the articles of manufacture or kits provided herein comprise T cells and/or T cell compositions, such as any T cells and/or T cell compositions described herein.

In some embodiments, the articles of manufacture or kits include polypeptides, nucleic acids, vectors, and/or polynucleotides used in performing the present methods. In some embodiments, the articles of manufacture or kits include one or more nucleic acid molecules, such as a plasmid or DNA fragment that contains one or more components of one or more agents capable of causing genetic disruption, and/or template polynucleotides, such as template polynucleotides containing a transgene encoding a recombinant receptor or an antigen-binding fragment or chain thereof, or one or more second template polynucleotides. In some embodiments, the articles of manufacture or kits provided herein contain control vectors.

In some embodiments, the articles of manufacture or kits provided herein comprise one or more agents, wherein each of the one or more agents is independently capable of causing genetic disruption of a target site in the T cell receptor constant alpha (TRAC) gene and/or the T cell receptor constant beta (TRBC) gene; and a template polynucleotide comprising a transgene encoding a recombinant TCR or an antigen-binding fragment or chain thereof, wherein the transgene encoding the recombinant TCR or an antigen-binding fragment or chain thereof is targeted for integration at or near the target site via homology-directed repair (HDR).

In some embodiments, the articles of manufacture or kits provided herein comprise T cells and/or T cell compositions, such as any T cells and/or T cell compositions described herein. In some embodiments, the T cells and/or T cell compositions of any modified T cells used in the screening methods described herein. In some embodiments, the articles of manufacture or kits provided herein comprise control T cells and/or T cell compositions.

In some embodiments, the articles of manufacture or kits include one or more components used to evaluate properties of a population and/or composition of engineered cells expressing a recombinant receptor. For example, the articles of manufacture or kits can include binding reagents, such as antibodies, MHC-peptide tetramers, and/or probes, used to evaluate specific properties of the introduced recombinant TCR, such as expression of the recombinant TCR on the cell surface, recognition of a peptide in the context of an MHC molecule, and/or a detectable signal produced by a reporter, such as a T cell activation reporter. In some embodiments, the articles of manufacture or kits can include components that are used to determine specific properties, such as labeled components, such as fluorescently labeled components, and/or components that can produce a detectable signal, such as substrates that can produce fluorescence or luminescence.

In some embodiments, the finished article or kits include one or more containers, typically a plurality of containers, packaging material, and a label or insert associated with the container or containers and/or the packaging, typically including instructions for use, such as instructions for introducing components into cells for constructing and/or evaluating populations of engineered cells and/or compositions. In some embodiments, the finished article or kits include one or more instructions for administering engineered cells and/or cell compositions for therapy.

The articles of manufacture provided herein comprise packaging materials. Packaging materials for use in packaging the materials provided herein are well known. See, for example, U.S. Patent Nos. 5,323,907, 5,052,558, and 5,033,252, each of which is incorporated herein by reference in its entirety. Examples of packaging materials include, but are not limited to, blister packs, bottles, test tubes, inhalers, pumps, bags, vials, containers, syringes, disposable laboratory supplies such as pipette tips, and/or plastic plates or bottles. The articles of manufacture or kits may include a device that facilitates dispensing of materials or facilitates high-throughput or large-scale use, such as facilitating use in robotic equipment. Typically, the packaging does not react with the composition contained therein.

In some embodiments, the agent capable of causing genetic destruction and/or template polynucleotides are packaged separately. In some embodiments, each container may have one compartment. In some embodiments, other components of the finished product or kits are packaged separately or together in one compartment.

VIII. DEFINITIONS

Unless otherwise specified, all terms in the art, designations and other technical and scientific terms or terminology used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a material difference from what is commonly understood in the art.

The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues, and are not limited by a minimum length. Polypeptides, including the provided antibodies and antibody chains and other peptides, such as linkers, may include amino acid residues, including natural and/or non-natural amino acid residues. The terms also include modifications of the polypeptide after expression, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. In some aspects, polypeptides may contain modifications relative to native or natural sequences, as long as the protein retains the desired activity. These modifications may be intentional, such as through site-directed mutagenesis, or may be accidental, such as due to mutations in hosts that produce the proteins, or errors due to PCR amplification.

An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid molecule includes a nucleic acid molecule that is contained in cells that normally contain a nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or in a chromosomal location that is different from its natural chromosomal location.

"Isolated nucleic acid encoding a TCR or antibody" refers to one or more nucleic acid molecules encoding a TCR alpha or β chain (or fragments thereof) or an antibody heavy and light chain (or fragments thereof), including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present in one or more locations in a host cell.

The terms "host cell," "host cell colony," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the original transformed cell and the progeny derived from it, regardless of passage number. Progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as those tested or selected for the originally transformed cell are included herein.

As used herein, "percent (%) amino acid sequence identity" and "percent identity" when used with respect to an amino acid sequence (a reference polypeptide sequence) are defined as the percentage of amino acid residues in a candidate sequence (e.g., an antibody or fragment) that are identical to amino acid residues in a reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without taking into account any conservative substitutions as part of the sequence identity. Alignment for the purposes of determining percent amino acid sequence identity can be achieved using various known, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Suitable parameters for sequence alignment can be determined, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

In some embodiments, "operably linked" may include assembling components such as a DNA sequence (e.g., heterologous nucleic acids) and regulatory sequences in a manner that permits gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are linked to the regulatory sequence. This therefore means that the components described are in a relationship that allows them to function in the intended manner.

An amino acid substitution may involve the substitution of one amino acid in a polypeptide with another amino acid. The substitution may be a conservative amino acid substitution or a non-conservative amino acid substitution. Amino acid substitutions may be introduced into the binding molecule of interest, e.g., an antibody, and products tested for a desired activity, e.g. , maintaining/improving antigen binding, reducing immunogenicity, or improving ADCC or CDC.

Amino acids can generally be grouped according to the following side chain properties:

(1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) alkaline: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

In some embodiments, conservative substitutions may involve exchanging a member of one of these classes for another member of the same class. In some embodiments, non-conservative amino acid substitutions may involve exchanging a member of one of these classes for another class.

As used herein, the term "vector" refers to a nucleic acid molecule capable of reproducing other nucleic acids to which it is linked. The term includes a vector as a self-replicating nucleic acid structure, as well as a vector incorporated into the genome of a host cell into which it has been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

The term "package insert" is used to refer to instructions typically included in commercial packages of therapeutic products that contain information regarding the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings regarding the use of such therapeutic products.

As used herein, the singular forms include the plural unless the context clearly dictates otherwise. For example, "a" or "an" means "at least one" or "one or more." It is understood that aspects and variants described herein include "consisting of" and/or "consisting essentially of" aspects and variants.

In this disclosure, various aspects of the claimed subject matter are presented in range format. It should be understood that the description in range format is merely for convenience and brevity, and is not to be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, a description of a range should be considered to specifically disclose all possible sub-ranges, as well as individual numerical values, within that range. For example, if a range of values is provided, each sub-range between the upper and lower limits of that range, and any other stated or sub-range values within that recited range, are intended to be encompassed by the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included within the smaller ranges, and are also included within the claimed subject matter subject to any specifically excluded limitation within the recited range. If a recited range includes one or both limitations, ranges excluding one or both included limitations are also included within the claimed subject matter. This applies regardless of the breadth of the range.

As used herein, the term "about" refers to a typical error range for the corresponding value, well known to those skilled in the art. Reference to "about" a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se . For example, a description referring to "about X" includes a description of "X". In some embodiments, "about" may refer to ±25%, ±20%, ±15%, ±10%, ±5%, or ±1%.

As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells. It may be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.

As used herein, a statement that a cell or population of cells is "positive" for a particular marker refers to the detectable presence on or in the cell of the particular marker, typically a surface marker. When referring to a surface marker, the term refers to the presence of surface expression detectable by flow cytometry, such as by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is detected by flow cytometry at a level substantially higher than the staining determined by performing the same procedure with an isotype-like control under otherwise identical conditions and/or at a level substantially similar to the level for a cell known to be positive for the marker and/or at a level substantially higher than for a cell known to be negative for the marker.

As used herein, a statement that a cell or population of cells is "negative" for a particular marker refers to the absence of significant detectable presence on or in the cell of the particular marker, typically a surface marker. When referring to a surface marker, the term refers to the absence of surface expression as determined by flow cytometry, such as by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is not detectable by flow cytometry at a level substantially greater than the staining determined by performing the same procedure with an isotype-like control under otherwise identical conditions and/or at a level substantially lower than the level for a cell known to be positive for the marker and/or at a level substantially similar to that of a cell known to be negative for the marker.

All publications, including patent documents, scientific articles, and databases, referenced in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication was individually incorporated by reference. If a definition set forth herein conflicts with or is otherwise inconsistent with a definition set forth in patents, applications, published applications, and other publications that are incorporated herein by reference, the definition set forth herein controls over the definition that is incorporated herein by reference.

The section headings used here are for organizational purposes only and should not be construed as limiting the subject matter described.

IX. TYPICAL VARIANTS

The presented embodiments include:

1. A method for producing a genetically engineered immune cell, comprising:

(a) administering to an immune cell one or more agents, wherein each of the one or more agents is independently capable of causing genetic disruption of a target site in the T cell receptor constant alpha (TRAC) gene and/or the T cell receptor constant beta (TRBC) gene, thereby causing genetic disruption of at least one target site; and

(b) introducing into an immune cell a matrix polynucleotide comprising a transgene encoding a recombinant T-cell receptor (TCR) or an antigen-binding fragment or chain thereof, wherein the transgene encoding the recombinant TCR or an antigen-binding fragment or chain thereof is targeted for integration into or near one of at least one target site via homology-directed repair (HDR).

2. A method for producing a genetically engineered immune cell, comprising:

introducing into an immune cell having a genetic disruption of at least one target site in a T cell receptor constant alpha (TRAC) gene and/or a T cell receptor constant beta (TRBC) gene, a matrix polynucleotide comprising a transgene encoding a recombinant T cell receptor (TCR) or an antigen-binding fragment or chain thereof, wherein the genetic disruption is caused by one or more agents, wherein each of the one or more agents is independently capable of causing the genetic disruption, and the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof is targeted for integration at or near one of the at least one target position via homology-directed repair (HDR).

3. The method of embodiment 1 or embodiment 2, wherein at least one of the one or more agents is capable of causing genetic disruption of a target site in the TRAC gene.

4. The method according to any one of embodiments 1-3, wherein at least one of the one or more agents is capable of causing genetic disruption of a target site in the TRBC gene.

5. The method according to any one of embodiments 1-4, wherein the one or more agents comprise at least one agent that is capable of causing genetic disruption of a target site in a TRAC gene and at least one agent that is capable of causing genetic disruption of a target site in a TRBC gene.

6. The method of embodiment 4 or embodiment 5, wherein the TRBC gene is one or both of the T cell receptor beta constant 1 ( TRBC1 ) or T cell receptor beta constant 2 ( TRBC2 ) genes.

7. A method for producing a genetically engineered immune cell, comprising:

(a) administering to an immune cell one or more agents, wherein each of the one or more agents is independently capable of causing genetic disruption of a target site in the T cell receptor constant alpha (TRAC) gene and the T cell receptor constant beta (TRBC) gene, thereby causing genetic disruption of the target sites; and

(b) introducing into an immune cell a matrix polynucleotide comprising a transgene encoding a recombinant T-cell receptor (TCR) or an antigen-binding fragment or chain thereof, wherein the transgene encoding the recombinant TCR or an antigen-binding fragment or chain thereof is targeted for integration at or near a target site via homology-directed repair (HDR).

8. A method for producing a genetically engineered immune cell, comprising:

introducing into an immune cell having a genetic disruption of at least one target site in a T cell receptor constant alpha (TRAC) gene and/or a T cell receptor constant beta (TRBC) gene, a matrix polynucleotide comprising a transgene encoding a recombinant T cell receptor (TCR) or an antigen-binding fragment or chain thereof, wherein the genetic disruption is caused by one or more agents, wherein each of the one or more agents is independently capable of causing the genetic disruption, and the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof is targeted for integration into or near one of the at least one target site via homology-directed repair (HDR).

9. The method of embodiment 7 or embodiment 8, wherein the TRBC gene is one or both of the T cell receptor beta constant 1 ( TRBC1 ) or T cell receptor beta constant 2 ( TRBC2 ) genes.

10. The method according to any one of embodiments 1-9, wherein the one or more agents capable of causing genetic disruption comprises a DNA binding protein or a DNA binding nucleic acid that specifically binds to or hybridizes to a target site.

11. The method of embodiment 10, wherein the one or more agents capable of causing genetic disruption comprises (a) a fusion protein comprising a DNA-directed protein and a nuclease or (b) an RNA-directed nuclease.

12. The method of embodiment 11, wherein the DNA-directed protein or RNA-directed nuclease comprises a zinc finger protein (ZFP), a TAL protein, or a clustered regularly intervening short paddledromic nucleic acid (CRISPR)-related nuclease (Cas) specific to the target site.

13. The method of any one of embodiments 10-12, wherein the one or more agents comprise a zinc finger nuclease (ZFN), a TAL effector nuclease (TALEN), or a CRISPR-Cas9 combination that specifically bind to, recognize, or hybridize to a target site.

14. The method according to any one of embodiments 1-13, wherein each of the one or more agents comprises a guide RNA (gRNA) having a targeting domain that is complementary to at least one target site.

15. The method of embodiment 14, wherein one or more agents are administered as a ribonucleoprotein (RNP) complex comprising gRNA and Cas9 protein.

16. The method of embodiment 15, wherein the RNP is administered via electroporation, gene gun, calcium phosphate transfection, cell condensation or cell compression.

17. The method of embodiment 15 or embodiment 16, wherein the RNP is administered via electroporation.

18. The method according to any one of embodiments 1-13, wherein one or more agents are administered as one or more polynucleotides encoding gRNA and/or Cas9 protein.

19. The method according to any one of embodiments 1-18, wherein at least one target site is within an exon of a TRAC , TRBC1 and/or TRBC2 gene.

20. The method of any one of embodiments 14-19, wherein the gRNA has a targeting domain that is complementary to a target site in the TRAC gene and comprises a sequence selected from UCUCUCAGCUGGUACACGGC (SEQ ID NO:28), UGGAUUUAGAGUCUCUCAGC (SEQ ID NO:29), ACACGGCAGGGUCAGGGUUC (SEQ ID NO:30), GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:31), GCUGGUACACGGCAGGGUCA (SEQ ID NO:32), CUCAGCUGGUACACGGC (SEQ ID NO:33), UGGUACACGGCAGGGUC (SEQ ID NO:34), GCUAGACAUGAGGUCUA (SEQ ID NO:35), GUCAGAUUUGUUGCUCC (SEQ ID NO:36), UCAGCUGGUACACGGCA (SEQ ID NO:37), GCAGACAGACUUGUCAC (SEQ ID NO:38), GGUACACGGCAGGGUCA (SEQ ID NO:39), CUUCAAGAGCAACAGUGCUG (SEQ ID NO:40), AGAGCAACAGUGCUGUGGCC (SEQ ID NO:41), AAAGUCAGAUUUGUUGCUCC (SEQ ID NO:42), ACAAAACUGUGCUAGACAUG (SEQ ID NO:43), AAACUGUGCUAGACAUG (SEQ ID NO:44), UGUGCUAGACAUGAGGUCUA (SEQ ID NO:45), GGCUGGGGAAGAAGGUGUCUUC (SEQ ID NO:46), GCUGGGGAAGAAGGUGUCUUC (SEQ ID NO:47), GGGGAAGAAGGUGUCUUC (SEQ ID NO:48), GUUUUGUCUGUGAUAUACACAU (SEQ ID NO:49), GGCAGACAGACUUGUCACUGGAUU (SEQ ID NO:50), GCAGACAGACUUGUCACUGGAUU (SEQ ID NO:51), GACAGACUUGUCACUGGAUU (SEQ ID NO:52), GUGAAUAGGCAGACAGACUUGUCA (SEQ ID NO:53), GAAUAGGCAGACAGACUUGUCA (SEQ ID NO:54), GAGUCUCUCAGCUGGUACACGG (SEQ ID NO:55), GUCUCUCAGCUGGUACACGG (SEQ ID NO:56), GGUACACGGCAGGGUCAGGGUU (SEQ ID NO:57) and GUACACGGCAGGGUCAGGGUU (SEQ ID NO:58).

21. The method of embodiment 20, wherein the gRNA has a targeting domain comprising the sequence GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:31).

22. The method of any one of embodiments 14-21, wherein the gRNA has a targeting domain that is complementary to a target site in one or more of the TRBC1 and TRBC2 genes and comprises a sequence selected from CACCCAGAUCGUCAGCGCCG (SEQ ID NO:59), CAAACACAGCGACCUCGGGU (SEQ ID NO:60), UGACGAGUGGACCCAGGAUA (SEQ ID NO:61), GGCUCUCGGAGAAUGACGAG (SEQ ID NO:62), GGCCUCGGCGCUGACGAUCU (SEQ ID NO:63), GAAAAACGUGUUCCCACCCG (SEQ ID NO:64), AUGACGAGUGGACCCAGGAU (SEQ ID NO:65), AGUCCAGUUCUACGGGCUCU (SEQ ID NO:66), CGCUGUCAAGUCCAGUUCUA (SEQ ID NO:67), CGCUGUCAAGUCCAGUUCUA (SEQ ID NO:68), CGCUGUCAAGUCCAGUUCUA (SEQ ID NO:69), CGCUGUCAAGUCCAGUUCUA (SEQ ID NO:70), CGCUGUCAAGUCCAGUUCUA (SEQ ID NO:71), CGCUGUCAAGUCCAGUUCUA (SEQ ID NO:72), CGCUGUCAAGUCCAGUUCUA (SEQ ID NO:73), CGCUGUCAAGUCCAGUUCUA (SEQ ID NO:74), CGCUGUCAAGUCCAGUUCUA (SEQ ID NO:75), CGCUGUCAAGUCCAGUUCUA (SEQ ID NO:76), CGCUGUCAAGUCCAGUUCUA (SEQ ID NO:77), CGCUGUCAAGUCCAGUUCUA (SEQ ID NO:78), CGCUGUCAAGUCCAGUUCUA (SEQ ID NO:79), CGCUGUCAAGUCCAGUUCUA (SEQ ID NO:80), CGCUGUCAAGUCCAGUUCUA (S NO:67), AUCGUCAGCGCCGAGGCCUG (SEQ ID NO:68), UCAAACACAGCGACCUCGGG (SEQ ID NO:69), CGUAGAACUGGACUUGACAG (SEQ ID NO:70), AGGCCUCGGCGCUGACGAUC (SEQ ID NO:71), UGACAGCGGAAGUGGUUGCG (SEQ ID NO:72), UUGACAGCGGAAGUGGUUGC (SEQ ID NO:73), UCUCCGAGAGCCCGUAGAAC (SEQ ID NO:74), CGGGUGGGAACACGUUUUUC (SEQ ID NO:75), GACAGGUUUGGCCCUAUCCU (SEQ ID NO:76), GAUCGUCAGCGCCGAGGCCU (SEQ ID NO:77), GGCUCAAACACAGCGACCUC (SEQ ID NO:78), UGAGGGUCUCGGCCACCUUC (SEQ ID NO:79), AGGCUUCUACCCCGACCACG (SEQ ID NO:80), CCGACCACGUGGAGCUGAGC (SEQ ID NO:81), UGACAGGUUUGGCCCUAUCC (SEQ ID NO:82), CUUGACAGCGGAAGUGGUUG (SEQ ID NO:83), AGAUCGUCAGCCCGAGGCC (SEQ ID NO:84), GCGCUGACGAUCUGGGUGAC (SEQ ID NO:85), UGAGGGCGGGGCUGCUCCUUG (SEQ ID NO:86), GUUGCGGGGGUUCUGCCAGA (SEQ ID NO:87), AGCUCAGCUCCACGUGGUCG (SEQ ID NO:88), GCGGCUGCUCAGGCAGUAUC (SEQ ID NO:89), GCGGGGGUUCUGCCAGAAGG (SEQ ID NO:90), UGGCUCAAACACAGCGACCU (SEQ ID NO:91), ACUGGACUUGACAGCGGAAG (SEQ ID NO:92), GACAGCGGAAGUGGUUGCGG (SEQ ID NO:93), GCUGUCAAGUCCAGUUCUAC (SEQ ID NO:94), GUAUCUGGAGUCAUUGAGGG (SEQ ID NO:95), CUCGGCGCUGACGAUCU (SEQ ID NO:96), CCUCGGCGCUGACGAUC (SEQ ID NO:97), CCGAGAGCCCGUAGAAC (SEQ ID NO:98), CCAGAUCGUCAGCGCCG (SEQ ID NO:99), GAAUGACGAGUGGACCC (SEQ ID NO:100), GGGUGACAGGUUUGGCCCUAUC (SEQ ID NO:101), GGUGACAGGUUUGGCCCUAUC (SEQ ID NO:102), GUGACAGGUUUGGCCCUAUC (SEQ ID NO:103), GACAGGUUUGGCCCUAUC (SEQ ID NO:104), GAUACUGCCUGAGCAGCCGCCU (SEQ ID NO:105), GACCACGUGGAGCUGAGCUGGUGG (SEQ ID NO:106), GUGGAGCUGAGCUGGUGG (SEQ ID NO:107), GGGCGGGCUGCUCCUUGAGGGGCU (SEQ ID NO:108), GGCGGGCUGCUCCUUGAGGGGCU (SEQ ID NO:109), GCGGGCUGCUCCUUGAGGGGCU (SEQ ID NO:110), GGGCUGCUCCUUGAGGGGCU (SEQ ID NO:111), GGCUGCUCCUUGAGGGGCU (SEQ ID NO:112), GCUGCUCCUUGAGGGGCU (SEQ ID NO:113), GGUGAAUGGGAAGGAGGUGCACAG (SEQ ID NO:114), GUGAAUGGGAAGGAGGUGCACAG (SEQ ID NO:115) and GAAUGGGAAGGAGGUGCACAG (SEQ ID NO:116).

23. The method of embodiment 22, wherein the gRNA has a targeting domain comprising the sequence GGCCUCGGCGCUGACGAUCU (SEQ ID NO:63).

24. The method according to any one of embodiments 1-23, wherein the template polynucleotide comprises the structure [5' homology arm]-[transgene]-[3' homology arm].

25. The method of embodiment 24, wherein the 5' homology arm and the 3' homology arm comprise nucleic acid sequences homologous to nucleic acid sequences surrounding the at least one target site.

26. The method of embodiment 24 or embodiment 25, wherein the 5' homology arm comprises nucleic acid sequences that are homologous to nucleic acid sequences 5' of the target site.

27. The method of embodiment 24 or embodiment 25, wherein the 3' homology arm comprises nucleic acid sequences that are homologous to nucleic acid sequences of the 3' target site.

28. The method of any one of embodiments 24-27, wherein the 5' homology arm and the 3' homology arm independently have at least, or at least about, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 base pairs, or less, or less than about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 base pairs.

29. The method of embodiment 28, wherein the 5' homology arm and the 3' homology arm independently have between about 50 and 100, 100 and 250, 250 and 500, 500 and 750, 750 and 1000, 1000 and 2000 base pairs.

30. The method of embodiment 29, wherein the 5' homology arm and the 3' homology arm independently have about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 base pairs.

31. The method of any one of embodiments 1-30, wherein the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof is targeted for integration at or near a target site in the TRAC gene.

32. The method of any one of embodiments 1-32, wherein the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof is targeted for integration at or near a target site in one or both of the TRBC1 and TRBC2 genes .

33. The method of any one of embodiments 1-32, further comprising introducing into the immune cell one or more second template polynucleotides comprising one or more second transgenes, wherein the second transgene is targeted for integration at or near one of the at least one target site via homology-directed repair (HDR).

34. The method of embodiment 33, wherein the second template polynucleotide comprises the structure [second 5' homology arm]-[one or more second transgenes]-[second 3' homology arm].

35. The method of embodiment 34, wherein the second 5' homology arm and the second 3' homology arm comprise nucleic acid sequences homologous to nucleic acid sequences surrounding the at least one target site.

36. The method of embodiment 34 or embodiment 35, wherein the second 5' homology arm comprises nucleic acid sequences that are homologous to nucleic acid sequences of the second 5' target site.

37. The method of embodiment 34 or embodiment 35, wherein the second 3' homology arm comprises nucleic acid sequences that are homologous to nucleic acid sequences of the second 3' target site.

38. The method of any one of embodiments 34-37, wherein the second 5' homology arm and the second 3' homology arm independently have at least or at least about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 base pairs, or less or less than about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 base pairs.

39. The method of embodiment 38, wherein the second 5' homology arm and the second 3' homology arm independently have between about 50 and 100, 100 and 250, 250 and 500, 500 and 750, 750 and 1000, 1000 and 2000 base pairs.

40. The method of embodiment 39, wherein the second 5' homology arm and the second 3' homology arm independently have about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 base pairs.

41. The method of any one of embodiments 33-40, wherein the one or more second transgenes are targeted for integration at or near a target site in the TRAC gene.

42. The method according to any one of embodiments 33-41, wherein the one or more second transgenes are targeted for integration at or near a target site in the TRBC1 or TRBC2 gene.

43. The method of any one of embodiments 33-42, wherein the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof is targeted for integration at or near a target site in the TRAC gene, TRBC1 gene, or TRBC2 gene, and one or more second transgenes are targeted for integration at or near one or more target sites that are not targeted by the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof.

44. The method of any one of embodiments 33-43, wherein the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof is targeted for integration at or near a target site in the TRAC gene, and one or more second transgenes are targeted for integration at or near one or more target sites in the TRBC1 gene and/or the TRBC2 gene.

45. The method of any one of embodiments 33-44, wherein the one or more second transgenes encode a molecule selected from a costimulatory ligand, a cytokine, a soluble single-strand variable fragment (scFv), an immunomodulatory fusion protein, a chimeric switch receptor (CSR), or a coreceptor.

46. The method of embodiment 45, wherein the encoded molecule is a costimulatory ligand, optionally selected from a tumor necrosis factor (TNF) ligand selected from 4-1BBL, OX40L, CD70, LIGHT, and CD30L or an immunoglobulin (Ig) superfamily ligand selected from CD80 and CD86.

47. The method of embodiment 45, wherein the encoded molecule is a cytokine optionally selected from IL-2, IL-3, IL-6, IL-11, IL-12, IL-7, IL-15, IL-21, granulocyte monocyte colony stimulating factor (GM-CSF), interferon alpha (IFN-α), interferon beta (IFN-β) or interferon gamma (IFN-γ), and erythropoietin.

48. The method of embodiment 45, wherein the encoded molecule is a soluble single-stranded variable fragment (scFv) that optionally binds a polypeptide that has an immunosuppressive effect or an immunostimulatory effect selected from CD47, PD-1, CTLA-4 and their ligands or CD28, OX-40, 4-1BB and their ligands.

49. The method of embodiment 45, wherein the encoded molecule is an immunomodulatory fusion protein optionally comprising (a) an extracellular binding domain that specifically binds an antigen derived from CD200R, SIRPα, CD279 (PD-1), CD2, CD95 (Fas), CD152 (CTLA4), CD223 (LAG3), CD272 (BTLA), A2aR, KIR, TIM3, CD300, or LPA5; (b) an intracellular binding domain derived from CD3ε, CD3δ, CD3ζ, CD25, CD27, CD28, CD40, CD47, CD79A, CD79B, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD278 (ICOS), CD357 (GITR), XAPD11, DAP10, DAP12, FcRα, FcRβ, FcRγ, Fyn, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, ROR2, Ryk, Slp76, pTα, TCRα, TCRβ, TRFM, Zap70, PTCH2, or any combination thereof; and (c) a hydrophobic transmembrane domain derived from CD2, CD3ε, CD3δ, CD3ζ, CD25, CD27, CD28, CD40, CD79A, CD79B, CD80, CD86, CD95 (Fas), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD200R, CD223 (LAG3), CD270 (HVEM), CD272 (BTLA), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), CD279 (PD-1), CD300, CD357 (GITR), A2aR, DAP10, FcRα, FcRβ, FcRγ, Fyn, GAL9, KIR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPα, pTα, TCRα, TCRβ, TIM3, TRIM, LPA5 or Zap70.

50. The method of embodiment 45, wherein the encoded molecule is a chimeric switch receptor (CSR) that optionally comprises a truncated extracellular domain of PD1 and transmembrane and cytoplasmic signaling domains of CD28.

51. The method of embodiment 45, wherein the encoded molecule is a co-receptor, optionally selected from CD4 or CD8.

52. The method of any one of embodiments 33-44, wherein the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof encodes one chain of the recombinant TCR and the second transgene encodes another chain of the recombinant TCR.

53. The method of embodiment 52, wherein the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof encodes the alpha (TCRα) chain of the recombinant TCR and the second transgene encodes the beta (TCRβ) chain of the recombinant TCR.

54. The method according to any one of embodiments 1-53, wherein the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof and/or one or more second transgenes independently further comprise a regulatory or control element.

55. The method of embodiment 54, wherein the regulatory or control element comprises a promoter, an enhancer, an intron, a polyadenylation signal, a Kozak consensus sequence, an internal ribosome entry site (IRES), a sequence encoding ribosome skip sequences, a splice acceptor sequence, or a splice donor sequence.

56. The method according to embodiment 55, wherein the regulating or controlling element comprises a promoter.

57. The method of embodiment 56, wherein the promoter is selected from a constitutive promoter, an inducible promoter, a repressible promoter, and/or a tissue-specific promoter.

58. The method of embodiment 56 or embodiment 57, wherein the promoter is selected from an RNA pol I, pol II, or pol III promoter.

59. The method according to embodiment 58, wherein the promoter is selected from:

pol III promoter, which is the U6 or H1 promoter; or

pol II promoter, which is the CMV, SV40 early region or adenovirus major late region promoter.

60. The method of any one of embodiments 56-58, wherein the promoter is or comprises the human elongation factor 1 alpha (EF1α) promoter or the MND promoter or a variant thereof.

61. The method according to any one of embodiments 56-58, wherein the promoter is an inducible promoter or a repressible promoter.

62. The method of embodiment 61, wherein the promoter comprises a Lac operator sequence, a tetracycline operator sequence, a galactose operator sequence, or a doxycycline operator sequence, or is an analog thereof, or is capable of binding to or being recognized by a Lac repressor or a tetracycline repressor, or an analog thereof.

63. The method of any one of embodiments 1-62, wherein the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof and/or one or more second transgenes independently comprise one or more multicistronic elements.

64. The method of embodiment 63, wherein the one or more multicistronic elements are located upstream of the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof, and/or one or more second transgenes.

65. The method of embodiment 63 or embodiment 64, wherein the multicistronic elements are located between the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof and one or more second transgenes.

66. The method according to any one of embodiments 63-65, wherein the multicistronic elements are located between the nucleic acid sequence encoding TCRα or a portion thereof and the nucleic acid sequence encoding TCRβ or a portion thereof.

67. The method of embodiment 66, wherein the multicistronic elements comprise a sequence encoding a ribosome skip element selected from T2A, P2A, E2A, or F2A or an internal ribosome entry site (IRES).

68. The method of embodiment 67, wherein the sequence encoding the ribosome skip element is targeted to be in-frame with the gene at the target site.

69. The method according to any one of embodiments 1-53, wherein the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof and/or one or more second transgenes are independently operably linked to an endogenous gene promoter at a target site.

70. The method of any one of embodiments 1-69, wherein the recombinant TCR is capable of binding to an antigen that is associated with, specific for, and/or expressed on a cell or tissue of a disease, disorder, or condition.

71. The method of embodiment 70, wherein the disease, disorder or condition is an infectious disease, an autoimmune disease, an inflammatory disease or a tumor or cancer.

72. The method of embodiment 70 or embodiment 71, wherein the antigen is a tumor antigen or a pathogenic antigen.

73. The method of embodiment 72, wherein the pathogenic antigen is a bacterial antigen or a viral antigen.

74. The method of embodiment 73, wherein the antigen is a viral antigen, and the viral antigen is hepatitis A, hepatitis B, hepatitis C virus (HCV), human papillomavirus (HPV), hepatitis viral infections, Epstein-Barr virus (EBV), human herpes virus 8 (HHV-8), human T-cell leukemia virus-1 (HTLV-1), human T-cell leukemia virus-2 (HTLV-2), or cytomegalovirus (CMV).

75. The method of embodiment 74, wherein the antigen is an antigen from an HPV selected from HPV-16, HPV-18, HPV-31, HPV-33, and HPV-35.

76. The method of embodiment 75, wherein the antigen is an HPV-16 antigen, which is an HPV-16 E6 or HPV-16 E7 antigen.

77. The method of embodiment 73, wherein the viral antigen is an EBV antigen selected from Epstein-Barr nuclear antigen (EBNA)-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA leader protein (EBNA-LP), latent membrane proteins LMP-1, LMP-2A and LMP-2B, EBV-EA, EBV-MA and EBV-VCA.

78. The method of embodiment 73, wherein the viral antigen is an HTLV antigen, which is TAX.

79. The method of embodiment 73, wherein the viral antigen is an HBV antigen, which is a hepatitis B core antigen or a hepatitis B envelope antigen.

80. The method according to any one of embodiments 70-72, wherein the antigen is a tumor antigen.

81. The method of embodiment 80, wherein the antigen is selected from glioma-associated antigen, β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, Melanin-A/MART-1, WT-1, S-100, MBP, CD63, MUC1 ( e.g., MUC1-8), p53, Ras, cyclin B1, HER-2/neu, carcinoembryonic antigen (CEA), gp100, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A11, MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-C1, BAGE, GAGE-1, GAGE-2, pl5, tyrosinase, tyrosinase-related protein 1 (TRP-1), tyrosinase-related protein 2 (TRP-2), β-catenin, NY-ESO-1, LAGE-1a, PP1, MDM2, MDM4, EGVFvIII, Tax, SSX2, telomerase, TARP, pp65, CDK4, vimentin, S100, eIF-4A1, IFN-inducible p78, melanotransferrin (p97), Uroplakin II, prostate-specific antigen (PSA), human kallikrein (huK2), prostate-specific membrane antigen (PSM) and prostatic acid phosphatase (PAP), neutrophil elastase, ephrin B2, BA-46, Bcr-abl, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Caspase 8, FRα, CD24, CD44, CD133, CD 166, epCAM, CA-125, HE4, Oval, estrogen receptor, progesterone receptor, uPA, PAI-1, CD19, CD20, CD22, ROR1, CD33/IL3Ra, c-Met, PSMA, Glycolipid F77, GD-2, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin.

82. The method according to any one of embodiments 1-81, wherein the immune cell is a T cell.

83. The method of embodiment 82, wherein the T cell is a CD8+ T cell or subtypes thereof.

84. The method of embodiment 82, wherein the T cell is a CD4+ T cell or subtypes thereof.

85. The method according to any one of embodiments 1-81, wherein the immune cell is obtained from a multipotent or pluripotent cell, which is optionally an iPSC.

86. The method according to any one of embodiments 1-85, wherein the immune cell comprises a T cell that is autologous to the subject.

87. The method of any one of embodiments 1-85, wherein the immune cell comprises a T cell that is autologous to the subject.

88. The method according to any one of embodiments 1-87, wherein the first template polynucleotide, one or more second template polynucleotides and/or one or more polynucleotides encoding gRNA and/or a Cas9 protein are contained in one or more vectors, which are optionally viral vectors.

89. The method of embodiment 88, wherein the vector is an AAV vector.

90. The method of embodiment 89, wherein the AAV vector is selected from an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, or AAV8 vector.

91. The method of embodiment 90, wherein the AAV vector is an AAV2 or AAV6 vector.

92. The method of embodiment 88, wherein the viral vector is a retroviral vector.

93. The method of embodiment 92, wherein the viral vector is a lentiviral vector.

94. The method according to any one of embodiments 1-93, wherein the administration of one or more agents capable of causing genetic destruction and the administration of the template polynucleotide are carried out simultaneously or sequentially, in any order.

95. The method according to any one of embodiments 1-94, wherein the administration of the template polynucleotide is carried out after the administration of one or more agents capable of causing genetic destruction.

96. The method of embodiment 95, wherein the template polynucleotide is administered immediately after or within about 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 6 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, or 4 hours after administration of one or more agents capable of causing genetic destruction.

97. The method according to any one of embodiments 33-96, wherein the administration of the template polynucleotide and the administration of one or more second template polynucleotides are carried out simultaneously or sequentially, in any order.

98. The method according to any one of embodiments 1-97, wherein the administration of one or more agents capable of causing genetic destruction and the administration of the template polynucleotide are carried out in a single experimental reaction.

99. The method according to any one of embodiments 33-98, wherein the administration of one or more agents capable of causing genetic disruption and the administration of the template polynucleotide and the second template polynucleotides are carried out in a single experimental reaction.

100. The method of any one of embodiments 1-99, wherein at least or more than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the cells in the plurality of engineered cells comprise a genetic disruption of at least one target site in a gene encoding a domain or region of the T cell receptor constant alpha (TRAC) gene and/or the T cell receptor constant beta (TRBC) gene.

101. The method of any one of embodiments 1-99, wherein at least or more than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the cells in the plurality of engineered cells express the recombinant receptor or antigen-binding fragment thereof and/or exhibit antigen binding.

102. The method of any one of embodiments 1-99, wherein the coefficient of variation of expression and/or antigen binding of the recombinant receptor or antigen-binding fragment thereof among the plurality of engineered cells is lower than 0.70, 0.65, 0.60, 0.55, 0.50, 0.45, 0.40, 0.35, or 0.30 or less.

103. The method of any one of embodiments 1-99, wherein the coefficient of variation of expression and/or antigen binding of the recombinant receptor or antigen-binding fragment thereof among the plurality of engineered cells is at least 100%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% lower than the coefficient of variation of expression and/or antigen binding of the same recombinant receptor that is integrated into the genome by random integration.

104. The method according to any one of embodiments 100-103, wherein the expression and/or antigen binding of the recombinant receptor or antigen-binding fragment thereof is assessed by contacting cells in the composition with a binding reagent specific for the TCRα chain or TCRβ chain and assessing binding of the reagent to the cells.

105. The method of embodiment 104, wherein the binding reagent is an anti-TCR Vβ antibody or is an anti-TCR Vα antibody that specifically recognizes a particular family of Vβ or Vα chains.

106. The method of embodiment 104, wherein the binding agent is a peptide antigen-MHC complex, which is optionally a tetramer.

107. An engineered cell or a plurality of engineered cells created using the method of any one of embodiments 1-106.

108. A composition comprising engineered cells or a plurality of engineered cells according to embodiment 107.

109. The composition of embodiment 108, wherein at least or more than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the cells in the composition comprise a genetic disruption of at least one target site in a gene encoding a domain or region of the T cell receptor constant alpha (TRAC) gene and/or the T cell receptor constant beta (TRBC) gene.

110. The composition of embodiment 108, wherein at least or more than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the cells in the composition express the recombinant receptor or antigen-binding fragment thereof and/or exhibit antigen binding.

111. The composition of embodiment 108, wherein the coefficient of variation of expression and/or antigen binding of the recombinant receptor or antigen-binding fragment thereof among the plurality of cells is lower than 0.70, 0.65, 0.60, 0.55, 0.50, 0.45, 0.40, 0.35, or 0.30 or less.

112. The composition of embodiment 108, wherein the coefficient of variation of expression and/or antigen binding of the recombinant receptor or antigen-binding fragment thereof among the plurality of cells is at least 100%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% lower than the coefficient of variation of expression and/or antigen binding of the same recombinant receptor that is integrated into the genome by random integration.

113. The composition of any one of embodiments 109-112, wherein the expression and/or antigen binding of the recombinant receptor or antigen-binding fragment thereof is assessed by contacting cells in the composition with a binding reagent specific for the TCRα chain or TCRβ chain and assessing binding of the reagent to the cells.

114. The composition of embodiment 113, wherein the binding reagent is an anti-TCR Vβ antibody or is an anti-TCR Vα antibody that specifically recognizes a particular family of Vβ or Vα chains.

115. The composition of embodiment 113, wherein the binding agent is a peptide antigen-MHC complex, which is optionally a tetramer.

116. The composition of any one of embodiments 108-115, further comprising a pharmaceutically acceptable carrier.

117. A composition comprising a plurality of engineered T cells comprising a recombinant receptor or antigen-binding fragment or chain thereof encoded by a transgene, and a genetic disruption of at least one target site in a T cell receptor constant alpha (TRAC) gene and/or a T cell receptor constant beta (TRBC) gene, wherein at least or more than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the cells in the composition comprise a genetic disruption of at least one target site in the TRAC gene and/or the TRBC gene; and

a transgene encoding a recombinant TCR or an antigen-binding fragment or chain thereof targeted for integration into or near one of at least one target site via homology-directed repair (HDR).

118. A composition comprising a plurality of engineered T cells comprising a recombinant receptor or antigen-binding fragment or chain thereof encoded by a transgene, and a genetic disruption of at least one target site in the T cell receptor constant alpha (TRAC) gene and/or the T cell receptor constant beta (TRBC) gene, wherein at least or more than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the cells in the composition express the recombinant receptor or antigen-binding fragment thereof and/or exhibit antigen binding; and

a transgene encoding a recombinant TCR or an antigen-binding fragment or chain thereof targeted for integration into or near one of at least one target site via homology-directed repair (HDR).

119. A composition comprising a plurality of engineered T cells comprising a recombinant receptor or an antigen-binding fragment or chain thereof encoded by a transgene, and a genetic disruption of at least one target site in the T cell receptor constant alpha (TRAC) gene and/or the T cell receptor constant beta (TRBC) gene, wherein the coefficient of variation of expression and/or antigen binding of the recombinant receptor among the plurality of cells is lower than 0.70, 0.65, 0.60, 0.55, 0.50, 0.45, 0.40, 0.35, or 0.30 or less.

120. A composition comprising a plurality of engineered T cells comprising a recombinant receptor or an antigen-binding fragment or chain thereof encoded by a transgene, and a genetic disruption of at least one target site in the T-cell receptor constant alpha (TRAC) gene and/or the T-cell receptor constant beta (TRBC) gene, wherein the coefficient of variation of expression and/or antigen binding of the recombinant receptor among the plurality of cells is at least 100%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% lower than the coefficient of variation of expression and/or antigen binding of the same recombinant receptor that is integrated into the genome by random integration.

121. The composition according to any one of embodiments 117-120, wherein the composition is obtained:

(a) administering to a plurality of T cells one or more agents, wherein each of the one or more agents is independently capable of causing genetic disruption of a target site in the T cell receptor constant alpha (TRAC) gene and/or the T cell receptor constant beta (TRBC) gene, thereby causing genetic disruption of at least one target site; and

(b) introducing into a plurality of T cells a template polynucleotide comprising a transgene encoding a recombinant T cell receptor (TCR) or an antigen-binding fragment or chain thereof, wherein the transgene encoding the recombinant TCR or an antigen-binding fragment or chain thereof is targeted for integration at or near one of at least one target site via homology-directed repair (HDR).

122. The composition of any one of embodiments 117-121, wherein the expression and/or antigen binding of the recombinant receptor or antigen-binding fragment thereof is assessed by contacting cells in the composition with a binding reagent specific for the TCRα chain or TCRβ chain and assessing binding of the reagent to the cells.

123. The composition of embodiment 122, wherein the binding reagent is an anti-TCR Vβ antibody or is an anti-TCR Vα antibody that specifically recognizes a particular family of Vβ or Vα chains.

124. The composition of embodiment 122, wherein the binding agent is a peptide antigen-MHC complex, which is optionally a tetramer.

125. The composition of any one of embodiments 121-124, wherein at least one of the one or more agents is capable of causing genetic disruption of a target site in the TRAC gene.

126. The composition of any one of embodiments 121-125, wherein at least one of the one or more agents is capable of causing genetic disruption of a target site in the TRBC gene.

127. The composition of any one of embodiments 121-126, wherein the one or more agents comprise at least one agent that is capable of causing genetic disruption of a target site in a TRAC gene and at least one agent that is capable of causing genetic disruption of a target site in a TRBC gene.

128. The composition of any one of embodiments 117-127, wherein the TRBC gene is one or both of the T cell receptor beta constant 1 ( TRBC1 ) or T cell receptor beta constant 2 ( TRBC2 ) genes.

129. The composition of any one of embodiments 121-128, wherein the one or more agents capable of causing genetic disruption comprise a DNA binding protein or a DNA binding nucleic acid that specifically binds to or hybridizes to a target site.

130. The composition of embodiment 129, wherein the one or more agents capable of causing genetic disruption comprise (a) a fusion protein comprising a DNA-directed protein and a nuclease or (b) an RNA-directed nuclease.

131. The composition of embodiment 130, wherein the DNA-directed protein or RNA-directed nuclease comprises a zinc finger protein (ZFP), a TAL protein, or a clustered regularly intervening short paddledromic nucleic acid (CRISPR)-related nuclease (Cas) specific to the target site.

132. The composition of any one of embodiments 129-131, wherein the one or more agents comprise a zinc finger nuclease (ZFN), a TAL effector nuclease (TALEN), and/or a CRISPR-Cas9 combination that specifically bind to, recognize, or hybridize to a target site.

133. The composition of any one of embodiments 121-132, wherein each of the one or more agents comprises a guide RNA (gRNA) having a targeting domain that is complementary to at least one target site.

134. The composition of embodiment 133, wherein one or more agents are administered as a ribonucleoprotein (RNP) complex comprising gRNA and Cas9 protein.

135. The composition of embodiment 134, wherein the RNP is administered via electroporation, gene gun, calcium phosphate transfection, cell condensation or cell compression.

136. The composition of embodiment 134 or embodiment 135, wherein the RNP is administered via electroporation.

137. The composition of any one of embodiments 121-132, wherein one or more agents are administered as one or more polynucleotides encoding gRNA and/or Cas9 protein.

138. The composition of any one of embodiments 121-137, wherein at least one target site is within an exon of a TRAC , TRBC1 and/or TRBC2 gene.

139. The composition of any one of embodiments 133-138, wherein the gRNA has a targeting domain that is complementary to a target site in the TRAC gene and comprises a sequence selected from UCUCUCAGCUGGUACACGGC (SEQ ID NO:28), UGGAUUUAGAGUCUCUCAGC (SEQ ID NO:29), ACACGGCAGGGUCAGGGUUC (SEQ ID NO:30), GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:31), GCUGGUACACGGCAGGGUCA (SEQ ID NO:32), CUCAGCUGGUACACGGC (SEQ ID NO:33), UGGUACACGGCAGGGUC (SEQ ID NO:34), GCUAGACAUGAGGUCUA (SEQ ID NO:35), GUCAGAUUUGUUGCUCC (SEQ ID NO:36), UCAGCUGGUACACGGCA (SEQ ID NO:37), GCAGACAGACUUGUCAC (SEQ ID NO:38), GGUACACGGCAGGGUCA (SEQ ID NO:39), CUUCAAGAGCAACAGUGCUG (SEQ ID NO:40), AGAGCAACAGUGCUGUGGCC (SEQ ID NO:41), AAAGUCAGAUUUGUUGCUCC (SEQ ID NO:42), ACAAAACUGUGCUAGACAUG (SEQ ID NO:43), AAACUGUGCUAGACAUG (SEQ ID NO:44), UGUGCUAGACAUGAGGUCUA (SEQ ID NO:45), GGCUGGGGAAGAAGGUGUCUUC (SEQ ID NO:46), GCUGGGGAAGAAGGUGUCUUC (SEQ ID NO:47), GGGGAAGAAGGUGUCUUC (SEQ ID NO:48), GUUUUGUCUGUGAUAUACACAU (SEQ ID NO:49), GGCAGACAGACUUGUCACUGGAUU (SEQ ID NO:50), GCAGACAGACUUGUCACUGGAUU (SEQ ID NO:51), GACAGACUUGUCACUGGAUU (SEQ ID NO:52), GUGAAUAGGCAGACAGACUUGUCA (SEQ ID NO:53), GAAUAGGCAGACAGACUUGUCA (SEQ ID NO:54), GAGUCUCUCAGCUGGUACACGG (SEQ ID NO:55), GUCUCUCAGCUGGUACACGG (SEQ ID NO:56), GGUACACGGCAGGGUCAGGGUU (SEQ ID NO:57) and GUACACGGCAGGGUCAGGGUU (SEQ ID NO:58).

140. The composition of embodiment 139, wherein the gRNA has a targeting domain comprising the sequence GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:31).

141. The composition of any one of embodiments 133-140, wherein the gRNA has a targeting domain that is complementary to a target site in one or more of the TRBC1 and TRBC2 genes and comprises a sequence selected from CACCCAGAUCGUCAGCGCCG (SEQ ID NO:59), CAAACACAGCGACCUCGGGU (SEQ ID NO:60), UGACGAGUGGACCCAGGAUA (SEQ ID NO:61), GGCUCUCGGAGAAUGACGAG (SEQ ID NO:62), GGCCUCGGCGCUGACGAUCU (SEQ ID NO:63), GAAAAACGUGUUCCCACCCG (SEQ ID NO:64), AUGACGAGUGGACCCAGGAU (SEQ ID NO:65), AGUCCAGUUCUACGGGCUCU (SEQ ID NO:66), CGCUGUCAAGUCCAGUUCUA (SEQ ID NO: NO:67), AUCGUCAGCGCCGAGGCCUG (SEQ ID NO:68), UCAAACACAGCGACCUCGGG (SEQ ID NO:69), CGUAGAACUGGACUUGACAG (SEQ ID NO:70), AGGCCUCGGCGCUGACGAUC (SEQ ID NO:71), UGACAGCGGAAGUGGUUGCG (SEQ ID NO:72), UUGACAGCGGAAGUGGUUGC (SEQ ID NO:73), UCUCCGAGAGCCCGUAGAAC (SEQ ID NO:74), CGGGUGGGAACACGUUUUUC (SEQ ID NO:75), GACAGGUUUGGCCCUAUCCU (SEQ ID NO:76), GAUCGUCAGCGCCGAGGCCU (SEQ ID NO:77), GGCUCAAACACAGCGACCUC (SEQ ID NO:78), UGAGGGUCUCGGCCACCUUC (SEQ ID NO:79), AGGCUUCUACCCCGACCACG (SEQ ID NO:80), CCGACCACGUGGAGCUGAGC (SEQ ID NO:81), UGACAGGUUUGGCCCUAUCC (SEQ ID NO:82), CUUGACAGCGGAAGUGGUUG (SEQ ID NO:83), AGAUCGUCAGCCCGAGGCC (SEQ ID NO:84), GCGCUGACGAUCUGGGUGAC (SEQ ID NO:85), UGAGGGCGGGGCUGCUCCUUG (SEQ ID NO:86), GUUGCGGGGGUUCUGCCAGA (SEQ ID NO:87), AGCUCAGCUCCACGUGGUCG (SEQ ID NO:88), GCGGCUGCUCAGGCAGUAUC (SEQ ID NO:89), GCGGGGGUUCUGCCAGAAGG (SEQ ID NO:90), UGGCUCAAACACAGCGACCU (SEQ ID NO:91), ACUGGACUUGACAGCGGAAG (SEQ ID NO:92), GACAGCGGAAGUGGUUGCGG (SEQ ID NO:93), GCUGUCAAGUCCAGUUCUAC (SEQ ID NO:94), GUAUCUGGAGUCAUUGAGGG (SEQ ID NO:95), CUCGGCGCUGACGAUCU (SEQ ID NO:96), CCUCGGCGCUGACGAUC (SEQ ID NO:97), CCGAGAGCCCGUAGAAC (SEQ ID NO:98), CCAGAUCGUCAGCGCCG (SEQ ID NO:99), GAAUGACGAGUGGACCC (SEQ ID NO:100), GGGUGACAGGUUUGGCCCUAUC (SEQ ID NO:101), GGUGACAGGUUUGGCCCUAUC (SEQ ID NO:102), GUGACAGGUUUGGCCCUAUC (SEQ ID NO:103), GACAGGUUUGGCCCUAUC (SEQ ID NO:104), GAUACUGCCUGAGCAGCCGCCU (SEQ ID NO:105), GACCACGUGGAGCUGAGCUGGUGG (SEQ ID NO:106), GUGGAGCUGAGCUGGUGG (SEQ ID NO:107), GGGCGGGCUGCUCCUUGAGGGGCU (SEQ ID NO:108), GGCGGGCUGCUCCUUGAGGGGCU (SEQ ID NO:109), GCGGGCUGCUCCUUGAGGGGCU (SEQ ID NO:110), GGGCUGCUCCUUGAGGGGCU (SEQ ID NO:111), GGCUGCUCCUUGAGGGGCU (SEQ ID NO:112), GCUGCUCCUUGAGGGGCU (SEQ ID NO:113), GGUGAAUGGGAAGGAGGUGCACAG (SEQ ID NO:114), GUGAAUGGGAAGGAGGUGCACAG (SEQ ID NO:115) and GAAUGGGAAGGAGGUGCACAG (SEQ ID NO:116).

142. The composition of embodiment 141, wherein the gRNA has a targeting domain comprising the sequence GGCCUCGGCGCUGACGAUCU (SEQ ID NO:63).

143. The composition of any one of embodiments 121-142, wherein the template polynucleotide comprises the structure [5' homology arm]-[transgene]-[3' homology arm].

144. The composition of embodiment 143, wherein the 5' homology arm and the 3' homology arm comprise nucleic acid sequences homologous to nucleic acid sequences surrounding the at least one target site.

145. The composition of embodiment 143 or embodiment 144, wherein the 5' homology arm comprises nucleic acid sequences that are homologous to nucleic acid sequences 5' of the target site.

146. The composition of embodiment 143 or embodiment 144, wherein the 3' homology arm comprises nucleic acid sequences that are homologous to nucleic acid sequences of the 3' target site.

147. The composition of any one of embodiments 143-146, wherein the 5' homology arm and the 3' homology arm independently have at least, or at least about, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides, or less or less than about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides.

148. The composition of embodiment 147, wherein the 5' homology arm and the 3' homology arm independently have between about 50 and 100, 100 and 250, 250 and 500, 500 and 750, 750 and 1000, 1000 and 2000 nucleotides.

149. The composition of embodiment 148, wherein the 5' homology arm and the 3' homology arm independently have from or about 100 to 1000 nucleotides, 100 to 750 nucleotides, 100 to 600 nucleotides, 100 to 400 nucleotides, 100 to 300 nucleotides, 100 to 200 nucleotides, 200 to 1000 nucleotides, 200 to 750 nucleotides, 200 to 600 nucleotides, 200 to 400 nucleotides, 200 to 300 nucleotides, 300 to 1000 nucleotides, 300 to 750 nucleotides, 300 to 600 nucleotides, 300 to 400 nucleotides, 400 to 1000 nucleotides, 400 to 750 nucleotides, 400 to 600 nucleotides, 600 to 1000 nucleotides, 600 to 750 nucleotides, or 750 to 1000 nucleotides.

150. The composition of any one of embodiments 117-149, wherein the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof is targeted for integration at or near a target site in the TRAC gene.

151. The composition of any one of embodiments 117-150, wherein the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof is targeted for integration at or near a target site in one or both of the TRBC1 and TRBC2 genes .

152. The composition of any one of embodiments 117-151, wherein the composition is obtained by further introducing into the immune cell one or more second template polynucleotides comprising one or more second transgenes, wherein the second transgene is targeted for integration at or near one of at least one target site via homology-directed repair (HDR).

153. The composition of embodiment 152, wherein the second template polynucleotide comprises the structure [second 5' homology arm]-[one or more second transgenes]-[second 3' homology arm].

154. The composition of embodiment 153, wherein the second 5' homology arm and the second 3' homology arm comprise nucleic acid sequences homologous to nucleic acid sequences surrounding the at least one target site.

55. The composition of embodiment 153 or embodiment 154, wherein the second 5' homology arm comprises nucleic acid sequences that are homologous to nucleic acid sequences of the second 5' target site.

156. The composition of embodiment 153 or embodiment 154, wherein the second 3' homology arm comprises nucleic acid sequences that are homologous to nucleic acid sequences of the second 3' target site.

157. The composition of any one of embodiments 153-156, wherein the second 5' homology arm and the second 3' homology arm independently have at least, or at least about, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides, or less or less than about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides.

158. The composition of embodiment 157, wherein the second 5' homology arm and the second 3' homology arm independently have between about 50 and 100, 100 and 250, 250 and 500, 500 and 750, 750 and 1000, 1000 and 2000 nucleotides.

159. The composition of embodiment 158, wherein the second 5' homology arm and the second 3' homology arm independently have from or about 100 to 1000 nucleotides, 100 to 750 nucleotides, 100 to 600 nucleotides, 100 to 400 nucleotides, 100 to 300 nucleotides, 100 to 200 nucleotides, 200 to 1000 nucleotides, 200 to 750 nucleotides, 200 to 600 nucleotides, 200 to 400 nucleotides, 200 to 300 nucleotides, 300 to 1000 nucleotides, 300 to 750 nucleotides, 300 to 600 nucleotides, 300 up to 400 nucleotides, from 400 to 1000 nucleotides, from 400 to 750 nucleotides, from 400 to 600 nucleotides, from 600 to 1000 nucleotides, from 600 to 750 nucleotides, or from 750 to 1000 nucleotides.

160. The composition of any one of embodiments 152-159, wherein the one or more second transgenes are targeted for integration at or near a target site in the TRAC gene.

161. The composition of any one of embodiments 152-160, wherein the one or more second transgenes are targeted for integration at or near a target site in the TRBC1 or TRBC2 gene.

162. The composition of any one of embodiments 152-161, wherein the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof is targeted for integration at or near a target site in the TRAC gene, TRBC1 gene, or TRBC2 gene, and one or more second transgenes are targeted for integration at or near one or more target sites that are not targeted by the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof.

163. The composition of any one of embodiments 152-162, wherein the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof is targeted for integration at or near a target site in the TRAC gene, and one or more second transgenes are targeted for integration at or near one or more target sites in the TRBC1 gene and/or the TRBC2 gene.

164. The composition of any one of embodiments 152-163, wherein the one or more second transgenes encode a molecule selected from a costimulatory ligand, a cytokine, a soluble single-strand variable fragment (scFv), an immunomodulatory fusion protein, a chimeric switch receptor (CSR), or a coreceptor.

165. The composition of embodiment 164, wherein the encoded molecule is a costimulatory ligand, optionally selected from a tumor necrosis factor (TNF) ligand selected from 4-1BBL, OX40L, CD70, LIGHT, and CD30L, or an immunoglobulin (Ig) superfamily ligand selected from CD80 and CD86.

166. The composition of embodiment 164, wherein the encoded molecule is a cytokine optionally selected from IL-2, IL-3, IL-6, IL-11, IL-12, IL-7, IL-15, IL-21, granulocyte monocyte colony stimulating factor (GM-CSF), interferon alpha (IFN-α), interferon beta (IFN-β) or interferon gamma (IFN-γ), and erythropoietin.

167. The composition of embodiment 164, wherein the encoded molecule is a soluble single-stranded variable fragment (scFv) that optionally binds a polypeptide that has an immunosuppressive effect or an immunostimulatory effect selected from CD47, PD-1, CTLA-4 and their ligands or CD28, OX-40, 4-1BB and their ligands.

168. The composition of embodiment 164, wherein the encoded molecule is an immunomodulatory fusion protein, optionally comprising:

(a) an extracellular binding domain that specifically binds an antigen derived from CD200R, SIRPα, CD279 (PD-1), CD2, CD95 (Fas), CD152 (CTLA4), CD223 (LAG3), CD272 (BTLA), A2aR, KIR, TIM3, CD300, or LPA5;

(b) an intracellular binding domain derived from CD3ε, CD3δ, CD3ζ, CD25, CD27, CD28, CD40, CD47, CD79A, CD79B, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD278 (ICOS), CD357 (GITR), XAPD11, DAP10, DAP12, FcRα, FcRβ, FcRγ, Fyn, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, ROR2, Ryk, Slp76, pTα, TCRα, TCRβ, TRFM, Zap70, PTCH2, or any combination thereof; and

(c) hydrophobic transmembrane domain derived from CD2, CD3ε, CD3δ, CD3ζ, CD25, CD27, CD28, CD40, CD79A, CD79B, CD80, CD86, CD95 (Fas), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD200R, CD223 (LAG3), CD270 (HVEM), CD272 (BTLA), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), CD279 (PD-1), CD300, CD357 (GITR), A2aR, DAP10, FcRα, FcRβ, FcRγ, Fyn, GAL9, KIR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPα, pTα, TCRα, TCRβ, TIM3, TRIM, LPA5 or Zap70.

169. The composition of embodiment 164, wherein the encoded molecule is a chimeric switch receptor (CSR), which optionally comprises a truncated extracellular domain of PD1 and transmembrane and cytoplasmic signaling domains of CD28.

170. The composition of embodiment 164, wherein the encoded molecule is a co-receptor, optionally selected from CD4 or CD8.

171. The composition of any one of embodiments 152-163, wherein the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof encodes one chain of the recombinant TCR and the second transgene encodes another chain of the recombinant TCR.

172. The composition of embodiment 171, wherein the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof encodes the alpha (TCRα) chain of the recombinant TCR and the second transgene encodes the beta (TCRβ) chain of the recombinant TCR.

173. The composition of any one of embodiments 117-172, wherein the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof and/or one or more second transgenes independently further comprise a regulatory or control element.

174. The composition of embodiment 173, wherein the regulatory or control element comprises a promoter, an enhancer, an intron, a polyadenylation signal, a Kozak consensus sequence, a splice acceptor sequence, or a splice donor sequence.

175. The composition of embodiment 174, wherein the regulatory or control element comprises a promoter.

176. The composition of embodiment 175, wherein the promoter is selected from a constitutive promoter, an inducible promoter, a repressible promoter, and/or a tissue-specific promoter.

177. The composition of embodiment 175 or embodiment 176, wherein the promoter is selected from an RNA pol I, pol II, or pol III promoter.

178. The composition according to embodiment 177, wherein the promoter is selected from:

pol III promoter, which is the U6 or H1 promoter; or

pol II promoter, which is CMV, the SV40 early region promoter, or the major late region of adenovirus.

179. The composition of any one of embodiments 175-177, wherein the promoter is or comprises the human elongation factor 1 alpha (EF1α) promoter or the MND promoter or a variant thereof.

180. The composition of any one of embodiments 175-177, wherein the promoter is an inducible promoter or a repressible promoter.

181. The composition of embodiment 180, wherein the promoter comprises a Lac operator sequence, a tetracycline operator sequence, a galactose operator sequence, or a doxycycline operator sequence, or is an analog thereof, or is capable of binding to or being recognized by a Lac repressor or a tetracycline repressor, or an analog thereof.

182. The composition of any one of embodiments 108-181, wherein the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof and/or one or more second transgenes independently comprise one or more multicistronic elements.

183. The composition of embodiment 182, wherein one or more multicistronic elements are located upstream of the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof, and/or one or more second transgenes.

184. The composition of embodiment 182 or embodiment 183, wherein the multicistronic elements are located between the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof and one or more second transgenes.

185. The composition of any one of embodiments 182-184, wherein the multicistronic elements are located between the nucleic acid sequence encoding TCRα or a portion thereof and the nucleic acid sequence encoding TCRβ or a portion thereof.

186. The composition of any one of embodiments 182-185, wherein the multicistronic elements comprise a sequence encoding a ribosome skip element selected from T2A, P2A, E2A, or F2A, or an internal ribosome entry site (IRES).

187. The composition of embodiment 186, wherein the sequence encoding the ribosome skip element is targeted to be in-frame with the gene at the target site.

188. The composition of any one of embodiments 117-172, wherein in HDR, the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof, and/or one or more second transgenes, is independently operably linked to an endogenous gene promoter at a target site.

189. The composition of any one of embodiments 117-188, wherein the recombinant TCR is capable of binding to an antigen that is associated with, specific for, and/or expressed on a cell or tissue of a disease, disorder, or condition.

190. The composition of embodiment 189, wherein the disease, disorder or condition is an infectious disease, an autoimmune disease, an inflammatory disease or a tumor or cancer.

191. The composition of embodiment 189 or embodiment 190, wherein the antigen is a tumor antigen or a pathogenic antigen.

192. The composition of embodiment 191, wherein the pathogenic antigen is a bacterial antigen or a viral antigen.

193. The composition of embodiment 192, wherein the antigen is a viral antigen, and the viral antigen is hepatitis A, hepatitis B, hepatitis C virus (HCV), human papillomavirus (HPV), hepatitis viral infections, Epstein-Barr virus (EBV), human herpes virus 8 (HHV-8), human T-cell leukemia virus-1 (HTLV-1), human T-cell leukemia virus-2 (HTLV-2), or cytomegalovirus (CMV).

194. The composition of embodiment 193, wherein the antigen is an antigen from HPV selected from HPV-16, HPV-18, HPV-31, HPV-33, and HPV-35.

195. The composition of embodiment 194, wherein the antigen is an HPV-16 antigen that is an HPV-16 E6 or HPV-16 E7 antigen.

196. The composition of embodiment 192, wherein the viral antigen is an EBV antigen selected from Epstein-Barr nuclear antigen (EBNA)-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA leader protein (EBNA-LP), latent membrane proteins LMP-1, LMP-2A and LMP-2B, EBV-EA, EBV-MA and EBV-VCA.

197. The composition of embodiment 192, wherein the viral antigen is an HTLV antigen, which is TAX.

198. The composition of embodiment 192, wherein the viral antigen is an HBV antigen, which is a hepatitis B core antigen or a hepatitis B envelope antigen.

199. The composition of any one of embodiments 189-191, wherein the antigen is a tumor antigen.

200. The composition of embodiment 199, wherein the antigen is selected from glioma-associated antigen, β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, Melanin-A/MART-1, WT-1, S-100, MBP, CD63, MUC1 ( e.g., MUC1-8), p53, Ras, cyclin B1, HER-2/neu, carcinoembryonic antigen (CEA), gp100, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A11, MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-C1, BAGE, GAGE-1, GAGE-2, pl5, tyrosinase, tyrosinase-related protein 1 (TRP-1), tyrosinase-related protein 2 (TRP-2), β-catenin, NY-ESO-1, LAGE-1a, PP1, MDM2, MDM4, EGVFvIII, Tax, SSX2, telomerase, TARP, pp65, CDK4, vimentin, S100, eIF-4A1, IFN-inducible p78, melanotransferrin (p97), Uroplakin II, prostate-specific antigen (PSA), human kallikrein (huK2), prostate-specific membrane antigen (PSM), and prostate-specific acid phosphatase (PAP), neutrophil elastase, ephrin B2, BA-46, Bcr-abl, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Caspase 8, FRα, CD24, CD44, CD133, CD 166, epCAM, CA-125, HE4, Oval, estrogen receptor, progesterone receptor, uPA, PAI-1, CD19, CD20, CD22, ROR1, CD33/IL3Ra, c-Met, PSMA, Glycolipid F77, GD-2, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin.

201. The composition of any one of embodiments 117-200, wherein the T cell is a CD8+ T cell or subtypes thereof.

202. The composition of any one of embodiments 117-200, wherein the T cell is a CD4+ T cell or subtypes thereof.

203. The composition of any one of embodiments 117-202, wherein the T cell is autologous to the subject.

204. The composition of any one of embodiments 117-202, wherein the T cell is allogeneic to the subject.

205. The composition of any one of embodiments 117-204, wherein the first template polynucleotide, one or more second template polynucleotides and/or one or more polynucleotides encoding gRNA and/or Cas9 protein are contained in one or more vectors, which are optionally viral vectors.

206. The composition of embodiment 205, wherein the vector is an AAV vector.

207. The composition of embodiment 206, wherein the AAV vector is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, or AAV8 vector.

208. The composition of embodiment 207, wherein the AAV vector is an AAV2 or AAV6 vector.

209. The composition of embodiment 205, wherein the viral vector is a retroviral vector.

210. The composition of embodiment 209, wherein the viral vector is a lentiviral vector.

211. The composition of any one of embodiments 117-210, wherein the administration of one or more agents capable of causing genetic destruction and the administration of the template polynucleotide are carried out simultaneously or sequentially, in any order.

212. The composition of any one of embodiments 117-211, wherein the administration of the template polynucleotide is carried out after the administration of one or more agents capable of causing genetic destruction.

213. The composition of embodiment 212, wherein the template polynucleotide is administered immediately after or within about 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 6 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, or 4 hours after administration of one or more agents capable of causing genetic destruction.

214. The composition of any one of embodiments 152-213, wherein the administration of the template polynucleotide and the administration of one or more second template polynucleotides are carried out simultaneously or sequentially, in any order.

215. The composition of any one of embodiments 117-214, wherein the administration of one or more agents capable of causing genetic disruption and the administration of the template polynucleotide are carried out in a single experimental reaction.

216. The composition of any one of embodiments 152-215, wherein the administration of one or more agents capable of causing genetic disruption and the administration of the template polynucleotide and the second template polynucleotides are carried out in a single experimental reaction.

217. The composition of any one of embodiments 108-216, further comprising a pharmaceutically acceptable carrier.

218. A method of treatment comprising administering an engineered cell, a plurality of engineered cells, or a composition of any one of embodiments 107-216 to a subject.

219. Use of an engineered cell, a plurality of engineered cells, or a composition of any one of embodiments 107-216 for treating cancer.

220. Use of an engineered cell, a plurality of engineered cells, or a composition of any one of embodiments 107-216 in the manufacture of a medicament for the treatment of cancer.

221. An engineered cell, a plurality of engineered cells, or a composition of any one of embodiments 107-216 for use in treating cancer.

222. A set containing:

one or more agents, wherein each of the one or more agents is independently capable of causing genetic disruption of a target site in the T cell receptor alpha constant (TRAC) gene and/or the T cell receptor beta constant (TRBC) gene; and

a template polynucleotide comprising a transgene encoding a recombinant TCR or an antigen-binding fragment or chain thereof, wherein the transgene encoding the recombinant TCR or an antigen-binding fragment or chain thereof is targeted for integration at or near a target site via homology-directed repair (HDR).

223. The kit of embodiment 222, wherein the one or more agents capable of causing genetic disruption comprises a DNA binding protein or a DNA binding nucleic acid that specifically binds to or hybridizes to a target site.

224. The kit of embodiment 223, wherein the one or more agents capable of causing genetic disruption comprises (a) a fusion protein comprising a DNA-directed protein and a nuclease or (b) an RNA-directed nuclease.

225. The kit of embodiment 224, wherein the DNA-directed protein or RNA-directed nuclease comprises a zinc finger protein (ZFP), a TAL protein, or a clustered regularly intervening short paddledromic nucleic acid (CRISPR)-related nuclease (Cas) specific to the target site.

226. The kit of any one of embodiments 222-225, wherein the one or more agents comprise a zinc finger nuclease (ZFN), a TAL effector nuclease (TALEN), and/or a CRISPR-Cas9 combination that specifically bind to, recognize, or hybridize to a target site.

227. The kit of any one of embodiments 222-226, wherein each of the one or more agents comprises a guide RNA (gRNA) having a targeting domain that is complementary to at least one target site.

228. The kit of embodiment 227, wherein one or more agents are administered as a ribonucleoprotein (RNP) complex comprising gRNA and Cas9 protein.

229. The kit of embodiment 228, wherein the RNP is administered via electroporation, gene gun, calcium phosphate transfection, cell condensation or squeezing.

230. The kit of embodiment 228 or embodiment 229, wherein the RNP is administered via electroporation.

231. The kit according to any one of embodiments 222-230, wherein one or more agents are administered as one or more polynucleotides encoding gRNA and/or Cas9 protein.

232. The kit of any one of embodiments 222-231, wherein at least one target site is within an exon of a TRAC , TRBC1 and/or TRBC2 gene.

233. The kit of any one of embodiments 227-232, wherein the gRNA has a targeting domain that is complementary to a target site in the TRAC gene and comprises a sequence selected from UCUCUCAGCUGGUACACGGC (SEQ ID NO:28), UGGAUUUAGAGUCUCUCAGC (SEQ ID NO:29), ACACGGCAGGGUCAGGGUUC (SEQ ID NO:30), GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:31), GCUGGUACACGGCAGGGUCA (SEQ ID NO:32), CUCAGCUGGUACACGGC (SEQ ID NO:33), UGGUACACGGCAGGGUC (SEQ ID NO:34), GCUAGACAUGAGGUCUA (SEQ ID NO:35), GUCAGAUUUGUUGCUCC (SEQ ID NO:36), UCAGCUGGUACACGGCA (SEQ ID NO:37), GCAGACAGACUUGUCAC (SEQ ID NO:38), GGUACACGGCAGGGUCA (SEQ ID NO:39), CUUCAAGAGCAACAGUGCUG (SEQ ID NO:40), AGAGCAACAGUGCUGUGGCC (SEQ ID NO:41), AAAGUCAGAUUUGUUGCUCC (SEQ ID NO:42), ACAAAACUGUGCUAGACAUG (SEQ ID NO:43), AAACUGUGCUAGACAUG (SEQ ID NO:44), UGUGCUAGACAUGAGGUCUA (SEQ ID NO:45), GGCUGGGGAAGAAGGUGUCUUC (SEQ ID NO:46), GCUGGGGAAGAAGGUGUCUUC (SEQ ID NO:47), GGGGAAGAAGGUGUCUUC (SEQ ID NO:48), GUUUUGUCUGUGAUAUACACAU (SEQ ID NO:49), GGCAGACAGACUUGUCACUGGAUU (SEQ ID NO:50), GCAGACAGACUUGUCACUGGAUU (SEQ ID NO:51), GACAGACUUGUCACUGGAUU (SEQ ID NO:52), GUGAAUAGGCAGACAGACUUGUCA (SEQ ID NO:53), GAAUAGGCAGACAGACUUGUCA (SEQ ID NO:54), GAGUCUCUCAGCUGGUACACGG (SEQ ID NO:55), GUCUCUCAGCUGGUACACGG (SEQ ID NO:56), GGUACACGGCAGGGUCAGGGUU (SEQ ID NO:57) and GUACACGGCAGGGUCAGGGUU (SEQ ID NO:58).

234. The kit of embodiment 233, wherein the gRNA has a targeting domain comprising the sequence GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:31).

235. The kit of any one of embodiments 227-234, wherein the gRNA has a targeting domain that is complementary to a target site in one or more of the TRBC1 and TRBC2 genes and comprises a sequence selected from CACCCAGAUCGUCAGCGCCG (SEQ ID NO:59), CAAACACAGCGACCUCGGGU (SEQ ID NO:60), UGACGAGUGGACCCAGGAUA (SEQ ID NO:61), GGCUCUCGGAGAAUGACGAG (SEQ ID NO:62), GGCCUCGGCGCUGACGAUCU (SEQ ID NO:63), GAAAAACGUGUUCCCACCCG (SEQ ID NO:64), AUGACGAGUGGACCCAGGAU (SEQ ID NO:65), AGUCCAGUUCUACGGGCUCU (SEQ ID NO:66), CGCUGUCAAGUCCAGUUCUA (SEQ ID NO: NO:67), AUCGUCAGCGCCGAGGCCUG (SEQ ID NO:68), UCAAACACAGCGACCUCGGG (SEQ ID NO:69), CGUAGAACUGGACUUGACAG (SEQ ID NO:70), AGGCCUCGGCGCUGACGAUC (SEQ ID NO:71), UGACAGCGGAAGUGGUUGCG (SEQ ID NO:72), UUGACAGCGGAAGUGGUUGC (SEQ ID NO:73), UCUCCGAGAGCCCGUAGAAC (SEQ ID NO:74), CGGGUGGGAACACGUUUUUC (SEQ ID NO:75), GACAGGUUUGGCCCUAUCCU (SEQ ID NO:76), GAUCGUCAGCGCCGAGGCCU (SEQ ID NO:77), GGCUCAAACACAGCGACCUC (SEQ ID NO:78), UGAGGGUCUCGGCCACCUUC (SEQ ID NO:79), AGGCUUCUACCCCGACCACG (SEQ ID NO:80), CCGACCACGUGGAGCUGAGC (SEQ ID NO:81), UGACAGGUUUGGCCCUAUCC (SEQ ID NO:82), CUUGACAGCGGAAGUGGUUG (SEQ ID NO:83), AGAUCGUCAGCCCGAGGCC (SEQ ID NO:84), GCGCUGACGAUCUGGGUGAC (SEQ ID NO:85), UGAGGGCGGGGCUGCUCCUUG (SEQ ID NO:86), GUUGCGGGGGUUCUGCCAGA (SEQ ID NO:87), AGCUCAGCUCCACGUGGUCG (SEQ ID NO:88), GCGGCUGCUCAGGCAGUAUC (SEQ ID NO:89), GCGGGGGUUCUGCCAGAAGG (SEQ ID NO:90), UGGCUCAAACACAGCGACCU (SEQ ID NO:91), ACUGGACUUGACAGCGGAAG (SEQ ID NO:92), GACAGCGGAAGUGGUUGCGG (SEQ ID NO:93), GCUGUCAAGUCCAGUUCUAC (SEQ ID NO:94), GUAUCUGGAGUCAUUGAGGG (SEQ ID NO:95), CUCGGCGCUGACGAUCU (SEQ ID NO:96), CCUCGGCGCUGACGAUC (SEQ ID NO:97), CCGAGAGCCCGUAGAAC (SEQ ID NO:98), CCAGAUCGUCAGCGCCG (SEQ ID NO:99), GAAUGACGAGUGGACCC (SEQ ID NO:100), GGGUGACAGGUUUGGCCCUAUC (SEQ ID NO:101), GGUGACAGGUUUGGCCCUAUC (SEQ ID NO:102), GUGACAGGUUUGGCCCUAUC (SEQ ID NO:103), GACAGGUUUGGCCCUAUC (SEQ ID NO:104), GAUACUGCCUGAGCAGCCGCCU (SEQ ID NO:105), GACCACGUGGAGCUGAGCUGGUGG (SEQ ID NO:106), GUGGAGCUGAGCUGGUGG (SEQ ID NO:107), GGGCGGGCUGCUCCUUGAGGGGCU (SEQ ID NO:108), GGCGGGCUGCUCCUUGAGGGGCU (SEQ ID NO:109), GCGGGCUGCUCCUUGAGGGGCU (SEQ ID NO:110), GGGCUGCUCCUUGAGGGGCU (SEQ ID NO:111), GGCUGCUCCUUGAGGGGCU (SEQ ID NO:112), GCUGCUCCUUGAGGGGCU (SEQ ID NO:113), GGUGAAUGGGAAGGAGGUGCACAG (SEQ ID NO:114), GUGAAUGGGAAGGAGGUGCACAG (SEQ ID NO:115) and GAAUGGGAAGGAGGUGCACAG (SEQ ID NO:116).

236. The kit of embodiment 235, wherein the gRNA has a targeting domain comprising the sequence GGCCUCGGCGCUGACGAUCU (SEQ ID NO:63).

237. The kit of any one of embodiments 222-236, wherein the template polynucleotide comprises the structure [5' homology arm]-[transgene]-[3' homology arm].

238. The kit of embodiment 237, wherein the 5' homology arm and the 3' homology arm comprise nucleic acid sequences homologous to nucleic acid sequences surrounding at least one target site.

239. The kit of embodiment 237 or embodiment 238, wherein the 5' homology arm comprises nucleic acid sequences that are homologous to nucleic acid sequences 5' of the target site.

240. The kit of embodiment 237 or embodiment 238, wherein the 3' homology arm comprises nucleic acid sequences that are homologous to nucleic acid sequences of the 3' target site.

241. The kit of any one of embodiments 237-240, wherein the 5' homology arm and the 3' homology arm independently have at least, or at least about, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides, or less, or less than about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides.

242. The kit of embodiment 241, wherein the 5' homology arm and the 3' homology arm independently have between about 50 and 100, 100 and 250, 250 and 500, 500 and 750, 750 and 1000, 1000 and 2000 nucleotides.

243. The kit of embodiment 242, wherein the 5' homology arm and the 3' homology arm independently have from or about 100 to 1000 nucleotides, 100 to 750 nucleotides, 100 to 600 nucleotides, 100 to 400 nucleotides, 100 to 300 nucleotides, 100 to 200 nucleotides, 200 to 1000 nucleotides, 200 to 750 nucleotides, 200 to 600 nucleotides, 200 to 400 nucleotides, 200 to 300 nucleotides, 300 to 1000 nucleotides, 300 to 750 nucleotides, 300 to 600 nucleotides, 300 to 400 nucleotides, 400 to 1000 nucleotides, 400 to 750 nucleotides, 400 to 600 nucleotides, 600 to 1000 nucleotides, 600 to 750 nucleotides, or 750 to 1000 nucleotides.

244. The kit of any one of embodiments 222-243, wherein the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof is targeted for integration at or near a target site in the TRAC gene.

245. The kit of any one of embodiments 222-244, wherein the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof is targeted for integration at or near a target site in one or both of the TRBC1 and TRBC2 genes .

246. The kit of any one of embodiments 222-245, further comprising one or more second template polynucleotides comprising one or more second transgenes, wherein the second transgene is targeted for integration at or near one of the at least one target site via homology-directed repair (HDR).

247. The kit of embodiment 246, wherein the second template polynucleotide comprises the structure [second 5' homology arm]-[one or more second transgenes]-[second 3' homology arm].

248. The kit of embodiment 247, wherein the second 5' homology arm and the second 3' homology arm comprise nucleic acid sequences homologous to nucleic acid sequences surrounding at least one target site.

249. The kit of embodiment 247 or embodiment 248, wherein the second 5' homology arm comprises nucleic acid sequences that are homologous to nucleic acid sequences of the second 5' target site.

250. The kit of embodiment 247 or embodiment 248, wherein the second 3' homology arm comprises nucleic acid sequences that are homologous to nucleic acid sequences of the second 3' target site.

251. The kit of any one of embodiments 247-250, wherein the second 5' homology arm and the second 3' homology arm independently have at least, or at least about, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides, or less, or less than about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides.

252. The kit of embodiment 251, wherein the second 5' homology arm and the second 3' homology arm independently have between about 50 and 100, 100 and 250, 250 and 500, 500 and 750, 750 and 1000, 1000 and 2000 nucleotides.

253. The kit of embodiment 252, wherein the second 5' homology arm and the second 3' homology arm independently have from or about 100 to 1000 nucleotides, 100 to 750 nucleotides, 100 to 600 nucleotides, 100 to 400 nucleotides, 100 to 300 nucleotides, 100 to 200 nucleotides, 200 to 1000 nucleotides, 200 to 750 nucleotides, 200 to 600 nucleotides, 200 to 400 nucleotides, 200 to 300 nucleotides, 300 to 1000 nucleotides, 300 to 750 nucleotides, 300 to 600 nucleotides, 300 to 400 nucleotides, 400 to 1000 nucleotides, 400 to 750 nucleotides, 400 to 600 nucleotides, 600 to 1000 nucleotides, 600 to 750 nucleotides, or 750 to 1000 nucleotides.

254. The kit of any one of embodiments 246-253, wherein the one or more second transgenes are targeted for integration at or near a target site in the TRAC gene.

255. The kit of any one of embodiments 246-253, wherein the one or more second transgenes are targeted for integration at or near a target site in the TRBC1 or TRBC2 gene.

256. The kit of any one of embodiments 246-255, wherein the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof is targeted for integration at or near a target site in the TRAC gene, TRBC1 gene, or TRBC2 gene, and one or more second transgenes are targeted for integration at or near one or more target sites that are not targeted by the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof.

257. The kit of any one of embodiments 246-256, wherein the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof is targeted for integration at or near a target site in the TRAC gene, and one or more second transgenes are targeted for integration at or near one or more target sites in the TRBC1 gene and/or the TRBC2 gene.

258. The kit of any one of embodiments 246-257, wherein the one or more second transgenes encode a molecule selected from a costimulatory ligand, a cytokine, a soluble single-stranded variable fragment (scFv), an immunomodulatory fusion protein, a chimeric switch receptor (CSR), or a coreceptor.

259. The kit of embodiment 258, wherein the encoded molecule is a costimulatory ligand, optionally selected from a tumor necrosis factor (TNF) ligand selected from 4-1BBL, OX40L, CD70, LIGHT, and CD30L, or an immunoglobulin (Ig) superfamily ligand selected from CD80 and CD86.

260. The kit of embodiment 258, wherein the encoded molecule is a cytokine optionally selected from IL-2, IL-3, IL-6, IL-11, IL-30, IL-7, IL-24, IL-30, granulocyte monocyte colony stimulating factor (GM-CSF), interferon alpha (IFN-α), interferon beta (IFN-β) or interferon gamma (IFN-γ), and erythropoietin.

261. The kit of embodiment 258, wherein the encoded molecule is a soluble single-stranded variable fragment (scFv) that optionally binds a polypeptide that has an immunosuppressive effect or an immunostimulatory effect selected from CD47, PD-1, CTLA-4 and their ligands or CD28, OX-40, 4-1BB and their ligands.

262. The kit of embodiment 258, wherein the encoded molecule is an immunomodulatory fusion protein, optionally comprising:

(a) an extracellular binding domain that specifically binds an antigen derived from CD290R, SIRPα, CD279 (PD-1), CD2, CD95 (Fas), CD242 (CTLA4), CD223 (LAG3), CD272 (BTLA), A2aR, KIR, TIM3, CD300, or LPA5;

(b) an intracellular binding domain derived from CD3ε, CD3δ, CD3ζ, CD25, CD27, CD28, CD40, CD47, CD79A, CD79B, CD224 (OX40), CD227 (4-1BB), CD240 (SLAMF1), CD278 (ICOS), CD357 (GITR), XAPD11, DAP10, DAP30, FcRα, FcRβ, FcRγ, Fyn, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, ROR2, Ryk, Slp76, pTα, TCRα, TCRβ, TRFM, Zap70, PTCH2, or any combination thereof; and

(c) hydrophobic transmembrane domain derived from CD2, CD3ε, CD3δ, CD3ζ, CD25, CD27, CD28, CD40, CD79A, CD79B, CD80, CD86, CD95 (Fas), CD224 (OX40), CD227 (4-1BB), CD240 (SLAMF1), CD242 (CTLA4), CD290R, CD223 (LAG3), CD270 (HVEM), CD272 (BTLA), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), CD279 (PD-1), CD300, CD357 (GITR), A2aR, DAP10, FcRα, FcRβ, FcRγ, Fyn, GAL9, KIR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPα, pTα, TCRα, TCRβ, TIM3, TRIM, LPA5 or Zap70.

263. The kit of embodiment 258, wherein the encoded molecule is a chimeric switch receptor (CSR), which optionally comprises a truncated extracellular domain of PD1 and transmembrane and cytoplasmic signaling domains of CD28.

264. The kit of embodiment 258, wherein the encoded molecule is a co-receptor, optionally selected from CD4 or CD8.

265. The kit of any one of embodiments 246-257, wherein the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof encodes one chain of the recombinant TCR and the second transgene encodes another chain of the recombinant TCR.

266. The kit of embodiment 265, wherein the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof encodes the alpha (TCRα) chain of the recombinant TCR and the second transgene encodes the beta (TCRβ) chain of the recombinant TCR.

267. The kit of any one of embodiments 222-266, wherein the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof and/or one or more second transgenes independently further comprise a regulatory or control element.

268. The kit of embodiment 267, wherein the regulatory or control element comprises a promoter, an enhancer, an intron, a polyadenylation signal, a Kozak consensus sequence, a splice acceptor sequence, or a splice donor sequence.

269. The kit according to embodiment 268, wherein the regulatory or control element comprises a promoter.

270. The kit of embodiment 269, wherein the promoter is selected from a constitutive promoter, an inducible promoter, a repressible promoter, and/or a tissue-specific promoter.

271. The kit of embodiment 269 or embodiment 270, wherein the promoter is selected from an RNA pol I, pol II, or pol III promoter.

272. The kit according to embodiment 271, wherein the promoter is selected from:

pol III promoter, which is the U6 or H1 promoter; or

pol II promoter, which is CMV, SV40 early region promoter or adenovirus major late region promoter.

273. The kit of any one of embodiments 269-271, wherein the promoter is or comprises the human elongation factor 1 alpha (EF1α) promoter or the MND promoter or a variant thereof.

274. The kit according to any one of embodiments 269-271, wherein the promoter is an inducible promoter or a repressible promoter.

275. The kit of embodiment 274, wherein the promoter comprises a Lac operator sequence, a tetracycline operator sequence, a galactose operator sequence, or a doxycycline operator sequence, or is an analog thereof, or is capable of binding to or being recognized by a Lac repressor or a tetracycline repressor, or an analog thereof.

276. The kit of any one of embodiments 222-275, wherein the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof and/or one or more second transgenes independently comprise one or more multicistronic elements.

277. The kit of embodiment 276, wherein the one or more multicistronic elements are located upstream of the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof, and/or one or more second transgenes.

278. The kit of embodiment 276 or embodiment 277, wherein the multicistronic elements are located between the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof and one or more second transgenes.

279. The kit of any one of embodiments 276-278, wherein the multicistronic elements are located between the nucleic acid sequence encoding TCRα or a portion thereof and the nucleic acid sequence encoding TCRβ or a portion thereof.

280. The kit of any one of embodiments 276-279, wherein the multicistronic elements comprise a sequence encoding a ribosome skip element selected from T2A, P2A, E2A or F2A or an internal ribosome entry site (IRES).

281. The kit of embodiment 280, wherein the sequence encoding the ribosome skip element is targeted to be in-frame with the gene at the target site.

282. The kit of any one of embodiments 222-266, wherein in HDR, the transgene encoding the recombinant TCR or antigen-binding fragment or chain thereof and/or one or more second transgenes are independently operably linked to an endogenous gene promoter at a target site.

283. The kit of any one of embodiments 222-282, wherein the recombinant TCR is capable of binding to an antigen that is associated with, specific for, and/or expressed on a cell or tissue of a disease, disorder, or condition.

284. The kit of embodiment 283, wherein the disease, disorder or condition is an infectious disease, an autoimmune disease, an inflammatory disease or a tumor or cancer.

285. The kit of embodiment 283 or embodiment 284, wherein the antigen is a tumor antigen or a pathogenic antigen.

286. The kit according to embodiment 285, wherein the pathogenic antigen is a bacterial antigen or a viral antigen.

287. The kit of embodiment 286, wherein the antigen is a viral antigen, and the viral antigen is hepatitis A, hepatitis B, hepatitis C virus (HCV), human papillomavirus (HPV), hepatitis viral infections, Epstein-Barr virus (EBV), human herpes virus 8 (HHV-8), human T-cell leukemia virus-1 (HTLV-1), human T-cell leukemia virus-2 (HTLV-2), or cytomegalovirus (CMV).

288. The kit of embodiment 287, wherein the antigen is an antigen from HPV selected from HPV-25, HPV-27, HPV-31, HPV-33, and HPV-35.

289. The kit of embodiment 288, wherein the antigen is an HPV-25 antigen that is an HPV-25 E6 or HPV-25 E7 antigen.

290. The kit of embodiment 286, wherein the viral antigen is an EBV antigen selected from Epstein-Barr nuclear antigen (EBNA)-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA leader protein (EBNA-LP), latent membrane proteins LMP-1, LMP-2A, and LMP-2B, EBV-EA, EBV-MA and EBV-VCA.

291. The kit according to embodiment 286, wherein the viral antigen is an HTLV antigen, which is TAX.

292. The kit of embodiment 286, wherein the viral antigen is an HBV antigen, which is a hepatitis B core antigen or a hepatitis B envelope antigen.

293. The kit according to any one of embodiments 283-285, wherein the antigen is a tumor antigen.

294. The kit of embodiment 293, wherein the antigen is selected from glioma-associated antigen, β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, Melanin-A/MART-1, WT-1, S-100, MBP, CD63, MUC1 ( e.g., MUC1-8), p53, Ras, cyclin B1, HER-2/neu, carcinoembryonic antigen (CEA), gp100, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A11, MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-C1, BAGE, GAGE-1, GAGE-2, pl5, tyrosinase, tyrosinase-related protein 1 (TRP-1), tyrosinase-related protein 2 (TRP-2), β-catenin, NY-ESO-1, LAGE-1a, PP1, MDM2, MDM4, EGVFvIII, Tax, SSX2, telomerase, TARP, pp65, CDK4, vimentin, S100, eIF-4A1, IFN-inducible p78, melanotransferrin (p97), Uroplakin II, prostate-specific antigen (PSA), human kallikrein (huK2), prostate-specific membrane antigen (PSM), and prostate-specific acid phosphatase (PAP), neutrophil elastase, ephrin B2, BA-46, Bcr-abl, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Caspase 8, FRα, CD24, CD44, CD223, CD 256, epCAM, CA-224, HE4, Oval, estrogen receptor, progesterone receptor, uPA, PAI-1, CD28, CD29, CD22, ROR1, CD33/IL3Ra, c-Met, PSMA, Glycolipid F77, GD-2, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin.

295. The kit according to any one of embodiments 222-294, wherein the first template polynucleotide, one or more second template polynucleotides and/or one or more polynucleotides encoding gRNA and/or Cas9 protein are contained in one or more vectors, which are optionally viral vectors.

296. The kit according to embodiment 295, wherein the vector is an AAV vector.

297. The kit of embodiment 296, wherein the AAV vector is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, or AAV8 vector.

298. The kit according to embodiment 297, wherein the AAV vector is an AAV2 or an AAV6 vector.

299. The kit according to embodiment 295, wherein the viral vector is a retroviral vector.

300. The kit according to embodiment 299, wherein the viral vector is a lentiviral vector.

X. EXAMPLES

The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1: Generation and evaluation of engineered T cells expressing recombinant T cell receptor (TCR) by targeted knockin or random integration of TCR encoding sequences

Polynucleotides encoding generic recombinant T-cell receptors (TCRs) are introduced into T cells by genetic disruption at the locus of the endogenous gene encoding the T-cell receptor alpha (TCRα) chain, CRISPR/Cas9-mediated genome editing and targeted integration at the site of genetic disruption via homology-directed repair (HDR) or random integration via lentiviral transduction.

A. Recombinant TCR transgene constructs

Exemplary template polynucleotides are designed for targeted integration by HDR of a transgene containing nucleic acid sequences encoding one of two exemplary recombinant TCRs. The general structure of exemplary template polynucleotides is as follows: [5' homology arm]-[transgene sequences]-[3' homology arm]. The homology arms include approximately 600 bp of nucleic acid sequences homologous to sequences surrounding the target integration site in exon 1 of the human TCR α constant region ( TRAC ) gene (5' homology arm, sequence shown in SEQ ID NO:124; 3' homology arm, sequence shown in SEQ ID NO:125).

The transgene comprises nucleic acid sequences encoding the α and β chains of one or more exemplary recombinant TCRs that recognize an epitope of the E7 oncoprotein of human papillomavirus (HPV) 16 (TCR #1 and TCR #2), in which the sequences encoding the TCRα and TCRβ chains are separated by the 2A ribosomal skip element. The nucleotide sequences encoding TCR #1 and the constant region for TCR #2 are also modified by codon optimization and/or mutations to promote the formation of a non-native disulfide bond at the interface between the constant domains of the TCR to enhance TCR pairing and stability. Non-native disulfide bonds are strengthened by modification of TCR chains at residue 48 of the TCR alpha chain constant region (Cα) region from Thr to Cys, and residue 57 of the TCR beta chain constant region (Cβ) region from Ser to Cys (see Kuball et al. (2007) Blood, 109:2331–2338).

The transgene also comprises either a) a human elongation factor 1 alpha (EF1α) promoter to drive expression of recombinant TCR encoding sequences (the sequence set forth in SEQ ID NO:127); or b) sequences encoding the P2A ribosome skip element (the sequence set forth in SEQ ID NO:128) upstream of the recombinant TCR encoding sequences to drive expression of recombinant TCR from the endogenous TCRα locus by HDR-mediated in-frame targeted integration into the human TCRα gene constant region ( TRAC ).

For targeted integration of HDR, adeno-associated virus (AAV) vector constructs containing the template polynucleotides described above are generated. An AAV stock solution is prepared by triple transfecting an AAV vector that includes a template polynucleotide, a serotype helper plasmid, and an adenoviral helper plasmid into a colony of 293T cells. Transfected cells are harvested, lysed, and the AAV stock solution is collected for cell transduction.

As a control for random integration, nucleic acid sequences encoding the exemplary recombinant TCR transgene constructs described above, or sequences encoding a reference TCR capable of binding to HPV 16 E7 but containing murine Cα and Cβ regions, under the control of the EF1α promoter, were introduced into a exemplary HIV-1-derived lentiviral vector. Pseudotypic lentiviral vector particles were produced by standard methods by transiently transfecting HEK-293T cells with the resulting vectors, helper plasmids (containing gagpol plasmids and rev plasmids), and a pseudotyping plasmid, and used to transduce the cells.

B. Creation of engineered T cells

Primary human CD4+ and CD8+ T cells from 2 different human donors were isolated by immunoaffinity-based selection from human peripheral blood mononuclear cells (PBMC) obtained from healthy donors. CD8+ cells (for TCR #1) or CD4+ and CD8+ cells combined at a 1:1 ratio (for TCR #2) were stimulated for 72 h at 37ºC by culturing with anti-CD3/anti-CD28 reagent at a 1:1 bead:cell ratio in medium containing human serum, IL-2, IL-7, and IL-15. To introduce genetic disruption at the endogenous TRAC locus by CRISPR/Cas9-mediated genome editing, the anti-CD3/anti-CD28 reagent is removed and cells are electroporated with 2 μM ribonucleoprotein (RNP) complexes containing Streptococcus pyogenes Cas9 and guide RNA (gRNA) with the targeting domain sequence GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:31), which targets genetic disruption within exon 1 of the endogenous TCR α constant region ( TRAC ) gene. Following electroporation, cells are mixed with medium containing an AAV preparation containing the HDR template polynucleotide encoding a type recombinant TCR under the control of the EF1α promoter for transduction (HDR KO). As a control, cells were treated with the same conditions as those used for electroporation but without the addition of RNP (mock KO), transduced with a lentiviral vector encoding a recombinant TCR (Lenti) or a reference TCR capable of binding to HPV 16 E7 but containing murine Cα and Cβ regions (Lenti Ref), or transduced with a lentiviral vector encoding a recombinant TCR and also electroporated with RNP complexes directed at genetic disruption of the TRAC locus (Lenti KO). Following transduction, cells were cultured for approximately 7 days in medium containing human serum and IL-2, IL-7, and IL-15.

C. TCR expression

At day 7 post-electroporation, cells were assessed by flow cytometry for staining with anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-Vbeta antibody specific for each recombinant TCR, and peptide-MHC tetramer complexed with HPV16 E7 peptide.

Results for TCR #1-expressing CD8+ cells are shown in FIGS. 1A-1C . As shown, cells expressing TCR #1 via HDR-mediated targeted integration at the TRAC locus exhibit a higher proportion of tetramer-bound cells ( FIG. 1A ) and the highest mean fluorescence intensity of tetramer staining in CD8+ cells ( FIG. 1B ). Among tetramer-bound CD8+ cells, the extent of tetramer binding is generally more uniform in cells that have undergone HDR-mediated integration compared to random integration by lentiviral transduction, as indicated by the lower coefficient of variation (standard deviation of signal in a population of cells divided by the mean signal in the respective population; see FIG. 1C ). Results for TCR #2-expressing cells are shown in FIGS. 2A-2B . As shown, CD4+ and CD8+ cells expressing TCR #2 via HDR-mediated integration at the TRAC locus exhibited a higher proportion of tetramer-bound cells ( FIG. 2A ) and the highest mean fluorescence intensity of tetramer staining ( FIG. 2B ).

D. Cytolytic activity and cytokine formation

Cytolytic activity and cytokine production of cells engineered to express recombinant TCR #1 or recombinant TCR #2 via HDR or random integration as described above were assessed following incubation with target cells expressing HPV 16 E7 in vitro .

Cytolytic activity is assessed by co-culture of recombinant TCR-expressing effector cells with target cells expressing HPV 16 E7 labeled with NucLight Red (NLR) at effector to target (E:T) ratios of 10:1, 5:1, and 2.5:1. The ability of T cells to induce antigen-specific lysis of target cells is assessed by measuring the loss of labeled target cells every 2 hours for up to 44 hours after co-culture. Cytolytic activity is determined from the average of the 2 donors in each group, from the area under the curve (AUC) of % death normalized to Vbeta expression for each recombinant TCR, and compared to a sham-transduced control. Cytokine production is measured after incubation of recombinant TCR-expressing effector cells with target cells. Interferon-gamma (IFNγ) and interleukin-2 (IL-2) secretion in the supernatant was determined by ELISA, normalized to Vbeta expression, and averaged for the 2 donors in each group.

The results for TCR #1 are shown in FIGS. 3A-3B . The extent of killing and IFNγ secretion were higher in cells into which recombinant TCR #1 driven by the EF1α promoter was introduced by HDR-mediated targeted integration at the TRAC locus (EF1α-TCR #1 HDR KO), compared to random integration, with (TCR #1 Lenti) or without knockout of the TRAC locus (TCR #1 Lenti KO).

Representative results for TCR #2 are shown in FIGS. 4A-4G. Secretion of AUC and IFNγ and IL-2 by target cells at 24 hours was similar or higher in cells into which recombinant TCR #2 driven by the EF1α promoter was introduced by HDR-mediated targeted integration (EF1α-TCR #2 HDR KO) compared to random integration, with (TCR #2 Lenti) or without TRAC locus knockout (TCR #2 Lenti KO), and the reference TCR introduced by random integration (Lenti Ref) ( FIGS. 4A-4C ). CD8+ and CD4+ cells expressing TCR #2 introduced by HDR at the TRAC locus exhibited higher target cell killing compared to random integration, with or without TRAC locus knockout, and reference TCR introduced by random integration ( FIGS. 4D-4E ). IFNγ production was observed by CD8+ cells at an E:T ratio of 2.5:1, or by CD4+ cells at an E:T ratio of 10:1, the same as for cells expressing TCR #2 introduced by HDR at the TRAC locus or by random integration ( FIGS. 4F-4G ).

E. Viability of cells expressing recombinant TCRs

The viability of CD4+ and CD8+ cells engineered to express TCR #2 introduced by different routes was assessed during cryopreservation and after thawing of preserved cells by acridine orange (AO) and propidium iodide (PI) staining. As shown in FIGS. 5A-5B , cell viability was substantially unaffected by introduction of TCR #2 coding sequences via HDR, compared to sample-treated cells or cells expressing the reference TCR.

F. Conclusion

Overall, the results are consistent with the observation that targeted integration via HDR of nucleic acid sequences encoding typical recombinant TCRs results in higher expression of recombinant human TCR in human T cells compared to introduction of TCR by random integration, thereby resulting in higher functional activity of cells expressing recombinant TCR.

Example 2. Generation and expression evaluation of recombinant T-cell receptors (TCRs) by targeted knockin or random integration of TCR coding sequences into T cells with knockout of endogenous TCRα and TCRβ chains

A polynucleotide encoding one of the exemplary recombinant TCRs is targeted for integration at one of the sites of genetic disruption via homology-directed repair (HDR), or introduced by random integration via lentiviral transduction, into cells engineered to genetically disrupt endogenous gene loci that encode T cell receptor alpha (TCRα) and beta (TCRβ) chains, or both, via CRISPR/Cas9-mediated genome editing.

For targeted integration via HDR, AAV preparations containing template polynucleotide constructs encoding a representative recombinant TCR #1 are generated essentially as described in Examples 1, except for the following differences: an additional AAV construct is generated containing the MND promoter (the sequence set forth in SEQ ID NO:126), which is a synthetic promoter that contains the U3 region of a modified MoMuLV LTR with a myeloproliferative sarcoma virus enhancer, to control expression of the recombinant TCR encoding sequences.

For random integration, lentiviral preparations are created containing nucleic acid sequences encoding a representative recombinant TCR #1, generally as described in Example 1. For these studies, the lentiviral transduction construct further comprises a polynucleotide encoding a truncated receptor derived from a recombinant TCR transgene, with a sequence encoding a T2A ribosome skip sequence for expression of both the recombinant TCR and the truncated receptor from the same construct; the truncated receptor serves as a surrogate marker for transduction. As a control, a polynucleotide encoding a chimeric TCR is generated wherein the Cα and Cβ of the recombinant TCR are replaced by constant regions from the mouse TCR (the mouse Cα sequence shown in SEQ ID NO:122; the mouse Cβ sequence shown in SEQ ID NO:123), or the cells are mock transduced.

Primary human CD4+ and CD8+ T cells are isolated and engineered to introduce genetic disruption at the endogenous TRAC and TRBC loci via CRISPR/Cas9-mediated genome editing and transduced with AAV preparations containing template polynucleotides for HDR or lentiviral preparations for random integration, generally as described in Example 1B above. The cells are electroporated with ribonucleoprotein (RNP) complexes containing the TRAC -targeting guide RNA (gRNA) described in Example 1B and RNPs containing gRNA directed to the target site of the consensus sequence common to exon 1 or both TCRβ constant regions 1 and 2 (with the targeting domain sequence GGCCUCGGCGCUGACGAUCU (SEQ ID NO:63)) (TCRαβ KO). As a control, cells were treated under the same conditions used for electroporation but without the addition of RNP (mock KO; also referred to as TCRαβ WT).

Cells are then cultured for four (4) days, then assessed by flow cytometry for staining with anti-CD3 antibody, anti-Vbeta antibody that recognizes recombinant TCR #1, and with peptide-MHC tetramer complexed with the antigen recognized by the recombinant TCR (HPV16 E7 peptide). Cells are also co-stained for CD8 or CD4.

The results are shown in FIGS. 6A-6E . As shown in FIGS. 6A and 6B , CRISPR/Cas9-mediated knockout (KO) of TRAC and TRBC (panel labeled “TCRαβ KO” in the figures) resulted in nearly complete ablation of TCR expression in CD8+ cells, as evidenced by the absence of CD3 staining in KOed and mock-transduced cells (panel labeled TCRαβ KO/mock transd.). Recombinant TCR expression (as evidenced by cells staining with a specific Vbeta antibody or cells positive for tetramer staining among CD8+ cells) was slightly improved following lentiviral transduction into cells that had KOed the endogenous TCR (TCRαβ KO/lenti human) compared to cells that retained endogenous TCR expression (TCRαβ WT/lenti human). In cells retaining endogenous TCR, expression of recombinant TCR was improved by lentiviral transduction of recombinant TCR containing the murine constant domain compared to lentiviral transduction of fully human recombinant TCR (comparison of TCRαβ WT/lenti human and TCRαβ WT/lenti mouse).

HDR-mediated targeted knockin of recombinant TCR and KO of endogenous TCR yielded a substantially higher proportion of cells expressing recombinant TCR than observed following lentiviral transduction (first two left panels, labeled “HDR” versus TCRαβ KO/lenti human in FIGS. 6A and 6B ). The geometric mean fluorescence intensity (gMFI) of recombinant TCR expression, assessed by Vbeta or tetramer staining in CD8+ cells ( FIG. 6C ) or Vbeta staining in CD4+ cells ( FIG. 6D ), was also significantly higher in HDR-treated cells compared to lentiviral transduction. The extent of recombinant TCR expression by HDR was similar regardless of whether the recombinant TCR was under the control of the EF1α or MND promoter.

Among the cells that were positive for recombinant TCR expression, the extent of expression was generally more uniform or dense in cells that had undergone HDR-mediated targeted integration compared to random integration by lentiviral transduction (see FIGS. 6A and 6B ). As shown in FIGS. 6E and 6F , a lower coefficient of variation (standard deviation of the signal in a population of cells divided by the mean signal in the respective population) of recombinant TCR expression, as determined by peptide-MHC tetramer binding, and Vbeta expression, respectively, was observed in CD8+ cells that had undergone HDR-mediated integration compared to random integration. The results are consistent with the finding that targeted knockin of recombinant TCR into the endogenous TCRα locus, combined with knockout of endogenous TCRαβ chains, yields higher and more uniform expression levels in a population of cells engineered to express recombinant TCR compared to other methods.

Example 3. Evaluation of HDR-mediated knock-in of recombinant T cell receptor (TCR) encoding sequences in T cells with knockout of endogenous TCRα or TCRβ chains or both

To further evaluate recombinant TCR expression via HDR, nucleic acid sequences encoding a typical recombinant TCR at the TRAC locus were targeted for integration into cells containing a double knockout of the TRAC and TRBC loci, a knockout of the TRAC locus alone, or a knockout of the TRBC locus alone.

A. Recombinant TCR transgene constructs and generation of engineered cells

These studies were performed using AAV (for HDR) and lentiviral constructs (for random integration) encoding type TCR #1 essentially as described in Examples 1 and 2, except for the following differences: lentiviral constructs were generated containing a polynucleotide encoding recombinant TCR under the functional control of the EF1α or MND promoter. A lentiviral construct containing a polynucleotide encoding recombinant TCR #1 under the functional control of the EF1α promoter and a truncated receptor separated from the recombinant TCR transgene by a sequence encoding the T2A ribosome skip sequence were also generated for comparison.

For targeted integration via HDR, primary human CD4+ and CD8+ T cells are stimulated, cultured, and electroporated with ribonucleoprotein (RNP) complexes containing only TRAC -targeted gRNA, only TRBC -targeted gRNA, or both TRAC- and TRBC -targeted gRNAs as generally described in Examples 1 and 2. Following electroporation, the cells are transduced with AAV preparations containing polynucleotides that encode a recombinant TCR to target the endogenous TRAC locus, generally as described above.

For random integration, primary human CD4+ and CD8+ T cells are thawed, stimulated, and cultured essentially as described in Examples 1 and 2, followed by transduction with a lentiviral preparation that encodes a recombinant TCR. In this assay, lentiviral preparations are transduced into primary T cells that retain the endogenous TCR. As a control, cells are treated under the same conditions as used for lentiviral transduction, but without the addition of lentivirus (mock transduction).

B. TCR expression

Cells are then cultured for 4-10 additional days and assessed by flow cytometry after staining with anti-CD3 antibody, anti-Vbeta antibody specific for recombinant TCR #1, and peptide-MHC tetramer complexed with the antigen recognized by the TCR (HPV16 E7 peptide). Cells are also co-stained for CD8 or CD4.

The results for CD3 staining are shown in FIGS. 7A-7C , for tetramer staining are shown in FIGS. 8A-8C , and for Vbeta staining are shown in FIGS. 9A-9D .

As shown in FIGS. 7A and 7C, electroporation with RNPs complexed with gRNAs targeting TRAC and TRBC resulted in efficient knockout of endogenous TCR, as evidenced by the absence of surface CD3 expression (see the panel labeled KO/mock transd. in FIG. 7A ; see also the panel labeled KO mock in FIG. 7C , which shows the proportion of CD3+CD8+ cells among CD8+ cells). The extent of KO with gRNAs targeting both TRAC and TRBC was greater than for cells electroporated with RNPs complexed with gRNAs targeting TRAC alone or TRBC alone, consistent with the observation that dual targeting of both constant domains of the TCR α and β chains improves the efficiency of interruption of endogenous TCR expression. In cells transduced with lentiviral vectors that do not disrupt endogenous TCR, CD3 expression was the same as in all conditions tested ( FIGS. 7B and 7C ). As shown in FIGS. 7A and 7C , CD3 expression was also similar among cells into which recombinant TCR was introduced via HDR, consistent with TCR/CD3 surface expression in cells into which recombinant TCR was introduced.

As shown in FIGS. 8A and 8C , the proportion of CD8+ cells that bound the peptide-MHC tetramer indicated that recombinant TCR expression was higher under conditions in which HDR was performed in cells knocked out for both TRAC and TRBC compared to TRAC alone (top and middle rows in FIG. 8A ; TRAC & TRBC vs. TRAC alone in FIG. 8C ). As shown in FIG. 8C, similar results were observed at days 7 and 13. Similar recombinant TCR expression was observed in cells where HDR was performed with an integration construct under the control of the exogenous EF1α or MND promoter or under the control of the endogenous TCR promoter (P2A-containing construct). As shown in FIG. 8B and FIG. 8C , fewer cells express recombinant TCR, as assessed by tetramer staining, following lentiviral-mediated transduction, regardless of the presence of the truncated receptor in the lentiviral constructs.

As shown in FIGS. 9A - 9C , similar to the above results, expression of recombinant TCR on CD8+ T cells was observed by direct staining for recombinant TCR with an antibody that specifically recognizes the Vbeta chain of recombinant TCR. Anti-Vbeta staining, which is also able to recognize recombinant TCR on CD4+ T cells (CD8 negative population), also showed that expression of recombinant TCR was observed in CD4+ cells ( FIG. 9A and FIG. 9D ).

For all methods of assessing recombinant TCR expression shown above (anti-CD3, tetramer, and anti-Vbeta), the above results also show that targeted integration of recombinant TCR into TRAC via HDR is specific for nuclease-induced DNA breakage at the TRAC locus, since cells electroporated with TRBC -targeted RNP do not express recombinant TCR (see, e.g., the “ TRBC only” condition in FIGS. 8A, 8C, 9A, 9C , and 9D ).

C. Cytolytic activity and cytokine formation

Cytolytic activity and cytokine production of CD8+ cells engineered to express recombinant TCR #1 by HDR or random integration as described above were assessed following incubation with target cells expressing HPV 16 E7 in vitro . In addition to the cells described above, primary human CD8+ cells transduced with a lentivirus encoding a reference TCR capable of binding to HPV 16 E7 but containing murine Cα and Cβ regions were also assessed.

Cytolytic activity was assessed by incubating recombinant TCR-expressing cells with HPV 16 E7-expressing target cells at effector-to-target (E:T) ratios of 10:1, 5:1, and 2.5:1. The ability of T cells to cause antigen-specific lysis of target cells was assessed 4 hours after co-culture. Cytolytic activity was determined from the area under the curve (AUC) of % killing normalized to Vbeta expression for each recombinant TCR and compared to a mock-transduced control. The results are shown in FIG. 10 . The extent of killing was higher in cells into which recombinant TCR was introduced by HDR-mediated targeted integration compared to random integration, consistent with the finding that higher recombinant TCR expression in cells confers higher functional activity.

Cytokine production was also monitored following incubation of recombinant TCR-expressing CD8+ effector cells with target cells expressing HPV 16 E7 at E:T ratios of 10:1 and 2.5:1 for 48 hours. IFNγ secretion in the supernatant was determined by ELISA and normalized to Vbeta expression for each group. The results are shown in FIG. 11. Similar to the results for cytolytic activity presented above, greater IFNγ production was observed in cells subjected to HDR-mediated integration compared to random integration. In the cytolytic activity assay and IFNγ secretion assessment, the functional activity of cells expressing recombinant TCR via HDR-mediated integration was similar to that of cells expressing a reference TCR containing murine constant domains via lentiviral transduction.

Proliferation of recombinant TCR-expressing cells was assessed after incubation with SCC152 target cells or T2 target cells challenged with the antigen peptide. Cells were labeled with CellTrace™ violet (ThermoFisher) dye. Dividing viable T cells was indicated by dilution of CellTrace™ violet dye as assessed by flow cytometry.

The results of various functional analyses are shown in FIG. 12 . As shown in the heat map depicting the relative activity of the recombinant TCR expressing cell population in various functional activities (AUC % death at E:T ratios of 10:1, 5:1, and 2.5:1 (labeled “AUC”), tetramer binding in CD8+ cells on days 7 and 13 (labeled “CD8 tetramer”), proliferation assay (labeled “CTV pulse”) using SCC152 cells or T2 target cells injected with the antigen peptide, and IFNγ secretion from CD8+ cells (labeled “CD8 IFNγ secreting”)), the functional activity of cells with the recombinant TCR directed to knockin at the constant chain domain locus of endogenous TCRα and knockout of the endogenous TCRαβ gene or TCRα gene is generally observed to be higher compared to cells where the polynucleotide encoding the recombinant TCR TCR was integrated by accident.

Overall, the results are consistent with the observation that targeted integration via HDR results in higher expression of recombinant human TCR in human T cells compared to TCR introduction by random integration, thereby leading to higher functional activity of recombinant TCR-expressing cells.

Example 4. Grade in vivo antitumor effects in mice of T cells engineered to express a recombinant T cell receptor (TCR) generated by an HDR-mediated knockin

The antitumor activity and pharmacokinetics of CD4+ and CD8+ cells expressing a typical TCR, generated by HDR-mediated knockin or random integration via lentiviral transduction of recombinant TCR encoding sequences, are assessed by introducing the engineered cells into a mouse tumor model.

A. Tumor mass and survival

The mouse tumor model was created by subcutaneous injection of 4× ¹⁰⁶ UPCI:SCC152 squamous cell carcinoma cell lines (ATCC® CRL-3240™) into female NOD/SCID/IL-2Rγ ^null (NSG) mice. Tumor engraftment occurred within approximately 25 days and tumor stage was determined based on tumor volume (P > 0.95) prior to injection of engineered cells.

Primary CD4+ cells and CD8+ cells engineered to express type 2 recombinant TCR#2 by targeted knockin or random integration of the TCR coding sequence are generated essentially as described above in Example 1 and administered 26 days after tumor cell injection. Two different total doses of recombinant TCR expressing cells (3× ¹⁰⁶ or 6× ¹⁰⁶ TCR expressing cells) are administered, where the dose is based on the proportion of cells positive for staining with an anti-Vbeta antibody that recognizes recombinant TCR#2. The following groups are compared: TCR#2 driven by the human elongation factor 1 alpha (EF1α) promoter targeted for integration at the TRAC locus by HDR (TCR#2 HDR KO EF1α); TCR#2 driven by the endogenous TRAC promoter (located upstream of the in-frame P2A ribosome skipping element) targeted for integration at the TRAC locus via HDR (TCR#2 HDR KO P2A); TCR#2 randomly integrated using a lentiviral construct (TCR#2 Lenti); TCR#2 randomly integrated using a lentiviral construct into cells containing a knockout of endogenous TRAC (TCR#2 Lenti KO); and a reference TCR capable of binding HPV 16 E7 but containing murine Cα and Cβ regions, randomly integrated using a lentiviral construct (Lenti Ref). Mice that did not receive the engineered cells (tumor only) or that received cells treated under the same conditions as for electroporation but without the addition of RNP (mock KO) were used as controls. Table E1 also lists the mouse numbers and the total number of T cells injected for each dose in each group.

Table E1. Study design for in vivo evaluation of antitumor activity in a mouse tumor model.

Group No. Group Description CD4 (Vbeta) CD8
(Vbeta) TCR per mouse (1:1 TCR+ CD4:CD8) Total T cells/mouse Total mice/group SCC152 on mouse 1 Just a tumor 0 0 7 4.00E+06 2 Mock KO 0,00E+00 1.98E+07 7 4.00E+06 3 Lenti Ref 62.6% 59.7% 6.00E+06 9.82E+06 7 4.00E+06 4 Lenti Ref 3.00E+06 4.91E+06 7 4.00E+06 5 TCR #2 Lenti 79.2% 88.7% 6.00E+06 7.17E+06 7 4.00E+06 6 TCR #2 Lenti 3.00E+06 3.59E+06 7 4.00E+06 7 TCR #2 Lenti KO EF1α 91.0% 88.4% 6.00E+06 6.69E+06 7 4.00E+06 8 TCR #2 Lenti KO EF1α 3.00E+06 3.35E+06 7 4.00E+06 9 TCR #2 HDR KO EF1α 33.5% 30.3% 6.00E+06 1.89E+07 7 4.00E+06 10 TCR #2 HDR KO EF1α 3.00E+06 9.43E+06 7 4.00E+06 11 TCR #2 HDR KO P2A 39.3% 41.1% 6.00E+06 1.49E+07 7 4.00E+06 12 TCR #2 HDR KO P2A 3.00E+06 7.47E+06 7 4.00E+06

Average tumor size is assessed twice weekly for up to 58 days after infusion of engineered cells. Changes in body weight, mouse survival, and the number of tumor-free mice at day 58 are also monitored.

The results of antitumor activity (tumor mass reduction) and survival of mice treated with TCR #2 expressing cells at two different doses are shown in FIGS. 13A-13B and FIGS. 14A-14B . As shown in FIGS. 13A-13B , the tumor volume reduction in mice treated with TCR #2 expressing cells via HDR-mediated targeted integration under the control of either EF1α (TCR #2HDR KO EF1α) or the endogenous TRAC promoter (TCR #2HDR KO P2A) was greater than that in mice treated with TCR #2 expressing cells by random integration via lentiviral transduction (TCR #2 Lenti or TCR #2 Lenti KO). At both doses, tumor volume reduction in mice treated with TCR #2 expressing cells via HDR-mediated targeted integration of TCR coding sequences under the control of the EF1α promoter (TCR #2HDR KO EF1α) was comparable to or greater than that in mice treated with cells expressing the reference TCR (Lenti Ref). As shown in FIGS. 14A-14B , the % survival of mice treated with TCR #2 expressing cells via HDR-mediated targeted integration was greater than that in mice treated with cells expressing TCR generated by random integration (TCR #2 Lenti or TCR #2 Lenti KO). As shown in Table E2, the number of mice that were tumor-free at day 58 post-cell administration was also greater in the groups that received TCR #2-expressing cells via HDR-mediated targeted integration, under the control of either EF1α (TCR #2HDR KO EF1α) or the endogenous TRAC promoter (TCR #2HDR KO P2A), compared to the other groups. Changes in body weight over the course of the study were also monitored, as shown in FIGS. 15A-15B .

Table E2. Number of tumor-free mice at day 58 after administration of engineered cells.

Group Number of tumor-free mice on day 59 58 (out of 7), dose 6×10 ⁶ Number of tumor-free mice on day 59 58 (out of 7), dose 3×10 ⁶ Just a tumor 0 0 Mock KO 0 0 Lenti Ref 5 0 TCR #2 Lenti 4 0 TCR #2 Lenti KO EF1α 1 0 TCR #2 HDR KO EF1α 7 4 TCR #2 HDR KO P2A 6 2

These results are consistent with the observation that administration of cells generated by targeted integration of recombinant TCR encoding sequences via HDR yields greater in vivo antitumor activity compared to cells generated by random integration of TCR encoding sequences.

B. Pharmacokinetic (PK) assessment

Cell viability and expansion are assessed in the mouse model described above in Example 4A. Mice in each group listed in Table E1 are alternately selected every 2 weeks (first cohort of 4 mice on days 7 and 21; second cohort of 3 mice on days 14 and 28), and each treatment group is assessed once a week (on days 7, 14, 21, and 28). To assess the pharmacokinetics of the injected cells, the cells are counted and the expression of various markers (CD3, CD4, CD8, CD45, CD45RA, CCR7, PD-1) and recombinant TCR (using recombinant TCR-specific anti-Vbeta) and peptide-MHC tetramer binding are determined by flow cytometry at each time point.

Cells expressing TCR #2 via HDR-mediated integration under the control of either the EF1α or P2 promoter showed an early initial increase followed by a rapid decline in the blood count of TCR-expressing cells between days 14 and 21. Cells expressing the reference TCR showed different expansion and survival kinetics in vivo , with a smaller decline in circulating TCR-expressing cells. From analysis of the proportion of CD4 or CD8 cells in total TCR+ cells over time, cells retaining endogenous TCR expression exhibited a higher proportion of CD4 at early time points compared to cells containing a knockout of the endogenous TRAC locus . Overall, an increase in the proportion of CD4 was observed over time for most groups.

Staining for cellular phenotypic markers such as CD45RA and CCR7 showed that the phenotype of the majority of circulating cells expressing recombinant TCR changed from CCR7+CD45RA+ (associated with a more naive phenotype) to CCR7-CD45RA- (associated with a more mature phenotype) between day 7 and day 14. The proportion of PD-1 expressing TCR+ cells also increased over time, from virtually 0% staining to around 60% of cells showing PD-1 staining in most groups.

Overall, pharmacokinetic analysis demonstrates increased peripheral proliferation and increased numbers of cells expressing recombinant TCR#2 via HDR-mediated integration under the control of the EF1α promoter, consistent with the observation of high antitumor activity at a low dose. Furthermore, cells expressing recombinant TCR#2 generally exhibit a more mature phenotype over time, and the overall kinetics of proliferation and viability are seen to differ from cells expressing the reference TCR.

Example 5 . Estimation of Homology Arm Lengths for Efficient HDR-Mediated Integration of Transgene Sequences

Polynucleotides containing a generic transgene flanked by a homology arm of varying length are introduced into T cells to target the transgene integration via homology-directed repair (HDR) and the integration efficiency is assessed.

A. Recombinant transgenic constructs and creation of engineered T cells

Generic HDR template polynucleotides containing a transgene sequence encoding green fluorescent protein (GFP) flanked by homology arms of varying lengths for targeted integration into the human TCRα constant region ( TRAC ) locus are introduced into T cells. More specifically, for integration via HDR, AAV preparations containing template nucleotide constructs encoding GFP are generated essentially as described in Examples 1-3, except for the following differences: AAV constructs containing a polynucleotide encoding GFP under the functional control of the MND promoter are generated and linked to an SV40 poly(A) sequence flanked by 50, 100, 200, 300, 400, 500, or 600 base pairs of the 5' and 3' homology arms (SEQ ID NOS: 227-233 and 234-240, respectively) homologous to sequences surrounding the target integration site in the human TCRα constant region ( TRAC ) gene. The full length of the AAV construct is made constant by using filler DNA sequences between the SV40 poly(A) sequence and the 3' homology arm.

For targeted integration via HDR, primary human CD4+ and CD8+ T cells from four human donors (Donors 1-4) are pooled at a 1:1 ratio, stimulated, and electroporated with ribonucleoprotein (RNP) complexes containing TRAC -targeted gRNA essentially as described in Examples 1-3. Following electroporation, cells are transduced with AAV preparations containing HDR template polynucleotides containing different lengths of the homology arms described above. GFP expression is measured by flow cytometry at 24, 48, 72, 96 hours, and 7 days after transduction with AAV, to determine the integration ratio (representing the proportion of all AAV within a cell that is integrated into the genome) based on the following formula:

% High MFI and % Low MFI indicate the proportion of cells that are above or below the MFI threshold as determined by flow cytometry, representing cells containing the integrated GFP transgene or the non-integrated AAV construct, respectively. Changes in integration ratio with increasing homology arm length are estimated by subtracting the integration ratio of the next shortest arm length from the integration ratio of a particular arm length.

A. Estimation of the integration coefficient

As shown in FIGS. 16A-16B , a constant or increased integration ratio observed at various time points was assessed for each homology arm length, consistent with non-integrated AAV constructs decaying over time. Evaluation of changes in GFP expression patterns over time revealed that the proportion of cells with an integrated GFP transgene (high MFI) generally remained constant after 72 hours, but the proportion of cells containing only non-integrated AAV (low MFI) continued to decline.

As shown in FIGS. 17A-17B , it appears that the most significant increase in integration efficiency occurs at the 200 bp homology arm, with little increase at 300 bp. No significant increase is observed between 300 bp and 500 bp, and an increased integration efficiency is observed between 500 bp and 600 bp, across all donors. The results support the use of a minimum of 300 bp homology arms for high integration efficiency, with 600 bp homology arms yielding even greater integration efficiency.

Example 6. Generation and evaluation of engineered T cells expressing a chimeric antigen receptor (CAR) by targeted knockin or random integration of TCR encoding sequences

Polynucleotides encoding generic chimeric antigen receptors (CARs) are introduced into T cells with a genetic disruption at the endogenous gene locus encoding the T cell receptor alpha (TCRα) chain via CRISPR/Cas9-mediated genome editing and targeted integration at the site of genetic disruption via homology-directed repair (HDR) or random integration.

A. Generic anti-CD19 CAR

a. Expression of generic anti-CD19 CARs

Nucleic acid sequences encoding a generic CAR specific for antigen cluster of differentiation 19 (anti-CD19 CAR) are targeted for integration into one of the sites of genetic disruption via homology-directed repair (HDR), or introduced by random integration via retroviral transduction, into cells engineered to genetically disrupt the endogenous gene locus that encodes the T-cell receptor alpha (TCRα) chain via CRISPR/Cas9-mediated genome editing, generally as described in Examples 1 and 2.

Three studies were conducted using AAV (for HDR) and retroviral constructs (for random integration) essentially as described in Examples 1 and 2, except for the following differences: the transgene sequences included nucleic acid sequences encoding a generic anti-CD19 CAR and an EF1α promoter to drive expression of the generic anti-CD19 CAR sequences (TRAC HDR EF1α promoter) or sequences encoding a P2A ribosome skip element upstream of the generic anti-CD19 CAR sequences (TRAC HDR P2A) to drive expression of the generic anti-CD19 CAR from the endogenous TCRα locus by HDR-mediated targeted in-frame integration into the human TCRα constant region ( TRAC ) gene.

For targeted integration via HDR, primary human CD4+ and CD8+ T cells are stimulated, cultured, and electroporated with ribonucleoprotein (RNP) complexes containing TRAC -targeted gRNA, generally as described in Examples 1 and 2. Following electroporation, the cells are transduced with AAV preparations containing polynucleotides that encode generic anti-CD19 CARs to target the endogenous TRAC locus, generally as described above. For random integration, primary human CD4+ and CD8+ T cells are transduced with a retroviral preparation that encodes a generic anti-CD19 CAR, with (TRAC RNP retrovirus or TCR KO retrovirus) or without (retrovirus only) electroporation of RNP complexes containing TRAC -targeted gRNA. As a control, cells were subjected to HDR targeting the TRAC locus after electroporation with RNP complexes containing TRBC -targeted gRNA, or mock-treated. On day 9 after cell thawing, engineered cells were assessed by flow cytometry for staining with anti-CD3 antibody or anti-idiotypic antibody (anti-ID), which specifically recognizes the anti-CD19 CAR.

As shown in FIG. 18A , anti-CD19 CAR T cells expressing HDR-mediated targeted integration at the TRAC locus exhibit the highest proportion of cells bound by the anti-ID antibody. As shown in FIG. 18B , the integration and expression efficiency of the exemplary anti-CD19 CAR generated by HDR-mediated targeted integration at the TRAC locus is comparable to or greater than that observed in cells engineered using random integration, with or without genome editing of the endogenous TRAC locus. The results are consistent with the observation of similar or improved expression of the exemplary anti-CD19 CAR in cells engineered by HDR-mediated targeted integration at the TRAC locus compared to cells engineered by random integration.

b. Expression of generic anti-CD19 CARs after serial re-stimulation

Cell surface expression of CARs is assessed after multiple rounds of treatment with the target antigen. The ability of CAR T cells to proliferate and demonstrate antigen-specific function ex vivo after repeated rounds of antigen stimulation may correlate with in vivo function and/or the ability of the cell to survive in vivo (e.g., after administration and initial activation in response to antigen challenge) (Zhao et al. (2015) Cancer Cell, 28:415–28).

Primary human T cells expressing a type-A anti-CD19 CAR generated by HDR-mediated targeted integration or random integration as described above are incubated with K562 human chronic myelogenous leukemia (CML) cells engineered to express CD19 (K562-CD19). Irradiated K562-CD19 target cells are added at an effector to target (E:T) ratio of 2.5:1. Every 4 days (start of each new round), CAR T cells are counted, harvested, and replated at the initial seeding density with fresh medium with freshly thawed, freshly irradiated target cells for a total of 3 rounds. At each round, the proportion of CAR-expressing cells, mean fluorescence intensity (MFI), and coefficient of variation (CV) of CAR expression are assessed by flow cytometry.

As shown inFIG. 19A, share of cells expressing anti-CD19 CAR was generally increased or stable in all groups tested. As shown inFIG. 19B and 19C, during repeated stimulations, exemplary anti-CD19 CAR-expressing cells engineered by HDR-mediated targeted integration of a nucleic acid sequence at the endogenous locusTRAC, under the control of the EF1α promoter or P2A (endogenousTRACpromoter), exhibit more uniform and less variable CAR expression compared to cells engineered by random integration using a retroviral vector. MFI is generally higher in cells engineered by random integration (FIG. 19B). Anti-CD19 CAR-expressing cells engineered using HDR-mediated targetingTRAClocus, exhibit a lower coefficient of variation (the standard deviation of the signal in a population of cells divided by the mean signal in the corresponding population;FIG. 19C), showing less fluctuation in expression during repeated stimulation.

c. Cytolytic activity and cytokine formation

Cytokine (interferon-gamma; IFNγ) production and cytolytic activity were monitored following incubation of anti-CD19 CAR+ T effector cells with K562 target cells engineered to express CD19 (K562-CD19) or a non-engineered control (K562 naive) at an E:T ratio of 2:1 for 48 hours, generally as described in Example 1.D above.

As shown in FIG. 20A , anti-CD19 CAR-expressing cells engineered using HDR-mediated targeting of the TRAC locus exhibit higher production of antigen-specific IFNγ, with cells engineered via HDR with the EF1α promoter exhibiting the highest production of antigen-specific IFNγ (left panel) and lower production of non-specific or off-target cytokine (right panel). As shown in FIG. 20B , the degree of target cell killing was similar across all groups of anti-CD19 CAR-expressing cells (left panel). Non-specific cell killing varied, with cells engineered via HDR with P2A (the endogenous TRAC promoter) exhibiting the least non-specific cell killing (right panel).

B. Typical anti-BCMA CARs

a. Expression of typical Anti-BCMA CAR

Nucleic acid sequences encoding different exemplary CARs specific for the B cell maturation antigen (anti-BCMA CAR) are introduced by targeted integration mediated by HDR at the TRAC locus under the control of EF1α or the endogenous TRAC promoter (integration of the P2A sequence), or by random integration using a lentiviral vector, with (LV-TRAC or LV-KO) or without (LV only) introduction of RNP complexes containing TRAC -targeted gRNA, generally as described in Examples 1, 2 and 6A above. As a control, cells were subjected to HDR targeting of the TRAC locus after electroporation with RNP complexes containing TRBC -targeted gRNA, or were mock treated (Mock). On day 9 post-thawing of the cells, engineered cells were assessed by flow cytometry to determine expression of the anti-BCMA CAR thawed with a BCMA-Fc fusion polypeptide containing soluble human BCMA fused at its C-terminus to the Fc region of IgG that specifically binds to the anti-BCMA CAR, and CD3 expression.

As shown in FIG. 21A , anti-BCMA CAR-T cells expressing HDR-mediated targeted integration at the TRAC locus exhibit a comparable proportion of BCMA-Fc-bound cells to cells engineered using random integration. As shown in FIG. 21B , the proportion of cells expressing the generic anti-BCMA CAR generated by HDR-mediated targeted integration at the TRAC locus was comparable to that observed in cells engineered using random integration, with or without genome editing of the endogenous TRAC locus. The results are consistent with the observation of similar or improved expression of the generic anti-BCMA CAR in cells engineered by HDR-mediated targeted integration at the TRAC locus compared to cells engineered using random integration.

b. Expression and activity of typical BCMA CAR after serial re-stimulation

Cell surface expression of CAR and antigen-specific activity of engineered cells are assessed after multiple rounds of target antigen treatment. Anti-BCMA CAR-expressing cells generated as described above are incubated with irradiated BCMA-expressing RPMI-8226 (BCMA+ multiple myeloma cell line) target cells at an effector to target (E:T) ratio of 1:1. Every 3-7 days after the start of each new round, CAR T cells are counted, harvested, and replated at the initial seeding density with fresh medium and freshly thawed, freshly irradiated target cells for a total of 4 rounds. The proportion of cells expressing anti-BCMA CAR is determined by flow cytometry. To assess cytokine production, cells from the first, second, or third round of serial stimulation were harvested 24 hours after stimulation and assessed for interferon gamma (IFNγ) and interleukin-2 (IL-2) production.

As shown in FIG. 22A , it is generally observed that the proportion of anti-BCMA CAR expressing cells is the same across multiple rounds of stimulation in cells engineered by different methods. As shown in FIG. 22B , it is observed that IFNγ production (upper panel) is the same in cells engineered by different methods. Cells engineered by targeted integration at the TRAC locus via HDR, under the functional control of the EF1α promoter, exhibit the highest IL-2 production after stimulation with target cells.

C. Conclusion

As shown, cells engineered to express generic CARs through targeted integration of a nucleic acid sequence into the endogenous TRAC locus exhibit similar or improved expression and antigen-specific activity and more uniform expression, including in the context of multiple rounds of stimulation.

The present invention is not intended to be limited in scope to the specific embodiments disclosed, which are provided, for example, to illustrate various aspects of the invention. Various modifications of the described compositions and methods will become apparent from the description and teachings provided herein. Such changes can be practiced without departing from the true scope and spirit of the disclosure, and are intended to be included within the scope of the present disclosure.

SEQUENCES

# SUBSEQUENCE ANNOTATION 1 atatccagaaccctgaccctgccgtgtaccagctgagagactctaaatccagtgacaagtctgtctgcctattcaccgattttgattctcaaacaaatgtgtcacaaagtaaggattctgatgtgtatatcacagacaaaactg tgctagacatgaggtctatggacttcaagagcaacagtgctgtggcctggagcaacaaatctgactttgcatgtgcaaacgccttcaacaacagcattattccagaagacaccttcttccccagcccaggtaagggcagctttgg tgccttcgcaggctgtttccttgcttcaggaatggccaggttctgcccagagctctggtcaatgatgtctaaaactcctctgattggtggtctcggccttatccattgccaccaaaaccctctttttactaagaaacagtgagc cttgttctggcagtccagagaatgacacgggaaaaaagcagatgaagagaaggtggcaggagagggcacgtggcccagcctcagtctctccaactgagttcctgcctgcctgcctttgctcagactgtttgccccttactgctct tctaggcctcattctaagccccttctccaagttgcctctccttatttctccctgtctgccaaaaaatctttcccagctcactaagtcagtctcacgcagtcactcattaacccaccaatcactgattgtgccggcacatgaatg caccaggtgttgaagtggaggaattaaaaagtcagatgaggggtgtgcccagaggaagcaccattctagttgggggagcccatctgtcagctgggaaaagtccaaataacttcagattggaatgtgttttaactcagggttgaga aaacagctaccttcaggacaaaagtcagggaagggctctctgaagaaatgctacttgaagataccagccctaccaagggcagggagaggaccctatagaggcctgggacaggagctcaatgagaaaggagaagagcagcaggca tgagttgaatgaaggaggcagggccgggtcacagggccttctaggccatgagagggtagacagtattctaagggacgccagaaagctgttgatcggcttcaagcaggggagggacacctaatttgcttttcttttttttttttttt tttttttttttttttgagatggagttttgctcttgttgcccaggctggagtgcaatggtgcatcttggctcactgcaacctccgcctcccaggttcaagtgattctcctgcctcagcctcccgagtagctgagattacaggcac ccgccaccatgcctggctaattttttgtatttttagtagagacagggtttcactatgttggccaggctggtctcgaactcctgacctcaggtgatccacccgcttcagcctcccaaagtgctgggattacaggcgtgagccacca cacccggcctgcttttcttaaagatcaatctgagtgctgtacggagagtgggttgtaagccaagagtagaagcagaaagggagcagttgcagcagagagatgatggaggcctgggcagggtggtggcagggaggtaaccaacac cattcaggtttcaaaggtagaaccatgcagggatgagaaagcaaagaggggatcaaggaaggcagctggattttggcctgagcagctgagtcaatgatagtgccgtttactaagaagaaaccaaggaaaaaatttggggtgcagg gatcaaaactttttggaacatatgaaagtacgtgtttatactctttatggcccttgtcactatgtatgcctcgctgcctccattggactctagaatgaagccaggcaagagcagggtctatgtgtgatggcacatgtggccagg gtcatgcaacatgtactttgtacaaacagtgtatattgagtaaatagaaatggtgtccaggagccgaggtatcggtcctgccagggccaggggctctccctagcaggtgctcatatgctgtaagttccctccagatctctccaca aggaggcatggaaaggctgtagttgttcacctgcccaagaactaggaggtctggggtgggagagtcagcctgctctggatgctgaaagaatgtctgtttttccttttagaaagttcctgtgatgtcaagctggtcgagaaaagct ttgaaacaggtaagacaggggtctagcctgggtttgcacaggattgcggaagtgatgaacccgcaataaccctgcctggatgagggagtgggaagaaattagtagatgtgggaatgaatgatgaggaatggaaacagcggttcaa gacctgcccagagctgggtggggtctctcctgaatccctctcaccatctctgactttccattctaagcactttgaggatgagtttctagcttcaatagaccaaggactctctcctaggcctctgtattcctttcaacagctcca ctgtcaagagagccagagagagcttctgggtggcccagctgtgaaatttctgagtcccttagggatagccctaaacgaaccagatcatcctgaggacagccaagaggttttgccttcttttcaagacaagcaacagtactcacata ggctgtgggcaatggtcctgtctctcaagaatcccctgccactcctcacacccaccctgggcccatattcatttccatttgagttgttcttattgagtcatccttcctgtggtagcggaactcactaaggggcccatctggacc cgaggtattgtgatgataaattctgagcacctaccccatccccagaagggctcagaaataaaataagagccaagtctagtcggtgtttcctgtcttgaaacacaatactgttggccctggaagaatgcacagaatctgtttgtaa ggggatatgcacagaagctgcaagggacaggaggtgcaggagctgcaggcctcccccacccagcctgctctgccttggggaaaaccgtgggtgtgtcctgcaggccatgcaggcctgggacatgcaagcccataaccgctgtgg cctcttggttttacagatacgaacctaaactttcaaaacctgtcagtgattgggttccgaatcctcctcctgaaagtggccgggtttaatctgctcatgacgctgcggctgtggtccagctgaggtgaggggccttgaagctggg agtggggtttagggacgcgggtctctgggtgcatcctaagctctgagagcaaacctccctgcagggtcttgcttttaagtccaaagcctgagcccaccaaactctcctacttcttcctgttacaaattcctcttgtgcaataata atggcctgaaacgctgtaaaatatcctcatttcagccgcctcagttgcacttctcccctatgaggtaggaagaacagttgtttagaaacgaagaaactgaggccccacagctaatgagtggaggaagagagacacttgtgtacac cacatgccttgtgttgtacttctctcaccgtgtaacctcctcatgtcctctctccccagtacggctctcttagctcagtagaaagaagacattacactcatattacaccccaatcctggctagagtctccgcaccctcctcccc cagggtccccagtcgtcttgctgacaactgcatcctgttccatcaccatcaaaaaaaaactccaggctgggtgcgggggctcacacctgtaatcccagcactttgggaggcagaggcaggaggagcacaggagctggagaccagc ctgggcaacacagggagaccccgcctctacaaaaagtgaaaaaattaaccaggtgtggtgctgcacacctgtagtcccagctacttaagaggctgagatgggaggatcgcttgagccctggaatgttgaggctacaatgagctg tgattgcgtcactgcactccagcctggaagacaaagcaagatcctgtctcaaataataaaaaaaataagaactccagggtacatttgctcctagaactctaccacatagccccaaacagagccatcaccatcacatccctaacag tcctgggtcttcctcagtgtccagcctgacttctgttcttcctcattccagatctgcaagattgtaagacagcctgtgctccctcgctccttcctctgcattgcccctcttctccctctccaaacagagggaactctcctaccc ccaaggaggtgaaagctgctaccacctctgtgcccccccggcaatgccaccaactggatcctacccgaatttatgattaagattgctgaagagctgccaaacactgctgccaccccctctgttcccttattgctgcttgtcactg cctgacattcacggcagaggcaaggctgctgcagcctcccctggctgtgcacattccctcctgctccccagagactgcctccgccatcccacagatgatggatcttcagtgggttctcttgggctctaggtcctgcagaatgttg tgaggggtttatttttttttaatagtgttcataaagaaatacatagtattcttcttctcaagacgtggggggaaattatctcattatcgaggccctgctatgctgtgtatctgggcgtgttgtatgtcctgctgccgatgccttc Human TCR alpha constant ( TRAC ) NCBI Reference Sequence: NG_001332,3, TRAC 2 aggacctgaacaaggtgttcccacccgaggtcgctgtgtttgagccatcagaagcagagatctcccacacccaaaaggccacactggtgtgcctggccacaggcttcttccccgaccacgtggagctgagctggtgggtgaatgggaaggaggtgcacagtggggtcagcacagacccgca gcccctcaagggagcagcccgccctcaatgactccagatactgcctgagcagccgcctgagggtctcggccaccttctggcagaacccccgcaaccacttccgctgtcaagtccagttctacgggctctcggagaatgacgagtggacccaggatagggccaaacccgtcacccagatcgtc agcgccgaggcctggggtagagcaggtgagtggggcctggggagatgcctggaggagattaggtgagaccagctaccagggaaaatggaaagatccaggtagcagacaagactagatccaaaaagaaaggaaccagcgcacaccatgaaggagaattgggcacctgtggttcattcttctc ccagattctcagcccaacagagccaagcagctgggtcccctttctatgtggcctgtgtaactctcatctgggtggtgccccccatccccctcagtgctgccacatgccatggattgcaaggacaatgtggctgacatctgcatggcagaagaaaggaggtgctgggctgtcagaggaagct ggtctgggcctgggagtctgtgccaactgcaaatctgactttacttttaattgcctatgaaaataaggtctctcatttattttcctctccctgctttctttcagactgtggctttacctcgggtaagtaagcccttccttttcctctccctctctcatggttcttgacctagaaccaaggc atgaagaactcacagacactggagggtggagggtgggagagaccagagctacctgtgcacaggtacccacctgtccttcctccgtgccaacagtgtcctaccagcaaggggtcctgtctgccaccatcctctatgagatcctgctagggaaggccaccctgtatgctgtgctggtcagcgc ccttgtgttgatggccatggtaagcaggagggcaggatggggccagcaggctggaggtgacacactgacaccaagcacccagaagtatagagtccctgccaggattggagctgggcagtagggagggaagagatttcattcaggtgcctcagaagataacttgcacctctgtaggatcaca gtggaagggtcatgctgggaaggagaagctggagtcaccagaaaacccaatggatgttgtgatgagccttactatttgtgtggtcaatgggccctactactttctctcaatcctcacaactcctggctcttaataacccccaaaactttctcttctgcaggtcaagagaaaggatttctga Human TCR beta constant 1 ( TRBC1 )
NCBI Reference Sequence: NG_001333,2, TRBC1 3 aggacctgaaaaacgtgttcccacccgaggtcgctgtgtttgagccatcagaagcagagatctcccacacccaaaaggccacactggtatgcctggccacaggcttctaccccgaccacgtggagctgagctggtgggtgaatgggaaggaggtgcacagtggggtcagcacagacccgcagcccc tcaaggagcagcccgccctcaatgactccagatactgcctgagcagccgcctgagggtctcggccaccttctggcagaacccccgcaaccacttccgctgtcaagtccagttctacgggctctcggagaatgacgagtggacccaggatagggccaaacccgtcacccagatcgtcagcgccgagg cctggggtagagcaggtgagtggggcctggggagatgcctggaggagattaggtgagaccagctaccagggaaaatggaaagatccaggtagcggacaagactagatccagaagaaagccagagtggacaaggtgggatgatcaaggttcacaggtcagcaaagcacggtgtgcacttcccccac caagaagcatagaggctgaatggagcacctcaagctcattcttccttcagatcctgacaccttagagctaagctttcaagtctccctgaggaccagccatacagctcagcatctgagtggtgtgcatcccattctcttctggggtcctggtttcctaagatcatagtgaccacttcgctggcactg gagcagcatgagggagacagaaccagggctatcaaaggaggctgactttgtactatctgatatgcatgtgtttgtggcctgtgagtctgtgatgtaaggctcaatgtccttacaaagcagcattctctcatccatttttcttcccctgttttctttcagactgtggcttcacctccggtaagtgag tctctccttttctctctctatctttcgccgtctctgctctcgaaccagggcatggagaatccacggacacaggggcgtgagggaggccagagccacctgtgcacaggtgcctacatgctctgttcttgtcaacagagtcttaccagcaaggggtcctgtctgccaccatcctctatgagatcttgct agggaaggccaccttgtatgccgtgctggtcagtgccctcgtgctgatggccatggtaaggaggagggtgggatagggcagatgatgggggcaggggatggaacatcacacatgggcataaaggaatctcagagccagagcacagcctaatatatcctatcacctcaatgaaaccataatgaagcc agactggggagaaaatgcagggaatatcacagaatgcatcatgggaggatggagacaaccagcgagccctactcaaattaggcctcagagcccgcctcccctgccctactcctgctgtgccatagcccctgaaaccctgaaaatgttctctcttccacaggtcaagagaaaggattccagaggctag Human TCR beta constant 2 ( TRBC2 )
NCBI Reference Sequence: NG_001333,2, TRBC2 4 cgtgaggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcct ttttcccgaggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtgc cttgaattacttccacgcccctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtggggagagttcgaggccttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcctgggcgctggggcc gccgcgtgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctgctgcgacgctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtatt tcggtttttggggccgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggccaccgagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgc cgtgtatcgccccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagatggccgcttcccggccctgctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaa aggaaaagggcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctcgattagttctcgagcttttggagtacgtcgtctttaggttggggggaggggttttatgcgatggagtttcccca cactgagtgggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggatcttggttcattctcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtcgtgaa EF1 alpha promoter (GenBank: J04617,1) 5 CGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTT TTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCACTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTG AATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGTGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGTGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCG AATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCAGCACACTGGTATTTCGGTTTTTGGG GCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGCCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCG CCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCACAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTC CGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCCAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGAC TGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAAAACTACCCCTAAAAGCCAAA EF1 alpha promoter 6 LEGGGEGRGSLLTCGDVEENPGPR T2A 7 EGRGSLLTCGDVEENPGP T2A 8 GSGATNFSLLKQAGDVEENPGP P2A 9 ATNFSLLKQAGDVEENPGP P2A 10 QCTNYALLKLAGDVESNPGP E2A 11 VKQTLNFDLLKLAGDVESNPGP F2A 12 MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTK IISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM EGFRt 13 RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCCK ATGQVCHALCSPEGCWGPEPRDCVSCRNNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM EGFRt 14 DIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS Mouse alpha constant TCR 15 NIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS Mouse alpha constant TCR 16 EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS Mouse Beta TCR Constant (Uniprot P01852) 17 DLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKRKNS Mouse TCR Beta constant 18 gggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagca MND promoter 19 PNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS Human Alpha TCR Constant (Uniprot P01848) 20 EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF Human Beta TCR Constant 1 (Uniprot P01850) 21 DLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG Human Beta TCR constant 2 (Uniprot A0A5B9) 22 -PGGG-(SGGGG)n-P- where P is proline, G is glycine and S is serine Linker 23 GSADDAKKDAAKKDGKS Linker 24 NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS Human Alpha TCR constant (Genbank Accession #CAA26636.1) 25 EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG Human TCR Beta Constant (Uniprot Sample #A0A0G2JNG9) 26 AGCGCTCTCGTACAGAGTTGGCATTATAATACGACTCACTATAGGGGAGAATCAAAATCGGTGAATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT TRAC nRNA transcriptional sequence 27 GAGAAUCAAAAUCGGUGAAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU TRAC nRNA sequence 28 UCUCUCAGCUGGUACACGGC TRAC -10 nRNA targeting domain 29 UGGAUUUAGAGUCUCUCAGC TRAC -110 nRNA targeting domain 30 ACACGGCAGGGUCAGGGUUC TRAC -116 nRNA targeting domain 31 GAGAAUCAAAAUCGGUGAAU TRAC -16 nRNA targeting domain 32 GCUGGUACACGGCAGGGUCA TRAC -4 nRNA targeting domain 33 CUCAGCUGGUACACGGC TRAC -49 nRNA targeting domain 34 UGGUACACGGCAGGGUC TRAC -2 nRNA targeting domain 35 GCUAGACAUGAGGUCUA TRAC -30 nRNA targeting domain 36 GUCAGAUUUGUUGCUCC TRAC -43 nRNA targeting domain 37 UCAGCUGGUACACGGCA TRAC -23 nRNA targeting domain 38 GCAGACAGACUUGUCAC TRAC -34 nRNA targeting domain 39 GGUACACGGCAGGGUCA TRAC -25 nRNA targeting domain 40 CUUCAAGAGCAACAGUGCUG TRAC -128 nRNA targeting domain 41 AGAGCAACAGUGCUGUGGCC TRAC -105 nRNA targeting domain 42 AAAGUCAGAUUUGUUGCUCC TRAC -106 nRNA targeting domain 43 ACAAACUGUGCUAGACAUG TRAC -123 nRNA targeting domain 44 AAACUGUGCUAGACAUG TRAC -64 nRNA targeting domain 45 UGUGCUAGACAUGAGGUCUA TRAC -97 nRNA targeting domain 46 GGCUGGGGAAGAAGGUGUCUUC TRAC -148 nRNA targeting domain 47 GCGGGGAAGAAGGUGUCUUC TRAC -147 nRNA targeting domain 48 GGGGAAGAAGGUGUCUUC TRAC -234 nRNA targeting domain 49 GUUUUGUCUGUGAUAUACACAU TRAC -167 nRNA targeting domain 50 GGCAGACAGACUUGUCACUGGAUU TRAC -177 nRNA targeting domain 51 GCAGACAGACUUGUCACUGGAUU TRAC -176 nRNA targeting domain 52 GACAGACUUGUCACUGGAUU TRAC -257 nRNA targeting domain 53 GUGAAUAGGCAGACAGACUUGUCA TRAC -233 nRNA targeting domain 54 GAAUAGGCAGACAGACUUGUCA TRAC -231 nRNA targeting domain 55 GAGUCUCAGCUGGUACACGG TRAC -163 nRNA targeting domain 56 GUCUCUCAGCUGGUACACGG TRAC -241 nRNA targeting domain 57 GGUACACGGCAGGGUCAGGGUU TRAC -179 nRNA targeting domain 58 GUACACGGCAGGGUCAGGGUU TRAC -178 nRNA targeting domain 59 CACCCAGAUCGUCAGCGCCG TRBC -40 nRNA targeting domain 60 CAAACACAGCGACCUCGGGU TRBC -52 nRNA targeting domain 61 UGACGAGUGGACCCAGGAUA TRBC -25 nRNA targeting domain 62 GGCUCUCGGAGAAUGACGAG TRBC -35 nRNA targeting domain 63 GGCCUCGGCGCUGACGAUCU TRBC -50 nRNA targeting domain 64 GAAAAACGUGUUCCCACCCG TRBC -39 nRNA targeting domain 65 AUGACGAGUGGACCCAGGAU TRBC -49 nRNA targeting domain 66 AGUCCAGUUCUACGGGCUCU TRBC -51 nRNA targeting domain 67 CGCUGUCAAGUCCAGUUCUA TRBC -26 nRNA targeting domain 68 AUCGUCAGCGCCGAGGCCUG TRBC -47 nRNA targeting domain 69 UCAAACACAGCGACCUCGGG TRBC -45 nRNA targeting domain 70 CGUAGAACUGGACUUGACAG TRBC -34 nRNA targeting domain 71 AGGCCUCGGCGCUGACGAUC TRBC -227 nRNA targeting domain 72 UGACAGCGGAAGUGGUUGCG TRBC -41 nRNA targeting domain 73 UUGACAGCGGAAGUGGUUGC TRBC -30 nRNA targeting domain 74 UCUCCGAGAGCCCGUAGAAC TRBC -206 nRNA targeting domain 75 CGGGUGGGAACACGUUUUUC TRBC -32 nRNA targeting domain 76 GACAGGUUUGGCCCUAUCCU TRBC -276 nRNA targeting domain 77 GAUCGUCAGCGCCGAGGCCU TRBC -274 nRNA targeting domain 78 GGCUCAAACACAGCGACCUC TRBC -230 nRNA targeting domain 79 UGAGGGUCUCGGCCACCUUC TRBC -235 nRNA targeting domain 80 AGGCUUCUACCCCGACCACG TRBC -38 nRNA targeting domain 81 CCGACCACGUGGAGCUGAGC TRBC -223 nRNA targeting domain 82 UGACAGGUUUGGCCCUAUCC TRBC -221 nRNA targeting domain 83 CUUGACAGCGGAAGUGGUUG TRBC -48 nRNA targeting domain 84 AGAUCGUCAGCGCCGAGGCC TRBC -216 nRNA targeting domain 85 GCGCGUGACGAUCUGGGUGAC TRBC -210 nRNA targeting domain 86 UGAGGGCGGGCUGCUCCUUG TRBC -268 nRNA targeting domain 87 GUUGCGGGGGUUCUGCCAGA TRBC -193 nRNA targeting domain 88 AGCUCAGCUCCACGUGGUCG TRBC -246 nRNA targeting domain 89 GCGGCUGCUCAGGCAGUAUC TRBC -228 nRNA targeting domain 90 GCGGGGGUUCUGCCAGAAGG TRBC -43 nRNA targeting domain 91 UGGCUCAAACACAGCGACCU TRBC -272 nRNA targeting domain 92 ACUCGACUGACCGGAAG TRBC -33 nRNA targeting domain 93 GACAGCGGAAGUGGUUGCGG TRBC -44 nRNA targeting domain 94 GCUGUCAAGUCCAGUUCUAC TRBC -211 nRNA targeting domain 95 GUAUCUGGAGUCAUUGAGGG TRBC -253 nRNA targeting domain 96 CUCGGCCGCUGACGAUCU TRBC -18 nRNA targeting domain 97 CCUCGGCGCUGACGAUC TRBC -6 nRNA targeting domain 98 CCGAGAGCCCGUAGAAC TRBC -85 nRNA targeting domain 99 CCAGAUCGUCAGCGCCG TRBC -129 nRNA targeting domain 100 GAUGAGAGUGGACCC TRBC -93 nRNA targeting domain 101 GGGUGACAGGUUUGGCCCUAUC TRBC -415 nRNA targeting domain 102 GGUGACAGGUUUGGCCCUAUC TRBC -414 nRNA targeting domain 103 GUGACAGGUUUGGCCCUAUC TRBC -310 nRNA targeting domain 104 GACAGGUUUGGCCCUAUC TRBC -308 nRNA targeting domain 105 GAUACUGCCUGAGCAGCCGCCU TRBC -401 nRNA targeting domain 106 GACCACGUGGAGCUGAGCUGGUGG TRBC -468 nRNA targeting domain 107 GUGGAGCUGAGCUGGUGG TRBC -462 nRNA targeting domain 108 GGGCGGGCUGCUCCUUGAGGGGCU TRBC -424 nRNA targeting domain 109 GGCGGGCUGCUCCUUGAGGGGCU TRBC -423 nRNA targeting domain 110 GCGGGCUGCUCCUUGAGGGGCU TRBC -422 nRNA targeting domain 111 GGGCUGCUCCUUGAGGGGCU TRBC -420 nRNA targeting domain 112 GGCUGCUCCUUGAGGGGCU TRBC -419 nRNA targeting domain 113 GCUGCUCCUUGAGGGGCU TRBC -418 nRNA targeting domain 114 GGUGAAUGGGAAGGAGGUGCACAG TRBC -445 nRNA targeting domain 115 GUGAAUGGGAAGGAGGUGCACAG TRBC -444 nRNA targeting domain 116 GAAUGGGAAGGAGGUGCACAG TRBC -442 nRNA targeting domain 117 ATTCACCGATTTTGATTCTC TRAC target sequence 1 118 AGATCGTCAGCGCCGAGGCC TRBC target sequence 2 119 CTGACCTCTTCTCTTCCTCCCACAG HBB splice site acceptor 120 TTTCTCTCCACAG IgG splice site acceptor 121 PYIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS Mouse Alpha TCR Constant (Uniprot P01849) 122 IQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS Modified mouse alpha constant TCR 123 EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS Modified mouse TCR beta constant 124 CTCTATCAATGAGAGAGCAATCTCCTGGTAATGTGATAGATTTCCCAACTTAATGCCAACATACCATAAACCTCCCATTCTGCTAATGCCCAGCCTAAGTTGGGGAGACCACTCCAGATTCCAAGATGTACAGTTTGCTTTGCTGGGCCTTT TTCCCATGCCTGCCTTTACTCTGCCAGAGTTATATTGCTGGGGTTTTGAAGAAGATCCTATTAAATAAAAGAATAAGCAGTATTATTAAGTAGCCCTGCATTTCAGGTTTCCTTGAGTGGCAGGCCAGGCCTGGCCGTGAACGTTCACTGAAA TCATGGCCTCTTGGCCAAGATTGATAGCTTGTGCCTGTCCCTGAGTCCCAGTCCATCACGAGCAGCTGGTTTCTAAGATGCTATTTCCCGTATAAAGCATGAGACCGTGACTTGCCAGCCCCACAGAGCCCCGCCCTTGTCCATCACTGGCAT CTGGACTCCAGCCTGGGTTGGGGCAAAGAGGGAAATGAGATCATGTCCTAACCCTGATCCTCTTGTCCCACAGATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGAT ACTR 5' homology arm 125 TTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACA GCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGGTAAGGGCAGCTTTGGTGCCTTCGCAGGCTGTTTCCTTGCTTCAGGAATGGCCAGGTTCTGCCCAGAGCTCTGGTCAATGATGTCTAAAACTCCCTCTGATTGGTGGTCTCGGCCTTATCCA TTGCCACCAAAACCCTCTTTTTACTAAGAAACAGTGAGCCTTGTTCTGGCAGTCCAGAGAATGACACGGGAAAAAAGCAGATGAAGAGAAGGTGGCAGGAGAGGGCACGTGGCCCAGCCTCAGTCTCTCCAACTGAGTTCCTGCCTGCCTGCCTTTG CTCAGACTGTTTGCCCCTTACTGCTCTTCTAGGCCTCATTCTAAGCCCCTCTCCAAGTTGCCTCTCCTTATTTCTCCCTGTCTGCCAAAAAATCTTTCCCAGCTCACTAAGTCAGTCTCACGCAGTCACTCATTAACCCACCAATCACTGATTGTG ACTR 3' homology arm 126 GAACAGAGAAACAGGAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGCCAAGAACAGTTGGAACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGCCAAGAACAGATGGTCCCCAGATGCGGT CCCGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATC MND promoter 127 GGATCTGCGATCGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGT CGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCATCTCTCCT TCACGCGCCCGCCGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTTAAAGCTCAGGTCGAGACCGGGCCTTT GTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTAC Ef1alpha promoter with HTLV1 enhancer 128 GGATCTGGAGCGACGAATTTTAGTCTACTGAAACAAGCGGGGAGACGTGGAGGAAAACCCTGGACCT P2A nucleotide sequence 129 RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD3 zeta 130 RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD3 zeta 131 ESKYGPPCPPCP spacer (IgG4 hinge) 132 GAATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCT spacer (IgG4 hinge) 133 ESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Hinge-CH3 spacer 134 ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Hinge-CH2-CH3 spacer 135 RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLLTLPRS LWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDH IgD-hinge-Fc 136 FWVLVVVGGVLACYSLLVTVAFIIFWV CD28 (amino acids 153-179 of sample no. P10747) 137 IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWV CD28 (amino acids 114-179 sample no. P10747) 138 RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS CD28 (amino acids 180-220 P10747) 139 RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS CD28 (LL to GG) 140 KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 4-1BB (amino acids 214-255 Q07011.1) 141 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD3 zeta 142 NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAGUGGCACCGAGUCGGUGC typical gRNA complementary domain 143 NNNNNNNNNNNNNNNNNNNN GUUUUAG A GCUAUGCU GAAA AGCAUAGC AAG UUAAAAU AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC typical gRNA complementary domain 144 NNNNNNNNNNNNNNNNNNNN GUUUUAG A GCUAUGCUG GAAA CAGCAUAGC AAG UUAAAAU AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC typical gRNA complementary domain 145 NNNNNNNNNNNNNNNNNNNN GUUUUAG A GCUAUGCUGUUUUG GAAA CAAAACAGCAUAGC AAG UUAAAAU AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC typical gRNA complementary domain 146 NNNNNNNNNNNNNNNNNNNNGU A UUAGAGCUAGAAAUAGCAAGUUAA U AUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC typical gRNA 147 NNNNNNNNNNNNNNNNNNNNGUUU A AGAGCUAGAAAUAGCAAGUU U AAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC typical gRNA 148 NNNNNNNNNNNNNNNNNNNNGU A UUAGAGCUAUGCUGU A UUGGAAACAA U ACAGCAUAGCAAGUUAA U AUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC typical gRNA 149 AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU typical proximal and tail domain 150 AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGGUGC typical proximal and tail domain 151 AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGGAUC typical proximal and tail domain 152 AAGGCUAGUCCGUUAUCAACUUGAAAAAGUG typical proximal and tail domain 153 AAGGCUAGUCCGUUAUCA typical proximal and tail domain 154 AAGGCUAGUCCG typical proximal and tail domain 155 NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU typical chimeric gRNA 156 NNNNNNNNNNNNNNNNNNNNGUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUUUU typical chimeric gRNA 157 KKPYSIGLDIGTNSVGWAVVTDDYKVPAKKMKVLGNTDKSHIEKNLLGALLFDSGNTAEDRRLKRTARRRYTRRRNRILYLQEIFSEEMGKVDDSFFHRLEDSFLVTEDKRGERHPIFGNLEEEVKYHENFPTIYHLRQYLADNPEKVDLRLVYLALAHIIKFRGHFL IEGKFDTRNNDVQRLFQEFLAVYDNTFENSSLQEQNVQVEEILTDKISKSAKKDRVLKLFPNEKSNGRFAEFLKLIVGNQADFKKHFELEEKAPLQFSKDTYEEELEVLLAQIGDNYAELFLSAKKLYDSILLSGILTVTDVGTKAPLSASMIQRYNEHQMDLAQLKQ FIRQKLSDKYNEVFSDVSKDGYAGYIDGKTNQEAFYKYLKGLLNKIEGSGYFLDKIEREDFLRKQRTFDNGSIPHQIHLQEMRAIIRRQAEFYPFLADNQDRIEKLLTFRIPYYVGPLARGKSDFAWLSRKSADKITPWNFDEIVDKEONOEAFINRMTNYDLYLPNQ KVLPKHSLLYEKFTVYNELTKVKYKTEQGKTAFFDANMKQEIFDGVFKVYRKVTKDKLMDFLEKEFDEFRIVDLTGLDKENKVFNASYGTYHDLCKILDKDFLDNSKNEKILEDIVLTLTLFEDREMIRKRLENYSDLLTKEQVKKLERRHYTGWGRLSAELIHGIRN KESRKTILDYLIDDGNSNRNFMQLINDDALSFKEEIAKAQVIGETDNLNQVVSDIAGSPAIKKGILQSLKIVDELVKIMGHQPENIVVEMARENQFTNQGRRNSQQRLKGLTDSIKEFGSQILKEHPVENSQLQNDRLFLYYLQNGRDMYTGEELDIDYLSQYDIDHI IPQAFIKDNSIDNRVLTSSKENRGKSDDVPSKDVVRKMKSYWSKLLSAKLITQRKFDNLTKAERGGLTDDDKAGFIKRQLVETRQITKHVARILDERFNTETDENNKKIRQVKIVTLKSNLVSNFRKEFELYKVREINDYHHAHDAYLNAVIGKALLGVYPQLEPEFV YGDYPHFHGHKENKATAKKFFYSNIMNFFKKDDVRTDKNGEIIWKKDEHISNIKKVLSYPQVNIVKKVEEQTGGFSKESILPKGNSDKLIPRKTKKFYWDTKKYGGFDSPIVAYSILVIADIEKGKSKKLKTVKALVGVTIMEKMTFERDPVAFLERKGYRNVQEENI IKLPKYSLFKLENGRKRLLASARELQKGNEIVLPNHLGTLLYHAKNIHKVDEPKHLDYVDKHKDEFKELLDVVSNFSKKYTLAEGNLEKIKELYAQNNGEDLKELASSFINLLTFTAIGAPATFKFFDKNIDRKRYTSTTEILNATLIHQSITGLYETRIDLNKLGGD Streptococcus mutans Cas9 158 DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIE GDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKPYALDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQ LPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMФНОDKNLPNEKVLPKHSL LYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLK SDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNK VDKPSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSE QEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIK LPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD Streptococcus pyogenes Cas9 159 TKPYSIGLDIGTNSVGWAVTTDNYKVPSKKMKVLGNTSKKYIKKNLLGVLLFDSGITAEGRRLKRTARRRYTRRRRNRILYLQEIFSTEMATLDDAFFQRLDDSFLVPDDKRDSKYPIFGNLVEEKAYHDEFPTIYHLRKYLADSTKKADLRLVYLALAHMIKYRGHFLIEGEF NSKNNDIQKNFQDFLDTYNAIFESDLSLENSKQLEEIVKDKISKLEKKDRILKLFPGEKNSGIFSEFLKLIVGNQADFRKCFNLDEKASLHFSKESYDEDLETLLGYIGDDYSDVFLKAKKLYDAILLSGFLTVTDNETEAPLONOMIKRYNEHKEDLALLKEYIRNISLKTY NEVFKDDTKNGYAGYIDGKTNQEDFYVYLKKLLAEFEGADYFLEKIDREDFLRKQRTFDNGSIPYQIHLQEMRAILDKQAKFYPFLAKNKERIEKILTFRIPYYVGPLARGNSDFAWSIRKRNEKITPWNFEDVIDKEONOEAFINRMTSFDLYLPEEKVLPKHSLLYETFNV YNELTKVRFIAESMRDYQFLDSKQKKDIVRLYFKDKRKVTDKDIIEYLHAIYGYDGIELKGIEKQFNSSLSTYHDLLNIINDKEFLDDSSNEAIIEEIIHTLTIFEDREMIKQRLSKFENIFDKSVLKKLSRRHYTGWGKLSAKLINGIRDEKSGNTILDYLIDDGISNRNFMQ LIHDDALSFKKKIQKAQIIGDEDKGNIKEVVKSLPGSPAIKKGILQSIKIVDELVKVMGGRKPESIVVEMARENQYTNQGKSNSQQRLKRLEKSLKELGSKILKENIPAKLSKIDNNALQNDRLYLYYLQNGKDMYTGDDLDIDRLSNYDIDHIIPQAFLKDNSIDNKVLVON ОSNRGKSDDVPSLEVVKKRKTFWYQLLKSKLISQRKFDNLTKAERGGLSPEDKAGFIQRQLVETRQITKHVARLLDEKFNNKKDENNRAVRTVKIITLKSTLVSQFRKDFELYKVREINDFHHAHDAYLNAVVASALLKKYPKLEPEFVYGDYPKYNSFRERKSATEKVYFYSN IMNIFKKSISLADGRVIERPLIEVNEETGESVWNKESDLATVRRVLSYPQVNVVKKVEEQNHGLDRGKPKGLFNAPYALSKPKPNSNENLVGAKEYLDPKKYGGYAGISNSFTVLVKGTIEKGAKKKITNVLEFQGISILDRINYRKDKLNFLLEKGYKDIELIIELPKYSLFE LSDGSRRMLASILSTNNKRGEIHKGNQIFLSQKFVKLLYHAKRISNTINENHRKYVENHKKEFEELFYYILEFNENYVGAKKNGKLLNSAFQSWQNHSIDELCSSFIGPTGSERKGLFELTSRGSAADFEFLGVKIPRYRDYTPSSLLKDATLIHQSVTGLYETRIDLAKLGEG Streptococcus thermophilus Cas9 160 KKPYTIGLDIGTNSVGWAVLTDQYDLVKRKMKIAGDSEKKQIKKNFWGVRLFDEGQTAADRRMARTARRRIERRRNRISYLQGIFAEEMSKTDANFFCRLSDSFYVDNEKRNSRHPFFATIEEEVEYHKNYPTIYHLREELVNSSEKADLRLVYLALAHIIKYRGN FLIEGALDTQNTSVDGIYKQFIQTYNQVFASGIEDGSLKKLEDNKDVAKILVEKVTRKEKLERILKLYPGEKSAGMFAQFISLIVGSKGNFQKPFDLIEKSDIECAKDSYEEDLESLLALIGDEYAELFVAAKNAYSAVVLSSIITVAETETNAKLSASMIERFDTH EEDLGELKAFIKLHLPKHYEEIFSNTEKHGYAGYIDGKTKQADFYKYMKMTLENIEGADYFIAKIEKENFLRKQRTFDNGAIPHQLHLEELEAILHQQAKYYPFLKENYDKIKSLVTFRIPYFVGPLANGQSEFAWLTRKADGEIRPWNIEEKVDFGKSAVDFIEK MTNKDTYLPKENVLPKHSLCYQKYLVYNELTKVRYINDQGKTSYFSGQEKEQIFNDLFKQKRKVKKKDLELFLRNMSHVESPTIEGLEDSFNSSYSTYHDLLKVGIKQEILDNPVNTEMLENIVKILTVFEDKRMIKEQLQQFSDVLDGVVLKKLERRHYTGWGRLS AKLLMGIRDKQSHLTILDYLMNDDGLNRNLMQLINDSNLSFKSIIEKEQVTTADKDIQSIVADLAGSPAIKKGILQSLKIVDELVSVMGYPPQTIVVEMARENQTTGKGKNNSRPRYKSLEKAIKEFGSQILKEHPDTDNQELRNNRLYLYYLQNGKDMYTGQDLDI HNLSNYDIDHIVPQSFITDNSIDNLVLTSSAGNREKGDDVPPLEIVRKRKVFWEKLYQGNLMSKRKFDYLTKAERGGLTEADKARFIHRQLVETRQITKNVANILHQRFNYEKDDHGNTMKQVRIVTLKSALVSQFRKQFQLYKVRDVNDYHHAHDAYLNGVVANTL LKVYPQLEPEFVYGDYHQFDWFKANKATAKKQFYTNIMLFFAQKDRIIDENGEILWDKKYLDTVKKVMSYRQMNIVKKTEIQKGEFSKATIKPKGNSSKLIPRKTNWDPMKYGGLDSPNMAYAVVIEYAKGKNKLVFEKKIIRVTIMERKAFEKDEKAFLEEQGYRQ PKVLAKLPKYTLYECEEGRRRMLASANEAQKGNQQVLPNHLVTLLHHAANCEVSDGKSLDYIESNREMFAELLAHVSEFAKRYTLAEANLNKINQLFEQNKEGDIKAIAQSFVDLMAFNAMGAPASFKFFETTIERKRYNNLKELLNSTIIYQSITGLYESRKRLDD Listeria innocua Cas9 161 MAAFKPNSINYILGLDIGIASVGWAMVEIDEEENPIRLIDLGVRVFERAEVPKTGDSLAMARRLARSVRRLLTRRRAHRLLRTRRLLKREGVLQAANFDENGLIKSLPNTPWQLRAAALDRKLTPLEWSAVLLHLI KHRGYLSQRKNEGETADKELGALLKGVAGNAHALQTGDFRTPAELALNKFEKESGHIRNQRSDYSHTFSRKDLQAELILLFEKQKEFGNPHVSGGLKEGIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAKN TYTAERFIWLTKLNNLRILEQGSERPLTDTERATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKDNAEASTLMEMKAYHAISRALEKEGLKDKKSPLNLSPELQDEIGTAFSLFKTDEDITGRLKDRIQP EILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKNTEEKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPARIHIETAREVGKSFKDRKEIEKRQEENRKDREKAAAKFREYF PNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLGRLNEKGYVEIDHALPFSRTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEFKARVETSRFPRSKKQRILLQKFDEDGFKERNLNDTRYVNRF LCQFVADRMRLTGKGKKRVFASNGQITNLLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEMNAFDGKTIDKETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTLEKLRTLLA EKLSSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVKSAKRLDEGVSVLRVPLTQLKLKDLEKMVNREREPKLYEALKARLEAHKDDPAKAFAEPFYKYDKAGNRTQQVKAVRVEQVQKTGVWVRNHNGIADNAT MVRVDVFEKGDKYYLVPIYSWQVAKGILPDRAVVQGKDEEDWQLIDDSFNFKFSLHPNDLVEVITKKARMFGYFASCHRGTGNINIRIHDLDHKIGKNGILEGIGVKTALSFQKYQIDELGKEIRPCRLKKRPPVR Neisseria meningitidis Cas9 162 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIE GDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQ LPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSL LYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLK SDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSE QEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIK LPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD Streptococcus pyogenes Cas9 163 GAGAATCAAAATCGGTGAAT TRAC target sequence 2 164 GGCCTCGGCGCTGACGATCT TRBC target sequence 2 165 GAGAATCAAAATCGGTGAATAGG TRAC target sequence with PAM 3 166 TTCAAAACCTGTCAGTGATTGGG TRAC target sequence with PAM 4 167 TGTGCTAGACATGAGGTCTATGG TRAC target sequence with PAM 5 168 CGTCATGAGCAGATTAAACCCGG TRAC target sequence with PAM 6 169 TCAGGGTTCTGGATATCTGTGGG TRAC target sequence with PAM 7 170 GTCAGGGTTCTGGATATCTGTGG TRAC target sequence with PAM 8 171 TTCGGAACCCAATCACTGACAGG TRAC target sequence with PAM 9 172 TAAACCCGGCCACTTTCAGGAGG TRAC target sequence with PAM 10 173 AAAGTCAGATTTGTTGCTCCAGG TRAC target sequence with PAM 11 174 AACAAATGTGTCACAAAGTAAGG TRAC target sequence with PAM 12 175 TGGATTTAGAGTCTCTCAGCTGG TRAC target sequence with PAM 13 176 TAGGCAGACAGACTTGTCACTGG TRAC target sequence with PAM 14 177 AGCTGGTACACGGCAGGGTCAGG TRAC target sequence with PAM 15 178 GCTGGTACACGGCAGGGTCAGGG TRAC target sequence with PAM 16 179 TCTCTCAGCTGGTACACGGCAGG TRAC target sequence with PAM 17 180 TTTCAAAACCTGTCAGTGATTGG TRAC target sequence with PAM 18 181 GATTAAACCCGGCCACTTTCAGG TRAC target sequence with PAM 19 182 CTCGACCAGCTTGACATCACAGG TRAC target sequence with PAM 20 183 AGAGTCTCTCAGCTGGTACACGG TRAC target sequence with PAM 21 184 CTCTCAGCTGGTACACGGCAGGG TRAC target sequence with PAM 22 185 AAGTTCCTGTGATGTCAAGCTGG TRAC target sequence with PAM 23 186 ATCCTCCTCCTGAAAGTGGCCGG TRAC target sequence with PAM 24 187 TGCTCATGACGCTGCGGCTGTGG TRAC target sequence with PAM 25 188 ACAAAACTGTGCTAGACATGAGG TRAC target sequence with PAM 26 189 ATTTGTTTTGAGAATCAAAATCGG TRAC target sequence with PAM 27 190 CATCACAGGAACTTTCTAAAAGG TRAC target sequence with PAM 28 191 GTCGAGAAAAGCTTTGAAACAGG TRAC target sequence with PAM 29 192 CCACTTTCAGGAGGAGGATTCGG TRAC target sequence with PAM 30 193 CTGACAGGTTTTGAAAGTTTAGG TRAC target sequence with PAM 31 194 AGCTTTGAAACAGGTAAGACAGG TRAC target sequence with PAM 32 195 TGGAATAATGCTGTTGTTGAAGG TRAC target sequence with PAM 33 196 AGAGCAACAGTGCTGTGGCCTGG TRAC target sequence with PAM 34 197 CTGTGGTCCAGCTGAGGTGAGGG TRAC target sequence with PAM 35 198 CTGCGGCTGTGGTCCAGCTGAGG TRAC target sequence with PAM 36 199 TGTGGTCCAGCTGAGGTGAGGGG TRAC target sequence with PAM 37 200 CTTCTTCCCCAGCCCAGGTAAGG TRAC target sequence with PAM 38 201 ACACGGCAGGGTCAGGGTTCTGG TRAC target sequence with PAM 39 202 CTTCAAGAGCAACAGTGCTGTGG TRAC target sequence with PAM 40 203 CTGGGGAAGAAGGTGTCTTCTGG TRAC target sequence with PAM 41 204 TCCTCCTCCTGAAAGTGGCCGGG TRAC target sequence with PAM 42 205 TTAATCTGCTCATGACGCTGCGG TRAC target sequence with PAM 43 206 ACCCGGCCACTTTCAGGAGGAGG TRAC target sequence with PAM 44 207 TTCTTCCCCAGCCCAGGTAAGGG TRAC target sequence with PAM 45 208 CTTACCTGGGCTGGGGAAGAAGG TRAC target sequence with PAM 46 209 GACACCTTCTTCCCCAGCCCAGG TRAC target sequence with PAM 47 210 GCTGTGGTCCAGCTGAGGTGAGG TRAC target sequence with PAM 48 211 CCGAATCCTCCTCCTGAAAGTGG TRAC target sequence with PAM 49 212 GCTGCTAAGTCCAGTTCTACGGG TRBC target sequence with PAM 3 213 CTATGGACTTCAAGAGCAACAGTGCTGT TRAC ZFN target sequence 1 214 CTCATGTCTAGCACAGTTTTGTCTGTGA TRAC ZFN target sequence 2 215 GTGCTGTGGCCTGGAGCAACAAATCTGA TRAC ZFN target sequence 3 216 TTGCTCTTGAAGTCCATAGACCTCATGT TRAC ZFN target sequence 4 217 GCTGTGGCCTGGAGCAACAAATCTGACT TRAC ZFN target sequence 5 218 CTGTTGCTCTTGAAGTCCATAGACCTCA TRAC ZFN target sequence 6 219 CTGTGGCCTGGAGCAACAAATCTGACTT TRAC ZFN target sequence 7 220 CTGACTTTGCATGTGCAAACGCCTTCAA TRAC ZFN target sequence 8 221 TTGTTGCTCCAGGCCACAGCACTGTTGC TRAC ZFN target sequence 9 222 TGAAAGTGGCCGGGTTTAATCTGCTCAT TRAC ZFN target sequence 10 223 AGGAGGATTCGGAACCCAATCACTGACA TRAC ZFN target sequence 11 224 GAGGAGGATTCGGAACCCAATCACTGAC TRAC ZFN target sequence 12 225 CCGTAGAACTGGACTTGACAGCGGAAGT TRBC ZFN target sequence 1 226 TCTCGGAGAATGACGAGTGGACCCAGGA TRBC ZFN target sequence 2 227 AGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGAT TRAC 50 bp 5' homology arm 228 CTGATCCTCTTGTCCCACAGATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGAT TRAC 100 bp 5' homology arm 229 GTGACTTGCCAGCCCCACAGAGCCCCGCCCTTGTCCATCACTGGCATCTGGACTCCAGCCTGGGTTGGGGCAAAGAGGGAAATGAGATCATGTCCTAACCCTGATCCTCTTGTCCCACAGATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGAT TRAC 200 bp 5' homology arm 230 CCTCTTGGCCAAGATTGATAGCTTGTGCCTGTCCCTGAGTCCCAGTCCATCACGAGCAGCTGGTTTCTAAGATGCTATTTCCCGTATAAAGCATGAGACCGTGACTTGCCAGCCCCACAGAGCCCCGCCCTTGTCCATCACTGGCATCTG GACTCCAGCCTGGGTTGGGGCAAAGAGGGAAATGAGATCATGTCCTAACCCTGATCCTCTTGTCCCACAGATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGAT TRAC 300 bp 5' homology arm 231 ATTAAATAAAAGAATAAGCAGTATTATTAAGTAGCCCTGCATTTCAGGTTTCCTTGAGTGGCAGGCCAGGCCTGGCCGTGAACGTTCACTGAAATCATGGCCTCTTGGCCAAGATTGATAGCTTGTGCCTGTCCCTGAGTCCCAGTCCATCACGAGCAGCTGGTTTCTAAGATGCTATTTCCCGTATAAAGCATGAGACC GTGACTTGCCAGCCCCACAGAGCCCCGCCCTTGTCCATCACTGGCATCTGGACTCCAGCCTGGGTTGGGGCAAAGAGGGAAATGAGATCATGTCCTAACCCTGATCCTCTTGTCCCACAGATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGAT TRAC 400 bp 5' homology arm 232 CTCCAGATTCCAAGATGTACAGTTTGCTTTGCTGGGCCTTTTTCCCATGCCTGCCTTTACTCTGCCAGAGTTATATTGCTGGGGTTTTGAAGAAGATCCTATTAAATAAAAGAATAAGCAGTATT ATTAAGTAGCCCTGCATTTCAGGTTTCCTTGAGTGGCAGGCCAGGCCTGGCCGTGAACGTTCACTGAAATCATGGCCTCTTGGCCAAGATTGATAGCTTGTGCCTGTCCCTGAGTCCCAGTCCAT CACGAGCAGCTGGTTTCTAAGATGCTATTTCCCGTATAAAAGCATGAGACCGTGACTTGCCAGCCCCACAGAGCCCCGCCCTTGTCCATCACTGGCATCTGGACTCCAGCCTGGGTTGGGGCAAAG AGGGAAATGAGATCATGTCCTAACCCTGATCCTCTTGTCCCACAGATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGAT TRAC 500 bp 5' homology arm 233 AGAGAGCAATTCCTGGTAATGTGATAGATTTCCCAACTTAATGCCAACATACCATAAACCTCCCATTCTGCTAATGCCCAGCCTAAGTTGGGGAGACCACTCCAGATTCCAAGATGTACAGTTTGCTTTGCTGGGCCTTTTTCCCATGC CTGCCTTTACTCTGCCAGAGTTATATTGCTGGGGTTTTGAAGAAGATCCTATTAAATAAAAGAATAAGCAGTATTATTAAGTAGCCCTGCATTTCAGGTTTCCTTGAGTGGCAGGCCAGGCCTGGCCGTGAACGTTCACTGAAATCATGG CCTCTTGGCCAAGATTGATAGCTTGTGCCTGTCCCTGAGTCCCAGTCCATCACGAGCAGCTGGTTTCTAAGATGCTATTTCCCGTATAAAGCATGAGACCGTGACTTGCCAGCCCCACAGAGCCCCGCCCTTGTCCATCACTGGCATCTG GACTCCAGCCTGGGTTGGGGCAAAGAGGGAAATGAGATCATGTCCTAACCCTGATCCTCTTGTCCCACAGATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGAT TRAC 600 bp 5' homology arm 234 TTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATAT TRAC 50 bp 3' homology arm 235 TTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACA TRAC 100 bp 3' homology arm 236 TTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGGTAAGGG TRAC 200 bp 3' homology arm 237 TTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTC AACAACAGCATTATTCCCAGAAGACACCTTCTTCCCCAGCCCAGGTAAGGGCAGCTTTGGTGCCTTCGCAGGCTGTTTCCTTGCTTCAGGAATGGCCAGGTTCTGCCCAGAGCTCTGGTCAATGATGTCTAAAACTCCCTCTGATTGGTGGT TRAC 300 bp 3' homology arm 238 TTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGGTAAGGG CAGCTTTGGTGCCTTCGCAGGCTGTTTCCTTGCTTCAGGAATGGCCAGGTTCTGCCCAGAGCTCTGGTCAATGATGTCTAAAACTCCCTCTGATTGGTGGTCTCGGCCTTATCCATTGCCACCAAAACCCTCTTTTTACTAAGAAACAGTGAGCCTTGTTCTGGCAGTCCAGAGAATGACACGGGAAAAAAGCAGATGAAG TRAC 400 bp 3' homology arm 239 TTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATC TGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGGTAAGGGCAGCTTTGGTGCCTTCGCAGGCTGTTTCCTTGCTTCAGGAATGGCCAGGT TCTGCCCAGAGCTCTGGTCAATGATGTCTAAAACTCCCTCTGATTGGTGGTCTCGGCCTTATCCATTGCCACCAAAACCCTCTTTTTACTAAGAAACAGTGAGCCTTGTTCTGGCAGTCCAGAGAA TGACACGGGAAAAAAGCAGATGAAGAGAAGGTGGCAGGAGAGGGCACGTGGCCCAGCCTCAGTCTCTCCAACTGAGTTCCTGCCTGCCTGCCTTTGCTCAGACTGTTTGCCCCTTACTGCTCTTC TRAC 500 bp 3' homology arm 240 TTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTC AACAACAGCATTATTCCCAGAAGACACCTTCTTCCCCAGCCCAGGTAAGGGCAGCTTTGGTGCCTTCGCAGGCTGTTTCCTTGCTTCAGGAATGGCCAGGTTCTGCCCAGAGCTCTGGTCAATGATGTCTAAAACTCCCTCTGATTGGTGGT CTCGGCCTTATCCATTGCCACCAAAACCCTCTTTTTACTAAAGAAACAGTGAGCCTTGTTCTGGCAGTCCAGAGAATGACACGGGAAAAAAGCAGATGAAGAGAAGGTGGCAGGAGAGGGCACGTGGCCCAGCCTCAGTCTCTCCAACTGA GTTCCTGCCTGCCTGCCTTTGCTCAGACTGTTTGCCCCTTACTGCTCTTCTAGGCCTCATTCTAAGCCCCTTTCTCCAAGTTGCCTCTCCTTATTTCTCCCTGTCTGCCAAAAAATCTTTCCCAGCTCACTAAGTCAGTCTCACGCAGTCA TRAC 600 bp 3' homology arm 241 NIQKPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPADTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS Human Alpha TCR constant 242 HIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS Human Alpha TCR constant 243 YIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS Human Alpha TCR constant 244 DIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS Human Alpha TCR constant 245 PNIQKPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPADTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS Human Alpha TCR constant 246 NIQKPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPADTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS Human Alpha TCR constant 247 HIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS Human Alpha TCR constant 248 YIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS Human Alpha TCR constant 249 NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS Human Alpha TCR constant 250 DIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS Human Alpha TCR constant 251 EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF Human TCR Beta constant 252 TEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG Human TCR Beta constant 253 LEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG Human TCR Beta constant 254 EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG Human TCR Beta constant 255 TEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMMVKRKDSRG Human TCR Beta constant 256 LEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG Human TCR Beta constant

--->

LIST OF SEQUENCES

<110> JUNO THERAPEUTICS, INC.

EDITAS MEDICINE, INC.

SATHER, Blythe D.

BORGES, Christopher

BURLEIGH, Stephen Michael

NYE, Christopher Heath

VONG, Queenie

WELSTEAD, G. Grant

<120> METHODS FOR OBTAINING CELLS EXPRESSING RECOMBINANT

RECEPTOR AND RELATED COMPOSITIONS

<130> 735042012740

<140> Not yet transmitted

<141> At the same time,

<150> 62/653,522

<151> 2018-04-05

<160> 256

<170> FastSEQ for Windows Version 4.0

<210> 1

<211> 4627

<212> DNA

<213> Homo sapiens

<220>

<223> Human TCR alpha constant (TRAC)

<300>

<308> NG_001332.3

<309> 2019-02-26

<400> 1

atatccagaa ccctgaccct gccgtgtacc agctgagaga ctctaaatcc agtgacaagt 60

ctgtctgcct attcaccgat tttgattctc aaacaaatgt gtcacaaagt aaggattctg 120

atgtgtatat cacagacaaa actgtgctag acatgaggtc tatggacttc aagagcaaca 180

gtgctgtggc ctggagcaac aaatctgact ttgcatgtgc aaacgccttc aacaacagca 240

ttattccaga agacaccttc ttccccagcc caggtaaggg cagctttggt gccttcgcag 300

gctgtttcct tgcttcagga atggccaggt tctgcccaga gctctggtca atgatgtcta 360

aaactcctct gattggtggt ctcggcctta tccattgcca ccaaaaccct ctttttacta 420

agaaacagtg agccttgttc tggcagtcca gagaatgaca cgggaaaaaa gcagatgaag 480

agaaggtggc aggagagggc acgtggccca gcctcagtct ctccaactga gttcctgcct 540

gcctgccttt gctcagactg tttgcccctt actgctcttc taggcctcat tctaagcccc 600

ttctccaagt tgcctctcct tatttctccc tgtctgccaa aaaatctttc ccagctcact 660

aagtcagtct cacgcagtca ctcattaacc caccaatcac tgattgtgcc ggcacatgaa 720

tgcaccaggt gttgaagtgg aggaattaaa aagtcagatg aggggtgtgc cccaggaag 780

caccattcta gttgggggag cccatctgtc agctgggaaa agtccaaata acttcagatt 840

ggaatgtgtt ttaactcagg gttgagaaaa cagctacctt caggacaaaa gtcagggaag 900

ggctctctga agaaatgcta cttgaagata ccagccctac caagggcagg gagaggaccc 960

tatagaggcc tgggacagga gctcaatgag aaaggagaag agcagcaggc atgagttgaa 1020

tgaaggaggc agggccgggt cacaggggcct tctaggccat gagagggtag acagtattct 1080

aaggacgcca gaaagctgtt gatcggcttc aagcagggga gggacaccta atttgctttt 1140

cttttttttt tttttttttt tttttttttt tgagatggag ttttgctctt gttgcccagg 1200

ctggagtgca atggtgcatc ttggctcact gcaacctccg cctcccaggt tcaagtgatt 1260

ctcctgcctc agcctcccga gtagctgaga ttacaggcac ccgccaccat gcctggctaa 1320

ttttttgtat ttttagtaga gacagggttt cactatgttg gccaggctgg tctcgaactc 1380

ctgacctcag gtgatccacc cgcttcagcc tcccaaagtg ctgggattac aggcgtgagc 1440

caccacaccc ggcctgcttt tcttaaagat caatctgagt gctgtacgga gagtgggttg 1500

taagccaaga gtagaagcag aaagggagca gttgcagcag agagatgatg gaggcctggg 1560

cagggtggtg gcagggaggt aaccaacacc attcaggttt caaaggtaga accatgcagg 1620

gatgagaaag caaagagggg atcaaggaag gcagctggat tttggcctga gcagctgagt 1680

caatgatagt gccgtttact aagaagaaac caaggaaaaa atttggggtg cagggatcaa 1740

aactttttgg aacatatgaa agtacgtgtt tatactcttt atggcccttg tcactatgta 1800

tgcctcgctg cctccatgg actctagaat gaagccaggc aagagcagg tctatgtgtg 1860

atggcacatg tggccagggt catgcaacat gtactttgta caaacagtgt atattgagta 1920

aatagaaatg gtgtccagga gccgaggtat cggtcctgcc agggccaggg gctctcccta 1980

gcaggtgctc atatgctgta agttccctcc agatctctcc acaaggaggc atggaaaggc 2040

tgtagttgtt cacctgccca agaactagga ggtctggggt ggggagagtca gcctgctctg 2100

gatgctgaaa gaatgtctgt ttttcctttt agaaagttcc tgtgatgtca agctggtcga 2160

gaaaagcttt gaaacaggta agacaggggt ctagcctggg tttgcacagg attgcggaag 2220

tgatgaaccc gcaataaccc tgcctggatg agggagtggg aagaaattag tagatgtggg 2280

aatgaatgat gaggaatgga aacagcggtt caagacctgc cccagctgg gtggggtctc 2340

tcctgaatcc ctctcaccat ctctgacttt ccattctaag cactttgagg atgagtttct 2400

agcttcaata gaccaaggac tctctcctag gcctctgtat tcctttcaac agctccactg 2460

tcaagagagc cagagagagc ttctgggtgg cccagctgtg aaatttctga gtcccttagg 2520

gatagcccta aacgaaccag atcatcctga ggacagccaa gaggttttgc cttctttcaa 2580

gacaagcaac agtactcaca taggctgtgg gcaatggtcc tgtctctcaa gaatcccctg 2640

ccactcctca cacccaccct gggcccatat tcatttccat ttgagttgtt cttattgagt 2700

catccttcct gtggtagcgg aactcactaa ggggcccatc tggacccgag gtattgtgat 2760

gataaattct gagcacctac cccatcccca gaagggctca gaaataaaat aagagccaag 2820

tctagtcggt gtttcctgtc ttgaaacaca atactgttgg ccctggaaga atgcacagaa 2880

tctgtttgta aggggatatg cacagaagct gcaagggaca ggaggtgcag gagctgcagg 2940

cctcccccac ccagcctgct ctgccttggg gaaaaccgtg ggtgtgtcct gcaggccatg 3000

caggcctggg acatgcaagc ccataaccgc tgtggcctct tggttttaca gatacgaacc 3060

taaactttca aaacctgtca gtgattgggt tccgaatcct cctcctgaaa gtggccgggt 3120

ttaatctgct catgacgctg cggctgtggt ccagctgagg tgaggggcct tgaagctggg 3180

agtggggttt agggacgcgg gtctctgggt gcatcctaag ctctgagagc aaacctccct 3240

gcagggtctt gcttttaagt ccaaagcctg agcccaccaa actctcctac ttcttcctgt 3300

tacaaattcc tcttgtgcaa taataatggc ctgaaacgct gtaaaatatc ctcatttcag 3360

ccgcctcagt tgcacttctc ccctatgagg taggaagaac agttgtttag aaacgaagaa 3420

actgaggccc cacagctaat gagtggagga agagagacac ttgtgtacac cacatgcctt 3480

gtgttgtact tctctcaccg tgtaacctcc tcatgtcctc tctccccagt acggctctct 3540

tagctcagta gaaagaagac attacactca tattacaccc caatcctggc tagagtctcc 3600

gcaccctcct cccccagggt ccccagtcgt cttgctgaca actgcatcct gttccatcac 3660

catcaaaaaa aaactccagg ctgggtgcgg gggctcacac ctgtaatccc agcactttgg 3720

gaggcagagg caggaggagc acaggagctg gagaccagcc tgggcaacac agggagaccc 3780

cgcctctaca aaaagtgaaa aaattaacca ggtgtggtgc tgcacacctg tagtcccagc 3840

tacttaagag gctgagatgg gaggatcgct tgagccctgg aatgttgagg ctacaatgag 3900

ctgtgattgc gtcactgcac tccagcctgg aagacaaagc aagatcctgt ctcaaataat 3960

aaaaaaaata agaactccag ggtacatttg ctcctagaac tctaccacat agccccaaac 4020

agagccatca ccatcacatc cctaacagtc ctgggtcttc ctcagtgtcc agcctgactt 4080

ctgttcttcc tcattccaga tctgcaagat tgtaagacag cctgtgctcc ctcgctcctt 4140

cctctgcatt gcccctcttc tccctctcca aacagaggga actctcctac ccccaaggag 4200

gtgaaagctg ctaccacctc tgtgcccccc cggcaatgcc accaactgga tcctacccga 4260

atttatgatt aagattgctg aagagctgcc aaacactgct gccaccccct ctgttccctt 4320

attgctgctt gtcactgcct gacattcacg gcagaggcaa ggctgctgca gcctcccctg 4380

gctgtgcaca ttccctcctg ctccccagag actgcctccg ccatcccaca gatgatggat 4440

cttcagtggg ttctcttggg ctctaggtcc tgcagaatgt tgtgaggggt ttattttttt 4500

ttaatagtgt tcataaagaa atacatagta ttcttcttct caagacgtgg ggggaaatta 4560

tctcattatc gaggccctgc tatgctgtgt atctgggcgt gttgtatgtc ctgctgccga 4620

tgccttc 4627

<210> 2

<211> 1448

<212> DNA

<213> Homo sapiens

<220>

<223> Human TCR beta constant 1 (TRBC1)

<300>

<308> NG_001333.2

<309> 2018-09-23

<400> 2

aggacctgaa caaggtgttc ccacccgagg tcgctgtgtt tgagccatca gaagcagaga 60

tctcccacac ccaaaaggcc acactggtgt gcctggccac aggcttcttc cccgaccacg 120

tggagctgag ctggtgggtg aatgggaagg aggtgcacag tggggtcagc acagacccgc 180

agcccctcaa ggagcagccc gccctcaatg actccagata ctgcctgagc agccgcctga 240

gggtctcggc caccttctgg cagaaccccc gcaaccactt ccgctgtcaa gtccagttct 300

acgggctctc ggagaatgac gagtggaccc aggatagggc caaacccgtc acccagatcg 360

tcagcgccga ggcctggggt agagcaggtg agtggggcct ggggagatgc ctggaggaga 420

ttaggtgaga ccagctacca gggaaaatgg aaagatccag gtagcagaca agactagatc 480

caaaaagaaa ggaaccagcg cacaccatga aggagaattg ggcacctgtg gttcattctt 540

ctcccagatt ctcagcccaa cagagccaag cagctgggtc ccctttctat gtggcctgtg 600

taactctcat ctgggtggtg ccccccatcc ccctcagtgc tgccacatgc catggattgc 660

aaggacaatg tggctgacat ctgcatggca gaagaaagga ggtgctgggc tgtcagagga 720

agctggtctg ggcctgggag tctgtgccaa ctgcaaatct gactttactt ttaattgcct 780

atgaaaataa ggtctctcat ttattttcct ctccctgctt tctttcagac tgtggcttta 840

cctcgggtaa gtaagccctt ccttttcctc tccctctctc atggttcttg acctagaacc 900

aaggcatgaa gaactcacag acactggagg gtggagggtg ggagagacca gagctacctg 960

tgcacaggta cccacctgtc cttcctccgt gccaacagtg tcctaccagc aaggggtcct 1020

gtctgccacc atcctctatg agatcctgct agggaaggcc accctgtatg ctgtgctggt 1080

cagcgccctt gtgttgatgg ccatggtaag caggagggca ggatggggcc agcaggctgg 1140

aggtgacaca ctgacaccaa gcacccagaa gtatagagtc cctgccagga ttggagctgg 1200

gcagtaggga gggaagagat ttcattcagg tgcctcagaa gataacttgc acctctgtag 1260

gatcacagtg gaagggtcat gctgggaagg agaagctgga gtcaccagaa aacccaatgg 1320

atgttgtgat gagccttact atttgtgtgg tcaatgggcc ctactacttt ctctcaatcc 1380

tcacaactcc tggctcttaa taacccccaa aactttctct tctgcaggtc aagagaaagg 1440

atttctga 1448

<210> 3

<211> 1489

<212> DNA

<213> Homo sapiens

<220>

<223> Human TCR beta constant 2 (TRBC2)

<300>

<308> NG_001333.2

<309> 2018-09-23

<400> 3

aggacctgaa aaacgtgttc ccacccgagg tcgctgtgtt tgagccatca gaagcagaga 60

tctcccacac ccaaaaggcc acactggtat gcctggccac aggcttctac cccgaccacg 120

tggagctgag ctggtgggtg aatgggaagg aggtgcacag tggggtcagc acagacccgc 180

agcccctcaa ggagcagccc gccctcaatg actccagata ctgcctgagc agccgcctga 240

gggtctcggc caccttctgg cagaaccccc gcaaccactt ccgctgtcaa gtccagttct 300

acgggctctc ggagaatgac gagtggaccc aggatagggc caaacccgtc acccagatcg 360

tcagcgccga ggcctggggt agagcaggtg agtggggcct ggggagatgc ctggaggaga 420

ttaggtgaga ccagctacca gggaaaatgg aaagatccag gtagcggaca agactagatc 480

cagaagaaag cccagtgga caaggtggga tgatcaaggt tcacagggtc agcaaagcac 540

ggtgtgcact tcccccacca agaagcatag aggctgaatg gagcacctca agctcattct 600

tccttcagat cctgacacct tagagctaag ctttcaagtc tccctgagga ccagccatac 660

agctcagcat ctgagtggtg tgcatcccat tctcttctgg ggtcctggtt tcctaagatc 720

atagtgacca cttcgctggc actggagcag catgagggag acagaaccag ggctatcaaa 780

ggaggctgac tttgtactat ctgatatgca tgtgtttgtg gcctgtgagt ctgtgatgta 840

aggctcaatg tccttacaaa gcagcattct ctcatccatt tttcttcccc tgttttcttt 900

cagactgtgg cttcacctcc ggtaagtgag tctctccttt ttctctctat ctttcgccgt 960

ctctgctctc gaaccagggc atggagaatc cacggacaca ggggcgtgag ggaggccaga 1020

gccacctgtg cacaggtgcc tacatgctct gttcttgtca acagagtctt accagcaagg 1080

ggtcctgtct gccaccatcc tctatgagat cttgctaggg aaggccacct tgtatgccgt 1140

gctggtcagt gccctcgtgc tgatggccat ggtaaggagg agggtgggat agggcagatg 1200

atggggggcag gggatggaac atcacacatg ggcataaagg aatctcagag ccagagcaca 1260

gcctaatata tcctatcacc tcaatgaaac cataatgaag ccagactggg gagaaaatgc 1320

agggaatatc acagaatgca tcatgggagg atggagacaa ccagcgagcc ctactcaaat 1380

taggcctcag agcccgcctc ccctgcccta ctcctgctgt gccatagccc ctgaaaccct 1440

gaaaatgttc tctcttccac aggtcaagag aaaggattcc agaggctag 1489

<210> 4

<211> 1189

<212> DNA

<213> Artificial sequence

<220>

<223> EF1 alpha promoter

<300>

<308> GenBank: J04617.1

<309> 1994-11-07

<400> 4

cgtgaggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt 60

tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg 120

aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tggggggagaa ccgtatataa 180

gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtaa 240

gtgccgtgtg tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt 300

gaattacttc cacgcccctg gctgcagtac gtgattcttg atcccgagct tcgggttgga 360

agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt 420

gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt 480

ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt 540

tttttctggc aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt 600

tttggggccg cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg 660

ggcctgcgag cgcggccacc gagaatcgga cggggggtagt ctcaagctgg ccggcctgct 720

ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg 780

tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca 840

aaatggagga cgcggcgctc ggggagagcgg gcgggtgagt cacccacaca aaggaaaagg 900

gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg 960

cacctcgatt agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt 1020

tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac 1080

ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag 1140

cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtgaa 1189

<210> 5

<211> 1205

<212> DNA

<213> Artificial sequence

<220>

<223> EF1 alpha promoter

<400> 5

cgtgaggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt 60

tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg 120

aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tggggggagaa ccgtatataa 180

gtgcactagt cgccgtgaac gttctttttc gcaacggggtt tgccgccaga acacaggtaa 240

gtgccgtgtg tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt 300

gaattacttc cacctggctg cagtacgtga ttcttgatcc cgagcttcgg gttggaagtg 360

ggtggggagag ttcgtggcct tgcgcttaag gagccccttc gcctcgtgct tgagttgtgg 420

cctggcctgg gcgctggggc cgccgcgtgc gaatctggtg gcaccttcgc gcctgtctcg 480

ctgctttcga taagtctcta gccatttaaa atttttgatg acctgctgcg acgctttttt 540

tctggcaaga tagtcttgta aatgcgggcc aagatcagca cactggtatt tcggtttttg 600

gggccgcggg cggcgacggg gcccgtgcgt cccagcgcac atgttcggcg aggcggggcc 660

tgcgagcgcg gccaccgaga atcggacggg ggtagtctca agctgcccgg cctgctctgg 720

tgcctggcct cgcgccgccg tgtatcgccc cgccctgggc ggcaaggctg gcccggtcgg 780

caccagttgc gtgagcggaa agatggccgc ttcccggccc tgctgcaggg agcacaaaat 840

ggagaggacgcg gcgctcggga gagcgggcgg gtgagtcacc cacacaaagg aaaagggcct 900

ttccgtcctc agccgtcgct tcatgtgact ccacggagta ccgggcgccg tccaggcacc 960

tcgattagtt ctccagcttt tggagtacgt cgtctttagg ttggggggag gggttttatg 1020

cgatggagtt tccccacact gagtgggtgg agactgaagt taggccagct tggcacttga 1080

tgtaattctc cttggaattt gccctttttg agtttggatc ttggttcatt ctcaagcctc 1140

agacagtggt tcaaagtttt tttcttccat ttcaggtgtc gtgaaaacta cccctaaaag 1200

ccaaa 1205

<210> 6

<211> 24

<212> PRT

<213> Artificial sequence

<220>

<223> T2A

<400> 6

Leu Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp

1 5 10 15

Val Glu Glu Asn Pro Gly Pro Arg

20

<210> 7

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> T2A

<400> 7

Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro

1 5 10 15

Gly Pro

<210> 8

<211> 22

<212> PRT

<213> Artificial sequence

<220>

<223> P2A

<400> 8

Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val

1 5 10 15

Glu Glu Asn Pro Gly Pro

20

<210> 9

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> P2A

<400> 9

Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn

1 5 10 15

Pro Gly Pro

<210> 10

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> E2A

<400> 10

Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser

1 5 10 15

Asn Pro Gly Pro

20

<210> 11

<211> 22

<212> PRT

<213> Artificial sequence

<220>

<223> F2A

<400> 11

Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val

1 5 10 15

Glu Ser Asn Pro Gly Pro

20

<210> 12

<211> 357

<212> PRT

<213> Artificial sequence

<220>

<223>EGFRt

<400> 12

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly

20 25 30

Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe

35 40 45

Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala

50 55 60

Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu

65 70 75 80

Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile

85 90 95

Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu

100 105 110

Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala

115 120 125

Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu

130 135 140

Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr

145 150 155 160

Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys

165 170 175

Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly

180 185 190

Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu

195 200 205

Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys

210 215 220

Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu

225 230 235 240

Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met

245 250 255

Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala

260 265 270

His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val

275 280 285

Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His

290 295 300

Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro

305 310 315 320

Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala

325 330 335

Thr Gly Met Val Gly Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly

340 345 350

Ile Gly Leu Phe Met

355

<210> 13

<211> 335

<212> PRT

<213> Artificial sequence

<220>

<223>EGFRt

<400> 13

Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu

1 5 10 15

Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile

20 25 30

Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe

35 40 45

Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr

50 55 60

Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn

65 70 75 80

Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg

85 90 95

Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile

100 105 110

Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val

115 120 125

Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp

130 135 140

Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn

145 150 155 160

Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu

165 170 175

Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser

180 185 190

Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu

195 200 205

Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln

210 215 220

Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly

225 230 235 240

Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro

245 250 255

His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr

260 265 270

Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His

275 280 285

Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro

290 295 300

Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala

305 310 315 320

Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met

325 330 335

<210> 14

<211> 137

<212> PRT

<213> Mus musculus

<220>

<223> Mouse alpha constant TCR

<400> 14

Asp Ile Gln Asn Pro Glu Pro Ala Val Tyr Gln Leu Lys Asp Pro Arg

1 5 10 15

Ser Gln Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe Asp Ser Gln Ile

20 25 30

Asn Val Pro Lys Thr Met Glu Ser Gly Thr Phe Ile Thr Asp Lys Thr

35 40 45

Val Leu Asp Met Lys Ala Met Asp Ser Lys Ser Asn Gly Ala Ile Ala

50 55 60

Trp Ser Asn Gln Thr Ser Phe Thr Cys Gln Asp Ile Phe Lys Glu Thr

65 70 75 80

Asn Ala Thr Tyr Pro Ser Ser Asp Val Pro Cys Asp Ala Thr Leu Thr

85 90 95

Glu Lys Ser Phe Glu Thr Asp Met Asn Leu Asn Phe Gln Asn Leu Ser

100 105 110

Val Met Gly Leu Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu

115 120 125

Leu Met Thr Leu Arg Leu Trp Ser Ser

130 135

<210> 15

<211> 137

<212> PRT

<213> Mus musculus

<220>

<223> Mouse alpha constant TCR

<400> 15

Asn Ile Gln Asn Pro Glu Pro Ala Val Tyr Gln Leu Lys Asp Pro Arg

1 5 10 15

Ser Gln Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe Asp Ser Gln Ile

20 25 30

Asn Val Pro Lys Thr Met Glu Ser Gly Thr Phe Ile Thr Asp Lys Thr

35 40 45

Val Leu Asp Met Lys Ala Met Asp Ser Lys Ser Asn Gly Ala Ile Ala

50 55 60

Trp Ser Asn Gln Thr Ser Phe Thr Cys Gln Asp Ile Phe Lys Glu Thr

65 70 75 80

Asn Ala Thr Tyr Pro Ser Ser Asp Val Pro Cys Asp Ala Thr Leu Thr

85 90 95

Glu Lys Ser Phe Glu Thr Asp Met Asn Leu Asn Phe Gln Asn Leu Ser

100 105 110

Val Met Gly Leu Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu

115 120 125

Leu Met Thr Leu Arg Leu Trp Ser Ser

130 135

<210> 16

<211> 173

<212> PRT

<213> Mus musculus

<220>

<223> Mouse TCR beta constant

<300>

<308> Uniprot P01852

<309> 1986-07-21

<400> 16

Glu Asp Leu Arg Asn Val Thr Pro Pro Lys Val Ser Leu Phe Glu Pro

1 5 10 15

Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala Thr Leu Val Cys Leu

20 25 30

Ala Arg Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val Asn

35 40 45

Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Ala Tyr Lys

50 55 60

Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala

65 70 75 80

Thr Phe Trp His Asn Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe

85 90 95

His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly Ser Pro Lys Pro

100 105 110

Val Thr Gln Asn Ile Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly

115 120 125

Ile Thr Ser Ala Ser Tyr Gln Gln Gly Val Leu Ser Ala Thr Ile Leu

130 135 140

Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser

145 150 155 160

Thr Leu Val Val Met Ala Met Val Lys Arg Lys Asn Ser

165 170

<210> 17

<211> 172

<212> PRT

<213> Mus musculus

<220>

<223> Mouse TCR beta constant

<400> 17

Asp Leu Arg Asn Val Thr Pro Pro Lys Val Ser Leu Phe Glu Pro Ser

1 5 10 15

Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala Thr Leu Val Cys Leu Ala

20 25 30

Arg Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val Asn Gly

35 40 45

Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Ala Tyr Lys Glu

50 55 60

Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr

65 70 75 80

Phe Trp His Asn Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe His

85 90 95

Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly Ser Pro Lys Pro Val

100 105 110

Thr Gln Asn Ile Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Ile

115 120 125

Thr Ser Ala Ser Tyr His Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr

130 135 140

Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Gly

145 150 155 160

Leu Val Leu Met Ala Met Val Lys Arg Lys Asn Ser

165 170

<210> 18

<211> 181

<212> DNA

<213> Artificial sequence

<220>

<223> MND promoter

<400> 18

gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca 60

ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg 120

tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc 180

a 181

<210> 19

<211> 142

<212> PRT

<213> Homo sapiens

<220>

<223> Human alpha constant TCR

<300>

<308> Uniprot P01848

<309> 2018-07-18

<400> 19

Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser

1 5 10 15

Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln

20 25 30

Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys

35 40 45

Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val

50 55 60

Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn

65 70 75 80

Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys

85 90 95

Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn

100 105 110

Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val

115 120 125

Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

130 135 140

<210> 20

<211> 177

<212> PRT

<213> Homo sapiens

<220>

<223> Human TCR beta constant 1

<300>

<308> Uniprot P01850

<309> 2018-07-18

<400> 20

Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro

1 5 10 15

Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu

20 25 30

Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val Asn

35 40 45

Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys

50 55 60

Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu

65 70 75 80

Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys

85 90 95

Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp

100 105 110

Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg

115 120 125

Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln Gly Val Leu Ser

130 135 140

Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala

145 150 155 160

Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp

165 170 175

Phe

<210> 21

<211> 178

<212> PRT

<213> Homo sapiens

<220>

<223> Human TCR beta constant 2

<300>

<308> Uniprot A0A5B9

<309> 2018-07-18

<400> 21

Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro Ser

1 5 10 15

Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu Ala

20 25 30

Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn Gly

35 40 45

Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys Glu

50 55 60

Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu Arg

65 70 75 80

Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys Gln

85 90 95

Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp Arg

100 105 110

Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg Ala

115 120 125

Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val Leu Ser Ala

130 135 140

Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val

145 150 155 160

Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp Ser

165 170 175

Arg Gly

<210> 22

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Linker

<220>

<221> REPEAT

<222> (0)…(0)

<223> SGGGG is repeated 5 or 6 times

<400> 22

Pro Gly Gly Gly Ser Gly Gly Gly Gly Pro

1 5 10

<210> 23

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> Linker

<400> 23

Gly Ser Ala Asp Asp Ala Lys Lys Asp Ala Ala Lys Lys Asp Gly Lys

1 5 10 15

Ser

<210> 24

<211> 141

<212> PRT

<213> Homo sapiens

<220>

<223> Human alpha constant TCR

<300>

<308> CAA26636.1

<309> 2016-07-25

<400> 24

Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys

1 5 10 15

Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr

20 25 30

Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr

35 40 45

Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala

50 55 60

Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser

65 70 75 80

Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp

85 90 95

Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe

100 105 110

Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala

115 120 125

Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

130 135 140

<210> 25

<211> 179

<212> PRT

<213> Homo sapiens

<220>

<223> Human TCR beta constant

<300>

<308> A0A0G2JNG9

<400> 25

Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro

1 5 10 15

Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu

20 25 30

Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn

35 40 45

Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys

50 55 60

Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu

65 70 75 80

Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys

85 90 95

Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp

100 105 110

Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg

115 120 125

Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val Leu Ser

130 135 140

Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala

145 150 155 160

Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp

165 170 175

Ser Arg Gly

<210> 26

<211> 149

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC gRNA transcriptional sequence

<400> 26

agcgctctcg tacagagttg gcattataat acgactcact ataggggaga atcaaaatcg 60

gtgaatgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga 120

aaaagtggca ccgagtcggt gcttttttt 149

<210> 27

<211> 100

<212> RNA

<213> Artificial sequence

<220>

<223> TRAC gRNA sequence

<400> 27

gagaaucaaa aucggugaau guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210> 28

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRAC-10 gRNA targeting domain

<400> 28

ucucucagcu gguacacggc 20

<210> 29

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRAC-110 gRNA targeting domain

<400> 29

uggauuuaga gucucucagc 20

<210> 30

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRAC-116 gRNA targeting domain

<400> 30

acacggcagg gucaggguuc 20

<210> 31

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRAC-16 gRNA targeting domain

<400> 31

gagaaucaaa aucggugaau 20

<210> 32

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRAC-4 gRNA targeting domain

<400> 32

gcugguacac ggcaggguca 20

<210> 33

<211> 17

<212> RNA

<213> Artificial sequence

<220>

<223> TRAC-49 gRNA targeting domain

<400> 33

cucagcuggu acacggc 17

<210> 34

<211> 17

<212> RNA

<213> Artificial sequence

<220>

<223> TRAC-2 gRNA targeting domain

<400> 34

ugguacacgg caggguc 17

<210> 35

<211> 17

<212> RNA

<213> Artificial sequence

<220>

<223> TRAC-30 gRNA targeting domain

<400> 35

gcuagacaug aggucua 17

<210> 36

<211> 17

<212> RNA

<213> Artificial sequence

<220>

<223> TRAC-43 gRNA targeting domain

<400> 36

gucagauuug uugcucc 17

<210> 37

<211> 17

<212> RNA

<213> Artificial sequence

<220>

<223> TRAC-23 gRNA targeting domain

<400> 37

ucagcuggua cacggca 17

<210> 38

<211> 17

<212> RNA

<213> Artificial sequence

<220>

<223> TRAC-34 gRNA targeting domain

<400> 38

gcagacagac uugucac 17

<210> 39

<211> 17

<212> RNA

<213> Artificial sequence

<220>

<223> TRAC-25 gRNA targeting domain

<400> 39

gguacacggc aggguca 17

<210> 40

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRAC-128 gRNA targeting domain

<400> 40

cuucaagagc aacagugcug 20

<210> 41

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRAC-105 gRNA targeting domain

<400> 41

agagcaacag ugcuguggcc 20

<210> 42

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRAC-106 gRNA targeting domain

<400> 42

aaagucagau uuguugcucc 20

<210> 43

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRAC-123 gRNA targeting domain

<400> 43

acaaaacugu gcuagacaug 20

<210> 44

<211> 17

<212> RNA

<213> Artificial sequence

<220>

<223> TRAC-64 gRNA targeting domain

<400> 44

aaacugugcu agacaug 17

<210> 45

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRAC-97 gRNA targeting domain

<400> 45

ugugcuagac augaggucua 20

<210> 46

<211> 22

<212> RNA

<213> Artificial sequence

<220>

<223> TRAC-148 gRNA targeting domain

<400> 46

ggcuggggaa gaaggugucu uc 22

<210> 47

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> TRAC-147 gRNA targeting domain

<400> 47

gcuggggaag aaggugucuu from 21

<210> 48

<211> 18

<212> RNA

<213> Artificial sequence

<220>

<223> TRAC-234 gRNA targeting domain

<400> 48

ggggaagaag gugucuuc 18

<210> 49

<211> 22

<212> RNA

<213> Artificial sequence

<220>

<223> TRAC-167 gRNA targeting domain

<400> 49

guuuugucug ugauauacac au 22

<210> 50

<211> 24

<212> RNA

<213> Artificial sequence

<220>

<223> TRAC-177 gRNA targeting domain

<400> 50

ggcagacaga cuugucacug gauu 24

<210> 51

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> TRAC-176 gRNA targeting domain

<400> 51

gcagacagac uugucacugg auu 23

<210> 52

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRAC-257 gRNA targeting domain

<400> 52

gacagacuug ucacuggauu 20

<210> 53

<211> 24

<212> RNA

<213> Artificial sequence

<220>

<223> TRAC-233 gRNA targeting domain

<400> 53

gugaauaggc agacagacuu guca 24

<210> 54

<211> 22

<212> RNA

<213> Artificial sequence

<220>

<223> TRAC-231 gRNA targeting domain

<400> 54

gaauaggcag acagacuugu ca 22

<210> 55

<211> 22

<212> RNA

<213> Artificial sequence

<220>

<223> TRAC-163 gRNA targeting domain

<400> 55

gagucucuca gcugguacac gg 22

<210> 56

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRAC-241 gRNA targeting domain

<400> 56

gucucucagc ugguacacgg 20

<210> 57

<211> 22

<212> RNA

<213> Artificial sequence

<220>

<223> TRAC-179 gRNA targeting domain

<400> 57

gguacacggc agggucaggg uu 22

<210> 58

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> TRAC-178 gRNA targeting domain

<400> 58

guacacggca gggucagggu u 21

<210> 59

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-40 gRNA targeting domain

<400> 59

cacccagauc gucagcgccg 20

<210> 60

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-52 gRNA targeting domain

<400> 60

caaacacagc gaccucgggu 20

<210> 61

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-25 gRNA targeting domain

<400> 61

ugacgagugg acccaggaua 20

<210> 62

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-35 gRNA targeting domain

<400> 62

ggcucucgga gaaugacgag 20

<210> 63

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-50 gRNA targeting domain

<400> 63

ggccucggcg cugacgaucu 20

<210> 64

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-39 gRNA targeting domain

<400> 64

gaaaaacgug uucccacccg 20

<210> 65

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-49 gRNA targeting domain

<400> 65

augacgagug gacccaggau 20

<210> 66

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-51 gRNA targeting domain

<400> 66

aguccaguuc uacggggcucu 20

<210> 67

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-26 gRNA targeting domain

<400> 67

cgcugucaag uccaguucua 20

<210> 68

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-47 gRNA targeting domain

<400> 68

aucgucagcg ccgaggccug 20

<210> 69

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-45 gRNA targeting domain

<400> 69

ucaaacacag cgaccucgggg 20

<210> 70

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-34 gRNA targeting domain

<400> 70

cguagaacug gacuugacag 20

<210> 71

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-227 gRNA targeting domain

<400> 71

aggccucggc gcugacgauc 20

<210> 72

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-41 gRNA targeting domain

<400> 72

ugacagcgga agugguugcg 20

<210> 73

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-30 gRNA targeting domain

<400> 73

uugacagcgg aagugguugc 20

<210> 74

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-206 gRNA targeting domain

<400> 74

ucucccgagag cccguagaac 20

<210> 75

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-32 gRNA targeting domain

<400> 75

cggggugggaa cacguuuuuc 20

<210> 76

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-276 gRNA targeting domain

<400> 76

gacagguuug gcccuauccu 20

<210> 77

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-274 gRNA targeting domain

<400> 77

gaucgucagc gccgaggccu 20

<210> 78

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-230 gRNA targeting domain

<400> 78

ggcucaaaca cagcgaccuc 20

<210> 79

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-235 gRNA targeting domain

<400> 79

ugagggucuc ggccaccuuc 20

<210> 80

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-38 gRNA targeting domain

<400> 80

aggcuucuac cccgaccacg 20

<210> 81

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-223 gRNA targeting domain

<400> 81

ccgaccacgu ggagcugagc 20

<210> 82

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-221 gRNA targeting domain

<400> 82

ugacagguuu ggcccuaucc 20

<210> 83

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-48 gRNA targeting domain

<400> 83

cuugacagcg gaagugguug 20

<210> 84

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-216 gRNA targeting domain

<400> 84

agaucgucag cgccgaggcc 20

<210> 85

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-210 gRNA targeting domain

<400> 85

gcgcugacga ucugggugac 20

<210> 86

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-268 gRNA targeting domain

<400> 86

ugaggcggg cugcuccuug 20

<210> 87

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-193 gRNA targeting domain

<400> 87

guugcggggg uucugccaga 20

<210> 88

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-246 gRNA targeting domain

<400> 88

agcucagcuc cacguggucg 20

<210> 89

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-228 gRNA targeting domain

<400> 89

gcggcugcuc aggcaguauc 20

<210> 90

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-43 gRNA targeting domain

<400> 90

gcggggguuc ugccagaagg 20

<210> 91

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-272 gRNA targeting domain

<400> 91

uggcucaaac acagcgaccu 20

<210> 92

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-33 gRNA targeting domain

<400> 92

acuggacuug acagcggaag 20

<210> 93

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-44 gRNA targeting domain

<400> 93

gacagcggaa gugguugcgg 20

<210> 94

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-211 gRNA targeting domain

<400> 94

gcugucaagu ccaguucuac 20

<210> 95

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-253 gRNA targeting domain

<400> 95

guaucuggag ucauugaggg 20

<210> 96

<211> 17

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-18 gRNA targeting domain

<400> 96

cucggcgcug acgaucu 17

<210> 97

<211> 17

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-6 gRNA targeting domain

<400> 97

ccucggcgcu gacgauc 17

<210> 98

<211> 17

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-85 gRNA targeting domain

<400> 98

ccgagagccc guagaac 17

<210> 99

<211> 17

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-129 gRNA targeting domain

<400> 99

ccagaucguc agcgccg 17

<210> 100

<211> 17

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-93 gRNA targeting domain

<400> 100

gaaugacgag uggaccc 17

<210> 101

<211> 22

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-415 gRNA targeting domain

<400> 101

gggugacagg uuuggcccua uc 22

<210> 102

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-414 gRNA targeting domain

<400> 102

ggugacaggu uuggcccuau from 21

<210> 103

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-310 gRNA targeting domain

<400> 103

gugacagguu uggcccuauc 20

<210> 104

<211> 18

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-308 gRNA targeting domain

<400> 104

gacagguuug gcccuauc 18

<210> 105

<211> 22

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-401 gRNA targeting domain

<400> 105

gauacugccu gagcagccgc cu 22

<210> 106

<211> 24

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-468 gRNA targeting domain

<400> 106

gaccacgugg agcugagcug gugg 24

<210> 107

<211> 18

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-462 gRNA targeting domain

<400> 107

guggagcuga gcuggugg 18

<210> 108

<211> 24

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-424 gRNA targeting domain

<400> 108

gggcgggcug cuccuugagg ggcu 24

<210> 109

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-423 gRNA targeting domain

<400> 109

ggcgggcugc uccuugaggg gcu 23

<210> 110

<211> 22

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-422 gRNA targeting domain

<400> 110

gcgggcugcu ccuugagggg cu 22

<210> 111

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-420 gRNA targeting domain

<400> 111

gggcugcucc uugaggggcu 20

<210> 112

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-419 gRNA targeting domain

<400> 112

ggcugcuccu ugaggggcu 19

<210> 113

<211> 18

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-418 gRNA targeting domain

<400> 113

gcugcuccuu gaggggcu 18

<210> 114

<211> 24

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-445 gRNA targeting domain

<400> 114

ggugaauggg aaggaggugc acag 24

<210> 115

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-444 gRNA targeting domain

<400> 115

gugaauggga aggaggugca cag 23

<210> 116

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC-442 gRNA targeting domain

<400> 116

gaaugggaag gaggugcaca g 21

<210> 117

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence 1

<400> 117

attcaccgat tttgattctc 20

<210> 118

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> TRBC target sequence 2

<400> 118

agatcgtcag cgccgaggcc 20

<210> 119

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> HBB splice site acceptor

<400> 119

ctgacctctt ctcttcctcc cacag 25

<210> 120

<211> 13

<212> DNA

<213> Artificial sequence

<220>

<223> IgG splice site acceptor

<400> 120

tttctctcca cag 13

<210> 121

<211> 138

<212> PRT

<213>muscle muscle

<220>

<223> Mouse alpha constant TCR

<300>

<308> Uniprot P01849

<309> 1986-07-21

<400> 121

Pro Tyr Ile Gln Asn Pro Glu Pro Ala Val Tyr Gln Leu Lys Asp Pro

1 5 10 15

Arg Ser Gln Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe Asp Ser Gln

20 25 30

Ile Asn Val Pro Lys Thr Met Glu Ser Gly Thr Phe Ile Thr Asp Lys

35 40 45

Thr Val Leu Asp Met Lys Ala Met Asp Ser Lys Ser Asn Gly Ala Ile

50 55 60

Ala Trp Ser Asn Gln Thr Ser Phe Thr Cys Gln Asp Ile Phe Lys Glu

65 70 75 80

Thr Asn Ala Thr Tyr Pro Ser Ser Asp Val Pro Cys Asp Ala Thr Leu

85 90 95

Thr Glu Lys Ser Phe Glu Thr Asp Met Asn Leu Asn Phe Gln Asn Leu

100 105 110

Ser Val Met Gly Leu Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn

115 120 125

Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

130 135

<210> 122

<211> 136

<212> PRT

<213> Artificial sequence

<220>

<223> Modified mouse alpha constant TCR

<400> 122

Ile Gln Asn Pro Glu Pro Ala Val Tyr Gln Leu Lys Asp Pro Arg Ser

1 5 10 15

Gln Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe Asp Ser Gln Ile Asn

20 25 30

Val Pro Lys Thr Met Glu Ser Gly Thr Phe Ile Thr Asp Lys Cys Val

35 40 45

Leu Asp Met Lys Ala Met Asp Ser Lys Ser Asn Gly Ala Ile Ala Trp

50 55 60

Ser Asn Gln Thr Ser Phe Thr Cys Gln Asp Ile Phe Lys Glu Thr Asn

65 70 75 80

Ala Thr Tyr Pro Ser Ser Asp Val Pro Cys Asp Ala Thr Leu Thr Glu

85 90 95

Lys Ser Phe Glu Thr Asp Met Asn Leu Asn Phe Gln Asn Leu Leu Val

100 105 110

Ile Val Leu Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu

115 120 125

Met Thr Leu Arg Leu Trp Ser Ser

130 135

<210> 123

<211> 173

<212> PRT

<213> Artificial sequence

<220>

<223> Modified mouse beta constant TCR

<400> 123

Glu Asp Leu Arg Asn Val Thr Pro Pro Lys Val Ser Leu Phe Glu Pro

1 5 10 15

Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala Thr Leu Val Cys Leu

20 25 30

Ala Arg Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val Asn

35 40 45

Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Ala Tyr Lys

50 55 60

Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala

65 70 75 80

Thr Phe Trp His Asn Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe

85 90 95

His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly Ser Pro Lys Pro

100 105 110

Val Thr Gln Asn Ile Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly

115 120 125

Ile Thr Ser Ala Ser Tyr Gln Gln Gly Val Leu Ser Ala Thr Ile Leu

130 135 140

Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser

145 150 155 160

Thr Leu Val Val Met Ala Met Val Lys Arg Lys Asn Ser

165 170

<210> 124

<211> 611

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC 5' homology arm

<400> 124

ctctatcaat gagagagcaa tctcctggta atgtgataga tttcccaact taatgccaac 60

ataccataaa cctcccattc tgctaatgcc cagcctaagt tggggagacc actccagatt 120

ccaagatgta cagtttgctt tgctgggcct ttttcccatg cctgccttta ctctgccaga 180

gttatattgc tggggttttg aagaagatcc tattaaataa aagaataagc agtattatta 240

agtagccctg catttcaggt ttccttgagt ggcaggccag gcctggccgt gaacgttcac 300

tgaaatcatg gcctcttggc caagattgat agcttgtgcc tgtccctgag tcccagtcca 360

tcacgagcag ctggtttcta agatgctatt tcccgtataa agcatgagac cgtgacttgc 420

cagccccaca gagccccgcc cttgtccatc actggcatct ggactccagc ctgggttggg 480

gcaaagaggg aaatgagatc atgtcctaac cctgatcctc ttgtcccaca gatatccaga 540

accctgaccc tgccgtgtac cagctgagag actctaaatc cagtgacaag tctgtctgcc 600

tattcaccga t 611

<210> 125

<211> 628

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC 3' homology arm

<400> 125

tttgattctc aaacaaatgt gtcacaaagt aaggattctg atgtgtatat cacagacaaa 60

actgtgctag acatgaggtc tatggacttc aagagcaaca gtgctgtggc ctggagcaac 120

aaatctgact ttgcatgtgc aaacgccttc aacaacagca ttattccaga agacaccttc 180

ttccccagcc caggtaaggg cagctttggt gccttcgcag gctgtttcct tgcttcagga 240

atggccaggt tctgcccaga gctctggtca atgatgtcta aaactcctct gattggtggt 300

ctcggcctta tccattgcca ccaaaaccct ctttttacta agaaacagtg agccttgttc 360

tggcagtcca gagaatgaca cgggaaaaaa gcagatgaag agaaggtggc aggagagggc 420

acgtggccca gcctcagtct ctccaactga gttcctgcct gcctgccttt gctcagactg 480

tttgcccctt actgctcttc taggcctcat tctaagcccc ttctccaagt tgcctctcct 540

tatttctccc tgtctgccaa aaaatctttc ccagctcact aagtcagtct cacgcagtca 600

ctcattaacc caccaatcac tgattgtg 628

<210> 126

<211> 347

<212> DNA

<213> Artificial sequence

<220>

<223> MND promoter

<400> 126

gaacagagaa acaggagaat atgggccaaa caggatatct gtggtaagca gttcctgccc 60

cggctcaggg ccaagaacag ttggaacagc agaatatggg ccaaacagga tatctgtggt 120

aagcagttcc tgccccggct cagggccaag aacagatggt ccccagatgc ggtcccgccc 180

tcagcagttt ctagagaacc atcagatgtt tccagggtgc cccaaggacc tgaaatgacc 240

ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc gcgcttctgc 300

tccccgagct ctatataagc agagctcgtt tagtgaaccg tcagatc 347

<210> 127

<211> 544

<212> DNA

<213> Artificial sequence

<220>

<223> Ef1 alpha promoter with HTLV1 enhancer

<400> 127

ggatctgcga tcgctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg 60

agaagttggg gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa 120

actgggaaag tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt 180

atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac 240

agctgaagct tcgaggggct cgcatctctc cttcacgcgc ccgccgccct acctgaggcc 300

gccatccacg ccggttgagt cgcgttctgc cgcctcccgc ctgtggtgcc tcctgaactg 360

cgtccgccgt ctaggtaagt ttaaagctca ggtcgagacc gggcctttgt ccggcgctcc 420

cttggagcct acctagactc agccggctct ccacgctttg cctgaccctg cttgctcaac 480

tctacgtctt tgtttcgttt tctgttctgc gccgttacag atccaagctg tgaccggcgc 540

ctac 544

<210> 128

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> P2A nucleotide sequence

<400> 128

ggatctggag cgacgaattt tagtctactg aaacaagcgg gagacgtgga ggaaaaccct 60

ggacct 66

<210> 129

<211> 112

<212> PRT

<213> homo sapiens

<220>

<223> CD3 zeta

<400> 129

Arg Val Lys Phe Ser Arg Ser Ala Glu Pro Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 130

<211> 112

<212> PRT

<213> homo sapiens

<220>

<223> CD3 zeta

<400> 130

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 131

<211> 12

<212> PRT

<213> homo sapiens

<220>

<223> spacer (IgG4 hinge)

<400> 131

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro

1 5 10

<210> 132

<211> 36

<212> DNA

<213> homo sapiens

<220>

<223> spacer (IgG4 hinge)

<400> 132

gaatctaagt acggaccgcc ctgcccccct tgccct 36

<210> 133

<211> 119

<212> PRT

<213> homo sapiens

<220>

<223> Hinge-CH3 spacer

<400> 133

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gln Pro Arg

1 5 10 15

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

20 25 30

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

35 40 45

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

50 55 60

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

65 70 75 80

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

85 90 95

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

100 105 110

Leu Ser Leu Ser Leu Gly Lys

115

<210> 134

<211> 229

<212> PRT

<213> homo sapiens

<220>

<223> Hinge-CH2-CH3 spacer

<400> 134

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe

1 5 10 15

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

20 25 30

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

85 90 95

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

100 105 110

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

115 120 125

Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

130 135 140

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

145 150 155 160

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

180 185 190

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly Lys

225

<210> 135

<211> 282

<212> PRT

<213> homo sapiens

<220>

<223> IgD-hinge-Fc

<400> 135

Arg Trp Pro Glu Ser Pro Lys Ala Gln Ala Ser Ser Val Pro Thr Ala

1 5 10 15

Gln Pro Gln Ala Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro Ala

20 25 30

Thr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys

35 40 45

Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro

50 55 60

Ser His Thr Gln Pro Leu Gly Val Tyr Leu Leu Thr Pro Ala Val Gln

65 70 75 80

Asp Leu Trp Leu Arg Asp Lys Ala Thr Phe Thr Cys Phe Val Val Gly

85 90 95

Ser Asp Leu Lys Asp Ala His Leu Thr Trp Glu Val Ala Gly Lys Val

100 105 110

Pro Thr Gly Gly Val Glu Glu Gly Leu Leu Glu Arg His Ser Asn Gly

115 120 125

Ser Gln Ser Gln His Ser Arg Leu Thr Leu Pro Arg Ser Leu Trp Asn

130 135 140

Ala Gly Thr Ser Val Thr Cys Thr Leu Asn His Pro Ser Leu Pro Pro

145 150 155 160

Gln Arg Leu Met Ala Leu Arg Glu Pro Ala Ala Gln Ala Pro Val Lys

165 170 175

Leu Ser Leu Asn Leu Leu Ala Ser Ser Asp Pro Pro Glu Ala Ala Ser

180 185 190

Trp Leu Leu Cys Glu Val Ser Gly Phe Ser Pro Pro Asn Ile Leu Leu

195 200 205

Met Trp Leu Glu Asp Gln Arg Glu Val Asn Thr Ser Gly Phe Ala Pro

210 215 220

Ala Arg Pro Pro Pro Gln Pro Gly Ser Thr Thr Phe Trp Ala Trp Ser

225 230 235 240

Val Leu Arg Val Pro Ala Pro Pro Ser Pro Gln Pro Ala Thr Tyr Thr

245 250 255

Cys Val Val Ser His Glu Asp Ser Arg Thr Leu Leu Asn Ala Ser Arg

260 265 270

Ser Leu Glu Val Ser Tyr Val Thr Asp His

275 280

<210> 136

<211> 27

<212> PRT

<213> homo sapiens

<220>

<223> CD28

<300>

<308> No. P10747

<309> 1989-07-01

<400> 136

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

20 25

<210> 137

<211> 66

<212> PRT

<213> homo sapiens

<220>

<223> CD28

<300>

<308> No. P10747

<309> 1989-07-01

<400> 137

Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn

1 5 10 15

Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu

20 25 30

Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly

35 40 45

Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe

50 55 60

Trp Val

65

<210> 138

<211> 41

<212> PRT

<213> homo sapiens

<220>

<223> CD28

<300>

<308> P10747

<309> 1989-07-01

<400> 138

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 139

<211> 41

<212> PRT

<213> homo sapiens

<220>

<223> CD28 (LL to GG)

<400> 139

Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 140

<211> 42

<212> PRT

<213> homo sapiens

<220>

<223> 4-1BB

<300>

<308> Q07011.1

<309> 1995-02-01

<400> 140

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 141

<211> 112

<212> PRT

<213> homo sapiens

<220>

<223> CD3 zeta

<400> 141

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 142

<211> 96

<212> RNA

<213> Artificial sequence

<220>

<223> typical gRNA complementary domain

<220>

<221> modified_base

<222> (1)...(20)

<223> a, c, u, g, unknown or other

<220>

<221> misc_sign

<222> (1)...(20)

<223> n is a, c, g, or u

<400> 142

nnnnnnnnnn nnnnnnnnnn guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugc 96

<210> 143

<211> 104

<212> RNA

<213> Artificial sequence

<220>

<223> typical gRNA complementary domain

<220>

<221> modified_base

<222> (1)...(20)

<223> a, c, u, g, unknown or other

<220>

<221> misc_sign

<222> (1)...(20)

<223> n is a, c, g, or u

<400> 143

nnnnnnnnnn nnnnnnnnnn guuuuagagc uaugcugaaa agcauagcaa guuaaaauaa 60

ggcuaguccg uuaucaacuu gaaaaagugg caccgagucg gugc 104

<210> 144

<211> 106

<212> RNA

<213> Artificial sequence

<220>

<223> typical gRNA complementary domain

<220>

<221> modified_base

<222> (1)...(20)

<223> a, c, u, g, unknown or other

<220>

<221> misc_sign

<222> (1)...(20)

<223> n is a, c, g, or u

<400> 144

nnnnnnnnnn nnnnnnnnnn guuuuagagc uaugcuggaa acagcauagc aaguuaaaau 60

aaggcuaguc cguuaucaac uugaaaaagu ggcaccgagu cggugc 106

<210> 145

<211> 116

<212> RNA

<213> Artificial sequence

<220>

<223> typical gRNA complementary domain

<220>

<221> modified_base

<222> (1)...(20)

<223> a, c, u, g, unknown or other

<220>

<221> misc_sign

<222> (1)...(20)

<223> n is a, c, g, or u

<400> 145

nnnnnnnnnn nnnnnnnnnn guuuuagagc uaugcuguuu uggaaacaaa acagcauagc 60

aaguuaaaau aaggcuaguc cguuaucaac uugaaaaagu ggcaccgagu cggugc 116

<210> 146

<211> 96

<212> RNA

<213> Artificial sequence

<220>

<223> typical gRNA

<220>

<221> modified_base

<222> (1)...(20)

<223> a, c, u, g, unknown or other

<220>

<221> misc_sign

<222> (1)...(20)

<223> n is a, c, g, or u

<400> 146

nnnnnnnnnn nnnnnnnnnn guauuagagc uagaaauagc aaguuaauau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugc 96

<210> 147

<211> 96

<212> RNA

<213> Artificial sequence

<220>

<223> typical gRNA

<220>

<221> modified_base

<222> (1)...(20)

<223> a, c, u, g, unknown or other

<220>

<221> misc_sign

<222> (1)...(20)

<223> n is a, c, g, or u

<400> 147

nnnnnnnnnn nnnnnnnnnn guuuaagagc uagaaauagc aaguuuaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugc 96

<210> 148

<211> 116

<212> RNA

<213> Artificial sequence

<220>

<223> typical gRNA

<220>

<221> modified_base

<222> (1)...(20)

<223> a, c, u, g, unknown or other

<220>

<221> misc_sign

<222> (1)...(20)

<223> n is a, c, g, or u

<400> 148

nnnnnnnnnn nnnnnnnnnn guauuagagc uaugcuguau uggaaacaau acagcauagc 60

aaguuaauau aaggcuaguc cguuaucaac uugaaaaagu ggcaccgagu cggugc 116

<210> 149

<211> 47

<212> RNA

<213> Artificial sequence

<220>

<223> typical proximal and tail domain

<400> 149

aaggcuaguc cguuaucaac uugaaaaagu ggcaccgagu cggugcu 47

<210> 150

<211> 49

<212> RNA

<213> Artificial sequence

<220>

<223> typical proximal and tail domain

<400> 150

aaggcuaguc cguuaucaac uugaaaaagu ggcaccgagu cgguggugc 49

<210> 151

<211> 51

<212> RNA

<213> Artificial sequence

<220>

<223> typical proximal and tail domain

<400> 151

aaggcuaguc cguuaucaac uugaaaaagu ggcaccgagu cggugcggau c 51

<210> 152

<211> 31

<212> RNA

<213> Artificial sequence

<220>

<223> typical proximal and tail domain

<400> 152

aaggcuaguc cguuaucaac uugaaaaagu g 31

<210> 153

<211> 18

<212> RNA

<213> Artificial sequence

<220>

<223> typical proximal and tail domain

<400> 153

aaggcuaguc cguuauca 18

<210> 154

<211> 12

<212> RNA

<213> Artificial sequence

<220>

<223> typical proximal and tail domain

<400> 154

aaggcuaguc cg 12

<210> 155

<211> 102

<212> RNA

<213> Artificial sequence

<220>

<223> typical chimeric gRNA

<220>

<221> modified base

<222> (1)...(20)

<223> a, c, u, g, unknown or other

<220>

<221> misc_sign

<222> (1)...(20)

<223> n is a, c, g, or u

<400> 155

nnnnnnnnnn nnnnnnnnnn guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu uu 102

<210> 156

<211> 102

<212> RNA

<213> Artificial sequence

<220>

<223> typical chimeric gRNA

<220>

<221> modified base

<222> (1)...(20)

<223> a, c, u, g, unknown or other

<220>

<221> misc_sign

<222> (1)...(20)

<223> n is a, c, g, or u

<400> 156

nnnnnnnnnn nnnnnnnnnn guuuuaguac ucuggaaaca gaaucuacua aaacaaggca 60

aaaugccgug uuuaucucgu caacuuguug gcgagauuuu uu 102

<210> 157

<211> 1344

<212> PRT

<213> Artificial sequence

<220>

<223> Streptococcus mutans Cas9

<400> 157

Lys Lys Pro Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly

1. 5 10 15

Trp Ala Val Val Thr Asp Asp Tyr Lys Val Pro Ala Lys Lys Met Lys

20 25 30

Val Leu Gly Asn Thr Asp Lys Ser His Ile Glu Lys Asn Leu Leu Gly

35 40 45

Ala Leu Leu Phe Asp Ser Gly Asn Thr Ala Glu Asp Arg Arg Leu Lys

50 55 60

Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Arg Asn Arg Ile Leu Tyr

65 70 75 80

Leu Gln Glu Ile Phe Ser Glu Glu Met Gly Lys Val Asp Asp Ser Phe

85 90 95

Phe His Arg Leu Glu Asp Ser Phe Leu Val Thr Glu Asp Lys Arg Gly

100 105 110

Glu Arg His Pro Ile Phe Gly Asn Leu Glu Glu Glu Val Lys Tyr His

115 120 125

Glu Asn Phe Pro Thr Ile Tyr His Leu Arg Gln Tyr Leu Ala Asp Asn

130 135 140

Pro Glu Lys Val Asp Leu Arg Leu Val Tyr Leu Ala Leu Ala His Ile

145 150 155 160

Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Lys Phe Asp Thr Arg

165 170 175

Asn Asn Asp Val Gln Arg Leu Phe Gln Glu Phe Leu Ala Val Tyr Asp

180 185 190

Asn Thr Phe Glu Asn Ser Ser Leu Gln Glu Gln Asn Val Gln Val Glu

195 200 205

Glu Ile Leu Thr Asp Lys Ile Ser Lys Ser Ala Lys Lys Asp Arg Val

210 215 220

Leu Lys Leu Phe Pro Asn Glu Lys Ser Asn Gly Arg Phe Ala Glu Phe

225 230 235 240

Leu Lys Leu Ile Val Gly Asn Gln Ala Asp Phe Lys Lys His Phe Glu

245 250 255

Leu Glu Glu Lys Ala Pro Leu Gln Phe Ser Lys Asp Thr Tyr Glu Glu

260 265 270

Glu Leu Glu Val Leu Leu Ala Gln Ile Gly Asp Asn Tyr Ala Glu Leu

275 280 285

Phe Leu Ser Ala Lys Lys Leu Tyr Asp Ser Ile Leu Leu Ser Gly Ile

290 295 300

Leu Thr Val Thr Asp Val Gly Thr Lys Ala Pro Leu Ser Ala Ser Met

305 310 315 320

Ile Gln Arg Tyr Asn Glu His Gln Met Asp Leu Ala Gln Leu Lys Gln

325 330 335

Phe Ile Arg Gln Lys Leu Ser Asp Lys Tyr Asn Glu Val Phe Ser Asp

340 345 350

Val Ser Lys Asp Gly Tyr Ala Gly Tyr Ile Asp Gly Lys Thr Asn Gln

355 360 365

Glu Ala Phe Tyr Lys Tyr Leu Lys Gly Leu Leu Asn Lys Ile Glu Gly

370 375 380

Ser Gly Tyr Phe Leu Asp Lys Ile Glu Arg Glu Asp Phe Leu Arg Lys

385 390 395 400

Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gln

405 410 415

Glu Met Arg Ala Ile Ile Arg Arg Gln Ala Glu Phe Tyr Pro Phe Leu

420 425 430

Ala Asp Asn Gln Asp Arg Ile Glu Lys Leu Leu Thr Phe Arg Ile Pro

435 440 445

Tyr Tyr Val Gly Pro Leu Ala Arg Gly Lys Ser Asp Phe Ala Trp Leu

450 455 460

Ser Arg Lys Ser Ala Asp Lys Ile Thr Pro Trp Asn Phe Asp Glu Ile

465 470 475 480

Val Asp Lys Glu Ser Ser Ala Glu Ala Phe Ile Asn Arg Met Thr Asn

485 490 495

Tyr Asp Leu Tyr Leu Pro Asn Gln Lys Val Leu Pro Lys His Ser Leu

500 505 510

Leu Tyr Glu Lys Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr

515 520 525

Lys Thr Glu Gln Gly Lys Thr Ala Phe Phe Asp Ala Asn Met Lys Gln

530 535 540

Glu Ile Phe Asp Gly Val Phe Lys Val Tyr Arg Lys Val Thr Lys Asp

545 550 555 560

Lys Leu Met Asp Phe Leu Glu Lys Glu Phe Asp Glu Phe Arg Ile Val

565 570 575

Asp Leu Thr Gly Leu Asp Lys Glu Asn Lys Val Phe Asn Ala Ser Tyr

580 585 590

Gly Thr Tyr His Asp Leu Cys Lys Ile Leu Asp Lys Asp Phe Leu Asp

595 600 605

Asn Ser Lys Asn Glu Lys Ile Leu Glu Asp Ile Val Leu Thr Leu Thr

610 615 620

Leu Phe Glu Asp Arg Glu Met Ile Arg Lys Arg Leu Glu Asn Tyr Ser

625 630 635 640

Asp Leu Leu Thr Lys Glu Gln Val Lys Lys Leu Glu Arg Arg His Tyr

645 650 655

Thr Gly Trp Gly Arg Leu Ser Ala Glu Leu Ile His Gly Ile Arg Asn

660 665 670

Lys Glu Ser Arg Lys Thr Ile Leu Asp Tyr Leu Ile Asp Asp Gly Asn

675 680 685

Ser Asn Arg Asn Phe Met Gln Leu Ile Asn Asp Asp Ala Leu Ser Phe

690 695 700

Lys Glu Glu Ile Ala Lys Ala Gln Val Ile Gly Glu Thr Asp Asn Leu

705 710 715 720

Asn Gln Val Val Ser Asp Ile Ala Gly Ser Pro Ala Ile Lys Lys Gly

725 730 735

Ile Leu Gln Ser Leu Lys Ile Val Asp Glu Leu Val Lys Ile Met Gly

740 745 750

His Gln Pro Glu Asn Ile Val Val Glu Met Ala Arg Glu Asn Gln Phe

755 760 765

Thr Asn Gln Gly Arg Arg Asn Ser Gln Gln Arg Leu Lys Gly Leu Thr

770 775 780

Asp Ser Ile Lys Glu Phe Gly Ser Gln Ile Leu Lys Glu His Pro Val

785 790 795 800

Glu Asn Ser Gln Leu Gln Asn Asp Arg Leu Phe Leu Tyr Tyr Leu Gln

805 810 815

Asn Gly Arg Asp Met Tyr Thr Gly Glu Glu Leu Asp Ile Asp Tyr Leu

820 825 830

Ser Gln Tyr Asp Ile Asp His Ile Ile Pro Gln Ala Phe Ile Lys Asp

835 840 845

Asn Ser Ile Asp Asn Arg Val Leu Thr Ser Ser Lys Glu Asn Arg Gly

850 855 860

Lys Ser Asp Asp Val Pro Ser Lys Asp Val Val Arg Lys Met Lys Ser

865 870 875 880

Tyr Trp Ser Lys Leu Leu Ser Ala Lys Leu Ile Thr Gln Arg Lys Phe

885 890 895

Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Thr Asp Asp Asp Lys

900 905 910

Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys

915 920 925

His Val Ala Arg Ile Leu Asp Glu Arg Phe Asn Thr Glu Thr Asp Glu

930 935 940

Asn Asn Lys Lys Ile Arg Gln Val Lys Ile Val Thr Leu Lys Ser Asn

945 950 955 960

Leu Val Ser Asn Phe Arg Lys Glu Phe Glu Leu Tyr Lys Val Arg Glu

965 970 975

Ile Asn Asp Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Ile

980 985 990

Gly Lys Ala Leu Leu Gly Val Tyr Pro Gln Leu Glu Pro Glu Phe Val

995 1000 1005

Tyr Gly Asp Tyr Pro His Phe His Gly His Lys Glu Asn Lys Ala Thr

1010 1015 1020

Ala Lys Lys Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Lys Asp

1025 1030 1035 1040

Asp Val Arg Thr Asp Lys Asn Gly Glu Ile Ile Trp Lys Lys Asp Glu

1045 1050 1055

His Ile Ser Asn Ile Lys Lys Val Leu Ser Tyr Pro Gln Val Asn Ile

1060 1065 1070

Val Lys Lys Val Glu Glu Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile

1075 1080 1085

Leu Pro Lys Gly Asn Ser Asp Lys Leu Ile Pro Arg Lys Thr Lys Lys

1090 1095 1100

Phe Tyr Trp Asp Thr Lys Lys Tyr Gly Gly Phe Asp Ser Pro Ile Val

1105 1110 1115 1120

Ala Tyr Ser Ile Leu Val Ile Ala Asp Ile Glu Lys Gly Lys Ser Lys

1125 1130 1135

Lys Leu Lys Thr Val Lys Ala Leu Val Gly Val Thr Ile Met Glu Lys

1140 1145 1150

Met Thr Phe Glu Arg Asp Pro Val Ala Phe Leu Glu Arg Lys Gly Tyr

1155 1160 1165

Arg Asn Val Gln Glu Glu Asn Ile Ile Lys Leu Pro Lys Tyr Ser Leu

1170 1175 1180

Phe Lys Leu Glu Asn Gly Arg Lys Arg Leu Leu Ala Ser Ala Arg Glu

1185 1190 1195 1200

Leu Gln Lys Gly Asn Glu Ile Val Leu Pro Asn His Leu Gly Thr Leu

1205 1210 1215

Leu Tyr His Ala Lys Asn Ile His Lys Val Asp Glu Pro Lys His Leu

1220 1225 1230

Asp Tyr Val Asp Lys His Lys Asp Glu Phe Lys Glu Leu Leu Asp Val

1235 1240 1245

Val Ser Asn Phe Ser Lys Lys Tyr Thr Leu Ala Glu Gly Asn Leu Glu

1250 1255 1260

Lys Ile Lys Glu Leu Tyr Ala Gln Asn Asn Gly Glu Asp Leu Lys Glu

1265 1270 1275 1280

Leu Ala Ser Ser Phe Ile Asn Leu Leu Thr Phe Thr Ala Ile Gly Ala

1285 1290 1295

Pro Ala Thr Phe Lys Phe Phe Asp Lys Asn Ile Asp Arg Lys Arg Tyr

1300 1305 1310

Thr Ser Thr Thr Glu Ile Leu Asn Ala Thr Leu Ile His Gln Ser Ile

1315 1320 1325

Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Asn Lys Leu Gly Gly Asp

1330 1335 1340

<210> 158

<211> 1367

<212> PRT

<213> Artificial sequence

<220>

<223> Streptococcus pyogenes Cas9

<400> 158

Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly

1 5 10 15

Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys

20 25 30

Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly

35 40 45

Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys

50 55 60

Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr

65 70 75 80

Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe

85 90 95

Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His

100 105 110

Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His

115 120 125

Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser

130 135 140

Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met

145 150 155 160

Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp

165 170 175

Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn

180 185 190

Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys

195 200 205

Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu

210 215 220

Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu

225 230 235 240

Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp

245 250 255

Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp

260 265 270

Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu

275 280 285

Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile

290 295 300

Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met

305 310 315 320

Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala

325 330 335

Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp

340 345 350

Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln

355 360 365

Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly

370 375 380

Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys

385 390 395 400

Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly

405 410 415

Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu

420 425 430

Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro

435 440 445

Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met

450 455 460

Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val

465 470 475 480

Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn

485 490 495

Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu

500 505 510

Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr

515 520 525

Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys

530 535 540

Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val

545 550 555 560

Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser

565 570 575

Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr

580 585 590

Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn

595 600 605

Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu

610 615 620

Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His

625 630 635 640

Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr

645 650 655

Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys

660 665 670

Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala

675 680 685

Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys

690 695 700

Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His

705 710 715 720

Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile

725 730 735

Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg

740 745 750

His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr

755 760 765

Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu

770 775 780

Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val

785 790 795 800

Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln

805 810 815

Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu

820 825 830

Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp

835 840 845

Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly

850 855 860

Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn

865 870 875 880

Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe

885 890 895

Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys

900 905 910

Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys

915 920 925

His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu

930 935 940

Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys

945 950 955 960

Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu

965 970 975

Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val

980 985 990

Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val

995 1000 1005

Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser

1010 1015 1020

Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn

1025 1030 1035 1040

Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile

1045 1050 1055

Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val

1060 1065 1070

Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met

1075 1080 1085

Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe

1090 1095 1100

Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala

1105 1110 1115 1120

Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro

1125 1130 1135

Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys

1140 1145 1150

Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met

1155 1160 1165

Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys

1170 1175 1180

Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr

1185 1190 1195 1200

Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala

1205 1210 1215

Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val

1220 1225 1230

Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro

1235 1240 1245

Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr

1250 1255 1260

Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile

1265 1270 1275 1280

Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His

1285 1290 1295

Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe

1300 1305 1310

Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr

1315 1320 1325

Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala

1330 1335 1340

Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp

1345 1350 1355 1360

Leu Ser Gln Leu Gly Gly Asp

1365

<210> 159

<211> 1387

<212> PRT

<213> Artificial sequence

<220>

<223> Streptococcus thermophilus Cas9

<400> 159

Thr Lys Pro Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly

1 5 10 15

Trp Ala Val Thr Thr Asp Asn Tyr Lys Val Pro Ser Lys Lys Met Lys

20 25 30

Val Leu Gly Asn Thr Ser Lys Lys Tyr Ile Lys Lys Asn Leu Leu Gly

35 40 45

Val Leu Leu Phe Asp Ser Gly Ile Thr Ala Glu Gly Arg Arg Leu Lys

50 55 60

Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Arg Asn Arg Ile Leu Tyr

65 70 75 80

Leu Gln Glu Ile Phe Ser Thr Glu Met Ala Thr Leu Asp Asp Ala Phe

85 90 95

Phe Gln Arg Leu Asp Asp Ser Phe Leu Val Pro Asp Asp Lys Arg Asp

100 105 110

Ser Lys Tyr Pro Ile Phe Gly Asn Leu Val Glu Glu Lys Ala Tyr His

115 120 125

Asp Glu Phe Pro Thr Ile Tyr His Leu Arg Lys Tyr Leu Ala Asp Ser

130 135 140

Thr Lys Lys Ala Asp Leu Arg Leu Val Tyr Leu Ala Leu Ala His Met

145 150 155 160

Ile Lys Tyr Arg Gly His Phe Leu Ile Glu Gly Glu Phe Asn Ser Lys

165 170 175

Asn Asn Asp Ile Gln Lys Asn Phe Gln Asp Phe Leu Asp Thr Tyr Asn

180 185 190

Ala Ile Phe Glu Ser Asp Leu Ser Leu Glu Asn Ser Lys Gln Leu Glu

195 200 205

Glu Ile Val Lys Asp Lys Ile Ser Lys Leu Glu Lys Lys Asp Arg Ile

210 215 220

Leu Lys Leu Phe Pro Gly Glu Lys Asn Ser Gly Ile Phe Ser Glu Phe

225 230 235 240

Leu Lys Leu Ile Val Gly Asn Gln Ala Asp Phe Arg Lys Cys Phe Asn

245 250 255

Leu Asp Glu Lys Ala Ser Leu His Phe Ser Lys Glu Ser Tyr Asp Glu

260 265 270

Asp Leu Glu Thr Leu Leu Gly Tyr Ile Gly Asp Asp Tyr Ser Asp Val

275 280 285

Phe Leu Lys Ala Lys Lys Leu Tyr Asp Ala Ile Leu Leu Ser Gly Phe

290 295 300

Leu Thr Val Thr Asp Asn Glu Thr Glu Ala Pro Leu Ser Ser Ala Met

305 310 315 320

Ile Lys Arg Tyr Asn Glu His Lys Glu Asp Leu Ala Leu Leu Lys Glu

325 330 335

Tyr Ile Arg Asn Ile Ser Leu Lys Thr Tyr Asn Glu Val Phe Lys Asp

340 345 350

Asp Thr Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Lys Thr Asn Gln

355 360 365

Glu Asp Phe Tyr Val Tyr Leu Lys Lys Leu Leu Ala Glu Phe Glu Gly

370 375 380

Ala Asp Tyr Phe Leu Glu Lys Ile Asp Arg Glu Asp Phe Leu Arg Lys

385 390 395 400

Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro Tyr Gln Ile His Leu Gln

405 410 415

Glu Met Arg Ala Ile Leu Asp Lys Gln Ala Lys Phe Tyr Pro Phe Leu

420 425 430

Ala Lys Asn Lys Glu Arg Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro

435 440 445

Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Asp Phe Ala Trp Ser

450 455 460

Ile Arg Lys Arg Asn Glu Lys Ile Thr Pro Trp Asn Phe Glu Asp Val

465 470 475 480

Ile Asp Lys Glu Ser Ser Ala Glu Ala Phe Ile Asn Arg Met Thr Ser

485 490 495

Phe Asp Leu Tyr Leu Pro Glu Glu Lys Val Leu Pro Lys His Ser Leu

500 505 510

Leu Tyr Glu Thr Phe Asn Val Tyr Asn Glu Leu Thr Lys Val Arg Phe

515 520 525

Ile Ala Glu Ser Met Arg Asp Tyr Gln Phe Leu Asp Ser Lys Gln Lys

530 535 540

Lys Asp Ile Val Arg Leu Tyr Phe Lys Asp Lys Arg Lys Val Thr Asp

545 550 555 560

Lys Asp Ile Ile Glu Tyr Leu His Ala Ile Tyr Gly Tyr Asp Gly Ile

565 570 575

Glu Leu Lys Gly Ile Glu Lys Gln Phe Asn Ser Ser Leu Ser Thr Tyr

580 585 590

His Asp Leu Leu Asn Ile Ile Asn Asp Lys Glu Phe Leu Asp Asp Ser

595 600 605

Ser Asn Glu Ala Ile Ile Glu Glu Ile Ile His Thr Leu Thr Ile Phe

610 615 620

Glu Asp Arg Glu Met Ile Lys Gln Arg Leu Ser Lys Phe Glu Asn Ile

625 630 635 640

Phe Asp Lys Ser Val Leu Lys Lys Leu Ser Arg Arg His Tyr Thr Gly

645 650 655

Trp Gly Lys Leu Ser Ala Lys Leu Ile Asn Gly Ile Arg Asp Glu Lys

660 665 670

Ser Gly Asn Thr Ile Leu Asp Tyr Leu Ile Asp Asp Gly Ile Ser Asn

675 680 685

Arg Asn Phe Met Gln Leu Ile His Asp Asp Ala Leu Ser Phe Lys Lys

690 695 700

Lys Ile Gln Lys Ala Gln Ile Ile Gly Asp Glu Asp Lys Gly Asn Ile

705 710 715 720

Lys Glu Val Val Lys Ser Leu Pro Gly Ser Pro Ala Ile Lys Lys Gly

725 730 735

Ile Leu Gln Ser Ile Lys Ile Val Asp Glu Leu Val Lys Val Met Gly

740 745 750

Gly Arg Lys Pro Glu Ser Ile Val Val Glu Met Ala Arg Glu Asn Gln

755 760 765

Tyr Thr Asn Gln Gly Lys Ser Asn Ser Gln Gln Arg Leu Lys Arg Leu

770 775 780

Glu Lys Ser Leu Lys Glu Leu Gly Ser Lys Ile Leu Lys Glu Asn Ile

785 790 795 800

Pro Ala Lys Leu Ser Lys Ile Asp Asn Asn Ala Leu Gln Asn Asp Arg

805 810 815

Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Lys Asp Met Tyr Thr Gly Asp

820 825 830

Asp Leu Asp Ile Asp Arg Leu Ser Asn Tyr Asp Ile Asp His Ile Ile

835 840 845

Pro Gln Ala Phe Leu Lys Asp Asn Ser Ile Asp Asn Lys Val Leu Val

850 855 860

Ser Ser Ala Ser Asn Arg Gly Lys Ser Asp Asp Val Pro Ser Leu Glu

865 870 875 880

Val Val Lys Lys Arg Lys Thr Phe Trp Tyr Gln Leu Leu Lys Ser Lys

885 890 895

Leu Ile Ser Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly

900 905 910

Gly Leu Ser Pro Glu Asp Lys Ala Gly Phe Ile Gln Arg Gln Leu Val

915 920 925

Glu Thr Arg Gln Ile Thr Lys His Val Ala Arg Leu Leu Asp Glu Lys

930 935 940

Phe Asn Asn Lys Lys Asp Glu Asn Asn Arg Ala Val Arg Thr Val Lys

945 950 955 960

Ile Ile Thr Leu Lys Ser Thr Leu Val Ser Gln Phe Arg Lys Asp Phe

965 970 975

Glu Leu Tyr Lys Val Arg Glu Ile Asn Asp Phe His His Ala His Asp

980 985 990

Ala Tyr Leu Asn Ala Val Val Ala Ser Ala Leu Leu Lys Lys Tyr Pro

995 1000 1005

Lys Leu Glu Pro Glu Phe Val Tyr Gly Asp Tyr Pro Lys Tyr Asn Ser

1010 1015 1020

Phe Arg Glu Arg Lys Ser Ala Thr Glu Lys Val Tyr Phe Tyr Ser Asn

1025 1030 1035 1040

Ile Met Asn Ile Phe Lys Lys Ser Ile Ser Leu Ala Asp Gly Arg Val

1045 1050 1055

Ile Glu Arg Pro Leu Ile Glu Val Asn Glu Glu Thr Gly Glu Ser Val

1060 1065 1070

Trp Asn Lys Glu Ser Asp Leu Ala Thr Val Arg Arg Val Leu Ser Tyr

1075 1080 1085

Pro Gln Val Asn Val Val Lys Lys Val Glu Glu Gln Asn His Gly Leu

1090 1095 1100

Asp Arg Gly Lys Pro Lys Gly Leu Phe Asn Ala Asn Leu Ser Ser Lys

1105 1110 1115 1120

Pro Lys Pro Asn Ser Asn Glu Asn Leu Val Gly Ala Lys Glu Tyr Leu

1125 1130 1135

Asp Pro Lys Lys Tyr Gly Gly Tyr Ala Gly Ile Ser Asn Ser Phe Thr

1140 1145 1150

Val Leu Val Lys Gly Thr Ile Glu Lys Gly Ala Lys Lys Lys Ile Thr

1155 1160 1165

Asn Val Leu Glu Phe Gln Gly Ile Ser Ile Leu Asp Arg Ile Asn Tyr

1170 1175 1180

Arg Lys Asp Lys Leu Asn Phe Leu Leu Glu Lys Gly Tyr Lys Asp Ile

1185 1190 1195 1200

Glu Leu Ile Ile Glu Leu Pro Lys Tyr Ser Leu Phe Glu Leu Ser Asp

1205 1210 1215

Gly Ser Arg Arg Met Leu Ala Ser Ile Leu Ser Thr Asn Asn Lys Arg

1220 1225 1230

Gly Glu Ile His Lys Gly Asn Gln Ile Phe Leu Ser Gln Lys Phe Val

1235 1240 1245

Lys Leu Leu Tyr His Ala Lys Arg Ile Ser Asn Thr Ile Asn Glu Asn

1250 1255 1260

His Arg Lys Tyr Val Glu Asn His Lys Lys Glu Phe Glu Glu Leu Phe

1265 1270 1275 1280

Tyr Tyr Ile Leu Glu Phe Asn Glu Asn Tyr Val Gly Ala Lys Lys Asn

1285 1290 1295

Gly Lys Leu Leu Asn Ser Ala Phe Gln Ser Trp Gln Asn His Ser Ile

1300 1305 1310

Asp Glu Leu Cys Ser Ser Phe Ile Gly Pro Thr Gly Ser Glu Arg Lys

1315 1320 1325

Gly Leu Phe Glu Leu Thr Ser Arg Gly Ser Ala Ala Asp Phe Glu Phe

1330 1335 1340

Leu Gly Val Lys Ile Pro Arg Tyr Arg Asp Tyr Thr Pro Ser Ser Leu

1345 1350 1355 1360

Leu Lys Asp Ala Thr Leu Ile His Gln Ser Val Thr Gly Leu Tyr Glu

1365 1370 1375

Thr Arg Ile Asp Leu Ala Lys Leu Gly Glu Gly

1380 1385

<210> 160

<211> 1333

<212> PRT

<213> Artificial sequence

<220>

<223> Listeria innocua Cas9

<400> 160

Lys Lys Pro Tyr Thr Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly

1 5 10 15

Trp Ala Val Leu Thr Asp Gln Tyr Asp Leu Val Lys Arg Lys Met Lys

20 25 30

Ile Ala Gly Asp Ser Glu Lys Lys Gln Ile Lys Lys Asn Phe Trp Gly

35 40 45

Val Arg Leu Phe Asp Glu Gly Gln Thr Ala Ala Asp Arg Arg Met Ala

50 55 60

Arg Thr Ala Arg Arg Arg Ile Glu Arg Arg Arg Asn Arg Ile Ser Tyr

65 70 75 80

Leu Gln Gly Ile Phe Ala Glu Glu Met Ser Lys Thr Asp Ala Asn Phe

85 90 95

Phe Cys Arg Leu Ser Asp Ser Phe Tyr Val Asp Asn Glu Lys Arg Asn

100 105 110

Ser Arg His Pro Phe Phe Ala Thr Ile Glu Glu Glu Val Glu Tyr His

115 120 125

Lys Asn Tyr Pro Thr Ile Tyr His Leu Arg Glu Glu Leu Val Asn Ser

130 135 140

Ser Glu Lys Ala Asp Leu Arg Leu Val Tyr Leu Ala Leu Ala His Ile

145 150 155 160

Ile Lys Tyr Arg Gly Asn Phe Leu Ile Glu Gly Ala Leu Asp Thr Gln

165 170 175

Asn Thr Ser Val Asp Gly Ile Tyr Lys Gln Phe Ile Gln Thr Tyr Asn

180 185 190

Gln Val Phe Ala Ser Gly Ile Glu Asp Gly Ser Leu Lys Lys Leu Glu

195 200 205

Asp Asn Lys Asp Val Ala Lys Ile Leu Val Glu Lys Val Thr Arg Lys

210 215 220

Glu Lys Leu Glu Arg Ile Leu Lys Leu Tyr Pro Gly Glu Lys Ser Ala

225 230 235 240

Gly Met Phe Ala Gln Phe Ile Ser Leu Ile Val Gly Ser Lys Gly Asn

245 250 255

Phe Gln Lys Pro Phe Asp Leu Ile Glu Lys Ser Asp Ile Glu Cys Ala

260 265 270

Lys Asp Ser Tyr Glu Glu Asp Leu Glu Ser Leu Leu Ala Leu Ile Gly

275 280 285

Asp Glu Tyr Ala Glu Leu Phe Val Ala Ala Lys Asn Ala Tyr Ser Ala

290 295 300

Val Val Leu Ser Ser Ile Ile Thr Val Ala Glu Thr Glu Thr Asn Ala

305 310 315 320

Lys Leu Ser Ala Ser Met Ile Glu Arg Phe Asp Thr His Glu Glu Asp

325 330 335

Leu Gly Glu Leu Lys Ala Phe Ile Lys Leu His Leu Pro Lys His Tyr

340 345 350

Glu Glu Ile Phe Ser Asn Thr Glu Lys His Gly Tyr Ala Gly Tyr Ile

355 360 365

Asp Gly Lys Thr Lys Gln Ala Asp Phe Tyr Lys Tyr Met Lys Met Thr

370 375 380

Leu Glu Asn Ile Glu Gly Ala Asp Tyr Phe Ile Ala Lys Ile Glu Lys

385 390 395 400

Glu Asn Phe Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ala Ile Pro

405 410 415

His Gln Leu His Leu Glu Glu Leu Glu Ala Ile Leu His Gln Gln Ala

420 425 430

Lys Tyr Tyr Pro Phe Leu Lys Glu Asn Tyr Asp Lys Ile Lys Ser Leu

435 440 445

Val Thr Phe Arg Ile Pro Tyr Phe Val Gly Pro Leu Ala Asn Gly Gln

450 455 460

Ser Glu Phe Ala Trp Leu Thr Arg Lys Ala Asp Gly Glu Ile Arg Pro

465 470 475 480

Trp Asn Ile Glu Glu Lys Val Asp Phe Gly Lys Ser Ala Val Asp Phe

485 490 495

Ile Glu Lys Met Thr Asn Lys Asp Thr Tyr Leu Pro Lys Glu Asn Val

500 505 510

Leu Pro Lys His Ser Leu Cys Tyr Gln Lys Tyr Leu Val Tyr Asn Glu

515 520 525

Leu Thr Lys Val Arg Tyr Ile Asn Asp Gln Gly Lys Thr Ser Tyr Phe

530 535 540

Ser Gly Gln Glu Lys Glu Gln Ile Phe Asn Asp Leu Phe Lys Gln Lys

545 550 555 560

Arg Lys Val Lys Lys Lys Asp Leu Glu Leu Phe Leu Arg Asn Met Ser

565 570 575

His Val Glu Ser Pro Thr Ile Glu Gly Leu Glu Asp Ser Phe Asn Ser

580 585 590

Ser Tyr Ser Thr Tyr His Asp Leu Leu Lys Val Gly Ile Lys Gln Glu

595 600 605

Ile Leu Asp Asn Pro Val Asn Thr Glu Met Leu Glu Asn Ile Val Lys

610 615 620

Ile Leu Thr Val Phe Glu Asp Lys Arg Met Ile Lys Glu Gln Leu Gln

625 630 635 640

Gln Phe Ser Asp Val Leu Asp Gly Val Val Leu Lys Lys Leu Glu Arg

645 650 655

Arg His Tyr Thr Gly Trp Gly Arg Leu Ser Ala Lys Leu Leu Met Gly

660 665 670

Ile Arg Asp Lys Gln Ser His Leu Thr Ile Leu Asp Tyr Leu Met Asn

675 680 685

Asp Asp Gly Leu Asn Arg Asn Leu Met Gln Leu Ile Asn Asp Ser Asn

690 695 700

Leu Ser Phe Lys Ser Ile Ile Glu Lys Glu Gln Val Thr Thr Ala Asp

705 710 715 720

Lys Asp Ile Gln Ser Ile Val Ala Asp Leu Ala Gly Ser Pro Ala Ile

725 730 735

Lys Lys Gly Ile Leu Gln Ser Leu Lys Ile Val Asp Glu Leu Val Ser

740 745 750

Val Met Gly Tyr Pro Pro Gln Thr Ile Val Val Glu Met Ala Arg Glu

755 760 765

Asn Gln Thr Thr Gly Lys Gly Lys Asn Asn Ser Arg Pro Arg Tyr Lys

770 775 780

Ser Leu Glu Lys Ala Ile Lys Glu Phe Gly Ser Gln Ile Leu Lys Glu

785 790 795 800

His Pro Thr Asp Asn Gln Glu Leu Arg Asn Asn Arg Leu Tyr Leu Tyr

805 810 815

Tyr Leu Gln Asn Gly Lys Asp Met Tyr Thr Gly Gln Asp Leu Asp Ile

820 825 830

His Asn Leu Ser Asn Tyr Asp Ile Asp His Ile Val Pro Gln Ser Phe

835 840 845

Ile Thr Asp Asn Ser Ile Asp Asn Leu Val Leu Thr Ser Ser Ala Gly

850 855 860

Asn Arg Glu Lys Gly Asp Asp Val Pro Pro Leu Glu Ile Val Arg Lys

865 870 875 880

Arg Lys Val Phe Trp Glu Lys Leu Tyr Gln Gly Asn Leu Met Ser Lys

885 890 895

Arg Lys Phe Asp Tyr Leu Thr Lys Ala Glu Arg Gly Gly Leu Thr Glu

900 905 910

Ala Asp Lys Ala Arg Phe Ile His Arg Gln Leu Val Glu Thr Arg Gln

915 920 925

Ile Thr Lys Asn Val Ala Asn Ile Leu His Gln Arg Phe Asn Tyr Glu

930 935 940

Lys Asp Asp His Gly Asn Thr Met Lys Gln Val Arg Ile Val Thr Leu

945 950 955 960

Lys Ser Ala Leu Val Ser Gln Phe Arg Lys Gln Phe Gln Leu Tyr Lys

965 970 975

Val Arg Asp Val Asn Asp Tyr His His Ala His Asp Ala Tyr Leu Asn

980 985 990

Gly Val Val Ala Asn Thr Leu Leu Lys Val Tyr Pro Gln Leu Glu Pro

995 1000 1005

Glu Phe Val Tyr Gly Asp Tyr His Gln Phe Asp Trp Phe Lys Ala Asn

1010 1015 1020

Lys Ala Thr Ala Lys Lys Gln Phe Tyr Thr Asn Ile Met Leu Phe Phe

1025 1030 1035 1040

Ala Gln Lys Asp Arg Ile Ile Asp Glu Asn Gly Glu Ile Leu Trp Asp

1045 1050 1055

Lys Lys Tyr Leu Asp Thr Val Lys Lys Val Met Ser Tyr Arg Gln Met

1060 1065 1070

Asn Ile Val Lys Lys Thr Glu Ile Gln Lys Gly Glu Phe Ser Lys Ala

1075 1080 1085

Thr Ile Lys Pro Lys Gly Asn Ser Ser Lys Leu Ile Pro Arg Lys Thr

1090 1095 1100

Asn Trp Asp Pro Met Lys Tyr Gly Gly Leu Asp Ser Pro Asn Met Ala

1105 1110 1115 1120

Tyr Ala Val Val Ile Glu Tyr Ala Lys Gly Lys Asn Lys Leu Val Phe

1125 1130 1135

Glu Lys Lys Ile Ile Arg Val Thr Ile Met Glu Arg Lys Ala Phe Glu

1140 1145 1150

Lys Asp Glu Lys Ala Phe Leu Glu Glu Gln Gly Tyr Arg Gln Pro Lys

1155 1160 1165

Val Leu Ala Lys Leu Pro Lys Tyr Thr Leu Tyr Glu Cys Glu Glu Gly

1170 1175 1180

Arg Arg Arg Met Leu Ala Ser Ala Asn Glu Ala Gln Lys Gly Asn Gln

1185 1190 1195 1200

Gln Val Leu Pro Asn His Leu Val Thr Leu Leu His His Ala Ala Asn

1205 1210 1215

Cys Glu Val Ser Asp Gly Lys Ser Leu Asp Tyr Ile Glu Ser Asn Arg

1220 1225 1230

Glu Met Phe Ala Glu Leu Leu Ala His Val Ser Glu Phe Ala Lys Arg

1235 1240 1245

Tyr Thr Leu Ala Glu Ala Asn Leu Asn Lys Ile Asn Gln Leu Phe Glu

1250 1255 1260

Gln Asn Lys Glu Gly Asp Ile Lys Ala Ile Ala Gln Ser Phe Val Asp

1265 1270 1275 1280

Leu Met Ala Phe Asn Ala Met Gly Ala Pro Ala Ser Phe Lys Phe Phe

1285 1290 1295

Glu Thr Thr Ile Glu Arg Lys Arg Tyr Asn Asn Leu Lys Glu Leu Leu

1300 1305 1310

Asn Ser Thr Ile Ile Tyr Gln Ser Ile Thr Gly Leu Tyr Glu Ser Arg

1315 1320 1325

Lys Arg Leu Asp Asp

1330

<210> 161

<211> 1082

<212> PRT

<213> Artificial sequence

<220>

<223> Neisseria meningitidis Cas9

<400> 161

Met Ala Ala Phe Lys Pro Asn Ser Ile Asn Tyr Ile Leu Gly Leu Asp

1 5 10 15

Ile Gly Ile Ala Ser Val Gly Trp Ala Met Val Glu Ile Asp Glu Glu

20 25 30

Glu Asn Pro Ile Arg Leu Ile Asp Leu Gly Val Arg Val Phe Glu Arg

35 40 45

Ala Glu Val Pro Lys Thr Gly Asp Ser Leu Ala Met Ala Arg Arg Leu

50 55 60

Ala Arg Ser Val Arg Arg Leu Thr Arg Arg Arg Ala His Arg Leu Leu

65 70 75 80

Arg Thr Arg Arg Leu Leu Lys Arg Glu Gly Val Leu Gln Ala Ala Asn

85 90 95

Phe Asp Glu Asn Gly Leu Ile Lys Ser Leu Pro Asn Thr Pro Trp Gln

100 105 110

Leu Arg Ala Ala Ala Leu Asp Arg Lys Leu Thr Pro Leu Glu Trp Ser

115 120 125

Ala Val Leu Leu His Leu Ile Lys His Arg Gly Tyr Leu Ser Gln Arg

130 135 140

Lys Asn Glu Gly Glu Thr Ala Asp Lys Glu Leu Gly Ala Leu Leu Lys

145 150 155 160

Gly Val Ala Gly Asn Ala His Ala Leu Gln Thr Gly Asp Phe Arg Thr

165 170 175

Pro Ala Glu Leu Ala Leu Asn Lys Phe Glu Lys Glu Ser Gly His Ile

180 185 190

Arg Asn Gln Arg Ser Asp Tyr Ser His Thr Phe Ser Arg Lys Asp Leu

195 200 205

Gln Ala Glu Leu Ile Leu Leu Phe Glu Lys Gln Lys Glu Phe Gly Asn

210 215 220

Pro His Val Ser Gly Gly Leu Lys Glu Gly Ile Glu Thr Leu Leu Met

225 230 235 240

Thr Gln Arg Pro Ala Leu Ser Gly Asp Ala Val Gln Lys Met Leu Gly

245 250 255

His Cys Thr Phe Glu Pro Ala Glu Pro Lys Ala Ala Lys Asn Thr Tyr

260 265 270

Thr Ala Glu Arg Phe Ile Trp Leu Thr Lys Leu Asn Asn Leu Arg Ile

275 280 285

Leu Glu Gln Gly Ser Glu Arg Pro Leu Thr Asp Thr Glu Arg Ala Thr

290 295 300

Leu Met Asp Glu Pro Tyr Arg Lys Ser Lys Leu Thr Tyr Ala Gln Ala

305 310 315 320

Arg Lys Leu Leu Gly Leu Glu Asp Thr Ala Phe Phe Lys Gly Leu Arg

325 330 335

Tyr Gly Lys Asp Asn Ala Glu Ala Ser Thr Leu Met Glu Met Lys Ala

340 345 350

Tyr His Ala Ile Ser Arg Ala Leu Glu Lys Glu Gly Leu Lys Asp Lys

355 360 365

Lys Ser Pro Leu Asn Leu Ser Pro Glu Leu Gln Asp Glu Ile Gly Thr

370 375 380

Ala Phe Ser Leu Phe Lys Thr Asp Glu Asp Ile Thr Gly Arg Leu Lys

385 390 395 400

Asp Arg Ile Gln Pro Glu Ile Leu Glu Ala Leu Leu Lys His Ile Ser

405 410 415

Phe Asp Lys Phe Val Gln Ile Ser Leu Lys Ala Leu Arg Arg Ile Val

420 425 430

Pro Leu Met Glu Gln Gly Lys Arg Tyr Asp Glu Ala Cys Ala Glu Ile

435 440 445

Tyr Gly Asp His Tyr Gly Lys Lys Asn Thr Glu Glu Lys Ile Tyr Leu

450 455 460

Pro Pro Ile Pro Ala Asp Glu Ile Arg Asn Pro Val Val Leu Arg Ala

465 470 475 480

Leu Ser Gln Ala Arg Lys Val Ile Asn Gly Val Val Arg Arg Tyr Gly

485 490 495

Ser Pro Ala Arg Ile His Ile Glu Thr Ala Arg Glu Val Gly Lys Ser

500 505 510

Phe Lys Asp Arg Lys Glu Ile Glu Lys Arg Gln Glu Glu Asn Arg Lys

515 520 525

Asp Arg Glu Lys Ala Ala Ala Lys Phe Arg Glu Tyr Phe Pro Asn Phe

530 535 540

Val Gly Glu Pro Lys Ser Lys Asp Ile Leu Lys Leu Arg Leu Tyr Glu

545 550 555 560

Gln Gln His Gly Lys Cys Leu Tyr Ser Gly Lys Glu Ile Asn Leu Gly

565 570 575

Arg Leu Asn Glu Lys Gly Tyr Val Glu Ile Asp His Ala Leu Pro Phe

580 585 590

Ser Arg Thr Trp Asp Asp Ser Phe Asn Asn Lys Val Leu Val Leu Gly

595 600 605

Ser Glu Asn Gln Asn Lys Gly Asn Gln Thr Pro Tyr Glu Tyr Phe Asn

610 615 620

Gly Lys Asp Asn Ser Arg Glu Trp Gln Glu Phe Lys Ala Arg Val Glu

625 630 635 640

Thr Ser Arg Phe Pro Arg Ser Lys Lys Gln Arg Ile Leu Leu Gln Lys

645 650 655

Phe Asp Glu Asp Gly Phe Lys Glu Arg Asn Leu Asn Asp Thr Arg Tyr

660 665 670

Val Asn Arg Phe Leu Cys Gln Phe Val Ala Asp Arg Met Arg Leu Thr

675 680 685

Gly Lys Gly Lys Lys Arg Val Phe Ala Ser Asn Gly Gln Ile Thr Asn

690 695 700

Leu Leu Arg Gly Phe Trp Gly Leu Arg Lys Val Arg Ala Glu Asn Asp

705 710 715 720

Arg His His Ala Leu Asp Ala Val Val Val Ala Cys Ser Thr Val Ala

725 730 735

Met Gln Gln Lys Ile Thr Arg Phe Val Arg Tyr Lys Glu Met Asn Ala

740 745 750

Phe Asp Gly Lys Thr Ile Asp Lys Glu Thr Gly Glu Val Leu His Gln

755 760 765

Lys Thr His Phe Pro Gln Pro Trp Glu Phe Phe Ala Gln Glu Val Met

770 775 780

Ile Arg Val Phe Gly Lys Pro Asp Gly Lys Pro Glu Phe Glu Glu Ala

785 790 795 800

Asp Thr Leu Glu Lys Leu Arg Thr Leu Leu Ala Glu Lys Leu Ser Ser

805 810 815

Arg Pro Glu Ala Val His Glu Tyr Val Thr Pro Leu Phe Val Ser Arg

820 825 830

Ala Pro Asn Arg Lys Met Ser Gly Gln Gly His Met Glu Thr Val Lys

835 840 845

Ser Ala Lys Arg Leu Asp Glu Gly Val Ser Val Leu Arg Val Pro Leu

850 855 860

Thr Gln Leu Lys Leu Lys Asp Leu Glu Lys Met Val Asn Arg Glu Arg

865 870 875 880

Glu Pro Lys Leu Tyr Glu Ala Leu Lys Ala Arg Leu Glu Ala His Lys

885 890 895

Asp Asp Pro Ala Lys Ala Phe Ala Glu Pro Phe Tyr Lys Tyr Asp Lys

900 905 910

Ala Gly Asn Arg Thr Gln Gln Val Lys Ala Val Arg Val Glu Gln Val

915 920 925

Gln Lys Thr Gly Val Trp Val Arg Asn His Asn Gly Ile Ala Asp Asn

930 935 940

Ala Thr Met Val Arg Val Asp Val Phe Glu Lys Gly Asp Lys Tyr Tyr

945 950 955 960

Leu Val Pro Ile Tyr Ser Trp Gln Val Ala Lys Gly Ile Leu Pro Asp

965 970 975

Arg Ala Val Val Gln Gly Lys Asp Glu Glu Asp Trp Gln Leu Ile Asp

980 985 990

Asp Ser Phe Asn Phe Lys Phe Ser Leu His Pro Asn Asp Leu Val Glu

995 1000 1005

Val Ile Thr Lys Lys Ala Arg Met Phe Gly Tyr Phe Ala Ser Cys His

1010 1015 1020

Arg Gly Thr Gly Asn Ile Asn Ile Arg Ile His Asp Leu Asp His Lys

1025 1030 1035 1040

Ile Gly Lys Asn Gly Ile Leu Glu Gly Ile Gly Val Lys Thr Ala Leu

1045 1050 1055

Ser Phe Gln Lys Tyr Gln Ile Asp Glu Leu Gly Lys Glu Ile Arg Pro

1060 1065 1070

Cys Arg Leu Lys Lys Arg Pro Pro Val Arg

1075 1080

<210> 162

<211> 1368

<212> PRT

<213> Artificial sequence

<220>

<223> Streptococcus pyogenes Cas9

<400> 162

Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val

1 5 10 15

Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe

20 25 30

Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile

35 40 45

Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu

50 55 60

Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys

65 70 75 80

Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser

85 90 95

Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys

100 105 110

His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr

115 120 125

His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp

130 135 140

Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His

145 150 155 160

Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro

165 170 175

Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr

180 185 190

Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala

195 200 205

Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn

210 215 220

Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn

225 230 235 240

Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe

245 250 255

Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp

260 265 270

Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp

275 280 285

Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp

290 295 300

Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser

305 310 315 320

Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys

325 330 335

Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe

340 345 350

Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser

355 360 365

Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp

370 375 380

Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg

385 390 395 400

Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu

405 410 415

Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe

420 425 430

Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile

435 440 445

Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp

450 455 460

Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu

465 470 475 480

Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr

485 490 495

Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser

500 505 510

Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys

515 520 525

Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln

530 535 540

Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr

545 550 555 560

Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp

565 570 575

Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly

580 585 590

Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp

595 600 605

Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr

610 615 620

Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala

625 630 635 640

His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr

645 650 655

Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp

660 665 670

Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe

675 680 685

Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe

690 695 700

Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu

705 710 715 720

His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly

725 730 735

Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly

740 745 750

Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln

755 760 765

Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile

770 775 780

Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro

785 790 795 800

Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu

805 810 815

Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg

820 825 830

Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys

835 840 845

Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg

850 855 860

Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys

865 870 875 880

Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys

885 890 895

Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp

900 905 910

Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr

915 920 925

Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp

930 935 940

Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser

945 950 955 960

Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg

965 970 975

Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val

980 985 990

Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe

995 1000 1005

Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys

1010 1015 1020

Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser

1025 1030 1035 1040

Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu

1045 1050 1055

Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile

1060 1065 1070

Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser

1075 1080 1085

Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly

1090 1095 1100

Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile

1105 1110 1115 1120

Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser

1125 1130 1135

Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly

1140 1145 1150

Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile

1155 1160 1165

Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala

1170 1175 1180

Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys

1185 1190 1195 1200

Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser

1205 1210 1215

Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr

1220 1225 1230

Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser

1235 1240 1245

Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His

1250 1255 1260

Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val

1265 1270 1275 1280

Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys

1285 1290 1295

His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu

1300 1305 1310

Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp

1315 1320 1325

Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp

1330 1335 1340

Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile

1345 1350 1355 1360

Asp Leu Ser Gln Leu Gly Gly Asp

1365

<210> 163

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence 2

<400> 163

gagaatcaaa atcggtgaat 20

<210> 164

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> TRBC target sequence 2

<400> 164

ggcctcggcg ctgacgatct 20

<210> 165

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 3

<400> 165

gagaatcaaa atcggtgaat agg 23

<210> 166

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 4

<400> 166

ttcaaaacct gtcagtgatt ggg 23

<210> 167

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 5

<400> 167

tgtgctagac atgaggtcta tgg 23

<210> 168

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 6

<400> 168

cgtcatgagc agattaaacc cgg 23

<210> 169

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 7

<400> 169

tcagggttct ggatatctgt ggg 23

<210> 170

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 8

<400> 170

gtcagggttc tggatatctg tgg 23

<210> 171

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 9

<400> 171

ttcggaaccc aatcactgac agg 23

<210> 172

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 10

<400> 172

taaacccggc cactttcagg agg 23

<210> 173

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 11

<400> 173

aaagtcagat ttgttgctcc agg 23

<210> 174

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 12

<400> 174

aacaaatgtg tcacaaagta agg 23

<210> 175

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 13

<400> 175

tggatttaga gtctctcagc tgg 23

<210> 176

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 14

<400> 176

taggcagaca gacttgtcac tgg 23

<210> 177

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 15

<400> 177

agctggtaca cggcagggtc agg 23

<210> 178

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 16

<400> 178

gctggtacac ggcagggtca ggg 23

<210> 179

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 17

<400> 179

tctctcagct ggtacacggc agg 23

<210> 180

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 18

<400> 180

tttcaaaacc tgtcagtgat tgg 23

<210> 181

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 19

<400> 181

gattaaaccc ggccactttc agg 23

<210> 182

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 20

<400> 182

ctcgaccagc ttgacatcac agg 23

<210> 183

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 21

<400> 183

agagtctctc agctggtaca cgg 23

<210> 184

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 22

<400> 184

ctctcagctg gtacacggca ggg 23

<210> 185

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 23

<400> 185

aagttcctgt gatgtcaagc tgg 23

<210> 186

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 24

<400> 186

atcctcctcc tgaaagtggc cgg 23

<210> 187

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 25

<400> 187

tgctcatgac gctgcggctg tgg 23

<210> 188

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 26

<400> 188

acaaaactgt gctagacatg agg 23

<210> 189

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 27

<400> 189

atttgtttga gaatcaaaat cgg 23

<210> 190

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 28

<400> 190

catcacagga actttctaaa agg 23

<210> 191

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 29

<400> 191

gtcgagaaaa gctttgaaac agg 23

<210> 192

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 30

<400> 192

ccactttcag gaggaggatt cgg 23

<210> 193

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 31

<400> 193

ctgacaggtt ttgaaagttt agg 23

<210> 194

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 32

<400> 194

agctttgaaa caggtaagac agg 23

<210> 195

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 33

<400> 195

tggaataatg ctgttgttga agg 23

<210> 196

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 34

<400> 196

agagcaacag tgctgtggcc tgg 23

<210> 197

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 35

<400> 197

ctgtggtcca gctgaggtga ggg 23

<210> 198

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 36

<400> 198

ctgcggctgt ggtccagctg agg 23

<210> 199

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 37

<400> 199

tgtggtccag ctgaggtgag ggg 23

<210> 200

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 38

<400> 200

cttcttcccc agcccaggta agg 23

<210> 201

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 39

<400> 201

acacggcagg gtcagggttc tgg 23

<210> 202

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 40

<400> 202

cttcaagagc aacagtgctg tgg 23

<210> 203

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 41

<400> 203

ctggggaaga aggtgtcttc tgg 23

<210> 204

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 42

<400> 204

tcctcctcct gaaagtggcc ggg 23

<210> 205

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 43

<400> 205

ttaatctgct catgacgctg cgg 23

<210> 206

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 44

<400> 206

acccggccac tttcaggagg agg 23

<210> 207

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 45

<400> 207

ttcttcccca gcccaggtaa ggg 23

<210> 208

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 46

<400> 208

cttacctggg ctggggaaga agg 23

<210> 209

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 47

<400> 209

gacaccttct tccccagccc agg 23

<210> 210

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 48

<400> 210

gctgtggtcc agctgaggtg agg 23

<210> 211

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC target sequence with PAM 49

<400> 211

ccgaatcctc ctcctgaaag tgg 23

<210> 212

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> TRBC target sequence with PAM 3

<400> 212

gctgtcaagt ccagttctac ggg 23

<210> 213

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC ZFN target sequence 1

<400> 213

ctatggactt caagagcaac agtgctgt 28

<210> 214

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC ZFN target sequence 2

<400> 214

ctcatgtcta gcacagtttt gtctgtga 28

<210> 215

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC ZFN target sequence 3

<400> 215

gtgctgtggc ctggagcaac aaatctga 28

<210> 216

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC ZFN target sequence 4

<400> 216

ttgctcttga agtccataga cctcatgt 28

<210> 217

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC ZFN target sequence 5

<400> 217

ctgtggcctg gagcaacaaa tctgact 27

<210> 218

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC ZFN target sequence 6

<400> 218

ctgttgctct tgaagtccat agacctca 28

<210> 219

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC ZFN target sequence 7

<400> 219

ctgtggcctg gagcaacaaa tctgactt 28

<210> 220

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC ZFN target sequence 8

<400> 220

ctgactttgc atgtgcaaac gccttcaa 28

<210> 221

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC ZFN target sequence 9

<400> 221

ttgttgctcc aggccacagc actgttgc 28

<210> 222

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC ZFN target sequence 10

<400> 222

tgaaagtggc cgggtttaat ctgctcat 28

<210> 223

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC ZFN target sequence 11

<400> 223

aggaggattc ggaacccaat cactgaca 28

<210> 224

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC ZFN target sequence 12

<400> 224

gaggaggatt cggaacccaa tcactgac 28

<210> 225

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> TRBC ZFN target sequence 1

<400> 225

ccgtagaact ggacttgaca gcggaagt 28

<210> 226

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> TRBC ZFN target sequence 2

<400> 226

tctcggagaa tgacgagtgg acccagga 28

<210> 227

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC 50 bp 5' homology arm

<400> 227

agctgagaga ctctaaatcc agtgacaagt ctgtctgcct attcaccgat 50

<210> 228

<211> 100

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC 100 bp 5' homology arm

<400> 228

ctgatcctct tgtcccacag atatccagaa ccctgaccct gccgtgtacc agctgagaga 60

ctctaaatcc agtgacaagt ctgtctgcct attcaccgat 100

<210> 229

<211> 200

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC 200 bp 5' homology arm

<400> 229

gtgacttgcc agccccacag agccccgccc ttgtccatca ctggcatctg gactccagcc 60

tgggttgggg caaagaggga aatgagatca tgtcctaacc ctgatcctct tgtcccacag 120

atatccagaa ccctgaccct gccgtgtacc agctgagaga ctctaaatcc agtgacaagt 180

ctgtctgcct attcaccgat 200

<210> 230

<211> 300

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC 300 bp 5' homology arm

<400> 230

cctcttggcc aagattgata gcttgtgcct gtccctgagt cccagtccat cacgagcagc 60

tggtttctaa gatgctattt cccgtataaa gcatgagacc gtgacttgcc agccccacag 120

agccccgccc ttgtccatca ctggcatctg gactccagcc tgggttgggg caaagaggga 180

aatgagatca tgtcctaacc ctgatcctct tgtcccacag atatccagaa ccctgaccct 240

gccgtgtacc agctgagaga ctctaaatcc agtgacaagt ctgtctgcct attcaccgat 300

<210> 231

<211> 400

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC 400 bp 5' homology arm

<400> 231

attaaataaa agaataagca gtattattaa gtagccctgc atttcaggtt tccttgagtg 60

gcaggccagg cctggccgtg aacgttcact gaaatcatgg cctcttggcc aagattgata 120

gcttgtgcct gtccctgagt cccagtccat cacgagcagc tggtttctaa gatgctattt 180

cccgtataaa gcatgagacc gtgacttgcc agccccacag agccccgccc ttgtccatca 240

ctggcatctg gactccagcc tgggttgggg caaagaggga aatgagatca tgtcctaacc 300

ctgatcctct tgtcccacag atatccagaa ccctgaccct gccgtgtacc agctgagaga 360

ctctaaatcc agtgacaagt ctgtctgcct attcaccgat 400

<210> 232

<211> 500

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC 500 bp 5' homology arm

<400> 232

ctccagattc caagatgtac agtttgcttt gctgggcctt tttcccatgc ctgcctttac 60

tctgccagag ttatattgct ggggttttga agaagatcct attaaataaa agaataagca 120

gtattattaa gtagccctgc atttcaggtt tccttgagtg gcaggccagg cctggccgtg 180

aacgttcact gaaatcatgg cctcttggcc aagattgata gcttgtgcct gtccctgagt 240

cccagtccat cacgagcagc tggtttctaa gatgctattt cccgtataaa gcatgagacc 300

gtgacttgcc agccccacag agccccgccc ttgtccatca ctggcatctg gactccagcc 360

tgggttgggg caaagaggga aatgagatca tgtcctaacc ctgatcctct tgtcccacag 420

atatccagaa ccctgaccct gccgtgtacc agctgagaga ctctaaatcc agtgacaagt 480

ctgtctgcct attcaccgat 500

<210> 233

<211> 600

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC 600 bp 5' homology arm

<400> 233

agagagcaat ctcctggtaa tgtgatagat ttcccaactt aatgccaaca taccataaac 60

ctcccattct gctaatgccc agcctaagtt ggggagacca ctccagattc caagatgtac 120

agtttgcttt gctggggcctt tttcccatgc ctgcctttac tctgccagag ttatattgct 180

ggggttttga agaagatcct attaaataaa agaataagca gtattattaa gtagccctgc 240

atttcaggtt tccttgagtg gcaggccagg cctggccgtg aacgttcact gaaatcatgg 300

cctcttggcc aagattgata gcttgtgcct gtccctgagt cccagtccat cacgagcagc 360

tggtttctaa gatgctattt cccgtataaa gcatgagacc gtgacttgcc agccccacag 420

agccccgccc ttgtccatca ctggcatctg gactccagcc tgggttgggg caaagaggga 480

aatgagatca tgtcctaacc ctgatcctct tgtcccacag atatccagaa ccctgaccct 540

gccgtgtacc agctgagaga ctctaaatcc agtgacaagt ctgtctgcct attcaccgat 600

<210> 234

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC 50 bp 3' homology arm

<400> 234

tttgattctc aaacaaatgt gtcacaaagt aaggattctg atgtgtatat 50

<210> 235

<211> 100

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC 100 bp 3' homology arm

<400> 235

tttgattctc aaacaaatgt gtcacaaagt aaggattctg atgtgtatat cacagacaaa 60

actgtgctag acatgaggtc tatggacttc aagagcaaca 100

<210> 236

<211> 200

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC 200 bp 3' homology arm

<400> 236

tttgattctc aaacaaatgt gtcacaaagt aaggattctg atgtgtatat cacagacaaa 60

actgtgctag acatgaggtc tatggacttc aagagcaaca gtgctgtggc ctggagcaac 120

aaatctgact ttgcatgtgc aaacgccttc aacaacagca ttattccaga agacaccttc 180

ttccccagcc caggtaaggg 200

<210> 237

<211> 300

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC 300 bp 3' homology arm

<400> 237

tttgattctc aaacaaatgt gtcacaaagt aaggattctg atgtgtatat cacagacaaa 60

actgtgctag acatgaggtc tatggacttc aagagcaaca gtgctgtggc ctggagcaac 120

aaatctgact ttgcatgtgc aaacgccttc aacaacagca ttattccaga agacaccttc 180

ttccccagcc caggtaaggg cagctttggt gccttcgcag gctgtttcct tgcttcagga 240

atggccaggt tctgcccaga gctctggtca atgatgtcta aaactcctct gattggtggt 300

<210> 238

<211> 400

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC 400 bp 3' homology arm

<400> 238

tttgattctc aaacaaatgt gtcacaaagt aaggattctg atgtgtatat cacagacaaa 60

actgtgctag acatgaggtc tatggacttc aagagcaaca gtgctgtggc ctggagcaac 120

aaatctgact ttgcatgtgc aaacgccttc aacaacagca ttattccaga agacaccttc 180

ttccccagcc caggtaaggg cagctttggt gccttcgcag gctgtttcct tgcttcagga 240

atggccaggt tctgcccaga gctctggtca atgatgtcta aaactcctct gattggtggt 300

ctcggcctta tccattgcca ccaaaaccct ctttttacta agaaacagtg agccttgttc 360

tggcagtcca gagaatgaca cgggaaaaaa gcagatgaag 400

<210> 239

<211> 500

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC 500 bp 3' homology arm

<400> 239

tttgattctc aaacaaatgt gtcacaaagt aaggattctg atgtgtatat cacagacaaa 60

actgtgctag acatgaggtc tatggacttc aagagcaaca gtgctgtggc ctggagcaac 120

aaatctgact ttgcatgtgc aaacgccttc aacaacagca ttattccaga agacaccttc 180

ttccccagcc caggtaaggg cagctttggt gccttcgcag gctgtttcct tgcttcagga 240

atggccaggt tctgcccaga gctctggtca atgatgtcta aaactcctct gattggtggt 300

ctcggcctta tccattgcca ccaaaaccct ctttttacta agaaacagtg agccttgttc 360

tggcagtcca gagaatgaca cgggaaaaaa gcagatgaag agaaggtggc aggagagggc 420

acgtggccca gcctcagtct ctccaactga gttcctgcct gcctgccttt gctcagactg 480

tttgcccctt actgctcttc 500

<210> 240

<211> 600

<212> DNA

<213> Artificial sequence

<220>

<223> TRAC 600 bp 3' homology arm

<400> 240

tttgattctc aaacaaatgt gtcacaaagt aaggattctg atgtgtatat cacagacaaa 60

actgtgctag acatgaggtc tatggacttc aagagcaaca gtgctgtggc ctggagcaac 120

aaatctgact ttgcatgtgc aaacgccttc aacaacagca ttattccaga agacaccttc 180

ttccccagcc caggtaaggg cagctttggt gccttcgcag gctgtttcct tgcttcagga 240

atggccaggt tctgcccaga gctctggtca atgatgtcta aaactcctct gattggtggt 300

ctcggcctta tccattgcca ccaaaaccct ctttttacta agaaacagtg agccttgttc 360

tggcagtcca gagaatgaca cgggaaaaaa gcagatgaag agaaggtggc aggagagggc 420

acgtggccca gcctcagtct ctccaactga gttcctgcct gcctgccttt gctcagactg 480

tttgcccctt actgctcttc taggcctcat tctaagcccc ttctccaagt tgcctctcct 540

tatttctccc tgtctgccaa aaaatctttc ccagctcact aagtcagtct cacgcagtca 600

<210> 241

<211> 141

<212> PRT

<213> Artificial sequence

<220>

<223> Human alpha constant TCR

<400> 241

Asn Ile Gln Lys Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys

1 5 10 15

Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr

20 25 30

Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr

35 40 45

Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala

50 55 60

Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser

65 70 75 80

Ile Ile Pro Ala Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp

85 90 95

Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe

100 105 110

Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala

115 120 125

Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

130 135 140

<210> 242

<211> 141

<212> PRT

<213> Artificial sequence

<220>

<223> Human alpha constant TCR

<400> 242

His Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys

1 5 10 15

Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr

20 25 30

Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr

35 40 45

Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala

50 55 60

Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser

65 70 75 80

Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp

85 90 95

Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe

100 105 110

Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala

115 120 125

Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

130 135 140

<210> 243

<211> 141

<212> PRT

<213> Artificial sequence

<220>

<223> Human alpha constant TCR

<400> 243

Tyr Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys

1 5 10 15

Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr

20 25 30

Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr

35 40 45

Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala

50 55 60

Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser

65 70 75 80

Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp

85 90 95

Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe

100 105 110

Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala

115 120 125

Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

130 135 140

<210> 244

<211> 141

<212> PRT

<213> Artificial sequence

<220>

<223> Human alpha constant TCR

<400> 244

Asp Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys

1 5 10 15

Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr

20 25 30

Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr

35 40 45

Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala

50 55 60

Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser

65 70 75 80

Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp

85 90 95

Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe

100 105 110

Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala

115 120 125

Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

130 135 140

<210> 245

<211> 142

<212> PRT

<213> Artificial sequence

<220>

<223> Human alpha constant TCR

<400> 245

Pro Asn Ile Gln Lys Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser

1 5 10 15

Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln

20 25 30

Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys

35 40 45

Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val

50 55 60

Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn

65 70 75 80

Ser Ile Ile Pro Ala Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys

85 90 95

Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn

100 105 110

Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val

115 120 125

Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

130 135 140

<210> 246

<211> 141

<212> PRT

<213> Artificial sequence

<220>

<223> Human alpha constant TCR

<400> 246

Asn Ile Gln Lys Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys

1 5 10 15

Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr

20 25 30

Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Cys

35 40 45

Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala

50 55 60

Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser

65 70 75 80

Ile Ile Pro Ala Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp

85 90 95

Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe

100 105 110

Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala

115 120 125

Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

130 135 140

<210> 247

<211> 141

<212> PRT

<213> Artificial sequence

<220>

<223> Human alpha constant TCR

<400> 247

His Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys

1 5 10 15

Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr

20 25 30

Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Cys

35 40 45

Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala

50 55 60

Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser

65 70 75 80

Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp

85 90 95

Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe

100 105 110

Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala

115 120 125

Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

130 135 140

<210> 248

<211> 140

<212> PRT

<213> Artificial sequence

<220>

<223> Human alpha constant TCR

<400> 248

Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser

1 5 10 15

Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn

20 25 30

Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Cys Val

35 40 45

Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp

50 55 60

Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile

65 70 75 80

Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val

85 90 95

Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln

100 105 110

Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly

115 120 125

Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

130 135 140

<210> 249

<211> 141

<212> PRT

<213> Artificial sequence

<220>

<223> Human alpha constant TCR

<400> 249

Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys

5 10 15

Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr

20 25 30

Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Cys

35 40 45

Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala

50 55 60

Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser

65 70 75 80

Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp

85 90 95

Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe

100 105 110

Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala

115 120 125

Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

130 135 140

<210> 250

<211> 141

<212> PRT

<213> Artificial sequence

<220>

<223> Human alpha constant TCR

<400> 250

Asp Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys

1 5 10 15

Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr

20 25 30

Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Cys

35 40 45

Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala

50 55 60

Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser

65 70 75 80

Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp

85 90 95

Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe

100 105 110

Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala

115 120 125

Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

130 135 140

<210> 251

<211> 177

<212> PRT

<213> Artificial sequence

<220>

<223> Human TCR beta constant

<400> 251

Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro

1 5 10 15

Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu

20 25 30

Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val Asn

35 40 45

Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Pro Leu Lys

50 55 60

Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu

65 70 75 80

Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys

85 90 95

Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp

100 105 110

Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg

115 120 125

Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln Gly Val Leu Ser

130 135 140

Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala

145 150 155 160

Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp

165 170 175

Phe

<210> 252

<211> 180

<212> PRT

<213> Artificial sequence

<220>

<223> Human TCR beta constant

<400> 252

Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu

1 5 10 15

Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys

20 25 30

Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val

35 40 45

Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu

50 55 60

Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg

65 70 75 80

Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg

85 90 95

Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln

100 105 110

Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly

115 120 125

Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val Leu

130 135 140

Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr

145 150 155 160

Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys

165 170 175

Asp Ser Arg Gly

180

<210> 253

<211> 180

<212> PRT

<213> Artificial sequence

<220>

<223> Human TCR beta constant

<400> 253

Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu

1 5 10 15

Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys

20 25 30

Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val

35 40 45

Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu

50 55 60

Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg

65 70 75 80

Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg

85 90 95

Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln

100 105 110

Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly

115 120 125

Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val Leu

130 135 140

Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr

145 150 155 160

Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys

165 170 175

Asp Ser Arg Gly

180

<210> 254

<211> 179

<212> PRT

<213> Artificial sequence

<220>

<223> Human TCR beta constant

<400> 254

Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro

1 5 10 15

Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu

20 25 30

Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn

35 40 45

Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Pro Leu Lys

50 55 60

Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu

65 70 75 80

Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys

85 90 95

Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp

100 105 110

Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg

115 120 125

Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val Leu Ser

130 135 140

Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala

145 150 155 160

Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp

165 170 175

Ser Arg Gly

<210> 255

<211> 180

<212> PRT

<213> Artificial sequence

<220>

<223> Human TCR beta constant

<400> 255

Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu

1 5 10 15

Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys

20 25 30

Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val

35 40 45

Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Pro Leu

50 55 60

Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg

65 70 75 80

Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg

85 90 95

Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln

100 105 110

Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly

115 120 125

Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val Leu

130 135 140

Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr

145 150 155 160

Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys

165 170 175

Asp Ser Arg Gly

180

<210> 256

<211> 180

<212> PRT

<213> Artificial sequence

<220>

<223> Human TCR beta constant

<400> 256

Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu

1 5 10 15

Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys

20 25 30

Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val

35 40 45

Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Pro Leu

50 55 60

Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg

65 70 75 80

Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg

85 90 95

Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln

100 105 110

Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly

115 120 125

Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val Leu

130 135 140

Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr

145 150 155 160

Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys

165 170 175

Asp Ser Arg Gly

180

<---

Claims (86)

1. A method for producing genetically engineered T cells, comprising: (a) introducing into T cells a CRISPR agent comprising a guide RNA (gRNA) having a targeting domain that is complementary to at least one target site within exon 1 of the T cell receptor alpha constant (TRAC) gene, wherein said CRISPR agent is capable of causing genetic disruption of at least one target site, resulting in efficient knockout of an endogenous T cell receptor (TCR); and (b) introducing into said T cells a template polynucleotide, wherein said template polynucleotide comprises the structure [5' homology arm]-[transgene]-[3' homology arm], wherein: said transgene encodes a recombinant TCR comprising an alpha (TCRα) chain and a beta (TCRβ) chain, wherein said transgene comprises a nucleic acid sequence encoding a TCRα chain and a nucleic acid sequence encoding a TCRβ chain, and wherein said recombinant TCR is capable of binding to an antigen that is associated with, specific for and/or expressed on a cell or tissue of a disease, disorder or condition, And the homology arms are approximately 600 base pairs each on the 5' and 3' side of the nucleic acid sequence encoding said recombinant TCR for targeting exon 1 of the endogenous TRAC gene, wherein the transgene encoding said recombinant TCR is targeted for integration at or near one of at least one target site via homology-directed repair (HDR), and wherein the introduction of the matrix polynucleotide is carried out after the introduction of the said CRISPR agent. 2. The method according to claim 1, wherein the template polynucleotide is administered immediately after or within 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours or 4 hours after administration of said CRISPR agent, optionally within 2 hours after administration of said CRISPR agent. 3. The method according to claim 1 or 2, wherein the T cells comprise CD4+ T cells or their subtypes, or CD8+ T cells or their subtypes. 4. The method according to claim 3, wherein the T cells comprise CD4+ and CD8+ T cells, and the ratio of CD4+ to CD8+ T cells is from 1:3 to 3:1, optionally from 1:2 to 2:1, optionally 1:1. 5. The method according to any one of claims 1 to 4, wherein the CRISPR agent comprises a CRISPR-Cas9 combination. 6. The method according to claim 5, wherein the CRISPR-Cas9 combination is a ribonucleoprotein (RNP) complex containing gRNA and Cas9 protein. 7. The method according to claim 6, wherein the concentration of RNP is from 1 μM to 5 μM, optionally wherein the concentration of RNP is 2 μM. 8. The method according to any one of claims 1 to 7, wherein the introduction of said CRISPR agent is carried out by electroporation. 9. The method according to any one of claims 1 to 8, wherein the template polynucleotide is contained in a viral vector, and the introduction of the template polynucleotide is carried out by transduction. 10. The method according to claim 9, wherein said viral vector is an AAV vector. 11. The method according to any one of claims 1-10, wherein prior to the introduction of said CRISPR agent, the method comprises incubating said T cells, in vitro, with a stimulating agent(s) under conditions of stimulation or activation of said T cells. 12. The method according to claim 11, wherein said stimulating agent(s) comprises (contain) an anti-CD3 antibody and/or an anti-CD28 antibody, optionally wherein said stimulating agent(s) comprises (contain) anti-CD3/anti-CD28 spheres, optionally wherein the ratio of spheres to cells is 1:1. 13. The method according to claim 11 or 12, comprising removing said stimulatory agent(s) from said T cells prior to administering said CRISPR agent. 14. The method according to any one of claims 1-13, wherein the method further comprises incubating the cells before, during or after the administration of said CRISPR agent and/or the administration of the template polynucleotide with one or more recombinant cytokines, optionally wherein the one or more recombinant cytokines are selected from the group consisting of IL-2, IL-7, and IL-15. 15. The method according to claim 14, wherein said one or more recombinant cytokines are added at a concentration selected from IL-2 at a concentration of 10 U/ml to 200 U/ml, optionally at 50 IU/ml to 100 U/ml; IL-7 at a concentration of 0.5 ng/ml to 50 ng/ml, optionally at 5 ng/ml to 10 ng/ml and/or IL-15 at a concentration of 0.1 ng/ml to 20 ng/ml, optionally at 0.5 ng/ml to 5 ng/ml. 16. The method according to claim 14 or 15, wherein the incubation is carried out after the administration of said CRISPR agent and the administration of the template polynucleotide for up to about 24 hours, 36 hours, or 48 hours, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days, optionally up to 7 days. 17. The method according to any one of claims 1 to 16, wherein said transgene encoding a recombinant TCR further comprises a heterologous regulatory or control element, optionally wherein said heterologous regulatory or control element comprises a heterologous promoter. 18. A method for producing genetically engineered T cells, comprising: (a) introducing into T cells a CRISPR agent comprising a guide RNA (gRNA) having a targeting domain that is complementary to at least one target site within exon 1 of the T cell receptor alpha constant (TRAC) gene and/or the T cell receptor beta constant (TRBC) gene, wherein said CRISPR agent is capable of causing genetic disruption of at least one target site, resulting in efficient knockout of an endogenous T cell receptor (TCR); and (b) introducing into said T cells a template polynucleotide, wherein said template polynucleotide comprises the structure [5' homology arm]-[transgene]-[3' homology arm], wherein said transgene encodes a recombinant TCR comprising an alpha (TCRα) chain and a beta (TCRβ) chain, wherein said transgene comprises a nucleic acid sequence encoding a TCRα chain and a nucleic acid sequence encoding a TCRβ chain, and wherein the homology arms are approximately 600 base pairs each on the 5' and 3' side of a nucleic acid sequence encoding said recombinant TCR for targeting exon 1 of an endogenous TRAC gene, wherein said transgene comprises a heterologous promoter, and wherein the transgene is targeted for integration at or near one of at least one target site via homology-directed repair (HDR). 19. The method according to claim 18, wherein the introduction of said CRISPR agent and the introduction of the template polynucleotide are carried out simultaneously or sequentially, in any order. 20. The method according to claim 18 or 19, wherein the introduction of the matrix polynucleotide is carried out after the introduction of the CRISPR agent. 21. The method according to claim 20, wherein the template polynucleotide is administered immediately after or within 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours or 4 hours after administration of said CRISPR agent or within 2 hours after administration of said CRISPR agent. 22. The method according to claim 18 or 19, wherein the introduction of said CRISPR agent and the introduction of the template polynucleotide are carried out in a single experimental reaction. 23. The method according to any one of claims 18-22, wherein prior to the introduction of said CRISPR agent, the method comprises incubating cells, in vitro, with a stimulating agent(s) under conditions of stimulation or activation of said T cells. 24. The method according to claim 23, wherein said stimulating agent(s) comprises (comprise) an anti-CD3 antibody and/or an anti-CD28 antibody, optionally wherein said stimulating agent(s) comprises (comprise) anti-CD3/anti-CD28 beads, optionally wherein the ratio of beads to cells is about 1:1. 25. The method of claim 23 or 24, comprising removing said stimulatory agent(s) from said T cells prior to administering said CRISPR agent. 26. The method according to any one of claims 18-25, wherein the method further comprises incubating the cells before, during or after the administration of said CRISPR agent and/or the administration of the template polynucleotide with one or more recombinant cytokines, optionally wherein said one or more recombinant cytokines are selected from the group consisting of IL-2, IL-7, and IL-15. 27. The method according to claim 26, wherein said one or more recombinant cytokines are added at a concentration selected from IL-2 at a concentration of 10 U/ml to 200 U/ml, optionally at a concentration of 50 IU/ml to 100 U/ml; IL-7 at a concentration of 0.5 ng/ml to 50 ng/ml, optionally at a concentration of 5 ng/ml to 10 ng/ml and/or IL-15 at a concentration of 0.1 ng/ml to 20 ng/ml, optionally at a concentration of 0.5 ng/ml to 5 ng/ml. 28. The method according to claim 26 or 27, wherein the incubation is carried out after the administration of said CRISPR agent and the administration of the template polynucleotide for up to about 24 hours, 36 hours, or 48 hours, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days, optionally up to 7 days. 29. A method for producing genetically engineered T cells, comprising: introducing into T cells having a genetic disruption of at least one target site within exon 1 of the T cell receptor alpha constant (TRAC) gene, resulting in efficient knockout of the endogenous T cell receptor (TCR), a template polynucleotide, wherein said template polynucleotide comprises the structure [5' homology arm]-[transgene]-[3' homology arm], wherein: said transgene encodes a recombinant TCR comprising an alpha (TCRα) chain and a beta (TCRβ) chain, wherein said transgene comprises a nucleic acid sequence encoding a TCRα chain and a nucleic acid sequence encoding a TCRβ chain, and wherein the homology arms are approximately 600 base pairs each on the 5' and 3' side of the nucleic acid sequence encoding said recombinant TCR for targeting exon 1 of the endogenous TRAC gene, wherein said transgene encoding a recombinant TCR comprises a heterologous promoter, wherein the genetic disruption is caused by a CRISPR agent comprising a guide RNA (gRNA) having a targeting domain that is complementary to said at least one target site, and said transgene encoding a recombinant TCR is targeted for integration at or near one of the at least one target sites via homology-directed repair (HDR). 30. The method according to any one of claims 18-29, wherein said CRISPR agent comprises a CRISPR-Cas9 combination. 31. The method according to claim 30, wherein the CRISPR-Cas9 combination is a ribonucleoprotein (RNP) complex containing gRNA and Cas9 protein. 32. The method of claim 31, wherein the concentration of RNP is from 1 μM to 5 μM, optionally wherein the concentration of RNP is 2 μM. 33. The method according to claim 31 or 32, wherein the RNP is administered by electroporation. 34. The method according to any one of claims 18-33, wherein the T cells comprise CD8+ T cells or subtypes thereof. 35. The method according to any one of claims 18-33, wherein the T cells comprise CD4+ T cells or subtypes thereof. 36. The method according to any one of claims 18-33, wherein the T cells comprise CD4+ T cells or their subtypes and CD8+ T cells or their subtypes. 37. The method according to claim 36, wherein the ratio of CD4+ to CD8+ T cells is from 1:3 to 3:1, optionally from 1:2 to 2:1, optionally 1:1. 38. The method according to any one of claims 18 to 37, wherein the template polynucleotide is contained in a vector, optionally a viral vector. 39. The method according to claim 38, wherein said vector is a viral vector, and said viral vector is an AAV vector. 40. The method according to claim 39, wherein said AAV vector is selected from the group consisting of AAV1 vector, AAV2 vector, AAV3 vector, AAV4 vector, AAV5 vector, AAV6 vector, AAV7 vector and AAV8 vector. 41. The method according to claim 40, wherein said AAV vector is an AAV2 vector or an AAV6 vector. 42. The method according to claim 38, wherein said vector is a viral vector, and said viral vector is a retroviral vector, optionally a lentiviral vector. 43. The method according to any one of claims 1 to 42, wherein the gRNA comprises a sequence selected from the group consisting of UCUCUCAGCUGGUACACGGC (SEQ ID NO:28), UGGAUUUAGAGUCUCUCAGC (SEQ ID NO:29), ACACGGCAGGGUCAGGGUUC (SEQ ID NO:30), GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:31), GCUGGUACACGGCAGGGUCA (SEQ ID NO:32), CUCAGCUGGUACACGGC (SEQ ID NO:33), UGGUACACGGCAGGGUC (SEQ ID NO:34), GCUAGACAUGAGGUCUA (SEQ ID NO:35), GUCAGAUUUGUUGCUCC (SEQ ID NO:36), UCAGCUGGUACACGGCA (SEQ ID NO:37), GCAGACAGACUUGUCAC (SEQ ID NO:38), ID NO:38), GGUACACGGCAGGGUCA (SEQ ID NO:39), CUUCAAGAGCAACAGUGCUG (SEQ ID NO:40), AGAGCAACAGUGCUGUGGCC (SEQ ID NO:41), AAAGUCAGAUUUGUUGCUCC (SEQ ID NO:42), ACAAAACUGUGCUAGACAUG (SEQ ID NO:43), AAACUGUGCUAGACAUG (SEQ ID NO:44), UGUGCUAGACAUGAGGUCUA (SEQ ID NO:45), GGCUGGGGAAGAAGGUGUCUUC (SEQ ID NO:46), GCUGGGGAAGAAGGUGUCUUC (SEQ ID NO:47), GGGGAAGAAGGUGUCUUC (SEQ ID NO:48), GUUUUGUCUGUGAUAUACACAU (SEQ ID NO:49), GGCAGACAGACUUGUCACUGGAUU (SEQ ID NO:50), GCAGACAGACUUGUCACUGGAUU (SEQ ID NO:51), GACAGACUUGUCACUGGAUU (SEQ ID NO:52), GUGAAUAGGCAGACAGACUUGUCA (SEQ ID NO:53), GAAUAGGCAGACAGACUUGUCA (SEQ ID NO:54), GAGUCUCUCAGCUGGUACACGG (SEQ ID NO:55), GUCUCUCAGCUGGUACACGG (SEQ ID NO:56), GGUACACGGCAGGGUCAGGGUU (SEQ ID NO:57) and GUACACGGCAGGGUCAGGGUU (SEQ ID NO:58). 44. The method according to any one of claims 1 to 43, wherein the gRNA has a targeting domain comprising the sequence GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:31). 45. The method according to any one of claims 1 to 44, wherein the 5' homology arm and the 3' homology arm comprise nucleic acid sequences homologous to nucleic acid sequences surrounding at least one target site. 46. The method according to claim 45, wherein the 5’ homology arm comprises the sequence presented in SEQ ID NO: 124, and/or the 3’ homology arm comprises the sequence presented in SEQ ID NO: 125. 47. The method according to any one of claims 1 to 46, wherein the transgene further comprises one or more multicistronic elements, and the multicistronic elements are located between the nucleic acid sequence encoding TCRα or a portion thereof and the nucleic acid sequence encoding TCRβ or a portion thereof. 48. The method according to claim 47, wherein the multicistronic elements comprise a sequence encoding a ribosome skip element selected from T2A, P2A, E2A or F2A or an internal ribosome entry site (IRES). 49. The method according to any one of claims 1 to 48, wherein the method further comprises introducing into said T cells one or more second template polynucleotides comprising one or more second transgenes, wherein said one or more second transgenes are targeted for integration at or near one of at least one target site via homology-directed repair (HDR). 50. The method of claim 49, wherein said one or more second transgenes encode a molecule selected from a costimulatory ligand, a cytokine, a soluble single-chain variable fragment (scFv), an immunomodulatory fusion protein, a chimeric switch receptor (CSR), or a coreceptor. 51. The method according to claim 49 or 50, wherein said one or more second transgenes independently further comprise a heterologous regulatory or control element, optionally a promoter. 52. The method according to any one of claims 17 to 51, wherein said heterologous promoter is or comprises the human elongation factor 1 alpha (EF1α) promoter or the MND promoter or a variant thereof. 53. The method according to any one of claims 17 to 51, wherein said heterologous promoter is an inducible promoter or a repressible promoter. 54. The method according to any one of claims 1 to 53, wherein the TCRα chain comprises a constant region (Cα) region into which one or more cysteine residues have been introduced, and/or the TCRβ chain comprises a constant region (Cβ) region into which one or more cysteine residues have been introduced, wherein the one or more introduced cysteine residues are capable of forming one or more non-native disulfide bridges between the alpha chain and the beta chain. 55. The method of claim 54, wherein introducing one or more cysteine residues comprises replacing a non-cysteine residue with a cysteine residue. 56. The method according to claim 54 or 55, wherein the Cα region comprises a cysteine at a position corresponding to position 48 with the numbering indicated in any of SEQ ID NO: 24; and/or the Cβ region comprises a cysteine at a position corresponding to position 57 with the numbering indicated in SEQ ID NO: 20. 57. The method according to any one of claims 1 to 56, wherein said T cells are obtained from a multipotent or pluripotent cell, which is not necessarily an iPSC. 58. The method according to any one of claims 1-56, wherein said T cells are primary cells from the subject. 59. The method of claim 58, wherein the subject has or is suspected of having a disease, disorder or condition. 60. The method according to claim 59, wherein the subject is or is presumed to be healthy. 61. The method according to claim 58 or 59, wherein said T cells are autologous to the subject. 62. The method according to any one of claims 58-61, wherein said T cells are allogeneic to the subject. 63. The method according to any one of claims 1 to 62, wherein the template polynucleotide is at least 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4760, 5000, 5250, 5500, 5750, 6000, 7000, 7500, 8000, 9000 or 10000 nucleotides in length, or any value between any of the above. 64. The method according to any one of claims 1 to 63, wherein said template polynucleotide is between 2500 and 5000 nucleotides, 3500 and 4500 nucleotides, or 3750 nucleotides and 4250 nucleotides in length. 65. The method according to any one of claims 1 to 64, wherein at least or more than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or 90% of the T cells in the plurality of genetically engineered T cells obtained by said method comprise a genetic disruption of said at least one target site within exon 1 of the TRAC gene. 66. The method according to any one of claims 1 to 65, wherein at least or more than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or 90% of the cells in the plurality of genetically engineered T cells obtained by said method express said recombinant TCR. 67. The method according to any one of claims 1 to 66, wherein the coefficient of variation of expression of the recombinant TCR among the plurality of genetically engineered T cells obtained by said method is lower than 0.70, 0.65, 0.60, 0.55, 0.50, 0.45, 0.40, 0.35 or 0.30 or less. 68. The method according to any one of claims 1 to 67, wherein the coefficient of variation of the expression of the recombinant TCR among the plurality of genetically engineered T cells obtained by said method is at least 100%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% lower than the coefficient of variation of the expression of the same recombinant TCR among the plurality of genetically engineered T cells obtained by random integration. 69. A kit for implementing the method according to any one of claims 1 to 68, wherein said kit comprises: A CRISPR-Cas9 combination comprising a guide RNA (gRNA) having a targeting domain that is complementary to at least one target site in the T-cell receptor alpha constant (TRAC) gene, wherein said CRISPR-Cas9 combination is a ribonucleoprotein (RNP) complex comprising the gRNA and the Cas9 protein; and a template polynucleotide that is contained in a viral vector that is an AAV vector, wherein said template polynucleotide comprises the structure [5' homology arm]-[transgene]-[3' homology arm], wherein said transgene encodes a recombinant T cell receptor (TCR) comprising an alpha (TCRα) chain and a beta (TCRβ) chain, wherein said transgene comprises a nucleic acid sequence encoding a TCRα chain and a nucleic acid sequence encoding a TCRβ chain, and wherein said transgene is under the control of a constitutive promoter; and wherein the homology arms are approximately 600 base pairs each on the 5' and 3' side of a nucleic acid sequence encoding said recombinant TCR to be targeted to exon 1 of an endogenous TRAC gene, wherein said transgene encoding said recombinant TCR is targeted to integrate into or near said at least one target site via homology-directed repair (HDR), wherein said recombinant receptor is capable of binding to an antigen that is associated with, specific for, and/or expressed on a cell or tissue of a disease, disorder, or condition, wherein said disease, disorder, or condition is cancer; and instructions for carrying out the method according to any of paragraphs 1-68.

Images