Genetically modified t cells comprising modified intron of t cell receptor alpha gene

Abstract

To provide methods for producing genetically modified human T cells, genetically modified human T cells, and pharmaceutical compositions.SOLUTION: The invention provides genetically modified T cells comprising a modified human T cell receptor alpha gene in the genome. The modified T cell receptor alpha gene comprises a target exogenous sequence inserted into an intron in the modified T cell receptor alpha gene positioned 5' upstream of TRAC exon 1. The target exogenous sequence may contain an exogenous splice acceptor site and / or poly A signal, and inhibits the expression of T cell receptor alpha subunit. Additionally, the target sequence may contain a coding sequence of a polypeptide such as a chimeric antigen receptor. Further, the endogenous splice donor site and endogenous splice acceptor site adjacent to the intron are not modified and / or staying functional.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the fields of oncology, cancer immunotherapy, molecular biology, and recombinant nucleic acid technology. The present invention relates to a modified T cell receptor alpha gene located 5' upstream of TRAC exon 1. The present invention relates to genetically modified T cells containing introns as well as compositions and methods for producing the same. The invention further relates to the use of such cells to treat diseases, including cancer, in a subject. It concerns the method.

[0002] Reference to Sequence Listing Submitted as a Text File via EFS-WEB This application contains a sequence listing submitted in ASCII format via EFS-Web. , which is incorporated herein by reference in its entirety. The resulting ASCII copy is named P109070024US01-SEQ-HJD. and its size is 124,035 bytes. [Background technology]

[0003] T cell adoptive immunotherapy is a promising approach for cancer treatment. This strategy targets specific tumor-associated Using isolated human T cells genetically modified to enhance specificity for related antigens Genetic modifications include the use of chimeric antigen receptors or xenogeneic antigens to graft antigen specificity onto T cells. In contrast to exogenous T cell receptors, chimeric T cell receptors may be involved. Antigen receptors derive their specificity from the variable domains of monoclonal antibodies. T cells expressing antigen receptors (CAR T cells) can target major histocompatibility complexes in a non-restrictive manner. Induce tumor immune reactivity in combination with T cell adoptive immunotherapy. Acute lymphoblastic leukemia (ALL), B-cell non-Hodgkin lymphoma (NHL), and chronic lymphocytic leukemia), multiple myeloma, neuroblastoma, glioblastoma, progressive glioma, ovarian cancer, mesothelioma , melanoma, and pancreatic cancer have been used as clinical therapies for many cancers.

[0004] Despite their potential usefulness as cancer treatments, adoptive immunotherapies using CAR T cells are partially limited by the expression of endogenous T cell receptors on the cell surface. CAR T cells that express endogenous T cell receptors can recognize major and minor histocompatibility antigens after administration to allogeneic patients, leading to the development of graft-versus-host disease (GVHD). As a result, clinical trials have mainly focused on the use of autologous CAR T cells, where a patient's T cells are isolated, genetically modified to incorporate a chimeric antigen receptor, and then reinfused into the same patient. The autologous approach provides immune tolerance to the administered CAR T cells, but this approach is limited by both the time and cost required to produce patient-specific CAR T cells after a patient's cancer has been diagnosed.

[0005] Therefore, it would be advantageous to develop "off-the-shelf" CAR T cells prepared using T cells from healthy third-party donors with reduced expression of endogenous T cell receptors that do not initiate GVHD upon administration. Such products could be generated and validated before diagnosis and made immediately available to patients as needed. Therefore, there is a need to develop allogeneic CAR T cells lacking endogenous T cell receptors to prevent the occurrence of GVHD.

[0006] Genetic modification of genomic DNA is engineered to recognize the DNA sequence of the target locus It can be carried out using an endonuclease that is site-specific and has a low cleavage frequency. Methods for generating engineered site-specific endonucleases are known in the art. For example, zinc finger nucleases (ZFNs) can be engineered to recognize and cleave a predetermined site within the genome. A ZFN is a chimeric protein that includes a zinc finger DNA-binding domain fused to the nuclease domain of the FokI restriction enzyme. The zinc finger domain can be redesigned by rational or experimental means to generate a protein that binds to a predetermined DNA sequence of approximately 18 base pairs in length. By fusing this engineered protein domain to the FokI nuclease, it is possible to target DNA cleavage with genome-level specificity. ZFNs are widely used to target the addition, removal, and replacement of genes in a wide range of eukaryotes (reviewed in Non-Patent Document 1). Similarly, TAL effector nucleases (TALENs) can be generated to cleave specific sites in genomic DNA. Like ZFNs, TALENs include an engineered site-specific DNA-binding domain fused to the FokI nuclease domain (reviewed in Non-Patent Document 2). However, in this case, the DNA-binding domain includes a tandem array of TAL effector domains, each of which specifically recognizes a single DNA base pair. A limitation of ZFNs and TALENs with respect to the practice of the present invention is that they are heterodimers, and thus, co-expression of two protein monomers is required for the production of a single-functional nuclease in cells. Methods for generating engineered site-specific endonucleases are known in the art. For example, zinc finger nucleases (ZFNs) can be engineered to recognize and cleave a predetermined site within the genome. A ZFN is a chimeric protein that includes a zinc finger DNA-binding domain fused to the nuclease domain of the FokI restriction enzyme. The zinc finger domain can be redesigned by rational or experimental means to generate a protein that binds to a predetermined DNA sequence of approximately 18 base pairs in length. By fusing this engineered protein domain to the FokI nuclease, it is possible to target DNA cleavage with genome-level specificity. ZFNs are widely used to target the addition, removal, and replacement of genes in a wide range of eukaryotes (reviewed in Non-Patent Document 1). Similarly, TAL effector nucleases (TALENs) can be generated to cleave specific sites in genomic DNA. Like ZFNs, TALENs include an engineered site-specific DNA-binding domain fused to the FokI nuclease domain (reviewed in Non-Patent Document 2). However, in this case, the DNA-binding domain includes a tandem array of TAL effector domains, each of which specifically recognizes a single DNA base pair. A limitation of ZFNs and TALENs with respect to the practice of the present invention is that they are heterodimers, and thus, co-expression of two protein monomers is required for the production of a single-functional nuclease in cells. Similarly, TAL effector nucleases (TALENs) can be generated to cleave specific sites in genomic DNA. Like ZFNs, TALENs include an engineered site-specific DNA-binding domain fused to the FokI nuclease domain (reviewed in Non-Patent Document 2). However, in this case, the DNA-binding domain includes a tandem array of TAL effector domains, each of which specifically recognizes a single DNA base pair. A limitation of ZFNs and TALENs with respect to the practice of the present invention is that they are heterodimers, and thus, co-expression of two protein monomers is required for the production of a single-functional nuclease in cells. A limitation of ZFNs and TALENs with respect to the practice of the present invention is that they are heterodimers, and thus, co-expression of two protein monomers is required for the production of a single-functional nuclease in cells. A limitation of ZFNs and TALENs with respect to the practice of the present invention is that they are heterodimers, and thus, co-expression of two protein monomers is required for the production of a single-functional nuclease in cells. A limitation of ZFNs and TALENs with respect to the practice of the present invention is that they are heterodimers, and thus, co-expression of two protein monomers is required for the production of a single-functional nuclease in cells.

[0007] Compact TALENs have an alternative endonuclease structure that circumvents the need for dimerization (Non-Patent Document 3). Compact TALENs are engineered site-specific TAL effector DNA binding domains fused to a nuclease domain derived from the I-TevI homing endonuclease . Unlike FokI, I-TevI does not need to dimerize to generate single-strand DNA breaks, so compact TALENs function as monomers . . .

[0008] Engineered endonucleases based on the CRISPR system are also known in the art (Non-Patent Document 4; Non-Patent Document 5). CRISPR endonucleases consist of two components: (1) a caspase effector nuclease, typically the microbial Cas9, Cpf1, or another suitable nuclease; and (2) a short "guide RNA" containing a targeting sequence of about 20 nucleotides that directs the nuclease to the desired location within the genome . By expressing multiple guide RNAs, each with a different targeting sequence, in the same cell, DNA cleavage can be targeted simultaneously at multiple sites in the genome . Thus, CRISPR nucleases are suitable for the present invention. The main drawback of the CRISPR system is the reported high frequency of off-target DNA cleavage, which may limit the utility of the system for the treatment of human patients (Non-Patent Document 6). . . . . .

[0009] Homing endonucleases are a group of nucleases of natural origin that recognize cleavage sites of 15 to 40 base pairs commonly found in the genomes of plants and fungi . Homing The homing endonuclease is frequently associated with parasitic DNA elements such as group I self-splicing introns and inteins, etc. By generating double-strand breaks in chromosomes, the homing endonuclease naturally promotes homologous recombination or gene insertion at specific locations in the host genome and recruits the cellular DNA repair machinery (Non-Patent Document 7). The homing endonucleases are generally classified into four families: the LAGLIDADG (SEQ ID NO: 2) family, the GIY-YIG family, the His-Cys box family, and the HNH family. These families are characterized by structural motifs that affect catalytic activity and recognition sequences. For example, members of the LAGLIDADG (SEQ ID NO: 2) family are characterized by having one or two copies of the conserved LAGLIDADG (SEQ ID NO: 2) motif (see Non-Patent Document 8). LAGLIDADG (SEQ ID NO: 2) homing endonucleases having a single copy of the LAGLIDADG (SEQ ID NO: 2) motif form homodimers, while members having two copies of the LAGLIDADG (SEQ ID NO: 2) motif are found as monomers. The LAGLIDADG (SEQ ID NO: 2) family, the GIY-YIG family, the His-Cys box family, and the HNH family. These families are characterized by structural motifs that affect catalytic activity and recognition sequences. For example, members of the LAGLIDADG (SEQ ID NO: 2) family are characterized by having one or two copies of the conserved LAGLIDADG (SEQ ID NO: 2) motif (see Non-Patent Document 8). LAGLIDADG (SEQ ID NO: 2) homing endonucleases having a single copy of the LAGLIDADG (SEQ ID NO: 2) motif form homodimers, while members having two copies of the LAGLIDADG (SEQ ID NO: 2) motif are found as monomers. characterized by having one or two copies of the conserved LAGLIDADG (SEQ ID NO: 2) motif (see Non-Patent Document 8). LAGLIDADG (SEQ ID NO: 2) homing endonucleases having a single copy of the LAGLIDADG (SEQ ID NO: 2) motif form homodimers, while members having two copies of the LAGLIDADG (SEQ ID NO: 2) motif are found as monomers. characterized by having one or two copies of the conserved LAGLIDADG (SEQ ID NO: 2) motif (see Non-Patent Document 8). LAGLIDADG (SEQ ID NO: 2) homing endonucleases having a single copy of the LAGLIDADG (SEQ ID NO: 2) motif form homodimers, while members having two copies of the LAGLIDADG (SEQ ID NO: 2) motif are found as monomers. characterized by having one or two copies of the conserved LAGLIDADG (SEQ ID NO: 2) motif (see Non-Patent Document 8). LAGLIDADG (SEQ ID NO: 2) homing endonucleases having a single copy of the LAGLIDADG (SEQ ID NO: 2) motif form homodimers, while members having two copies of the LAGLIDADG (SEQ ID NO: 2) motif are found as monomers. LAGLIDADG (SEQ ID NO: 2) homing endonucleases having a single copy of the LAGLIDADG (SEQ ID NO: 2) motif form homodimers, while members having two copies of the LAGLIDADG (SEQ ID NO: 2) motif are found as monomers. LAGLIDADG (SEQ ID NO: 2) homing endonucleases having a single copy of the LAGLIDADG (SEQ ID NO: 2) motif form homodimers, while members having two copies of the LAGLIDADG (SEQ ID NO: 2) motif are found as monomers. LAGLIDADG (SEQ ID NO: 2) homing endonucleases having a single copy of the LAGLIDADG (SEQ ID NO: 2) motif form homodimers, while members having two copies of the LAGLIDADG (SEQ ID NO: 2) motif are found as monomers.

[0010] I-CreI (SEQ ID NO: 1) is a member of the LAGLIDADG (SEQ ID NO: 2) family of homing endonucleases that recognizes and cleaves a 22-base pair recognition sequence in the chloroplast chromosome of the alga Chlamydomonas reinhardtii. The selection tendency of the wild-type I-CreI cleavage site was changed using gene selection techniques (Non-Patent Documents 9 to 12). Recently, I-CreI and other homing I-CreI (SEQ ID NO: 1) is a member of the LAGLIDADG (SEQ ID NO: 2) family of homing endonucleases that recognizes and cleaves a 22-base pair recognition sequence in the chloroplast chromosome of the alga Chlamydomonas reinhardtii. The selection tendency of the wild-type I-CreI cleavage site was changed using gene selection techniques (Non-Patent Documents 9 to 12). Recently, I-CreI and other homing I-CreI (SEQ ID NO: 1) is a member of the LAGLIDADG (SEQ ID NO: 2) family of homing endonucleases that recognizes and cleaves a 22-base pair recognition sequence in the chloroplast chromosome of the alga Chlamydomonas reinhardtii. The selection tendency of the wild-type I-CreI cleavage site was changed using gene selection techniques (Non-Patent Documents 9 to 12). Recently, I-CreI and other homing I-CreI (SEQ ID NO: 1) is a member of the LAGLIDADG (SEQ ID NO: 2) family of homing endonucleases that recognizes and cleaves a 22-base pair recognition sequence in the chloroplast chromosome of the alga Chlamydomonas reinhardtii. The selection tendency of the wild-type I-CreI cleavage site was changed using gene selection techniques (Non-Patent Documents 9 to 12). Recently, I-CreI and other homing I-CreI (SEQ ID NO: 1) is a member of the LAGLIDADG (SEQ ID NO: 2) family of homing endonucleases that recognizes and cleaves a 22-base pair recognition sequence in the chloroplast chromosome of the alga Chlamydomonas reinhardtii. The selection tendency of the wild-type I-CreI cleavage site was changed using gene selection techniques (Non-Patent Documents 9 to 12). Recently, I-CreI and other homing An endonuclease can be comprehensively redesigned to target a wide variety of DNA sites, including sites in mammalian, yeast, plant, bacterial, and viral genomes , a method for rationally designing the monoLA GLIDADG (SEQ ID NO: 2) homing endonuclease has been described (Patent Document 1).

[0011] As first described in Patent Document 2, I-CreI and its engineered derivatives are usually dimers, but can be fused into a single polypeptide using a short peptide linker that binds the C-terminus of the first subunit to the N-terminus of the second subunit (Non-Patent Documents 1 3 and 14). Thus, a functional "single-stranded" meganuclease can be expressed from a single transcript .

[0012] The use of engineered meganucleases to cleave DNA targets of the human T cell receptor alpha gene has been previously disclosed. For example, in Patent Documents 3 and 4, the applicant disclosed engineered meganucleases having specificity for the recognition sequence of exon 1 of the T cell receptor alpha constant region (TRAC: T cell receptor alpha constant region) gene. Patent Documents 3 and 4 also disclosed methods for the targeted insertion of a CAR coding sequence into the meganuclease cleavage site. Furthermore, Patent Document 5 disclosed a mutant of the I-OnuI meganuclease engineered to target a recognition sequence (SEQ ID NO: 3 in the publication of Patent Document 5) within TRAC exon 1. Patent Document 5 examined the ability of a chimeric antigen receptor to be expressed in TCR knockout cells, but the authors did not disclose inserting a CAR co ding sequence into the meganuclease cleavage site. ded sequence into the meganuclease cleavage site.

[0013] Small hairpin RNA, zinc finger nuclease (ZFN), transcriptional activator -like effector nuclease (TALEN), megaTAL, and CRISPR system (e.g., Non-Patent Document 15; Patent Documents 6 to 8), the use of other nucleases and the mechanism of interfering with the expression of endogenous TCR are also disclosed. Furthermore, Non-Patent Document 16 discloses the use of the CRISPR / Cas9 system targeting the insertion of the CAR coding sequence into a site spanning both the 5'-end of TRAC exon 1 and the endogenous splice acceptor site located immediately upstream of the 5'-end of TRAC exon 1. The authors

[0014] describe that the predicted double-strand break site of Cas9 nuclease is within the splice acceptor site (see Eyquem, Supplementary Figure 1A). This splice acceptor site is necessary for TCR expression, as evidenced by the fact that disruption of the site by Cas9 causes TCR knockout in 70% of T cells in the absence of a donor template (see the second panel of Eyquem, Supplementary Figure 1C). In particular, the nucleases and CRISPR systems disclosed in the prior art each target the recognition sequence of the T cell receptor gene at a site important for gene expression and functional T cell receptor; for example, the TRAC exon, or a site or locus important for the formation of the endogenous splice acceptor site. Insertion of the CAR coding sequence into these cleavage sites may result in T cell receptor-negative (TCR-) cells that are positive for CAR (CAR+), but this approach (see Eyquem, Supplementary Figure 1A). This splice acceptor site is necessary for TCR expression, as evidenced by the fact that disruption of the site by Cas9 causes TCR knockout in 70% of T cells in the absence of a donor template (see the second panel of Eyquem, Supplementary Figure 1C). This splice acceptor site is proven to be necessary for TCR expression by the fact that interference with the site by Cas9 causes TCR knockout in 70% of T cells in the absence of a donor template. As such, it is necessary for TCR expression (see the second panel of Eyquem, Supplementary Figure 1C). (see Eyquem, Supplementary Figure 1C). ).

[0015] In particular, the nucleases and CRISPR systems disclosed in the prior art each target the recognition sequence of the T cell receptor gene at a site important for gene expression and functional T cell receptor; for example, the TRAC exon, or a site or locus important for the formation of the endogenous splice acceptor site. Insertion of the CAR coding sequence into these cleavage sites may result in T cell receptor-negative (TCR-) cells that are positive for CAR (CAR+), but this approach targets the recognition sequence of the T cell receptor gene at a site important for gene expression and functional T cell receptor; for example, the TRAC exon, or a site or locus important for the formation of the endogenous splice acceptor site. Insertion of the CAR coding sequence into these cleavage sites may result in T cell receptor-negative (TCR-) cells that are positive for CAR (CAR+), but this A major drawback of the approach is that, in the absence of a donor template, TCR expression can be knocked out by non-homologous end joining (NHEJ), which is error-prone at the cleavage site.

[0016] As a result, previous methods of producing CAR T cells yielded a mixed population of TCR- / CAR+ and TCR- / CAR- cells that required further enrichment for preclinical and clinical use. For example, as described above, Supplementary Figure 1C of Eyquem showed that when Cas9 disrupted the splice acceptor site upstream of the 5’ end of TRAC exon 1 in the absence of a donor template, approximately 70% of the cells were TCR- / CAR-. However, even in the presence of a CAR donor template (1e6 AAV6), a mixed population of TCR- / CAR- and TCR- / CAR+ cells was produced. Specifically, when template DNA was provided at a 1e6 AAV6 MOI, 45.6% of the T cells were TCR- / CAR+, but a significant proportion of the cells (30.7%) were TCR- / CAR- due to cleavage within the splice acceptor site by Cas9 and subsequent error-prone repair by NHEJ.

Prior Art Documents

Patent Documents

[0017]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Patent Document 7

Patent Document 8

Non-Patent Document

[0018]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

Summary of the Invention

Problems to be Solved by the Invention

[0019]

Means for Solving the Problems

[0020] In contrast, the present invention takes a counterintuitive approach for modifying the T gene and inserting a target sequence such as a CAR coding sequence. Instead of targeting the elements of the TCR gene essential for TCR expression, the present invention targets the intron of the TCR alpha gene upstream of the 5' of exon 1 of TRAC. As long as the endogenous splice donor site and the endogenous splice acceptor site adjacent to the intron are not modified, the double-strand break by the nuclease in this non-coding intron, even if NHEJ produces an indel at the cleavage site, the T ... ... ... ... ... Does not substantially affect CR expression.

[0021] Contrary to convention, by targeting the recognition sequence of the intron, at least exogenous The target sequence containing the price acceptor site and / or polyA signal is, for example, homologous TCR expression is only disrupted when inserted into the cleavage site, for example, by recombination. As a result, the TCR cells produced according to the present invention contain the target sequence inserted into the intron cleavage site. Expanding, when the inserted target sequence further contains a CAR coding sequence, most or all of the TCR- cells in the resulting cell population become TCR- / CAR+, which is completely contrary to previous methods, and the resulting population may also contain a substantial proportion of TRC- / CAR- cells. Thus, the present invention significantly advances this field by eliminating the tedious need to enrich CAR+ cells from a mixed population of TRC- cells. Furthermore, in some embodiments of the present invention, the target sequence inserted into the intron contains a 2A element

[0022] upstream of the 5' of the coding sequence (e.g., CAR coding sequence) (see Figure 1). Including this 2A element enables the expression of the coding sequence driven by the endogenous T cell receptor alpha gene promoter rather than an exogenous promoter. In this way, the expression of polypeptides such as CAR is controlled by the T cell feedback mechanism normally associated with TCR expression. The present invention provides genetically modified human

[0023] T cells or cells derived therefrom that contain a modified human T cell receptor alpha gene in their genome. The modified human T cell receptor alpha gene is It may contain an exogenous sequence of interest inserted into an intron within the T cell receptor alpha gene located upstream of the 5' of TRAC exon 1. The exogenous sequence of interest inserted into the intron can include an exogenous splice acceptor site and / or a polyA signal, which interferes with the expression of the T cell receptor alpha subunit. In some embodiments, the sequence of interest can also include a coding sequence of a polypeptide (e.g., a CAR coding sequence). Furthermore, the endogenous splice donor site and endogenous splice acceptor site adjacent to the intron are not modified and / or remain functional in the cell. Additionally, the cell surface expression of the endogenous T cell receptor is reduced when compared to unmodified control cells.

[0024] The present invention also provides compositions and methods for producing a population of T cells, along with the genetically modified T cells. The present invention further provides a method of immunotherapy for treating cancer by administering the genetically modified T cells, where the T cells express a receptor specific for a tumor-specific antigen (e.g., CAR).

[0025] Accordingly, in one aspect of the present invention, an engineered meganuclease that recognizes and cleaves a recognition sequence within an intron of the human T cell receptor alpha gene located upstream of the 5' of TRAC exon 1, wherein the engineered meganuclease comprises a first subunit and a second subunit, the first subunit binds to the first recognition half-site of the recognition sequence and includes a first hypervariable (HVR1) region, and the second subunit binds to the second recognition half-site of the recognition sequence and includes a second hypervariable (HVR2) region. Provides a crease. In some embodiments, the intron contains SEQ ID NO: 3 and is engineered The engineered meganuclease has no recognition sequence within the endogenous splice donor site or endogenous Splice acceptor site adjacent to the intron.

[0026] In certain embodiments, the recognition sequence comprises SEQ ID NO: 4 (i.e., the TRC11-12 recognition sequence) including.

[0027] In some such embodiments, the HVR1 region has at least 80%, at least 85%, at least 90%, at least 95%, or more sequence identity with the amino acid sequence corresponding to residues 215-270 of any one of SEQ ID NOs: 12-15 An amino acid sequence containing. including.

[0028] In some such embodiments, the HVR1 region is any one of SEQ ID NOs: 12-15 Residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 23 5, 237, 259, 261, 266, and 268.

[0029] In some such embodiments, the HVR1 region is any one of SEQ ID NOs: 12-15 Residues 215-270 are included.

[0030] In some such embodiments, the HVR2 region has at least 80%, at least 85%, at least 90%, at least 95%, or more sequence identity with the amino acid sequence corresponding to residues 24-79 of any one of SEQ ID NOs: 12-15 An amino acid sequence containing. .

[0031] In some such embodiments, the HVR2 region is any one of SEQ ID NOs: 12-15 Residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70 and residues corresponding to 75 and 77.

[0032] In some such embodiments, the HVR2 region comprises residues 24-79 of any one of SEQ ID NOs: 12-15. Residues 24-79 are included.

[0033] In some such embodiments, the first subunit comprises an amino acid sequence having at least 80%, at least 85%, at least 9 0%, at least 95%, or more sequence identity to residues 198-344 of any one of SEQ ID NOs: 12-15, and the second subunit comprises an amino acid sequence having at least 8 0%, at least 85%, at least 90%, at least 95%, or more sequence identity to residues 7-153 of any one of SEQ ID NOs: 12-15. 0%, at least 85%, at least 90%, at least 95%, or more sequence identity to residues 7-153 of any one of SEQ ID NOs: 12-15. sequence identity to residues 7-153 of any one of SEQ ID NOs: 12-15.

[0034] In some such embodiments, the first subunit comprises residues 198-344 of any one of SEQ ID NOs: 12-15. In some such embodiments, the second subunit comprises residues 7-153 of any one of SEQ ID NOs: 12-15. In some such embodiments, the second subunit comprises residues 7-153 of any one of SEQ ID NOs: 12-15.

[0035] In some such embodiments, the engineered meganuclease comprises a linker that covalently links the first subunit and the second subunit. The linker covalently couples the first subunit and the second subunit.

[0036] In some such embodiments, the engineered meganuclease comprises an amino acid sequence of any one of SEQ ID NOs: 12-15. In some such embodiments, the engineered meganuclease comprises an amino acid sequence of any one of SEQ ID NOs: 12-15.

[0037] In certain embodiments, the recognition sequence comprises SEQ ID NO: 6 (i.e., the TRC15-16 recognition sequence ).

[0038] In some such embodiments, the HVR1 region has at least 80%, at least 85%, at least 90%, at least 95%, or more sequence identity with the amino acid sequence corresponding to residues 24-79 of any one of SEQ ID NOs: 16-19 and comprises an amino acid sequence. .

[0039] In some such embodiments, the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70 of any one of SEQ ID NOs: 16-19. , 75, and 77.

[0040] In some such embodiments, the HVR1 region comprises the residue corresponding to residue 64 of any one of SEQ ID NOs: 16-19.

[0041] In some such embodiments, the HVR1 region comprises residues 24-79 of any one of SEQ ID NOs: 16-19.

[0042] In some such embodiments, the HVR2 region has at least 80%, at least 85%, at least 90%, at least 95%, or more sequence identity with the amino acid sequence corresponding to residues 215-270 of any one of SEQ ID NOs: 16-19 and comprises an amino acid sequence.

[0043] In some such embodiments, the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 23 5, 237, 259, 261, 266, and 268 of any one of SEQ ID NOs: 16-19.

[0044] In some such embodiments, the HVR2 region is any one of SEQ ID NOs: 16-19 and comprises residues 215-270.

[0045] In some such embodiments, the first subunit has at least 80%, at least 85%, at least 90% identity to an amino acid sequence corresponding to residues 7-153 of any one of SEQ ID NOs: 16-19, and the second subunit has at least 80% identity to an amino acid sequence corresponding to residues 198-344 of any one of SEQ ID NOs: 16-19, at least 85%, at least 90%, at least 95% or more. The amino acid sequence contains.

[0046] In some such embodiments, the first subunit comprises residues 7-153 of any one of SEQ ID NOs: 16-19.

[0047] In some such embodiments, the second subunit comprises residues 198-344 of any one of SEQ ID NOs: 16-19.

[0048] In some such embodiments, the engineered meganuclease comprises a linker that covalently links the first subunit and the second subunit.

[0049] In some such embodiments, the engineered meganuclease comprises an amino acid sequence of any one of SEQ ID NOs: 16-19.

[0050] In certain embodiments, the recognition sequence comprises SEQ ID NO: 8 (i.e., the TRC17-18 recognition sequence ).

[0051] In some such embodiments, the HVR1 region is any one of SEQ ID NOs: 20-23 An amino acid sequence corresponding to residues 24 to 79 and having at least 80%, at least 85%, at least 90%, at least 95%, or more sequence identity with the amino acid sequence is included .

[0052] In some such embodiments, the HVR1 region is any one of SEQ ID NOs: 20 to 23 Residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70 , 75, and 77 are included.

[0053] In some such embodiments, the HVR1 region is any one of SEQ ID NOs: 20 to 23 Residues corresponding to residue 66 are included.

[0054] In some such embodiments, the HVR1 region is any one of SEQ ID NOs: 20 to 23 Residues 24 to 79 are included.

[0055] In some such embodiments, the HVR2 region is any one of SEQ ID NOs: 20 to 23 An amino acid sequence corresponding to residues 215 to 270 and having at least 80%, at least 85%, at least 90%, at least 95%, or more sequence identity with the amino acid sequence is included.

[0056] In some such embodiments, the HVR2 region is any one of SEQ ID NOs: 20 to 23 Residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 23 5, 237, 259, 261, 266, and 268 are included.

[0057] In some such embodiments, the HVR2 region is any one of SEQ ID NOs: 20 to 23 Residues 215 to 270 are included.

[0058] In some such embodiments, the first subunit comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more sequence identity to any one of residues 7 to 153 of SEQ ID NO: 20-23, and the second subunit comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more sequence identity to residues 198 to 344 of any one of SEQ ID NO: 20-23. In some such embodiments, the first subunit comprises residues 7 to 153 of any one of SEQ ID NO: 20-23. In some such embodiments, the second subunit comprises residues 198 to 344 of any one of SEQ ID NO: 20-23. In some such embodiments, the engineered meganuclease comprises a linker that covalently links the first subunit and the second subunit.

[0059] In some such embodiments, the engineered meganuclease comprises an amino acid sequence of any one of SEQ ID NO: 20-23. In a particular embodiment, the recognition sequence comprises SEQ ID NO: 10 (i.e., the TRC19-20 recognition sequence).

[0060] In some such embodiments, the HVR1 region has at least 80%, at least 85%, at least 90%, at least 95%, or more sequence identity to the amino acid sequence corresponding to residues 24 to 79 of any one of SEQ ID NO: 24-27.

[0061] In some such embodiments, the engineered meganuclease comprises a linker that covalently links the first subunit and the second subunit. In some such embodiments, the engineered meganuclease comprises an amino acid sequence of any one of SEQ ID NO: 20-23.

[0062] In some such embodiments, the engineered meganuclease comprises an amino acid sequence of any one of SEQ ID NO: 20-23. In a particular embodiment, the recognition sequence comprises SEQ ID NO: 10 (i.e., the TRC19-20 recognition sequence).

[0063] In a particular embodiment, the recognition sequence comprises SEQ ID NO: 10 (i.e., the TRC19-20 recognition sequence). In some such embodiments, the HVR1 region has at least 80%, at least 85%, at least

[0064] 90%, at least 95%, or more sequence identity to the amino acid sequence corresponding to residues 24 to 79 of any one of SEQ ID NO: 24-27. In some such embodiments, the HVR1 region has at least 80%, at least 85%, at least comprising an amino acid sequence having at least 90%, at least 95%, or more sequence identity 。

[0065] In some such embodiments, the HVR1 region is any one of SEQ ID NOs: 24-27 residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70 , 75, and 77.

[0066] In some such embodiments, the HVR1 region is any one of SEQ ID NOs: 24-27 comprising residues 24-79.

[0067] In some such embodiments, the HVR2 region is any one of SEQ ID NOs: 24-27 having at least 80%, at least 85%, at least 90%, at least 95%, or more sequence identity with the amino acid sequence corresponding to residues 215-270 of any one of SEQ ID NOs: 24-27 and comprising.

[0068] In some such embodiments, the HVR2 region is any one of SEQ ID NOs: 24-27 residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 23 5, 237, 259, 261, 266, and 268.

[0069] In some such embodiments, the HVR2 region is any one of SEQ ID NOs: 24-27 comprising residues 215-270.

[0070] In some such embodiments, the first subunit has at least 80%, at least 85%, at least 90% sequence identity with the amino acid sequence corresponding to residues 7-153 of any one of SEQ ID NOs: 24-27, and the second subunit having at least 95%, or more sequence identity, and comprising The Bu unit contains at least 80 %, at least 85%, at least 90%, at least 95%, or more of the amino acid sequences having sequence identity with residues 198-344 of any one of SEQ ID NOs: 24-27.

[0071] In some such embodiments, the first subunit contains residues 7-153 of any one of SEQ ID NOs: 24-27.

[0072] In some such embodiments, the second subunit contains residues 198-344 of any one of SEQ ID NOs: 24-27.

[0073] In some such embodiments, the engineered meganuclease contains a linker that covalently links the first subunit and the second subunit.

[0074] In some such embodiments, the engineered meganuclease contains the amino acid sequence of any one of SEQ ID NOs: 24-27.

[0075] In another aspect, the present invention provides a polynucleotide containing a nucleic acid sequence encoding the engineered meganuclease described herein.

[0076] In certain embodiments, the polynucleotide is mRNA.

[0077] In a further aspect, the mRNA is a polycistronic mRNA encoding the engineered meganuclease described herein and at least one additional polypeptide or nucleic acid.

[0078] In another aspect, the present invention provides a recombinant DNA construct containing the polynucleotide described herein.

[0079] In certain embodiments, the recombinant DNA construct encodes a viral vector. In certain embodiments, the viral vector is an adenoviral vector, a lentiviral vector, a retroviral vector, or an adeno-associated virus (AAV) vector. In a specific embodiment, the viral vector is a recombinant AAV vector.

[0080] In another aspect, the present invention provides a viral vector comprising the polynucleotide described herein.

[0081] In certain embodiments, the viral vector is an adenoviral vector, a lentiviral vector, a retroviral vector, or an AAV vector. In certain embodiments, the viral vector is a recombinant AAV vector.

[0082] In another aspect, the present invention provides a method for producing a genetically modified T cell comprising an exogenous sequence of interest inserted into the chromosome of the T cell. The method comprises introducing into the T cell (a) a first nucleic acid sequence encoding an engineered meganuclease described herein, wherein the engineered meganuclease is expressed in the T cell; and (b) a second nucleic acid sequence comprising the sequence of interest, wherein the engineered meganuclease generates a chromosomal cleavage site at a recognition sequence within an intron of the human T cell receptor alpha gene located upstream of the 5' end of the TRAC exon 1; the sequence of interest is inserted into the chromosome at the cleavage site; the sequence of interest comprises an exogenous splice acceptor site and / or a polyA signal; and the endogenous splice donor site adjacent to the intron and the endogenous ​ The cis-splice acceptor site is unmodified and / or remains functional.

[0083] In some embodiments of the above method, the T cells are precursor T cells in which rearrangement of the V segment and J segment has not occurred.

[0084] In certain embodiments of the above method, the cell surface expression of the endogenous T cell receptor is reduced when compared to unmodified control cells.

[0085] In some embodiments of this method, the intron contains SEQ ID NO: 3.

[0086] In some embodiments of the above method, the recognition sequence contains SEQ ID NO: 4, and the engineered meganuclease is the engineered meganuclease described herein that recognizes and cleaves SEQ ID NO: 4. In some embodiments of the above method, the recognition sequence contains SEQ ID NO: 6, and the engineered meganuclease is the engineered meganuclease described herein that recognizes and cleaves SEQ ID NO: 6. In some embodiments of the above method, the recognition sequence contains SEQ ID NO: 8, and the engineered meganuclease is the engineered meganuclease described herein that recognizes and cleaves SEQ ID NO: 8. In some embodiments of the above method, the recognition sequence contains SEQ ID NO: 10, and the engineered meganuclease is the engineered meganuclease described herein that recognizes and cleaves SEQ ID NO: 10. In some embodiments of the above method, the recognition sequence contains SEQ ID NO: 8, and the engineered meganuclease is the engineered meganuclease described herein that recognizes and cleaves SEQ ID NO: 8. In some embodiments of the above method, the recognition sequence contains SEQ ID NO: 10, and the engineered meganuclease is the engineered meganuclease described herein that recognizes and cleaves SEQ ID NO: 10. In certain embodiments of the above method, the second nucleic acid sequence further contains a sequence identical to the sequence adjacent to the cleavage site, and the sequence of interest is inserted into the cleavage site by homologous recombination. is the engineered meganuclease described herein that recognizes and cleaves SEQ ID NO: 10.

[0087] In certain embodiments of the above method, the second nucleic acid sequence further contains a sequence identical to the sequence adjacent to the cleavage site, and the sequence of interest is inserted into the cleavage site by homologous recombination. is inserted into the cleavage site by homologous recombination.

[0088] In some embodiments of the above method, the T cells are human T cells or cells derived therefrom. .

[0089] In various embodiments of the above method, the sequence of interest comprises, from 5' to 3', an exogenous splice acceptor site, a 2A element or an IRES element, a coding sequence of the protein of interest, and a polyA signal. In certain specific embodiments of the above method, the 2A element is a T2A element, a P2A element, an E2A element, or an F2A element. In a specific embodiment of the above method, the 2A element is a T2A element.

[0090] In some embodiments of the above method, the sequence of interest further comprises an exogenous branch site located 5' upstream of the exogenous splice acceptor site.

[0091] In some embodiments of the above method, the sequence of interest comprises a coding sequence of a chimeric antigen receptor or an exogenous T cell receptor. In a specific embodiment of the above method, the chimeric antigen receptor or the exogenous T cell receptor comprises an extracellular ligand-binding domain having specificity for a tumor-specific antigen.

[0092] In some embodiments of the above method, at least the first nucleic acid sequence is introduced into T cells by mRNA.

[0093] In certain specific embodiments of the above method, at least the second nucleic acid sequence is introduced into T cells by a viral vector. In a specific embodiment of the above method, the viral vector is an adenovirus vector, a lentivirus vector, a retrovirus vector, or an AAV vector. In a specific embodiment of the above method, the viral vector is a recombinant AAV vector.

[0094] In another aspect, the present invention provides a gene comprising an exogenous sequence of interest inserted into a chromosome of a T cell. The present invention provides a method for producing modified T cells, the method comprising: (a) administering to a T cell a gene encoding a modified T cell as described herein; (b) introducing an engineered meganuclease comprising a nucleic acid sequence of interest; and transfecting the T cells with, wherein the engineered meganuclease is The endogenous TRAC exon 1 of the human T cell receptor alpha gene is located 5' upstream of the a recognition sequence in the chromosome that creates a cleavage site in the chromosome; the sequence of interest is stained at the cleavage site; the sequence of interest is inserted into the host; the sequence of interest may include an exogenous splice acceptor site and / or a polyA signal; and an endogenous splice donor site adjacent to the intron and an endogenous splice donor site adjacent to the intron. The rice acceptor site remains unaltered and / or functional.

[0095] In some embodiments of the above methods, the T cells are V and J segment rearrangements. These are precursor T cells that have not undergone

[0096] In some embodiments of the above methods, the endogenous T cell receptor is increased when compared to an unmodified control cell. The body has reduced cell surface expression.

[0097] In certain embodiments of the above methods, the intron comprises SEQ ID NO:3.

[0098] In some embodiments of the above method, the recognition sequence comprises SEQ ID NO:4, and the engineered meganucleotide The enzyme is an engineered meganucleotide sequence described herein that recognizes and cleaves SEQ ID NO:4. In some embodiments of the above method, the recognition sequence comprises SEQ ID NO:6 and is an engineered The engineered meganuclease described herein recognizes and cleaves SEQ ID NO: 6 and is the engineered meganuclease. In some embodiments of the above method, the recognition sequence includes SEQ ID NO: 8 and the engineered meganuclease is the engineered meganuclease described herein that recognizes and cleaves SEQ ID NO: 8. In some embodiments of the above method, the recognition sequence includes SEQ ID NO: 10 and the engineered meganuclease is the engineered meganuclease described herein that recognizes and cleaves SEQ ID NO: 10. In certain specific embodiments of the above method, the nucleic acid further includes a sequence homologous to the sequence adjacent to the cleavage site, and the target sequence is inserted into the cleavage site by homologous recombination.

[0099] In some embodiments of the above method, the T cell is a human T cell or a cell derived therefrom.

[0100] In some embodiments of the above method, the target sequence includes, from 5' to 3', an exogenous splice acceptor site, a 2A element or an IRES element, a coding sequence of the target protein, and a polyA signal. In certain specific embodiments of the above method, the 2A element is a T2A element, a P2A

[0101] element, an E2A element, or an F2A element. In specific embodiments of the above method, the 2A element is a T2A element. In some embodiments of the above method, the target sequence further includes an exogenous branch site located upstream of the 5' of the exogenous splice acceptor site.

[0102] In some embodiments of the above method, the target sequence is a chimeric antigen receptor or an exogenous T cell

[0103] ​​​​​​It contains the coding sequence of the receptor. In certain embodiments of the above method, the chimeric antigen receptor or exogenous T cell receptor has an extracellular ligand-binding domain with specificity for a tumor-specific antigen .

[0104] In certain specific embodiments of the above method, the nucleic acid is introduced into T cells by a viral vector . In certain embodiments of the above method, the viral vector is an adenoviral vector, a lentiviral vector, a retroviral vector, or an AAV vector. In a specific embodiment of the above method, the viral vector is a recombinant AAV vector.

[0105] In another aspect, the present invention provides a method for producing genetically modified T cells containing a modified human T cell receptor alpha gene. The method comprises: (a) introducing into T cells: (i) a first nucleic acid sequence encoding an engineered nuclease, the engineered nuclease being a first nucleic acid sequence expressed in T cells; or (ii) an engineered nuclease protein; and (b) introducing into the above cells a second nucleic acid sequence containing the exogenous sequence of interest, wherein the engineered nuclease generates a cleavage site at a recognition sequence within the intron of the human T cell receptor alpha gene located upstream of the 5' of exon 1 of TRAC; the sequence of interest is inserted into the human T cell receptor alpha gene at the cleavage site; the sequence of interest contains an exogenous splice acceptor site and / or a polyA signal; and the endogenous splice donor site and endogenous splice acceptor site adjacent to the intron remain unmodified and / or functional.

[0106] In some embodiments of the above method, the T cells are precursor T cells in which rearrangement of the V segment and J segment has not occurred.

[0107] In some embodiments of the above method, when compared with unmodified control cells, the cell surface expression of the endogenous T cell receptor is reduced.

[0108] In certain embodiments of the above method, the intron comprises SEQ ID NO: 3.

[0109] In some embodiments of the above method, the second nucleic acid sequence comprises, from 5' to 3': (a) a 5' homology arm homologous to the 5' upstream sequence adjacent to the cleavage site; (b) an exogenous sequence of interest; (c) a 3' homology arm homologous to the 3' downstream sequence adjacent to the cleavage site, wherein the exogenous sequence of interest is inserted into the human T cell receptor alpha gene at the cleavage site by homologous recombination.

[0110] In some embodiments of the above method, the sequence of interest further comprises an exogenous branch site located 5' upstream of the exogenous splice acceptor site.

[0111] In certain embodiments of the above method, the genetically modified T cells are genetically modified human T cells or cells derived therefrom.

[0112] In some embodiments of the above method, the exogenous sequence of interest comprises, from 5' to 3', an exogenous splice acceptor site, a 2A element or an IRES element, a coding sequence of the protein of interest, and a polyA signal. In certain embodiments of the above method, the 2A element is a T2A element, a P2A element, an E2A element, or an F2A element. In a particular embodiment of the above method, the 2A element is a T2A element.

[0113] In some embodiments of the above method, the target sequence comprises a coding sequence for a chimeric antigen receptor or an exogenous T cell receptor. In certain embodiments of the above method, the chimeric antigen receptor or exogenous T cell receptor comprises an extracellular ligand-binding domain with specificity for a tumor-specific antigen.

[0114] In some embodiments of the above method, at least the first nucleic acid sequence is introduced into T cells by mRNA cells.

[0115] In certain embodiments of the above method, at least the second nucleic acid sequence is introduced into T cells by a viral vector cells. In certain embodiments of the above method, the viral vector is an adenovirus vector, a lentivirus vector, a retrovirus vector, or an adeno associated virus (AAV) vector. In a specific embodiment of the above method, the viral vector is a recombinant AAV vector.

[0116] In some embodiments of the above method, the engineered nuclease is an engineered meganuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a compact TALEN, a CRISPR nuclease, or a me gaTAL. In certain embodiments of the above method, the engineered nuclease is an engineered meganuclease. In some embodiments of the above method, the engineered meganuclease has specificity for a recognition sequence comprising SEQ ID NO: 4. In some such embodiments of the above method, the engineered

[0117] meganuclease recognizes and cleaves SEQ ID NO: 4 as described herein engineered me ganuclease. It is a gRNA nuclease.

[0118] In some embodiments of the above method, the engineered meganuclease has a specificity for a recognition sequence containing SEQ ID NO: 6. In some such embodiments of the above method, the engineered meganuclease is the engineered meganuclease described herein that recognizes and cleaves SEQ ID NO: 6. It is a gRNA nuclease. In some embodiments of the above method, the engineered meganuclease has a specificity for a recognition sequence containing SEQ ID NO: 8.

[0119] In some embodiments of the above method, the engineered meganuclease has a specificity for a recognition sequence containing SEQ ID NO: 8. In some such embodiments of the above method, the engineered meganuclease is the engineered meganuclease described herein that recognizes and cleaves SEQ ID NO: 8. It is a gRNA nuclease. In some embodiments of the above method, the engineered meganuclease has a specificity for a recognition sequence containing SEQ ID NO: 10.

[0120] In some embodiments of the above method, the engineered meganuclease has a specificity for a recognition sequence containing SEQ ID NO: 10. In some such embodiments of the above method, the engineered meganuclease is the engineered meganuclease described herein that recognizes and cleaves SEQ ID NO: 10. It is a gRNA nuclease. In another aspect, the present invention provides a genetically modified T cell prepared by any of the methods described herein for producing a genetically modified T cell.

[0121] In another aspect, the present invention provides a genetically modified T cell containing a modified human T cell receptor alpha gene in its genome, where the modified human T cell receptor alpha gene contains an exogenous sequence of interest inserted into an intron within the T cell receptor alpha gene located 5' upstream of exon 1 of TRAC, and the exogenous sequence of interest contains an exogenous splice acceptor site and In another aspect, the present invention provides a genetically modified T cell containing a modified human T cell receptor alpha gene in its genome, where the modified human T cell receptor alpha gene contains an exogenous sequence of interest inserted into an intron within the T cell receptor alpha gene located 5' upstream of exon 1 of TRAC, and the exogenous sequence of interest contains an exogenous splice acceptor site and

[0122] In another aspect, the present invention provides a genetically modified T cell containing a modified human T cell receptor alpha gene in its genome, where the modified human T cell receptor alpha gene contains an exogenous sequence of interest inserted into an intron within the T cell receptor alpha gene located 5' upstream of exon 1 of TRAC, and the exogenous sequence of interest contains an exogenous splice acceptor site and In another aspect, the present invention provides a genetically modified T cell containing a modified human T cell receptor alpha gene in its genome, where the modified human T cell receptor alpha gene contains an exogenous sequence of interest inserted into an intron within the T cell receptor alpha gene located 5' upstream of exon 1 of TRAC, and the exogenous sequence of interest contains an exogenous splice acceptor site and In another aspect, the present invention provides a genetically modified T cell containing a modified human T cell receptor alpha gene in its genome, where the modified human T cell receptor alpha gene contains an exogenous sequence of interest inserted into an intron within the T cell receptor alpha gene located 5' upstream of exon 1 of TRAC, and the exogenous sequence of interest contains an exogenous splice acceptor site and In another aspect, the present invention provides a genetically modified T cell containing a modified human T cell receptor alpha gene in its genome, where the modified human T cell receptor alpha gene contains an exogenous sequence of interest inserted into an intron within the T cell receptor alpha gene located 5' upstream of exon 1 of TRAC, and the exogenous sequence of interest contains an exogenous splice acceptor site and / or includes a polyA signal, and the endogenous splice donor site and endogenous splice acceptor site adjacent to the intron are not modified and / or remain functional , and the cell surface expression of the endogenous T cell receptor is reduced when compared to unmodified control cells. .

[0123] In some embodiments, the intron includes SEQ ID NO: 3.

[0124] In certain embodiments, the genetically modified T cell is a genetically modified human T cell or a cell derived therefrom.

[0125] In some embodiments, the exogenous sequence of interest comprises, from 5' to 3', an exogenous splice acceptor site, a 2A element or an IRES element, a coding sequence of the protein of interest, and a polyA signal. In certain embodiments, the 2A element is a T2A element, a P2A element, an E2A element, or an F2A element. In a specific embodiment, the 2A element is a T2A element.

[0126] In some embodiments, the exogenous sequence of interest further comprises an exogenous branch site located 5' ' upstream of the exogenous splice acceptor site.

[0127] In certain embodiments, the sequence of interest comprises a coding sequence of a chimeric antigen receptor or an exogenous T cell receptor. In certain embodiments, the chimeric antigen receptor or exogenous T cell receptor comprises an extracellular ligand-binding domain having specificity for a tumor-specific antigen.

[0128] In some embodiments, the exogenous sequence of interest is an engineered meganuclease recognition site, a TALEN recognition site, a zinc finger nuclease recognition site, a CRISPR recognition site,​​ or inserted into the intron at the megaTAL recognition site. In certain embodiments, the exogenous sequence of interest is inserted into the intron at the engineered meganuclease recognition site. In a specific embodiment, the exogenous sequence of interest is inserted into the intron within SEQ ID No. 4. In other embodiments, the exogenous sequence of interest is inserted into the intron within SEQ ID No. 6. In a further embodiment, the exogenous sequence of interest

[0129] is inserted into the intron within SEQ ID No. 8. In other embodiments, the exogenous sequence of interest is inserted into the intron within SEQ ID No. 10.

[0130] In another aspect, the invention provides a population of genetically modified T cells comprising a plurality of the genetically modified T cells described herein. In some embodiments, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%

[0131] at least 98%, at least 99%, or up to 100% of the cells in the population are the genetically modified T cells described herein.

[0132] In certain embodiments, the genetically modified T cells are genetically modified human It comprises a coding sequence of a receptor or an exogenous T cell receptor. In certain embodiments, the chimeric antigen receptor or exogenous T cell receptor comprises an extracellular ligand binding domain having specificity for a tumor-specific antigen.

[0133] In some embodiments, the cell surface expression of the endogenous T cell receptor is reduced on the genetically modified T cells when compared to non-modified control cells.

[0134] In another aspect, the present invention provides a pharmaceutical composition useful for treating a disease in a subject in need thereof, the pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of the genetically modified T cells described herein.

[0135] In certain embodiments, the genetically modified T cells are genetically modified human T cells or cells derived therefrom.

[0136] In some embodiments, the exogenous sequence of interest present in the genetically modified T cells comprises a coding sequence of a chimeric antigen receptor or an exogenous T cell receptor. In certain embodiments, the chimeric antigen receptor or exogenous T cell receptor comprises an extracellular ligand binding domain having specificity for a tumor-specific antigen.

[0137] In some embodiments, the cell surface expression of the endogenous T cell receptor is reduced on the genetically modified T cells when compared to non-modified control cells.

[0138] In another aspect, the present invention provides a method of treating a disease in a subject in need thereof, the method comprising administering to the subject the genetically modified T cells described herein.

[0139] In some embodiments, the method comprises administering to a subject a pharmaceutical composition described herein thereof.

[0140] In certain embodiments, the method is an immunotherapy for the treatment of cancer in a subject in need thereof. In some such embodiments, the genetically modified T cells are genetically modified human T cells or cells derived therefrom, and the exogenous sequence of interest present in the genetically modified T cells comprises a coding sequence of an exogenous T cell receptor having a chimeric antigen receptor or an extracellular ligand-binding domain specific for a tumor-specific antigen, and the cell surface expression of the endogenous T cell receptor is reduced on the genetically modified T cells as compared to unmodified control cells.

[0141] In some embodiments of the method, the cancer is selected from the group consisting of carcinoma, lymphoma, sarcoma, blastoma, and leukemia cancer.

[0142] In certain embodiments of the method, the cancer is selected from the group consisting of B cell-originated cancer, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, melanoma, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, and Hodgkin lymphoma.

[0143] In certain embodiments of the method, the B cell-originated cancer is selected from the group consisting of B-lineage acute lymphoblastic leukemia , B cell chronic lymphocytic leukemia, B cell non-Hodgkin lymphoma, and multiple myeloma thereof.

[0144] In another aspect, the present invention provides a genetically modified cell described herein for use as a medicament. The present disclosure further relates to the use of a genetically modified cell described herein in need of ​Provided is the use of the genetically modified cells described herein in the manufacture of a medicament for treating a disease in a subject to be treated. In such an aspect, the medicament is useful for the treatment of cancer. In another aspect, the present invention provides a genetically modified cell described herein for use in the treatment of a disease, preferably cancer.

[0145] In another aspect, the present invention provides a genetically modified cell described herein for use in the treatment of a disease, preferably cancer. In another aspect, the present invention provides a genetically modified cell described herein for use in the treatment of a disease, preferably cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0146]

Figure 1

Figure 2

Figure 3

Figure 4

Figures 5A-D

Figures 6A-D

Figure 7

Figure 8

Figures 9A-C

Figures 10A-D

Figures 11A-C

[0147] Brief Description of the Sequence SEQ ID NO: 1 shows the amino acid sequence of the wild-type I-CreI meganuclease.

[0148] SEQ ID NO: 2 shows the amino acid sequence of LAGLIDADG.

[0149] SEQ ID NO: 3 shows the nucleic acid sequence of the human T cell receptor alpha gene intron.

[0150] SEQ ID NO: 4 shows the nucleic acid sequence of TRC11-12 (sense).

[0151] SEQ ID NO: 5 shows the nucleic acid sequence of TRC11-12 (antisense).

[0152] SEQ ID NO: 6 shows the nucleic acid sequence of TRC15-16 (sense).

[0153] SEQ ID NO: 7 shows the nucleic acid sequence of TRC15-16 (antisense).

[0154] SEQ ID NO: 8 shows the nucleic acid sequence of TRC17-18 (sense).

[0155] SEQ ID NO: 9 shows the nucleic acid sequence of TRC17-18 (antisense).

[0156] SEQ ID NO: 10 shows the nucleic acid sequence of TRC19-20 (sense).

[0157] SEQ ID NO: 11 shows the nucleic acid sequence of TRC19-20 (antisense).

[0158] SEQ ID NO: 12 shows the amino acid sequence of TRC11-12x.4 meganuclease.

[0159] SEQ ID NO: 13 shows the amino acid sequence of TRC11-12x.82 meganuclease.

[0160] SEQ ID NO: 14 shows the amino acid sequence of TRC11-12x.60 meganuclease.

[0161] SEQ ID NO: 15 shows the amino acid sequence of TRC11-12x.63 meganuclease.

[0162] SEQ ID NO: 16 shows the amino acid sequence of TRC15-16x.31 meganuclease.

[0163] SEQ ID NO: 17 shows the amino acid sequence of TRC15-16x.87 meganuclease.

[0164] SEQ ID NO: 18 shows the amino acid sequence of TRC15-16x.63 meganuclease.

[0165] SEQ ID NO: 19 shows the amino acid sequence of TRC15-16x.89 meganuclease.

[0166] SEQ ID NO: 20 shows the amino acid sequence of TRC17-18x.15 meganuclease.

[0167] Sequence number 21 shows the amino acid sequence of the TRC17-18x.82 meganuclease.

[0168] Sequence number 22 shows the amino acid sequence of the TRC17-18x.18 meganuclease.

[0169] Sequence number 23 shows the amino acid sequence of the TRC17-18x.71 meganuclease.

[0170] Sequence number 24 shows the amino acid sequence of the TRC19-20x.85 meganuclease.

[0171] Sequence number 25 shows the amino acid sequence of the TRC19-20x.74 meganuclease.

[0172] Sequence number 26 shows the amino acid sequence of the TRC19-20x.71 meganuclease.

[0173] Sequence number 27 shows the amino acid sequence of the TRC19-20x.87 meganuclease.

[0174] Sequence number 28 shows the amino acid sequence of the TRC11-12x.4 meganuclease TRC11 binding subunit to.

[0175] Sequence number 29 shows the amino acid sequence of the TRC11-12x.82 meganuclease TRC11 binding subunit unit.

[0176] Sequence number 30 shows the amino acid sequence of the TRC11-12x.60 meganuclease TRC11 binding subunit unit.

[0177] Sequence number 31 shows the amino acid sequence of the TRC11-12x.63 meganuclease TRC11 binding subunit unit.

[0178] Sequence number 32 shows the amino acid sequence of the TRC11-12x.4 meganuclease TRC12 binding subunit Shows the amino acid sequence of TRC11-12x.82 meganuclease TRC12-binding subunit.

[0179] SEQ ID NO: 33 shows the amino acid sequence of the TRC11-12x.82 meganuclease TRC12-binding subunit. Shows the amino acid sequence of the subunit.

[0180] SEQ ID NO: 34 shows the amino acid sequence of the TRC11-12x.60 meganuclease TRC12-binding subunit. Shows the amino acid sequence of the subunit.

[0181] SEQ ID NO: 35 shows the amino acid sequence of the TRC11-12x.63 meganuclease TRC12-binding subunit. Shows the amino acid sequence of the subunit.

[0182] SEQ ID NO: 36 shows the amino acid sequence of the TRC15-16x.31 meganuclease TRC15-binding subunit. Shows the amino acid sequence of the subunit.

[0183] SEQ ID NO: 37 shows the amino acid sequence of the TRC15-16x.87 meganuclease TRC15-binding subunit. Shows the amino acid sequence of the subunit.

[0184] SEQ ID NO: 38 shows the amino acid sequence of the TRC15-16x.63 meganuclease TRC15-binding subunit. Shows the amino acid sequence of the subunit.

[0185] SEQ ID NO: 39 shows the amino acid sequence of the TRC15-16x.89 meganuclease TRC15-binding subunit. Shows the amino acid sequence of the subunit.

[0186] SEQ ID NO: 40 shows the amino acid sequence of the TRC15-16x.31 meganuclease TRC16-binding subunit. Shows the amino acid sequence of the subunit.

[0187] SEQ ID NO: 41 shows the amino acid sequence of the TRC15-16x.87 meganuclease TRC16-binding subunit. Shows the amino acid sequence of the subunit.

[0188] SEQ ID NO: 42 shows the amino acid sequence of the TRC16 binding subunit of the TRC15-16x.63 meganuclease.

[0189] SEQ ID NO: 43 shows the amino acid sequence of the TRC16 binding subunit of the TRC15-16x.89 meganuclease.

[0190] SEQ ID NO: 44 shows the amino acid sequence of the TRC17 binding subunit of the TRC17-18x.15 meganuclease.

[0191] SEQ ID NO: 45 shows the amino acid sequence of the TRC17 binding subunit of the TRC17-18x.82 meganuclease.

[0192] SEQ ID NO: 46 shows the amino acid sequence of the TRC17 binding subunit of the TRC17-18x.18 meganuclease.

[0193] SEQ ID NO: 47 shows the amino acid sequence of the TRC17 binding subunit of the TRC17-18x.71 meganuclease.

[0194] SEQ ID NO: 48 shows the amino acid sequence of the TRC18 binding subunit of the TRC17-18x.15 meganuclease.

[0195] SEQ ID NO: 49 shows the amino acid sequence of the TRC18 binding subunit of the TRC17-18x.82 meganuclease.

[0196] SEQ ID NO: 50 shows the amino acid sequence of the TRC18 binding subunit of the TRC17-18x.18 meganuclease.

[0197] SEQ ID NO: 51 shows the amino acid sequence of the TRC18 binding subunit of the TRC17-18x.71 meganuclease. ​

[0198] SEQ ID NO: 52 shows the amino acid sequence of the TRC19-binding subunit of the TRC19-20x.85 meganuclease.

[0199] SEQ ID NO: 53 shows the amino acid sequence of the TRC19-binding subunit of the TRC19-20x.74 meganuclease.

[0200] SEQ ID NO: 54 shows the amino acid sequence of the TRC19-binding subunit of the TRC19-20x.71 meganuclease.

[0201] SEQ ID NO: 55 shows the amino acid sequence of the TRC19-binding subunit of the TRC19-20x.87 meganuclease.

[0202] SEQ ID NO: 56 shows the amino acid sequence of the TRC20-binding subunit of the TRC19-20x.85 meganuclease.

[0203] SEQ ID NO: 57 shows the amino acid sequence of the TRC20-binding subunit of the TRC19-20x.74 meganuclease.

[0204] SEQ ID NO: 58 shows the amino acid sequence of the TRC20-binding subunit of the TRC19-20x.71 meganuclease.

[0205] SEQ ID NO: 59 shows the amino acid sequence of the TRC20-binding subunit of the TRC19-20x.87 meganuclease.

[0206] SEQ ID NO: 60 shows the nucleic acid sequence of the donor template containing an anti-CD19 CAR that can be inserted into the TRC11-12 recognition sequence.

[0207] SEQ ID NO: 61 shows the nucleic acid sequence of a donor template containing an anti-CD19 CAR that can be inserted into the TRC15-16 recognition sequence.

[0208] SEQ ID NO: 62 shows the nucleic acid sequence of a donor template containing an anti-CD19 CAR that can be inserted into the TRC17-18 recognition sequence.

[0209] SEQ ID NO: 63 shows the nucleic acid sequence of a 7227 donor template encoding a GFP protein that can be inserted into the TRC11-12 recognition sequence.

[0210] SEQ ID NO: 64 shows the nucleic acid sequence of a 7225 donor template encoding an anti-CD19 CAR that can be inserted into the TRC11-12 recognition sequence.

[0211] SEQ ID NO: 65 shows the nucleic acid sequence of a 7228 donor template encoding a GFP protein that can be inserted into the TRC15-16 recognition sequence.

[0212] SEQ ID NO: 66 shows the nucleic acid sequence of a 7226 donor template encoding an anti-CD19 CAR that can be inserted into the TRC15-16 recognition sequence. DETAILED DESCRIPTION OF THE INVENTION

[0213] (Definitions) 1.1 References and Definitions The patents and scientific literature referred to herein establish the knowledge available to those skilled in the art. The issued U.S. patents, allowed applications, published foreign applications, and GenBank database sequences cited in this specification are hereby incorporated by reference in their entirety to the same extent as if each was specifically and individually indicated to be incorporated by reference. and are hereby made a part of this specification to the same extent as if each was specifically and individually indicated to be incorporated by reference.

[0214] The present invention can be embodied in various forms and is not to be construed as limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. For example, features illustrated with respect to one embodiment can be incorporated into other embodiments, and features illustrated with respect to a particular embodiment can be deleted from that embodiment. Further, numerous modifications and additions to the embodiments proposed herein that do not depart from the scope of the present invention will be apparent to those skilled in the art in light of this disclosure. It should not be interpreted as such. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. For example, features illustrated with respect to one embodiment can be incorporated into other embodiments, and features illustrated with respect to a particular embodiment can be deleted from that embodiment. Further, numerous modifications and additions to the embodiments proposed herein that do not depart from the scope of the present invention will be apparent to those skilled in the art in light of this disclosure. For example, features illustrated with respect to one embodiment can be incorporated into other embodiments, and features illustrated with respect to a particular embodiment can be deleted from that embodiment. Further, numerous modifications and additions to the embodiments proposed herein that do not depart from the scope of the present invention will be apparent to those skilled in the art in light of this disclosure. It should not be interpreted as such. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0215] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the present invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the present invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. It should not be interpreted as such. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0216] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety as part of this specification. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety as part of this specification.

[0217] As used herein, "a", "an", or "the" can mean one or more. For example, "a" cell can mean a single cell or a plurality of cells. As used herein, "a", "an", or "the" can mean one or more. For example, "a" cell can mean a single cell or a plurality of cells.

[0218] As used herein, unless otherwise specified, the word "or" is used in the inclusive sense of "and / or" rather than the exclusive sense of "either / or". As used herein, unless otherwise specified, the word "or" is used in the inclusive sense of "and / or" rather than the exclusive sense of "either / or".

[0219] As used herein, the term "meganuclease" refers to an endonuclease that binds to double-stranded DNA at a recognition sequence that is greater than 12 base pairs. Preferably, the recognition sequence of the meganuclease of the present invention is 22 base pairs. The meganuclease may be an endonuclease derived from I-CreI, for example, modified compared to native I-CreI with respect to DNA binding specificity, DNA cleavage activity, DNA binding affinity, or dimerization properties, and may refer to engineered variants of I-CreI. Methods for producing such modified variants of I-CreI are well known in the art (e.g., International Publication No. 2007 / 047859). The meganuclease used herein binds to double-stranded DNA as a heterodimer or as a "single-chain meganuclease" in which pairs of DNA binding domains are joined into a single polypeptide using a peptide linker. The term "homing endonuclease" is synonymous with the term "meganuclease". The meganuclease of the present invention is substantially non-toxic such that when expressed in cells, particularly human T cells, cells can be transfected and maintained at 37°C without observing a detrimental effect on cell viability or a significant decrease in meganuclease cleavage activity as measured using the methods described herein. As used herein, the term "single-chain meganuclease" refers to a polypeptide comprising a pair of nuclease subunits linked by a linker. The single-chain meganuclease has the structure: N-terminal subunit - linker - C-terminal subunit. The two meganuclease subunits generally have non-identical amino acid sequences. Preferably, the recognition sequence of the meganuclease of the present invention is 22 base pairs. The meganuclease may be an endonuclease derived from I-CreI, for example, modified compared to native I-CreI with respect to DNA binding specificity, DNA cleavage activity, DNA binding affinity, or dimerization properties, and may refer to engineered variants of I-CreI. Methods for producing such modified variants of I-CreI are well known in the art (e.g., International Publication No. 2007 / 047859). The meganuclease used herein binds to double-stranded DNA as a heterodimer or as a "single-chain meganuclease" in which pairs of DNA binding domains are joined into a single polypeptide using a peptide linker. The term "homing endonuclease" is synonymous with the term "meganuclease". The meganuclease of the present invention is substantially non-toxic such that when expressed in cells, particularly human T cells, cells can be transfected and maintained at 37°C without observing a detrimental effect on cell viability or a significant decrease in meganuclease cleavage activity as measured using the methods described herein. As used herein, the term "single-chain meganuclease" refers to a polypeptide comprising a pair of nuclease subunits linked by a linker. The single-chain meganuclease has the structure: N-terminal subunit - linker - C-terminal subunit. The two meganuclease subunits generally have non-identical amino acid sequences. Methods for producing such modified variants of I-CreI are well known in the art (e.g., International Publication No. 2007 / 047859). The meganuclease used herein binds to double-stranded DNA as a heterodimer or as a "single-chain meganuclease" in which pairs of DNA binding domains are joined into a single polypeptide using a peptide linker. The term "homing endonuclease" is synonymous with the term "meganuclease". The meganuclease of the present invention is substantially non-toxic such that when expressed in cells, particularly human T cells, cells can be transfected and maintained at 37°C without observing a detrimental effect on cell viability or a significant decrease in meganuclease cleavage activity as measured using the methods described herein. As used herein, the term "single-chain meganuclease" refers to a polypeptide comprising a pair of nuclease subunits linked by a linker. The single-chain meganuclease has the structure: N-terminal subunit - linker - C-terminal subunit. The two meganuclease subunits generally have non-identical amino acid sequences. Methods for producing such modified variants of I-CreI are well known in the art (e.g., International Publication No. 2007 / 047859). The meganuclease used herein binds to double-stranded DNA as a heterodimer or as a "single-chain meganuclease" in which pairs of DNA binding domains are joined into a single polypeptide using a peptide linker. The term "homing endonuclease" is synonymous with the term "meganuclease". The meganuclease of the present invention is substantially non-toxic such that when expressed in cells, particularly human T cells, cells can be transfected and maintained at 37°C without observing a detrimental effect on cell viability or a significant decrease in meganuclease cleavage activity as measured using the methods described herein. As used herein, the term "single-chain meganuclease" refers to a polypeptide comprising a pair of nuclease subunits linked by a linker. The single-chain meganuclease has the structure: N-terminal subunit - linker - C-terminal subunit. The two meganuclease subunits generally have non-identical amino acid sequences. Methods for producing such modified variants of I-CreI are well known in the art (e.g., International Publication No. 2007 / 047859). The meganuclease used herein binds to double-stranded DNA as a heterodimer or as a "single-chain meganuclease" in which pairs of DNA binding domains are joined into a single polypeptide using a peptide linker. The term "homing endonuclease" is synonymous with the term "meganuclease". The meganuclease of the present invention is substantially non-toxic such that when expressed in cells, particularly human T cells, cells can be transfected and maintained at 37°C without observing a detrimental effect on cell viability or a significant decrease in meganuclease cleavage activity as measured using the methods described herein.

[0220] As used herein, the term "single-chain meganuclease" refers to a polypeptide comprising a pair of nuclease subunits linked by a linker. The single-chain meganuclease has the structure: N-terminal subunit - linker - C-terminal subunit. The two meganuclease subunits generally have non-identical amino acid sequences. Methods for producing such modified variants of I-CreI are well known in the art (e.g., International Publication No. 2007 / 047859). The meganuclease used herein binds to double-stranded DNA as a heterodimer or as a "single-chain meganuclease" in which pairs of DNA binding domains are joined into a single polypeptide using a peptide linker. The term "homing endonuclease" is synonymous with the term "meganuclease". The meganuclease of the present invention is substantially non-toxic such that when expressed in cells, particularly human T cells, cells can be transfected and maintained at 37°C without observing a detrimental effect on cell viability or a significant decrease in meganuclease cleavage activity as measured using the methods described herein. recognizes the DNA sequence. Therefore, a single-stranded meganuclease usually cleaves a recognition sequence that is a pseudo-palindrome or is a non-palindrome. Although a single-stranded meganuclease is not actually a dimer, it may be referred to as a "single-stranded heterodimer" or "single-stranded heterodimer meganuclease". For clarity, unless otherwise specified, the term "meganuclease" may refer to a dimer or a single-stranded meganuclease. is not actually a dimer, but may be referred to as a "single-stranded heterodimer" or "single-stranded heterodimer meganuclease". For clarity, unless otherwise specified, the term "meganuclease" may refer to a dimer or a single-stranded meganuclease. is not actually a dimer, but may be referred to as a "single-stranded heterodimer" or "single-stranded heterodimer meganuclease". For clarity, unless otherwise specified, the term "meganuclease" may refer to a dimer or a single-stranded meganuclease. is not actually a dimer, but may be referred to as a "single-stranded heterodimer" or "single-stranded heterodimer meganuclease". For clarity, unless otherwise specified, the term "meganuclease" may refer to a dimer or a single-stranded meganuclease.

[0221] The term "linker" as used herein refers to an exogenous peptide sequence used to connect two meganuclease subunits into a single polypeptide. The linker may have a sequence found in natural proteins or may be an artificial sequence not found in any natural protein. The linker may be flexible and lack secondary structure, or may have a tendency to form a specific three-dimensional structure under physiological conditions. Examples of linkers include, but are not limited to, those included in U.S. Patent No. 8,445,251 and U.S. Patent No. 9,434,931. In some embodiments, the linker may have an amino acid sequence comprising residues 154-195 of any one of SEQ ID NOs: 12-27. The term "linker" as used herein refers to an exogenous peptide sequence used to connect two meganuclease subunits into a single polypeptide. The linker may have a sequence found in natural proteins or may be an artificial sequence not found in any natural protein. The linker may be flexible and lack secondary structure, or may have a tendency to form a specific three-dimensional structure under physiological conditions. Examples of linkers include, but are not limited to, those included in U.S. Patent No. 8,445,251 and U.S. Patent No. 9,434,931. In some embodiments, the linker may have an amino acid sequence comprising residues 154-195 of any one of SEQ ID NOs: 12-27. The term "linker" as used herein refers to an exogenous peptide sequence used to connect two meganuclease subunits into a single polypeptide. The linker may have a sequence found in natural proteins or may be an artificial sequence not found in any natural protein. The linker may be flexible and lack secondary structure, or may have a tendency to form a specific three-dimensional structure under physiological conditions. Examples of linkers include, but are not limited to, those included in U.S. Patent No. 8,445,251 and U.S. Patent No. 9,434,931. In some embodiments, the linker may have an amino acid sequence comprising residues 154-195 of any one of SEQ ID NOs: 12-27. The term "linker" as used herein refers to an exogenous peptide sequence used to connect two meganuclease subunits into a single polypeptide. The linker may have a sequence found in natural proteins or may be an artificial sequence not found in any natural protein. The linker may be flexible and lack secondary structure, or may have a tendency to form a specific three-dimensional structure under physiological conditions. Examples of linkers include, but are not limited to, those included in U.S. Patent No. 8,445,251 and U.S. Patent No. 9,434,931. In some embodiments, the linker may have an amino acid sequence comprising residues 154-195 of any one of SEQ ID NOs: 12-27. The term "linker" as used herein refers to an exogenous peptide sequence used to connect two meganuclease subunits into a single polypeptide. The linker may have a sequence found in natural proteins or may be an artificial sequence not found in any natural protein. The linker may be flexible and lack secondary structure, or may have a tendency to form a specific three-dimensional structure under physiological conditions. Examples of linkers include, but are not limited to, those included in U.S. Patent No. 8,445,251 and U.S. Patent No. 9,434,931. In some embodiments, the linker may have an amino acid sequence comprising residues 154-195 of any one of SEQ ID NOs: 12-27. Examples of linkers include, but are not limited to, those included in U.S. Patent No. 8,445,251 and U.S. Patent No. 9,434,931. In some embodiments, the linker may have an amino acid sequence comprising residues 154-195 of any one of SEQ ID NOs: 12-27. Examples of linkers include, but are not limited to, those included in U.S. Patent No. 8,445,251 and U.S. Patent No. 9,434,931. In some embodiments, the linker may have an amino acid sequence comprising residues 154-195 of any one of SEQ ID NOs: 12-27. Examples of linkers include, but are not limited to, those included in U.S. Patent No. 8,445,251 and U.S. Patent No. 9,434,931. In some embodiments, the linker may have an amino acid sequence comprising residues 154-195 of any one of SEQ ID NOs: 12-27. Examples of linkers include, but are not limited to, those included in U.S. Patent No. 8,445,251 and U.S. Patent No. 9,434,931. In some embodiments, the linker may have an amino acid sequence comprising residues 154-195 of any one of SEQ ID NOs: 12-27.

[0222] The term "zinc finger nuclease" or "ZFN" as used herein refers to a nuclease domain derived from an endonuclease or exonuclease including, but not limited to, restriction endonucleases, homing endonucleases, S1 nuclease, mung bean nuclease, pancreatic DNAse I, The term "zinc finger nuclease" or "ZFN" as used herein refers to a nuclease domain derived from an endonuclease or exonuclease including, but not limited to, restriction endonucleases, homing endonucleases, S1 nuclease, mung bean nuclease, pancreatic DNAse I, The term "zinc finger nuclease" or "ZFN" as used herein refers to a nuclease domain derived from an endonuclease or exonuclease including, but not limited to, restriction endonucleases, homing endonucleases, S1 nuclease, mung bean nuclease, pancreatic DNAse I, The term "zinc finger nuclease" or "ZFN" as used herein refers to a nuclease domain derived from an endonuclease or exonuclease including, but not limited to, restriction endonucleases, homing endonucleases, S1 nuclease, mung bean nuclease, pancreatic DNAse I, The term "zinc finger nuclease" or "ZFN" as used herein refers to a nuclease domain derived from an endonuclease or exonuclease including, but not limited to, restriction endonucleases, homing endonucleases, S1 nuclease, mung bean nuclease, pancreatic DNAse I, Refers to a chimeric protein containing a combined zinc finger DNA binding domain. Zinc fi Examples of nuclease domains useful for the design of zinc finger nucleases include, but are not limited to, restriction enzymes such as FokI, FoM, StsI, and those derived from type II restriction endonucleases. Additional type II restriction endonucleases are described in International Publication No. WO 200 7 / 014275, which is hereby incorporated by reference in its entirety to form a part of this specification. The structure of the zinc finger domain is stabilized by coordination of zinc ions. A DNA binding protein containing one or more zinc finger domains binds to DNA in a sequence-specific manner. The zinc finger domain may be a native sequence or may be redesigned by rational or experimental means to produce a protein that binds to a predetermined DNA sequence of approximately 18 base pairs in length. For example, U.S. Patent Nos. 5,789,538, 5,925,523, 6,007,988, 6,013,453, 6,200,759, and International Publication Nos. WO 95 / 194 31, WO 96 / 06166, WO 98 / 53057, WO 98 / 54311, WO 00 / 27878, WO 01 / 60970, WO 01 / 88197, and WO 02 / 099084, each of which is hereby incorporated by reference in its entirety to form a part of this specification, may be referred to. By fusing this engineered protein domain to a nuclease domain such as FokI nuclease, it is possible to target DNA cleavage with genomic-level specificity. The selection of target sites, zinc finger proteins, and methods for the design and construction of zinc finger nucleases is known to those of ordinary skill in the art. For example, see WO 01 / 88197, WO 02 / 099084, and WO 03 / 076574. Cleavage, and methods for the design and construction of zinc finger nucleases are known to those of ordinary skill in the art. By fusing this engineered protein domain to a nuclease domain such as FokI nuclease, it is possible to target DNA cleavage with genomic-level specificity. The selection of target sites, zinc finger proteins, and methods for the design and construction of zinc finger nucleases is known to those of ordinary skill in the art. For example, see WO 01 / 88197, WO 02 / 099084, and WO 03 / 076574. and are described in detail in US Patent Application Publication Nos. 20030232410, 20050208489, , 2005064474, 20050026157, 20060188 987, and International Publication No. 07 / 014275, each of which is hereby incorporated by reference in its entirety to form a part of this specification.

[0223] As used herein, the term "TALEN" refers to an endonuclease or exonuclease-derived nuclease domain or its active portion fused to a plurality of TAL domain repeats of the DNA binding domain containing restriction endonucleases, homing endonucleases, S1 nuclease, mung bean nuclease, pancreatic DNase I, Micrococcus nuclease, and yeast HO endonuclease, but not limited thereto. For example, see Christian et al. (2010) Genetics 186:757-761, which is hereby incorporated by reference in its entirety to form a part of this specification. Useful nuclease domains for TALEN design include, but are not limited to, those derived from type II restriction endonucleases such as FokI, Fo M, StsI, HhaI, HindIII, Nod, BbvCI, EcoRI, BglI , and AlwI. Additional type II restriction endonucleases are described in International Publication No. 2007 / 01 4275. In some embodiments, the nuclease domain of the TALEN is the FokI nuclease domain or its active portion. The TAL domain repeats may be derived from proteins of the TALE (transcription activator-like effector) family used in the infection process by plant pathogens of the genus Xanthomonas. T M, StsI, HhaI, HindIII, Nod, BbvCI, EcoRI, BglI , and AlwI. Additional type II restriction endonucleases are described in International Publication No. 2007 / 01 4275. In some embodiments, the nuclease domain of the TALEN is the FokI nuclease domain or its active portion. The TAL domain repeats may be derived from proteins of the TALE (transcription activator-like effector) family used in the infection process by plant pathogens of the genus Xanthomonas. T ALE (transcription activator-like effector) family of proteins. The TAL domain repeat is 33 to 34 amino acids with diverse 12th and 13th amino acids. These two positions, called repeat variable dipeptides (RVDs), are highly variable and show a strong correlation with specific nucleotide recognition. Each base pair of the DNA target sequence contacts a single TAL repeat due to the specificity of the RVD. In some embodiments, the TALEN contains 16 to 22 TAL domain repeats. DNA cleavage by TALENs requires two DNA recognition regions flanking a non-specific central region (i.e., the "spacer"). The term "spacer" with respect to TALENs refers to the nucleic acid sequence that separates the two nucleic acid sequences recognized and bound by each monomer that makes up the TALEN. The TAL domain repeat may be the native sequence of a naturally occurring TALE protein, or it can be redesigned by rational or experimental means to produce a protein that binds to a predetermined DNA sequence (see, for example, Boch et al. (2009) Science 326(5959):1509-1512 and Moscou and Bogdanove (2009) Science 326(5959):1501, each of which is incorporated herein by reference in its entirety). For examples of specific sequences and RVDs, and methods of engineering TALENs to recognize corresponding target nucleotides, see also U.S. Patent Application Publication No. 20110145940 and International Publication No. 2010 / 079430. In some embodiments, each nuclease (e.g., FokI) monomer is a TAL effector that recognizes a different DNA sequence. It may be fused to the factor array and is inactive only when two recognition sites are in proximity. The monomers come together to create a functional enzyme.

[0224] As used herein, the term "Compact TALEN" refers to any part of any of the endonucleases listed in Table 2 of U.S. Patent Application No. 20130117869, including but not limited to I-TevI homing endonuclease, or MmeI, EndA, End1, I-BasI, I-TevII, I-TevIII, I-TwoI, MspI, MvaI, NucA, NucM, etc. (incorporated herein by reference in its entirety), having a 16-22 TAL domain repeat fused in any direction to any part of the endonuclease. A Compact TALEN does not require dimerization for DNA processing activity and reduces the need for intervening DNA spacers at double target sites. In some embodiments, the Compact TALEN comprises 16 to 22 TAL domain repeats. homing endonuclease, or MmeI, EndA, End1, I-BasI, I-Tev II, I-TevIII, I-TwoI, MspI, MvaI, NucA, NucM, etc. listed in Table 2 of U.S. Patent Application No. 20130117869, including but not limited to these, incorporated herein by reference in its entirety and refers to an endonuclease containing a DNA binding domain having a 16-22 TAL domain repeat fused in any direction to any part of the endonuclease. A Compact TALEN does not require dimerization for DNA processing activity and reduces the need for intervening DNA spacers at double target sites. In some embodiments, the Compact TALEN comprises 16 to 22 TAL domain repeats. and refers to an endonuclease containing a DNA binding domain having a 16-22 TAL domain repeat fused in any direction to any part of the endonuclease. A Compact TALEN does not require dimerization for DNA processing activity and reduces the need for intervening DNA spacers at double target sites. In some embodiments, the Compact TALEN comprises 16 to 22 TAL domain repeats. and refers to an endonuclease containing a DNA binding domain having a 16-22 TAL domain repeat fused in any direction to any part of the endonuclease. A Compact TALEN does not require dimerization for DNA processing activity and reduces the need for intervening DNA spacers at double target sites. In some embodiments, the Compact TALEN comprises 16 to 22 TAL domain repeats. A Compact TALEN does not require dimerization for DNA processing activity and reduces the need for intervening DNA spacers at double target sites. In some embodiments, the Compact TALEN comprises 16 to 22 TAL domain repeats. A Compact TALEN does not require dimerization for DNA processing activity and reduces the need for intervening DNA spacers at double target sites. In some embodiments, the Compact TALEN comprises 16 to 22 TAL domain repeats. TAL domain repeats.

[0225] As used herein, the term "CRISPR" refers to a Caspase-based endonuclease, such as Cas9, Cpf1, or other suitable nucleases, and a guide RNA that hybridizes to a recognition site of genomic DNA to direct DNA cleavage by the Caspase. The Caspase component of CRISPR is an RNA-guided DNA endonuclease. In certain embodiments, the Caspase is a class II Cas enzyme. In some of these embodiments, the Caspase is a class II, type II enzyme such as Cas9. As used herein, the term "CRISPR" refers to a Caspase-based endonuclease, such as Cas9, Cpf1, or other suitable nucleases, and a guide RNA that hybridizes to a recognition site of genomic DNA to direct DNA cleavage by the Caspase. The Caspase component of CRISPR is an RNA-guided DNA endonuclease. In certain embodiments, the Caspase is a class II Cas enzyme. In some of these embodiments, the Caspase is a class II, type II enzyme such as Cas9. As used herein, the term "CRISPR" refers to a Caspase-based endonuclease, such as Cas9, Cpf1, or other suitable nucleases, and a guide RNA that hybridizes to a recognition site of genomic DNA to direct DNA cleavage by the Caspase. The Caspase component of CRISPR is an RNA-guided DNA endonuclease. In certain embodiments, the Caspase is a class II Cas enzyme. In some of these embodiments, the Caspase is a class II, type II enzyme such as Cas9. As used herein, the term "CRISPR" refers to a Caspase-based endonuclease, such as Cas9, Cpf1, or other suitable nucleases, and a guide RNA that hybridizes to a recognition site of genomic DNA to direct DNA cleavage by the Caspase. The Caspase component of CRISPR is an RNA-guided DNA endonuclease. In certain embodiments, the Caspase is a class II Cas enzyme. In some of these embodiments, the Caspase is a class II, type II enzyme such as Cas9. As used herein, the term "CRISPR" refers to a Caspase-based endonuclease, such as Cas9, Cpf1, or other suitable nucleases, and a guide RNA that hybridizes to a recognition site of genomic DNA to direct DNA cleavage by the Caspase. The Caspase component of CRISPR is an RNA-guided DNA endonuclease. In certain embodiments, the Caspase is a class II Cas enzyme. In some of these embodiments, the Caspase is a class II, type II enzyme such as Cas9. In certain embodiments, the Caspase is a class II Cas enzyme. In other embodiments, the caspase is a Class II, Type V enzyme such as Cpf1. The guide RNA comprises a direct repeat and a guide sequence complementary to the target recognition site (often referred to as a spacer with respect to the endogenous CRISPR system). In certain embodiments, CRISPR further comprises a tracrRNA (trans-activating CRISPR RNA) that is (fully or partially) complementary to the direct repeat sequence (sometimes referred to as the tracr mate sequence) present on the guide RNA. In certain embodiments, the caspase can be mutated with respect to the corresponding wild-type enzyme such that the enzyme lacks the ability to cleave a single strand of the target polynucleotide and functions as a nickase, cleaving only one strand of the target DNA. Non-limiting examples of caspase enzymes that function as nickases include the D10A mutation within the RuvC I catalytic domain, or Cas9 enzymes having mutations of H840A, N854A, or N863A. As used herein, the term "megaTAL" refers to a single-stranded nuclease comprising an engineered sequence-specific homing endonuclease with a transcription activator-like effector (TALE) DNA-binding domain. For a protein, the terms "recombinant" or "engineered" as used herein mean having an amino acid sequence that has been altered as a result of the application of genetic engineering techniques to the nucleic acid encoding the protein and to the cell or organism expressing the protein. For a nucleic acid, the terms "recombinant" or "engineered" mean as a result of the application of genetic engineering techniques.

[0226]

[0227] ​ means having a nucleic acid sequence that has been changed. Genetic engineering techniques include, but are not limited to, PCR and D NA cloning techniques; transfection, transformation, and other gene introduction techniques; homologous recombination; site -specific mutagenesis; and gene fusion. According to this definition, a protein having the same amino acid sequence as a protein of natural origin, but produced by cloning and expression in a heterologous host, is not considered a recombinant or engineered one. The term "wild-type" as used herein refers to the most common allele of natural origin in a population of alleles of the same type of gene (i.e., polynucleotide sequence), and the polypeptide encoded by the wild-type allele has its original function. The term "wild-type" also refers to the polypeptide encoded by the wild-type allele. A wild-type allele (i.e., polynucleotide) and polypeptide are distinguishable from a mutant or variant allele and polypeptide that contain one or more mutations and / or substitutions as compared to the wild-type sequence(s). While a wild-type allele or polypeptide can confer a normal phenotype on an organism, a mutant or variant allele or polypeptide can, in some cases, confer a change in phenotype. A wild-type nuclease can be distinguished from a recombinant, engineered, or non-naturally occurring nuclease. For a recombinant or engineered protein, the term "modification" as used herein means an insertion, deletion, or substitution of an amino acid residue in the recombinant sequence relative to a reference sequence (e.g., wild-type or native sequence).

[0228] The term "wild-type" as used herein refers to the most common allele of natural origin in a population of alleles of the same type of gene (i.e., polynucleotide sequence), and the polypeptide encoded by the wild-type allele has its original function. The term "wild-type" also refers to the polypeptide encoded by the wild-type allele. A wild-type allele (i.e., polynucleotide) and polypeptide are distinguishable from a mutant or variant allele and polypeptide that contain one or more mutations and / or substitutions as compared to the wild-type sequence(s). While a wild-type allele or polypeptide can confer a normal phenotype on an organism, a mutant or variant allele or polypeptide can, in some cases, confer a change in phenotype. A wild-type nuclease can be distinguished from a recombinant, engineered, or non-naturally occurring nuclease.

[0229] For a recombinant or engineered protein, the term "modification" as used herein means an insertion, deletion, or substitution of an amino acid residue in the recombinant sequence relative to a reference sequence (e.g., wild-type or native sequence). ​

[0230] As used herein, the term "recognition sequence" refers to a DNA sequence that is bound and cleaved by an endonuclease. In the case of a meganuclease, the recognition sequence contains a pair of "half-sites" of nine base pairs in reverse orientation separated by four base pairs. In the case of a single-strand meganuclease, the N-terminal domain of the protein contacts the first half-site and the C-terminal domain of the protein contacts the second half-site. Cleavage by the meganuclease results in a 3' "overhang" of four base pairs. An "overhang" or "sticky end" is a short single-stranded DNA segment that can be brought about by endonuclease cleavage of a double-stranded DNA sequence. In the case of a meganuclease derived from I-CreI and a single-strand meganuclease, the overhang contains 10 to 13 bases of the 22-base pair recognition sequence. For a compact TALEN, the recognition sequence may include a first CNNNGN sequence recognized by the I-TevI domain, followed by a non-specific spacer of 4 to 16 base pairs in length, followed by a second sequence of 16 to 22 bp in length recognized by the TAL effector domain (this sequence typically has a 5' T base). Cleavage by the compact TALEN results in a 3' overhang of two base pairs. In the case of CRISPR, the recognition sequence is a sequence for the guide RNA to bind and direct Cas9 cleavage, typically 16 to 24 base pairs. Complete complementarity between the guide sequence and the recognition sequence is not necessarily required for cleavage. Cleavage by CRISPR results in blunt ends (class II, type II caspases, etc.) or overhang ends (class II, type V caspases, etc.) depending on the caspase. In the case of a single-strand meganuclease, the N-terminal domain of the protein contacts the first half-site and the C-terminal domain of the protein contacts the second half-site. Cleavage by the meganuclease results in a 3' "overhang" of four base pairs. An "overhang" or "sticky end" is a short single-stranded DNA segment that can be brought about by endonuclease cleavage of a double-stranded DNA sequence. In the case of a meganuclease derived from I-CreI and a single-strand meganuclease, the overhang contains 10 to 13 bases of the 22-base pair recognition sequence. In the case of a single-strand meganuclease, the N-terminal domain of the protein contacts the first half-site and the C-terminal domain of the protein contacts the second half-site. Cleavage by the meganuclease results in a 3' "overhang" of four base pairs. An "overhang" or "sticky end" is a short single-stranded DNA segment that can be brought about by endonuclease cleavage of a double-stranded DNA sequence. In the case of a meganuclease derived from I-CreI and a single-strand meganuclease, the overhang contains 10 to 13 bases of the 22-base pair recognition sequence. In the case of a single-strand meganuclease, the N-terminal domain of the protein contacts the first half-site and the C-terminal domain of the protein contacts the second half-site. Cleavage by the meganuclease results in a 3' "overhang" of four base pairs. An "overhang" or "sticky end" is a short single-stranded DNA segment that can be brought about by endonuclease cleavage of a double-stranded DNA sequence. In the case of a meganuclease derived from I-CreI and a single-strand meganuclease, the overhang contains 10 to 13 bases of the 22-base pair recognition sequence. In the case of a single-strand meganuclease, the N-terminal domain of the protein contacts the first half-site and the C-terminal domain of the protein contacts the second half-site. Cleavage by the meganuclease results in a 3' "overhang" of four base pairs. An "overhang" or "sticky end" is a short single-stranded DNA segment that can be brought about by endonuclease cleavage of a double-stranded DNA sequence. In the case of a meganuclease derived from I-CreI and a single-strand meganuclease, the overhang contains 10 to 13 bases of the 22-base pair recognition sequence. In the case of a single-strand meganuclease, the N-terminal domain of the protein contacts the first half-site and the C-terminal domain of the protein contacts the second half-site. Cleavage by the meganuclease results in a 3' "overhang" of four base pairs. An "overhang" or "sticky end" is a short single-stranded DNA segment that can be brought about by endonuclease cleavage of a double-stranded DNA sequence. In the case of a meganuclease derived from I-CreI and a single-strand meganuclease, the overhang contains 10 to 13 bases of the 22-base pair recognition sequence. In the case of a single-strand meganuclease, the N-terminal domain of the protein contacts the first half-site and the C-terminal domain of the protein contacts the second half-site. Cleavage by the meganuclease results in a 3' "overhang" of four base pairs. An "overhang" or "sticky end" is a short single-stranded DNA segment that can be brought about by endonuclease cleavage of a double-stranded DNA sequence. In the case of a meganuclease derived from I-CreI and a single-strand meganuclease, the overhang contains 10 to 13 bases of the 22-base pair recognition sequence. In the case of a single-strand meganuclease, the N-terminal domain of the protein contacts the first half-site and the C-terminal domain of the protein contacts the second half-site. Cleavage by the meganuclease results in a 3' "overhang" of four base pairs. An "overhang" or "sticky end" is a short single-stranded DNA segment that can be brought about by endonuclease cleavage of a double-stranded DNA sequence. In the case of a meganuclease derived from I-CreI and a single-strand meganuclease, the overhang contains 10 to 13 bases of the 22-base pair recognition sequence. For a compact TALEN, the recognition sequence may include a first CNNNGN sequence recognized by the I-TevI domain, followed by a non-specific spacer of 4 to 16 base pairs in length, followed by a second sequence of 16 to 22 bp in length recognized by the TAL effector domain (this sequence typically has a 5' T base). Cleavage by the compact TALEN results in a 3' overhang of two base pairs. In the case of CRISPR, the recognition sequence is a sequence for the guide RNA to bind and direct Cas9 cleavage, typically 16 to 24 base pairs. Complete complementarity between the guide sequence and the recognition sequence is not necessarily required for cleavage. Cleavage by CRISPR results in blunt ends (class II, type II caspases, etc.) or overhang ends (class II, type V caspases, etc.) depending on the caspase. For a compact TALEN, the recognition sequence may include a first CNNNGN sequence recognized by the I-TevI domain, followed by a non-specific spacer of 4 to 16 base pairs in length, followed by a second sequence of 16 to 22 bp in length recognized by the TAL effector domain (this sequence typically has a 5' T base). Cleavage by the compact TALEN results in a 3' overhang of two base pairs. In the case of CRISPR, the recognition sequence is a sequence for the guide RNA to bind and direct Cas9 cleavage, typically 16 to 24 base pairs. Complete complementarity between the guide sequence and the recognition sequence is not necessarily required for cleavage. Cleavage by CRISPR results in blunt ends (class II, type II caspases, etc.) or overhang ends (class II, type V caspases, etc.) depending on the caspase. For a compact TALEN, the recognition sequence may include a first CNNNGN sequence recognized by the I-TevI domain, followed by a non-specific spacer of 4 to 16 base pairs in length, followed by a second sequence of 16 to 22 bp in length recognized by the TAL effector domain (this sequence typically has a 5' T base). Cleavage by the compact TALEN results in a 3' overhang of two base pairs. In the case of CRISPR, the recognition sequence is a sequence for the guide RNA to bind and direct Cas9 cleavage, typically 16 to 24 base pairs. Complete complementarity between the guide sequence and the recognition sequence is not necessarily required for cleavage. Cleavage by CRISPR results in blunt ends (class II, type II caspases, etc.) or overhang ends (class II, type V caspases, etc.) depending on the caspase. For a compact TALEN, the recognition sequence may include a first CNNNGN sequence recognized by the I-TevI domain, followed by a non-specific spacer of 4 to 16 base pairs in length, followed by a second sequence of 16 to 22 bp in length recognized by the TAL effector domain (this sequence typically has a 5' T base). Cleavage by the compact TALEN results in a 3' overhang of two base pairs. In the case of CRISPR, the recognition sequence is a sequence for the guide RNA to bind and direct Cas9 cleavage, typically 16 to 24 base pairs. Complete complementarity between the guide sequence and the recognition sequence is not necessarily required for cleavage. Cleavage by CRISPR results in blunt ends (class II, type II caspases, etc.) or overhang ends (class II, type V caspases, etc.) depending on the caspase. For a compact TALEN, the recognition sequence may include a first CNNNGN sequence recognized by the I-TevI domain, followed by a non-specific spacer of 4 to 16 base pairs in length, followed by a second sequence of 16 to 22 bp in length recognized by the TAL effector domain (this sequence typically has a 5' T base). Cleavage by the compact TALEN results in a 3' overhang of two base pairs. In the case of CRISPR, the recognition sequence is a sequence for the guide RNA to bind and direct Cas9 cleavage, typically 16 to 24 base pairs. Complete complementarity between the guide sequence and the recognition sequence is not necessarily required for cleavage. Cleavage by CRISPR results in blunt ends (class II, type II caspases, etc.) or overhang ends (class II, type V caspases, etc.) depending on the caspase. For a compact TALEN, the recognition sequence may include a first CNNNGN sequence recognized by the I-TevI domain, followed by a non-specific spacer of 4 to 16 base pairs in length, followed by a second sequence of 16 to 22 bp in length recognized by the TAL effector domain (this sequence typically has a 5' T base). Cleavage by the compact TALEN results in a 3' overhang of two base pairs. In the case of CRISPR, the recognition sequence is a sequence for the guide RNA to bind and direct Cas9 cleavage, typically 16 to 24 base pairs. Complete complementarity between the guide sequence and the recognition sequence is not necessarily required for cleavage. Cleavage by CRISPR results in blunt ends (class II, type II caspases, etc.) or overhang ends (class II, type V caspases, etc.) depending on the caspase. For a compact TALEN, the recognition sequence may include a first CNNNGN sequence recognized by the I-TevI domain, followed by a non-specific spacer of 4 to 16 base pairs in length, followed by a second sequence of 16 to 22 bp in length recognized by the TAL effector domain (this sequence typically has a 5' T base). Cleavage by the compact TALEN results in a 3' overhang of two base pairs. In the case of CRISPR, the recognition sequence is a sequence for the guide RNA to bind and direct Cas9 cleavage, typically 16 to 24 base pairs. Complete complementarity between the guide sequence and the recognition sequence is not necessarily required for cleavage. Cleavage by CRISPR results in blunt ends (class II, type II caspases, etc.) or overhang ends (class II, type V caspases, etc.) depending on the caspase. For a compact TALEN, the recognition sequence may include a first CNNNGN sequence recognized by the I-TevI domain, followed by a non-specific spacer of 4 to 16 base pairs in length, followed by a second sequence of 16 to 22 bp in length recognized by the TAL effector domain (this sequence typically has a 5' T base). Cleavage by the compact TALEN results in a 3' overhang of two base pairs. In the case of CRISPR, the recognition sequence is a sequence for the guide RNA to bind and direct Cas9 cleavage, typically 16 to 24 base pairs. Complete complementarity between the guide sequence and the recognition sequence is not necessarily required for cleavage. Cleavage by CRISPR results in blunt ends (class II, type II caspases, etc.) or overhang ends (class II, type V caspases, etc.) depending on the caspase. For a compact TALEN, the recognition sequence may include a first CNNNGN sequence recognized by the I-TevI domain, followed by a non-specific spacer of 4 to 16 base pairs in length, followed by a second sequence of 16 to 22 bp in length recognized by the TAL effector domain (this sequence typically has a 5' T base). Cleavage by the compact TALEN results in a 3' overhang of two base pairs. In the case of CRISPR, the recognition sequence is a sequence for the guide RNA to bind and direct Cas9 cleavage, typically 16 to 24 base pairs. Complete complementarity between the guide sequence and the recognition sequence is not necessarily required for cleavage. Cleavage by CRISPR results in blunt ends (class II, type II caspases, etc.) or overhang ends (class II, type V caspases, etc.) depending on the caspase. can result in, for example, spase, etc.). In those embodiments where CpfI caspase is utilized, cleavage by the CRISPR complex containing it results in a 5' overhang, and in certain embodiments, a 5-nucleotide 5' overhang. Each caspase enzyme also requires recognition of a PAM (protospacer adjacent motif) sequence near the recognition sequence complementary to the guide RNA. The exact sequence, length requirements for the PAM, and distance from the target sequence vary depending on the caspase enzyme, but the PAM is typically a sequence of 2 to 5 base pairs adjacent to the target / recognition sequence. The PAM sequences for specific caspase enzymes are known in the art (see, for example, U.S. Patent No. 8, 697,359 and U.S. Patent Application Publication No. 2016 / 0208243, each of which is incorporated herein by reference in its entirety). For a novel or engineered caspase enzyme, the PAM sequence can be identified using methods known in the art, such as a PAM depletion assay (see, for example, Karvelis et al. (2017) Methods 121-122:3-8, which is incorporated herein by reference in its entirety). For zinc fingers, the DNA binding domain typically recognizes an 18-bp recognition sequence containing pairs of 9-bp "half-sites" separated by 2 to 10 base pairs, and cleavage by the nuclease creates blunt ends or 5' overhangs of variable length (often 4 base pairs). The term "target site" or "target sequence" as used herein refers to the region of the chromosomal DNA of a cell that contains the recognition sequence of the nuclease. The term "DNA binding affinity" or "binding affinity" as used herein refers to the meganu

[0231]

[0232] ​ The tendency of the nuclease to bind non-covalently to a reference DNA molecule (e.g., a recognition sequence or any sequence) is meant. The binding affinity is the dissociation constant K d measured by. As used herein such that, the K of the nuclease for the reference recognition sequence d is increased or decreased by a statistically significant percent change compared to the reference nuclease, the nuclease has an "altered" binding affinity.

[0233] As used herein, the term "homologous recombination" or "HR" refers to the natural cellular process by which a double-strand DNA break is repaired using a repair template and a homologous DNA sequence (see, e.g., Cahill et al. (2006), Front. Biosci. 11:1958- 1976). The homologous DNA sequence may be an endogenous chromosomal sequence or an exogenous nucleic acid delivered to the cell.

[0234] As used herein, the term "non-homologous end joining" or "NHEJ" refers to the natural cellular process by which a double-strand DNA break is repaired by directly ligating two non- homologous DNA segments (see, e.g., Cahill et al. (2006), Front. Biosci. 1 1:1958-1976). DNA repair by non-homologous end joining is error-prone and frequently results in template-independent additions or deletions of DNA sequences at the repair site . In some cases, cleavage at a target recognition sequence results in NHEJ at the target recognition site . NHEJ-mediated DNA repair after nuclease-induced cleavage at a target site in the coding sequence of a gene can introduce mutations such as frameshift mutations that disrupt gene function into the coding sequence . ​It is thus possible to effectively knockout genes in a cell population using the engineered nuclease.

[0235] As used herein, "chimeric antigen receptor" or "CAR" refers to an engineered receptor that confers specificity for an antigen to an immune effector cell (e.g., a human T cell) or grafts it. A chimeric antigen receptor typically includes at least an extracellular ligand binding domain or portion and an intracellular domain that includes one or more signaling domains and / or co-stimulatory domains.

[0236] In some embodiments, the extracellular ligand binding domain or portion is in the form of a single chain variable fragment (scFV) derived from a monoclonal antibody and provides specificity for a particular epitope or antigen (e.g., an epitope or antigen that is preferentially present on the surface of cancer cells, or cells or particles that cause other diseases). In some embodiments, the scFv is attached via a linker sequence. In various embodiments, the extracellular ligand binding domain is specific for any antigen or epitope of interest. In some embodiments, the scFv is murine, humanized, or fully human.

[0237] The extracellular domain of the chimeric antigen receptor may include a self - antigen that can be recognized by a self - antigen - specific B - cell receptor on a B lymphocyte (see Payne et al. (2016), Science 353(6295):179 - 184), and direct T cells to specifically target and kill autoreactive B lymphocytes in antibody - mediated autoimmune diseases. Such CARs may be referred to as chimeric autoantibody receptors (CAARs), and their use is included in the present invention. ​​​​​​​​​​​​​​​ It exists.

[0238] The extracellular domain of the chimeric antigen receptor may also contain a naturally occurring ligand for the antigen of interest, or a fragment of a naturally occurring ligand that retains the ability to bind to the antigen of interest. It is also acceptable.

[0239] The intracellular stimulating domain may include one or more cytoplasmic signaling domains that transmit an activation signal to immune effector cells after antigen binding. Such cytoplasmic signaling domains include, but are not limited to, CD3ζ.

[0240] The intracellular stimulating domain may also include one or more intracellular co-stimulatory domains that transmit a proliferation and / or cell survival signal after ligand binding. As used herein, "co-stimulatory domain" refers to a polypeptide domain that transmits an intracellular proliferation and / or cell survival signal upon activation. Activation of the co-stimulatory domain may occur following homodimerization of two co-stimulatory domain polypeptides. Activation may also occur, for example, after activation of a construct containing a co-stimulatory domain (e.g., a chimeric antigen receptor or an inducible regulatory construct). Generally, the co-stimulatory domain may be derived from a transmembrane co-stimulatory receptor, particularly the intracellular portion of a co-stimulatory receptor. Such intracellular co-stimulatory domains may be any of those known in the art and include, but are not limited to, CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, domain domains. Activation may occur following homodimerization of two co-stimulatory domain polypeptides. Activation may also occur, for example, after activation of a construct containing a co-stimulatory domain (e.g., a chimeric antigen receptor or an inducible regulatory construct). Generally, the co-stimulatory domain may be derived from a transmembrane co-stimulatory receptor, particularly the intracellular portion of a co-stimulatory receptor. Such intracellular co-stimulatory domains may be any of those known in the art and are not limited, but include CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, B(CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83, N1, N6 and ligands that specifically bind thereto, or any combination thereof may include.

[0241] Chimeric antigen receptors are characterized by their ability to bind extracellular ligand-binding domains via hinge or spacer sequences. It may further comprise additional structural elements, including an attached transmembrane domain. The polypeptide may be derived from any membrane-bound or transmembrane protein. For example, a transmembrane polypeptide. The peptide binds to a subunit of the T cell receptor (i.e., α, β, γ, or ζ), which constitutes the CD3 complex. polypeptides that form the IL2 receptor p55 (a chain), p75 (β chain or γ chain), Fc receptor subunit chain of the receptor (e.g., Fcγ receptor III), or CD8 alpha chain, Alternatively, the transmembrane domain may be synthetic and may contain leucine. and valine.

[0242] The hinge region functions to link the transmembrane domain to the extracellular ligand-binding domain. For example, the hinge region may be up to 300 amino acids long, Preferably, it contains 10 to 100 amino acids, and most preferably 25 to 50 amino acids. The hinge region may be a whole or part of the extracellular domain of CD8, CD4, or CD28. or may be derived from all or a portion of a naturally occurring molecule, such as all or a portion of an antibody constant region. Alternatively, the hinge region may be a synthetic sequence which corresponds to a naturally occurring hinge sequence, or It may also be a completely synthetic hinge sequence. In a particular example, the hinge domain is a human C It may include portions of the D8 alpha chain, the FcyRlla receptor, or IgG1.

[0243] As used herein, an "exogenous T cell receptor" or "exogenous TCR" refers to a T cell receptor whose sequence is Immune effector cells (e.g., human T cells) that may or may not endogenously express CR refers to a TCR introduced into the genome of. Expression of an exogenous TCR on an immune effector cell can confer specificity for a particular epitope or antigen (e.g., an epitope or antigen preferentially present on the surface of cancer cells, or cells or particles that cause other diseases) . Such an exogenous T cell receptor may include an alpha chain and a beta chain, or may include a gamma chain and a delta chain. An exogenous TCR useful in the present invention may have specificity for any antigen or epitope of interest.

[0244] As used herein, the term "decrease in expression" refers to a decrease in the expression of the endogenous T cell receptor on the cell surface of a genetically modified T cell when compared to control cells. The term decreased may also refer to a decrease in the proportion of cells in a population of cells expressing an endogenous polypeptide (i.e., the endogenous T cell receptor) on the cell surface when compared to a population of control cells. Such a decrease may be up to 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80 %, 90%, 95%, 96%, 97%, 98%, 99%, or up to 100% . Thus, the term "decreased" encompasses both partial knockdown and complete knockdown of the endogenous T cell receptor.

[0245] When used herein with respect to both amino acid sequences and nucleic acid sequences, the terms "percent identity", "sequence identity", "percentage similarity", "sequence similarity", etc. maximize the similarity between aligned amino acid residues or nucleotides, and the same or similar residues or nucleo The number of oligomers, the total number of residues or nucleotides, and the presence of gaps in the sequence alignment and refers to a measure of the similarity of two sequences based on the alignment of the sequences that is a function of length . To determine sequence similarity using standard parameters, various algorithms and computer programs can be used. The sequence similarity used herein is determined using the BLASTp program for amino acid sequences and the BLASTn program for nucleic acid sequences, both of which are available from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ), and are described, for example, in Altschul et al. (1990), J. Mol. Biol. 215:403-410; Gish and States (1993), Nat ure Genet. 3:266-272; Madden et al. (1996), Meth . Enzymol. 266:131-141; Altschul et al. (1997), Nuc leic Acids Res. 25:3389-3402); Zhang et al. (200 0), J. Comput. Biol. 7(1-2):203-14. The percent similarity of two amino acid sequences used herein is a score based on the following parameters for the BLASTp algorithm: word size = 3; gap opening penalty = -11; gap extension penalty = -1; scoring matrix = BL OSUM62. The percent similarity of two nucleic acid sequences used herein is a score based on the following parameters for the BLASTn algorithm: word size = 11; gap opening penalty = -5; gap extension penalty = -2; match reward = 1; mismatch penalty = -3; scoring matrix = identity matrix. penalty = -3; scoring matrix = identity matrix. 0), J. Comput. Biol. 7(1-2):203-14. The percent similarity of two amino acid sequences used herein is a score based on the following parameters for the BLASTp algorithm: word size = 3; gap opening penalty = -11; gap extension penalty = -1; scoring matrix = BL OSUM62. The percent similarity of two nucleic acid sequences used herein is a score based on the following parameters for the BLASTn algorithm: word size = 11; gap opening penalty = -5; gap extension penalty = -2; match reward = 1; mismatch penalty = -3; scoring matrix = identity matrix. start penalty = -11; gap extension penalty = -1; scoring matrix = BL OSUM62. The percent similarity of two nucleic acid sequences used herein is a score based on the following parameters for the BLASTn algorithm: word size = 11; gap opening penalty = -5; gap extension penalty = -2; match reward = 1; mismatch penalty = -3; scoring matrix = identity matrix. Penalty for a foul = -3.

[0246] As used herein with respect to modifications of two proteins or amino acid sequences, the term "corresponding" is used to indicate that a particular modification of a first protein is the same amino acid residue substitution as a modification of a second protein, and when the two proteins are subjected to a standard sequence alignment (e.g., when using the BLASTp program), the amino acid position of the modification of the first protein corresponds to or aligns with the amino acid position of the modification of the second protein. Thus, a modification of the amino acid "A" of residue "X" of the first protein corresponds to a modification of the amino acid "A" of residue "Y" of the second protein, even though the residues X and Y may be different numbers when they match each other in a sequence alignment. As used herein, the terms "recognition half-site", "recognition sequence half-site", or simply "half-site" refer to a nucleic acid sequence within a double-stranded DNA molecule that is recognized by the monomer of a homodimeric or heterodimeric meganuclease, or by one subunit of a single-stranded meganuclease. As used herein, the term "hypervariable region" refers to a localized sequence within a meganuclease monomer or subunit that contains amino acids with relatively high variability. The hypervariable region can contain about 50 - 60 contiguous residues, about 53 - 57 contiguous residues, or preferably about 56 residues. In some embodiments, the residues of the hypervariable region are of SEQ ID NO. alignment (e.g., when using the BLASTp program), the amino acid position of the modification of the first protein corresponds to or aligns with the amino acid position of the modification of the second protein. Thus, a modification of the amino acid "A" of residue "X" of the first protein corresponds to a modification of the amino acid "A" of residue "Y" of the second protein, even though the residues X and Y may be different numbers when they match each other in a sequence alignment. alignment (e.g., when using the BLASTp program), the amino acid position of the modification of the first protein corresponds to or aligns with the amino acid position of the modification of the second protein. Thus, a modification of the amino acid "A" of residue "X" of the first protein corresponds to a modification of the amino acid "A" of residue "Y" of the second protein, even though the residues X and Y may be different numbers when they match each other in a sequence alignment. alignment (e.g., when using the BLASTp program), the amino acid position of the modification of the first protein corresponds to or aligns with the amino acid position of the modification of the second protein. Thus, a modification of the amino acid "A" of residue "X" of the first protein corresponds to a modification of the amino acid "A" of residue "Y" of the second protein, even though the residues X and Y may be different numbers when they match each other in a sequence alignment. alignment (e.g., when using the BLASTp program), the amino acid position of the modification of the first protein corresponds to or aligns with the amino acid position of the modification of the second protein. Thus, a modification of the amino acid "A" of residue "X" of the first protein corresponds to a modification of the amino acid "A" of residue "Y" of the second protein, even though the residues X and Y may be different numbers when they match each other in a sequence alignment. alignment (e.g., when using the BLASTp program), the amino acid position of the modification of the first protein corresponds to or aligns with the amino acid position of the modification of the second protein. Thus, a modification of the amino acid "A" of residue "X" of the first protein corresponds to a modification of the amino acid "A" of residue "Y" of the second protein, even though the residues X and Y may be different numbers when they match each other in a sequence alignment. alignment (e.g., when using the BLASTp program), the amino acid position of the modification of the first protein corresponds to or aligns with the amino acid position of the modification of the second protein. Thus, a modification of the amino acid "A" of residue "X" of the first protein corresponds to a modification of the amino acid "A" of residue "Y" of the second protein, even though the residues X and Y may be different numbers when they match each other in a sequence alignment.

[0247] As used herein, the terms "recognition half-site", "recognition sequence half-site", or simply "half-site" refer to a nucleic acid sequence within a double-stranded DNA molecule that is recognized by the monomer of a homodimeric or heterodimeric meganuclease, or by one subunit of a single-stranded meganuclease. As used herein, the terms "recognition half-site", "recognition sequence half-site", or simply "half-site" refer to a nucleic acid sequence within a double-stranded DNA molecule that is recognized by the monomer of a homodimeric or heterodimeric meganuclease, or by one subunit of a single-stranded meganuclease. As used herein, the terms "recognition half-site", "recognition sequence half-site", or simply "half-site" refer to a nucleic acid sequence within a double-stranded DNA molecule that is recognized by the monomer of a homodimeric or heterodimeric meganuclease, or by one subunit of a single-stranded meganuclease. As used herein, the terms "recognition half-site", "recognition sequence half-site", or simply "half-site" refer to a nucleic acid sequence within a double-stranded DNA molecule that is recognized by the monomer of a homodimeric or heterodimeric meganuclease, or by one subunit of a single-stranded meganuclease.

[0248] As used herein, the term "hypervariable region" refers to a localized sequence within a meganuclease monomer or subunit that contains amino acids with relatively high variability. The hypervariable region can contain about 50 - 60 contiguous residues, about 53 - 57 contiguous residues, or preferably about 56 residues. In some embodiments, the residues of the hypervariable region are of SEQ ID NO. As used herein, the term "hypervariable region" refers to a localized sequence within a meganuclease monomer or subunit that contains amino acids with relatively high variability. The hypervariable region can contain about 50 - 60 contiguous residues, about 53 - 57 contiguous residues, or preferably about 56 residues. In some embodiments, the residues of the hypervariable region are of SEQ ID NO. As used herein, the term "hypervariable region" refers to a localized sequence within a meganuclease monomer or subunit that contains amino acids with relatively high variability. The hypervariable region can contain about 50 - 60 contiguous residues, about 53 - 57 contiguous residues, or preferably about 56 residues. In some embodiments, the residues of the hypervariable region are of SEQ ID NO. As used herein, the term "hypervariable region" refers to a localized sequence within a meganuclease monomer or subunit that contains amino acids with relatively high variability. The hypervariable region can contain about 50 - 60 contiguous residues, about 53 - 57 contiguous residues, or preferably about 56 residues. In some embodiments, the residues of the hypervariable region are of SEQ ID NO. It may correspond to any one of positions 24 to 79 or positions 215 to 270 of numbers 12 to 27. Super The variable region can include one or more residues that contact DNA bases within the recognition array and can be modified to alter the base selectivity of the monomer or subunit. The hypervariable region can also include one or more residues that bind to the DNA backbone when the meganuclease associates with the double-stranded DNA recognition array. Modifying such residues can alter the binding affinity of the meganuclease for the DNA backbone and the target recognition sequence. In different embodiments of the present invention, the hypervariable region can include 1 to 20 residues that exhibit variability and may be modified to affect base selectivity and / or DNA binding affinity. In certain embodiments, the hypervariable region includes approximately 15 to 18 residues that exhibit variability and may be modified to affect base selectivity and / or DNA binding affinity. In some embodiments, the variable residues within the hypervariable region correspond to one or more of positions 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of any one of SEQ ID NOs: 12 to 27 In other embodiments, the variable residues within the hypervariable region correspond to one or more of positions 215, 217, 219, 221, 223, 224, 229, 2 31, 233, 235, 237, 259, 261, 266, and 268 of any one of SEQ ID NOs: 12 to 2 7. As used herein, the terms "T cell receptor alpha gene" or "TCR alpha gene" are used interchangeably and refer to the locus within a T cell that encodes the T cell receptor alpha subunit. The T cell receptor alpha is the NCBI gene before or after rearrangement. before or after rearrangement. before or after rearrangement. before or after rearrangement.

[0249] The term "T cell receptor alpha gene" or "TCR alpha gene" as used herein has the same meaning and refers to the locus within a T cell that encodes the T cell receptor alpha subunit. The T cell receptor alpha is the NCBI gene before or after rearrangement. before or after rearrangement. It may refer to the sub - ID number 6955. After rearrangement, the T - cell receptor alpha gene consists of an endogenous promoter, a rearranged V segment and J segment, an endogenous splice donor site position, an intron, an endogenous splice acceptor site, and a subunit - coding exon comprising the TRAC locus. See, for example, Figure 1.

[0250] As used herein, the terms "intron within the T - cell receptor alpha gene" and "target 5' intron" refer to the intron located 5' upstream of exon 1 of TRAC in the rearranged T - cell receptor alpha gene downstream of the V segment and J segment as shown in Figure 1, adjacent to the endogenous splice donor site and the endogenous splice acceptor site. The target 5' intron can have a sequence comprising SEQ ID NO: 3 and its functional variants that retain the nuclease recognition sequence encompassed by the present invention.

[0251] As used herein, the terms "T - cell receptor alpha constant region" and "TRAC" are used interchangeably and refer to the coding sequence of the T - cell receptor alpha gene. TRAC includes the wild - type sequence identified by NCBI Gen ID NO.28755 and its functional variants.

[0252] As used herein, the term "endogenous splice donor site" refers to a naturally - occurring splice donor site located 3' downstream of the endogenous TCR alpha gene promoter, downstream of the rearranged V segment and J segment, and 5' upstream of the target intron. Similarly, the "endogenous splice acceptor site" refers to the site located 3' downstream of the target intron and upstream of TRA as well as 5' upstream of the target intron. Refers to a naturally occurring splice acceptor site immediately 5' upstream of exon 1 of C. Endogenous splice donor sites and endogenous splice acceptor sites can be identified within a gene by methods known in the art, such as those described by Desmet et al. (Nucleic Acid Research (2009) 37(9):e67). The term "functional" with respect to endogenous splice donor sites and endogenous splice acceptor sites refers to the ability to pair to effect splicing of an intervening intron sequence.

[0253] As used herein, the term "exogenous splice acceptor site" refers to a splice acceptor site that is included in a heterologous sequence and introduced into a target 5' intron. An exogenous splice acceptor site can include a sequence that naturally occurs in the human T cell receptor alpha gene, or can include a splice acceptor sequence that does not naturally occur in a gene (e.g., a consensus sequence or a heterologous sequence). An exogenous splice acceptor site may further include an exogenous branch site if it is necessary to promote splicing of the intron. Such a branch site can include a sequence that naturally occurs in the T cell receptor alpha gene, or can include a branch site sequence that does not naturally occur in a gene (e.g., a consensus sequence or a heterologous sequence).

[0254] The terms "recombinant DNA construct", "recombinant construct", "expression cassette", " expression construct", "chimeric construct", "construct", and "recombinant DNA fragment" are used herein interchangeably and refer to a single-stranded or double-stranded It is a nucleotide. The recombinant construct is an artificial combination of single-stranded or double-stranded polynucleotides, including but not limited to regulatory sequences and coding sequences that are not found together in nature. For example, a recombinant DNA construct may contain regulatory sequences and coding sequences derived from different sources, or regulatory sequences and coding sequences derived from the same source but arranged in a manner different from that found in nature. Such constructs may be used alone or in combination with vectors. As used herein, the term "vector" or "recombinant DNA vector" may be a construct containing a replication system and sequences capable of transcribing and translating a sequence encoding a polypeptide in a given host cell. If a vector is used, the choice of vector depends on the method used to transform the host cell, as is well known to those skilled in the art. Vectors include plasmid vectors and recombinant AAV vectors, or other vectors known in the art suitable for delivering the gene encoding the meganuclease of the present invention to target cells, but are not limited thereto. Those skilled in the art are familiar with the genetic elements that must be present on the vector in order to successfully transform, select, and propagate host cells containing any of the isolated nucleotides or nucleic acid sequences of the present invention. Also, as used herein, the term "vector" may refer to a viral vector. Viral vectors include, but are not limited to, retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors (AAV).

[0255] As used herein, the term "vector" or "recombinant DNA vector" may be a construct containing a replication system and sequences capable of transcribing and translating a sequence encoding a polypeptide in a given host cell. If a vector is used, the choice of vector depends on the method used to transform the host cell, as is well known to those skilled in the art. Vectors include plasmid vectors and recombinant AAV vectors, or other vectors known in the art suitable for delivering the gene encoding the meganuclease of the present invention to target cells, but are not limited thereto. Those skilled in the art are familiar with the genetic elements that must be present on the vector in order to successfully transform, select, and propagate host cells containing any of the isolated nucleotides or nucleic acid sequences of the present invention. As used herein, the term "vector" or "recombinant DNA vector" may be a construct containing a replication system and sequences capable of transcribing and translating a sequence encoding a polypeptide in a given host cell. If a vector is used, the choice of vector depends on the method used to transform the host cell, as is well known to those skilled in the art. Vectors include plasmid vectors and recombinant AAV vectors, or other vectors known in the art suitable for delivering the gene encoding the meganuclease of the present invention to target cells, but are not limited thereto. Those skilled in the art are familiar with the genetic elements that must be present on the vector in order to successfully transform, select, and propagate host cells containing any of the isolated nucleotides or nucleic acid sequences of the present invention. As used herein, the term "vector" or "recombinant DNA vector" may be a construct containing a replication system and sequences capable of transcribing and translating a sequence encoding a polypeptide in a given host cell. If a vector is used, the choice of vector depends on the method used to transform the host cell, as is well known to those skilled in the art. Vectors include plasmid vectors and recombinant AAV vectors, or other vectors known in the art suitable for delivering the gene encoding the meganuclease of the present invention to target cells, but are not limited thereto. Those skilled in the art are familiar with the genetic elements that must be present on the vector in order to successfully transform, select, and propagate host cells containing any of the isolated nucleotides or nucleic acid sequences of the present invention. As used herein, the term "vector" or "recombinant DNA vector" may be a construct containing a replication system and sequences capable of transcribing and translating a sequence encoding a polypeptide in a given host cell. If a vector is used, the choice of vector depends on the method used to transform the host cell, as is well known to those skilled in the art. Vectors include plasmid vectors and recombinant AAV vectors, or other vectors known in the art suitable for delivering the gene encoding the meganuclease of the present invention to target cells, but are not limited thereto. Those skilled in the art are familiar with the genetic elements that must be present on the vector in order to successfully transform, select, and propagate host cells containing any of the isolated nucleotides or nucleic acid sequences of the present invention. As used herein, the term "vector" or "recombinant DNA vector" may be a construct containing a replication system and sequences capable of transcribing and translating a sequence encoding a polypeptide in a given host cell. If a vector is used, the choice of vector depends on the method used to transform the host cell, as is well known to those skilled in the art. Vectors include plasmid vectors and recombinant AAV vectors, or other vectors known in the art suitable for delivering the gene encoding the meganuclease of the present invention to target cells, but are not limited thereto. Those skilled in the art are familiar with the genetic elements that must be present on the vector in order to successfully transform, select, and propagate host cells containing any of the isolated nucleotides or nucleic acid sequences of the present invention. As used herein, the term "vector" or "recombinant DNA vector" may be a construct containing a replication system and sequences capable of transcribing and translating a sequence encoding a polypeptide in a given host cell. If a vector is used, the choice of vector depends on the method used to transform the host cell, as is well known to those skilled in the art. Vectors include plasmid vectors and recombinant AAV vectors, or other vectors known in the art suitable for delivering the gene encoding the meganuclease of the present invention to target cells, but are not limited thereto. Those skilled in the art are familiar with the genetic elements that must be present on the vector in order to successfully transform, select, and propagate host cells containing any of the isolated nucleotides or nucleic acid sequences of the present invention. As used herein, the term "vector" or "recombinant DNA vector" may be a construct containing a replication system and sequences capable of transcribing and translating a sequence encoding a polypeptide in a given host cell. If a vector is used, the choice of vector depends on the method used to transform the host cell, as is well known to those skilled in the art. Vectors include plasmid vectors and recombinant AAV vectors, or other vectors known in the art suitable for delivering the gene encoding the meganuclease of the present invention to target cells, but are not limited thereto. Those skilled in the art are familiar with the genetic elements that must be present on the vector in order to successfully transform, select, and propagate host cells containing any of the isolated nucleotides or nucleic acid sequences of the present invention. As used herein, the term "vector" or "recombinant DNA vector" may be a construct containing a replication system and sequences capable of transcribing and translating a sequence encoding a polypeptide in a given host cell. If a vector is used, the choice of vector depends on the method used to transform the host cell, as is well known to those skilled in the art. Vectors include plasmid vectors and recombinant AAV vectors, or other vectors known in the art suitable for delivering the gene encoding the meganuclease of the present invention to target cells, but are not limited thereto. Those skilled in the art are familiar with the genetic elements that must be present on the vector in order to successfully transform, select, and propagate host cells containing any of the isolated nucleotides or nucleic acid sequences of the present invention. As used herein, the term "vector" or "recombinant DNA vector" may be a construct containing a replication system and sequences capable of transcribing and translating a sequence encoding a polypeptide in a given host cell. If a vector is used, the choice of vector depends on the method used to transform the host cell, as is well known to those skilled in the art. Vectors include plasmid vectors and recombinant AAV vectors, or other vectors known in the art suitable for delivering the gene encoding the meganuclease of the present invention to target cells, but are not limited thereto. Those skilled in the art are familiar with the genetic elements that must be present on the vector in order to successfully transform, select, and propagate host cells containing any of the isolated nucleotides or nucleic acid sequences of the present invention.

[0256] Also, as used herein, the term "vector" may refer to a viral vector. Viral vectors include, but are not limited to, retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors (AAV). For example, a recombinant DNA construct may contain regulatory sequences and coding sequences derived from different sources, or regulatory sequences and coding sequences derived from the same source but arranged in a manner different from that found in nature. Such constructs may be used alone or in combination with vectors. ​

[0257] As used herein, "polycistronic" mRNA refers to a single messenger RNA that contains two or more coding sequences (i.e., cistrons) and encodes two or more proteins. Polycistronic mRNA can include, but is not limited to, any element known in the art that enables the translation of two or more genes from the same mRNA molecule, including IRES elements, T2A elements, P2A elements, E2A elements, and F2A elements. As used herein, "human T cells" or "T cells" refers to T cells isolated from a donor, particularly a human donor. T cells and cells derived therefrom include isolated T cells that have not been passaged in culture, T cells that have been passaged and maintained under cell culture conditions without immortalization, and T cells that have been immortalized and can be maintained indefinitely under cell culture conditions. As used herein, "human natural killer cells" or "human NK cells" or "natural killer cells" or "NK cells" refers to a type of cytotoxic lymphocyte that is important for the innate immune system. The role played by NK cells is similar to that of cytotoxic T cells in the adaptive immune response of vertebrates. NK cells respond rapidly to virus-infected cells, respond to tumor formation, and act approximately 3 days after infection. As used herein, "control" or "control cell" refers to a cell that provides a reference point for measuring changes in the genotype or phenotype of a genetically modified cell. Control cells can be, for example: (a) wild-type cells, i.e., the starting material for the genetic changes that resulted in the genetically modified cells.

[0258]

[0259]

[0260] : (a) wild-type cells, i.e., the same as the starting material for the genetic changes that resulted in the genetically modified cells. cells of the same genotype as the genetically modified cells; (b) cells of the same genotype as the genetically modified cells but transformed with a null construct (i.e., a construct that is known not to have an effect on the property of interest); or (c) cells that are genetically identical to the genetically modified cells but that have not been exposed to conditions or stimuli that induce the expression of the altered genotype or phenotype, or to further genetic modification. The term "treatment" or "treating a subject" as used herein refers to the administration of the genetically modified T cells of the invention to a subject having a disease. For example, the subject may be at risk of having a disease such as cancer, and the treatment may be an immunotherapy for treating the disease. Desirable effects of the treatment include, but are not limited to, prevention of the occurrence or recurrence of the disease, alleviation of symptoms, reduction of the direct or indirect pathological consequences of the disease, reduction in the rate of progression of the disease, improvement or alleviation of the disease state, and remission or improvement of the prognosis. In some embodiments, the genetically

[0261] modified cells described herein are administered in the form of a pharmaceutical composition of the invention during treatment. The term "effective amount" or "therapeutically effective amount" refers to an amount sufficient to produce a beneficial or desired biological and / or clinical result. A therapeutically effective amount will vary depending on the formulation used, the disease and its severity, and the age, weight, physical condition, and responsiveness of the subject being treated. The term "cancer" as used herein is understood to encompass any neoplastic disease (whether invasive or metastatic) characterized by abnormal, uncontrolled cell division that causes malignant growth or tumors. The term "cancer" as used herein is understood to encompass any neoplastic disease (whether invasive or metastatic) characterized by abnormal, uncontrolled cell division that causes malignant growth or tumors. The term "cancer" as used herein is understood to encompass any neoplastic disease (whether invasive or metastatic) characterized by abnormal, uncontrolled cell division that causes malignant growth or tumors. The term "cancer" as used herein is understood to encompass any neoplastic disease (whether invasive or metastatic) characterized by abnormal, uncontrolled cell division that causes malignant growth or tumors.

[0262] The term "effective amount" or "therapeutically effective amount" refers to an amount sufficient to produce a beneficial or desired biological and / or clinical result. A therapeutically effective amount will vary depending on the formulation used, the disease and its severity, and the age, weight, physical condition, and responsiveness of the subject being treated. The term "cancer" as used herein is understood to encompass any neoplastic disease (whether invasive or metastatic) characterized by abnormal, uncontrolled cell division that causes malignant growth or tumors. The term "cancer" as used herein is understood to encompass any neoplastic disease (whether invasive or metastatic) characterized by abnormal, uncontrolled cell division that causes malignant growth or tumors.

[0263] The term "cancer" as used herein is understood to encompass any neoplastic disease (whether invasive or metastatic) characterized by abnormal, uncontrolled cell division that causes malignant growth or tumors. The term "cancer" as used herein is understood to encompass any neoplastic disease (whether invasive or metastatic) characterized by abnormal, uncontrolled cell division that causes malignant growth or tumors. The term "cancer" as used herein is understood to encompass any neoplastic disease (whether invasive or metastatic) characterized by abnormal, uncontrolled cell division that causes malignant growth or tumors.

[0264] As used herein, the term "carcinoma" refers to a malignant growth composed of epithelial cells .

[0265] As used herein, the term "leukemia" refers to a malignant tumor of the hematopoietic organs / system, and generally is characterized by abnormal proliferation and development of white blood cells and their precursors in the blood and bone marrow .

[0266] As used herein, the term "sarcoma" refers to a tumor composed of substances such as embryonic connective tissue and generally consists of densely packed cells embedded in a fibrous, heterogeneous or homogeneous substance .

[0267] As used herein, the term "melanoma" refers to a tumor arising from the melanocyte system of the skin and other organs .

[0268] As used herein, the term "lymphoma" refers to a group of blood cell tumors arising from lymphocytes .

[0269] As used herein, the term "blastoma" refers to a type of cancer caused by a malignant tumor of precursor cells or blast cells (immature or embryonic tissue).

[0270] When used herein, the recitation of a numerical range of a variable is intended to convey that the invention can be practiced with any value within that range equal to the variable . Thus, in the case of a variable that is essentially non continuous, the variable can be equal to any integer value within the numerical range including the endpoints of that range . Similarly, in the case of a variable that is essentially continuous, the variable can be equal to any real value within the numerical range including the endpoints of that range . By way of example, and without limitation, having a value between 0 and 2 The variables described hereinabove can take the value 0, 1, or 2 if the variable is essentially discontinuous, and can take the value 0.0, 0.1, 0.01, 0.001, or any other real value between 0 and 2 if the variable is essentially continuous. 1, or any other real value greater than or equal to 0 and less than or equal to 2.

[0271] 2.1 Principle of the Invention The present invention is based in part on the discovery that inserting a target sequence (including an exogenous splice acceptor site and / or a polyA signal) into the nuclease cleavage site of the target 5' intron of the T cell receptor alpha gene enables the production of TCR- cells only when the insert is present. When the insert is absent, the nuclease-modified intron is simply removed and the endogenous gene is expressed. Thus, in the example where the subject's sequence contains the CAR coding sequence, the present invention provides a method for producing a population in which most or all of the TCR- cells are TCR- / CAR+ cells. Any other desired peptide can be expressed from the target sequence in the same manner as the CAR. In contrast, conventional nuclease-based approaches for generating modified T cells target the coding sequence of the endogenous splice acceptor site upstream of 5' of TCRAC and / or TRAC exon 1. As a result, a highly mixed population of TCR- cells containing a significant proportion of TCR- / CAR- cells can be generated by NHEJ at the nuclease cleavage site, creating indels and disrupting protein expression.

[0272] Thus, by reducing the need to purify a mixed population of TCR- cells, the present invention

[0273] reduces ​​​​​​​​​​​, a method for producing a purified population of allogeneic CAR T cells that express an antigen-specific CAR and have reduced expression of the endogenous TCR is provided. Such cells, when administered to an allogeneic subject, may show a reduced induction of graft-versus-host disease (GVHD) or no induction thereof at all. Furthermore, by including a 2A element in the exogenous sequence of interest, expression of the coding sequence driven by the endogenous T cell receptor alpha gene promoter rather than the exogenous promoter becomes possible. In this way, the expression of polypeptides such as CAR is controlled by the T cell feedback mechanism normally associated with TCR expression. In the art, it is known that site-specific nucleases can be used to cause DNA cleavage in the genome of living cells, and such DNA cleavage can result in permanent modification of the genome through mutagenic NHEJ repair or homologous recombination with a transgenic DNA sequence. NHEJ causes mutagenesis at the cleavage site, resulting in inactivation of the allele. NHEJ-related mutagenesis can inactivate alleles through the generation of frameshift mutations that produce premature stop codons or abnormal non-functional proteins, or can induce mechanisms such as nonsense-mediated mRNA decay. The use of nucleases that induce mutagenesis via NHEJ can be used to target specific mutations or sequences present in the wild-type allele. The use of nucleases that induce double-strand breaks at the target locus is particularly useful when a sequence homologous to the genomic target is adjacent to the transgenic DNA sequence. When administered to an allogeneic subject, it may show a reduced induction of graft-versus-host disease (GVHD) or no induction thereof at all. Furthermore, by including a 2A element in the exogenous sequence of interest, expression of the coding sequence driven by the endogenous T cell receptor alpha gene promoter rather than the exogenous promoter becomes possible. In this way, the expression of polypeptides such as CAR is controlled by the T cell feedback mechanism normally associated with TCR expression.

[0274] 2.2 Nucleases that Recognize and Cleave Recognition Sequences within the Target 5' Intron of the T Cell Receptor Alpha Gene 2.3 Methods for Producing Genetically Modified Cells In the art, it is possible to cause DNA cleavage in the genome of living cells using site-specific nucleases, and such DNA cleavage can result in permanent modification of the genome through mutagenic NHEJ repair or homologous recombination with a transgenic DNA sequence. NHEJ causes mutagenesis at the cleavage site, resulting in inactivation of the allele. NHEJ-related mutagenesis can inactivate alleles through the generation of frameshift mutations that produce premature stop codons or abnormal non-functional proteins, or can induce mechanisms such as nonsense-mediated mRNA decay. The use of nucleases that induce mutagenesis via NHEJ can be used to target specific mutations or sequences present in the wild-type allele. Double-strand breaks at the target locus induced by nucleases can be used to target specific mutations or sequences present in the wild-type allele. The use of nucleases that induce double-strand breaks at the target locus is particularly useful when a sequence homologous to the genomic target is adjacent to the transgenic DNA sequence. induced by nucleases can be used to target specific mutations or sequences present in the wild-type allele. ​It is known to stimulate homologous recombination of nick DNA sequences. In this way, an exogenous nucleic acid sequence can be inserted into a target locus. Such exogenous nucleic acids can encode, for example, a chimeric antigen receptor, an exogenous TCR, or any sequence or polypeptide of interest. It may be good.

[0275] In different embodiments, various different types of nucleases are useful for practicing the present invention. In one embodiment, the present invention can be practiced using engineered meganucleases. In another embodiment, the present invention can be practiced using a CRISPR nuclease or a CRISPR nickase. Methods for making CRISPR and CRISPR nickases that recognize a given DNA site are known in the art, for example, Ran et al. (2013) Nat Protoc. 8:2281-308. In another embodiment, the present invention can be practiced using a TALEN or a compact TALEN. Methods for making TALE domains that bind to a given DNA site are known in the art, for example, Reyon et al. (2012) Nat Biotechnol. 30:460-5. In another embodiment, the present invention can be practiced using a zinc finger nuclease (ZFN). In a further embodiment, the present invention can be practiced using a megaTAL. In a preferred embodiment, the nuclease used to practice the present invention is a single-stranded meganuclease. The single-stranded meganuclease comprises an N-terminal subunit and a C-terminal subunit linked by a linker peptide. Each of the two domains has a recognition sequence. The method of making a TALE domain that binds to a given DNA site is known in the art, for example, Reyon et al. (2012) Nat Biotechnol. 30:460-5. It is known. In another embodiment, the present invention can be practiced using a zinc finger nuclease (ZFN). In a further embodiment, the present invention can be practiced using a megaTAL. It can be practiced.

[0276] In a preferred embodiment, the nuclease used to practice the present invention is a single-stranded meganuclease. The single-stranded meganuclease comprises an N-terminal subunit and a C-terminal subunit linked by a linker peptide. The two domains each have a recognition sequence. Recognize half of the column (i.e., the recognition half-site), and the site of DNA cleavage is in the center of the recognition sequence near the boundary of the two subunits. DNA strand cleavage is counteracted by a 4-base pair such that DNA cleavage by the meganuclease generates a 3'-single-stranded overhang of 4 base pairs. In some examples, the engineered meganucleases of the invention are engineered to recognize and cleave the TRC11-12 recognition sequence (SEQ ID NO: 4). Such engineered meganucleases are collectively referred to herein as "TRC11-12 meganucleases". Exemplary TRC11-12 meganucleases are provided in SEQ ID NOs: 12-15. In some examples, the engineered meganucleases of the invention are engineered to recognize and cleave the TRC11-12 recognition sequence (SEQ ID NO: 4). Such engineered meganucleases are collectively referred to herein as "TRC11-12 meganucleases". Exemplary TRC11-12 meganucleases are provided in SEQ ID NOs: 12-15. In some examples, the engineered meganucleases of the invention are engineered to recognize and cleave the TRC11-12 recognition sequence (SEQ ID NO: 4). Such engineered meganucleases are collectively referred to herein as "TRC11-12 meganucleases". Exemplary TRC11-12 meganucleases are provided in SEQ ID NOs: 12-15.

[0277] In some examples, the engineered meganucleases of the invention are engineered to recognize and cleave the TRC11-12 recognition sequence (SEQ ID NO: 4). Such engineered meganucleases are collectively referred to herein as "TRC11-12 meganucleases". Exemplary TRC11-12 meganucleases are provided in SEQ ID NOs: 12-15. In some examples, the engineered meganucleases of the invention are engineered to recognize and cleave the TRC11-12 recognition sequence (SEQ ID NO: 4). Such engineered meganucleases are collectively referred to herein as "TRC11-12 meganucleases". Exemplary TRC11-12 meganucleases are provided in SEQ ID NOs: 12-15. In some examples, the engineered meganucleases of the invention are engineered to recognize and cleave the TRC11-12 recognition sequence (SEQ ID NO: 4). Such engineered meganucleases are collectively referred to herein as "TRC11-12 meganucleases". Exemplary TRC11-12 meganucleases are provided in SEQ ID NOs: 12-15. In some examples, the engineered meganucleases of the invention are engineered to recognize and cleave the TRC11-12 recognition sequence (SEQ ID NO: 4). Such engineered meganucleases are collectively referred to herein as "TRC11-12 meganucleases". Exemplary TRC11-12 meganucleases are provided in SEQ ID NOs: 12-15.

[0278] In a further example, the engineered meganucleases of the invention are engineered to recognize and cleave the TRC15-16 recognition sequence (SEQ ID NO: 6). Such engineered meganucleases are collectively referred to herein as "TRC15-16 meganucleases". Exemplary TRC15-16 meganucleases are provided in SEQ ID NOs: 16-19. In a further example, the engineered meganucleases of the invention are engineered to recognize and cleave the TRC15-16 recognition sequence (SEQ ID NO: 6). Such engineered meganucleases are collectively referred to herein as "TRC15-16 meganucleases". Exemplary TRC15-16 meganucleases are provided in SEQ ID NOs: 16-19. In a further example, the engineered meganucleases of the invention are engineered to recognize and cleave the TRC15-16 recognition sequence (SEQ ID NO: 6). Such engineered meganucleases are collectively referred to herein as "TRC15-16 meganucleases". Exemplary TRC15-16 meganucleases are provided in SEQ ID NOs: 16-19. In a further example, the engineered meganucleases of the invention are engineered to recognize and cleave the TRC15-16 recognition sequence (SEQ ID NO: 6). Such engineered meganucleases are collectively referred to herein as "TRC15-16 meganucleases". Exemplary TRC15-16 meganucleases are provided in SEQ ID NOs: 16-19.

[0279] In a further example, the engineered meganucleases of the invention are engineered to recognize and cleave the TRC17-18 recognition sequence (SEQ ID NO: 8). Such engineered meganucleases are collectively referred to herein as "TRC17-18 meganucleases". Exemplary TRC17-18 meganucleases are provided in SEQ ID NOs: 20-23. In a further example, the engineered meganucleases of the invention are engineered to recognize and cleave the TRC17-18 recognition sequence (SEQ ID NO: 8). Such engineered meganucleases are collectively referred to herein as "TRC17-18 meganucleases". Exemplary TRC17-18 meganucleases are provided in SEQ ID NOs: 20-23. In a further example, the engineered meganucleases of the invention are engineered to recognize and cleave the TRC17-18 recognition sequence (SEQ ID NO: 8). Such engineered meganucleases are collectively referred to herein as "TRC17-18 meganucleases". Exemplary TRC17-18 meganucleases are provided in SEQ ID NOs: 20-23. In a further example, the engineered meganucleases of the invention are engineered to recognize and cleave the TRC17-18 recognition sequence (SEQ ID NO: 8). Such engineered meganucleases are collectively referred to herein as "TRC17-18 meganucleases". Exemplary TRC17-18 meganucleases are provided in SEQ ID NOs: 20-23.

[0280] In a further example, the engineered meganucleases of the invention are engineered to recognize and cleave the TRC19-20 recognition sequence (SEQ ID NO: 10). Such engineered meganucleases are collectively referred to herein as "TRC19-20 meganucleases". In a further example, the engineered meganucleases of the invention are engineered to recognize and cleave the TRC19-20 recognition sequence (SEQ ID NO: 10). Such engineered meganucleases are collectively referred to herein as "TRC19-20 meganucleases". The enzymes are collectively referred to herein as "TRC19-20 meganucleases". For example Exemplary TRC19-20 meganucleases are provided in SEQ ID NOs: 24-27.

[0281] The engineered meganucleases of the invention comprise a first subunit comprising a first hypervariable (HVR1) region and a second subunit comprising a second hypervariable (HVR2) region. Further the first subunit binds to a first recognition half-site (such as a TRC11, TRC15, TRC17, or TRC19 half-site etc.) of the recognition sequence, and the second subunit binds to a second recognition half-site (e.g., a TRC12, TRC16, TRC18, or TRC20 half-site) of the recognition sequence. In embodiments where the recombinant meganuclease is a single-stranded meganuclease, the first and second subunits are oriented such that the first subunit, which contains the HVR1 region and binds to the first half-site, is positioned as the N-terminal subunit, and the second subunit, which contains the HVR2 region and binds to the second half-site, is positioned as the C-terminal subunit. In another embodiment, the first and second subunits are oriented such that the first subunit, which contains the HVR1 region and binds to the first half-site, is positioned as the C-terminal subunit, and the second subunit, which contains the HVR2 region and binds to the second

[0282] Table 1: Exemplary engineered meganucleases engineered to recognize and cleave the TRC1-2 recognition sequence (SEQ ID NO: 4) [Table 1]

[0283] Table 2: Exemplary engineered meganucleases engineered to recognize and cleave the TRC15-16 recognition sequence (SEQ ID NO: 6) [Table 2]

[0284] Table 3: Exemplary engineered meganucleases engineered to recognize and cleave the TRC17-18 recognition sequence (SEQ ID NO: 8) [Table 3]

[0285] Table 4: Exemplary engineered meganucleases engineered to recognize and cleave the TRC19-20 recognition sequence (SEQ ID NO: 10) [Table 4]

[0286] 2.4 Pharmaceutical Compositions After rearrangement, the human T cell receptor alpha gene contains multiple elements. Generally, without being bound by a particular theory, these elements include, from 5' to 3', an endogenous promoter, rearranged V and J segments, an endogenous splice donor site, an intron (i.e., the target 5' intron), an endogenous splice acceptor site, and an exon, and ​​​The TRAC gene contains the alpha subunit encoding and interspaced introns See, for example, FIG.

[0287] The invention disclosed herein provides a method for the production of genetically modified T cells comprising a modified TCR alpha gene. The present invention provides a method for producing T cells from peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, and the like. from many sources, including thymus tissue, tissue from infection sites, ascites, pleural fluid, spleen tissue, and tumors. In certain embodiments of the present disclosure, any number of T-cells available in the art may be used. In some embodiments of the present disclosure, the T cells can be any cell line known to those of skill in the art. The antibody may be obtained from a unit of blood collected from a subject using any number of techniques currently available. In embodiments, cells from the circulating blood of an individual are obtained by apheresis.

[0288] The altered T-cell receptor alpha gene is located 5' upstream of TRAC exon 1 An exogenous sequence of interest inserted into an intron within the TCR alpha gene (i.e., the target 5' intron) More specifically, the exogenous sequence of interest comprises a rearranged V segment and and J segments, as well as the 3' downstream of the endogenous splice donor site and the endogenous splice In a specific embodiment, the target 5' insert may be inserted 5' upstream of the acceptor site. The nucleic acid sequence may be a sequence as set forth in SEQ ID NO:3 or a sequence having at least 7 sequences identical to the nucleic acid sequence as set forth in SEQ ID NO:3. 5%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity and includes sequences that contain recognition sequences for the engineered nucleases described herein.

[0289] In some embodiments, the exogenous sequence of interest is an engineered meganuclease, a zinc finger enzyme, Finger nuclease, TALEN, Compact TALEN, CRISPR nuclease or at the double-stranded breaks generated by engineered nucleases such as megaTAL. The cleavage site generated by such a nuclease can be inserted into an intron. It may allow for direct homologous recombination of exogenous sequences into the 5' intron.

[0290] The "endogenous splice donor site" is the 3' endogenous TCR alpha gene promoter. The 5′ upstream of the target intron was also identified as a stably expressed V segment and J segment. The term "endogenous splice acceptor" refers to a naturally occurring splice donor site. The "site" is located 3' downstream of the target intron and immediately 5' upstream of TRAC exon 1. Refers to the naturally occurring splice acceptor site. See FIG. 1.

[0291] In specific embodiments, the engineered nucleases disclosed herein inhibit endogenous splices. It does not modify either the Rice donor site or the endogenous splice acceptor site, but since both sites should retain functionality for practicing the invention. In some embodiments, the endogenous splice donor site and / or the endogenous splice acceptor site are The -sites retain the ability of each site to pair with the other and splice the intron (i.e., As used herein, the term "polypeptide" can be used interchangeably with "polypeptide", so long as the polypeptide remains functional (i.e., retains functionality). In this case, a functional endogenous splice donor site is paired with an endogenous splice acceptor site. Similarly, as used herein, functional The endogenous splice acceptor site of the gene is paired with the endogenous splice donor site to form an endogenous splice It has the ability to remove Ron.

[0292] In certain embodiments, the target sequence can include an exogenous splice acceptor site. As used herein, the term "exogenous" or "heterologous" with respect to a nucleotide sequence means a sequence that is either purely synthetic, derived from a foreign species, or, when derived from the same species, is substantially modified from its natural form by intentional human intervention in terms of its composition and / or genomic locus. Thus, an exogenous splice acceptor site can be a purely synthetic splice acceptor site or a splice acceptor site from a human genome where the sequence or genomic locus has been modified.

[0293] In a specific embodiment, the exogenous splice acceptor site can cooperate with an endogenous splice donor site to splice out intervening intron sequences. In this way, the exogenous splice acceptor site may compete with the endogenous splice acceptor site to cooperate with the endogenous splice donor site, thereby potentially interfering with the natural splicing of the target 5' intron.

[0294] In various embodiments, the target exogenous sequence can include a coding sequence for the target protein. The coding sequence can be assumed to be for any target protein.

[0295] In certain specific embodiments, the target exogenous sequence includes a nucleic acid sequence encoding a CAR. Generally, the CARs of the present disclosure include at least an extracellular domain and an intracellular domain. In some embodiments, the extracellular domain is also a ligand-binding domain or a ligand-binding moiety. ​ comprises a so-called target-specific binding element. In some embodiments, the intracellular domain or cytoplasmic domain comprises at least one co-stimulatory domain and one or more signal transduction domains such as, for example, CD3ζ. In other embodiments, the CAR may comprise only a signal transduction domain such as CD3ζ, and the cell may comprise one or more co-stimulatory domains on another construct expressed intracellularly. In some embodiments, the CAR useful in the present invention comprises an extracellular target-specific binding element, also referred to as a ligand-binding domain or ligand-binding moiety. The choice of ligand-binding domain depends on the type and number of ligands that define the surface of the target cell. For example, the ligand-binding domain may be selected to recognize a ligand that acts as a cell surface marker on a target cell associated with a particular disease state. Thus, examples of cell surface markers that can act as ligands for the ligand-binding domain in a CAR include those associated with viral, bacterial, and parasitic infections, autoimmune diseases, and cancer cells.

[0296] In some embodiments, the CAR useful in the present invention comprises an extracellular target-specific binding element, also referred to as a ligand-binding domain or ligand-binding moiety. The choice of ligand-binding domain depends on the type and number of ligands that define the surface of the target cell. For example, the ligand-binding domain may be selected to recognize a ligand that acts as a cell surface marker on a target cell associated with a particular disease state. Thus, examples of cell surface markers that can act as ligands for the ligand-binding domain in a CAR include those associated with viral, bacterial, and parasitic infections, autoimmune diseases, and cancer cells. In some embodiments, the CAR is engineered to target a desired tumor-specific antigen by engineering the ligand-binding moiety that specifically binds to an antigen on a tumor cell. As used herein, "tumor antigen" refers to an antigen common to a particular hyperproliferative disorder such as cancer. In some embodiments, the extracellular ligand-binding domain of the CAR is specific for any antigen or epitope of interest, particularly any tumor antigen or epitope of interest. By way of non-limiting example, in some embodiments, the target antigen is ErbB2 (HER2 / neu), carcinoembryonic In some embodiments, the CAR is engineered to target a desired tumor-specific antigen by engineering the ligand-binding moiety that specifically binds to an antigen on a tumor cell. As used herein, "tumor antigen" refers to an antigen common to a particular hyperproliferative disorder such as cancer.

[0297] In some embodiments, the extracellular ligand-binding domain of the CAR is specific for any antigen or epitope of interest, particularly any tumor antigen or epitope of interest. By way of non-limiting example, in some embodiments, the target antigen is ErbB2 (HER2 / neu), carcinoembryonic antigen, Carcinoembryonic antigen (CEA), Epithelial cell adhesion molecule (EpCAM), Epidermal growth factor receptor (EGFR ), EGFR variant III (EGFRvIII), CD19, CD20, CD22, CD 30, CD40, CLL1, Disialoganglioside GD2, Mucin of ductal epithelium, gp36, TAG-72, Sphingolipid, Glioma-associated antigen, B-human chorionic gonadotropin, Alpha-fetoprotein (AFP), Lectin-reactive AFP, Thyroglobulin, RA GE-1, MN-CA IX, Human telomerase reverse transcriptase, RU1, RU2(AS), Intestinal carboxylesterase, mut hsp70-2, M-CSF, Prostase, Prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGA-la, p5 3, Prostain, PSMA, Survivin, and Telomerase, Prostate carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, Neutrophil elastase, Ephrin B2, Insulin-like growth factor (IGF1)-1, IGF-II, IGFI receptor, Mesothelin, Major histocompatibility complex (MHC) molecules presenting tumor-specific peptide epitopes, 5T4, ROR1, Nkp30, NKG2D, Tumor stromal antigen, Fibronectin extra domain A (EDA) and extra domain B (EDB) and Al domain of tenascin C (TnC Al), and tumor-associated surface antigens such as fibroblast activation protein (FAP); CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD38, CD1 23, CD133, CD138, CTLA-4, B7-1 (CD80), B7-2 (CD 86), etc. lineage-specific or tissue-specific antigens, Endoglin, Major histocompatibility complex (MHC ) Molecules, BCMA (CD269, TNFRSF17), CS1, or viral specific surface antigens such as HIV specific antigen (H IV gpl20, etc.); EBV specific antigen, CMV specific antigen, E 6 or HPV specific antigens such as E7 oncoproteins, Lassa virus specific antigen, influenza virus specific antigen, and any derivatives or variants of these surface markers . In certain embodiments of the present disclosure, the ligand binding domain is specific for CD19.

[0298] In some embodiments, the extracellular domain of the chimeric antigen receptor further includes an autoantigen that can be recognized by an antigen specific B cell receptor on B lymphocytes (see Payne et al. (201 6) Science, Vol.353(6295):179-184), and instructs T cells to specifically target and kill autoreactive B lymphocytes in antibody-mediated autoimmune diseases. Such CARs may be referred to as chimeric autoantibody receptors (CAARs).

[0299] In some embodiments, the extracellular domain of the chimeric antigen receptor may include a naturally occurring ligand for the antigen of interest, or a fragment of a naturally occurring ligand that retains the ability to bind to the antigen of interest.

[0300] In some embodiments, the CAR includes a transmembrane domain that links an extracellular ligand binding domain or autoantigen to an intracellular signaling and co-stimulatory domain via a hinge or spacer sequence. The transmembrane domain can be derived from any membrane-bound or transmembrane protein. For example, the transmembrane polypeptide can be a subunit of a T cell receptor (i.e., α, β, γ or ζ, a polypeptide constituting the CD3 complex), IL2 receptor p55 (α chain), p7 5 (β chain) or γ chain, a subunit chain of the Fc receptor (e.g., Fcγ receptor III), or it may be a CD protein such as the CD8 alpha chain. Alternatively, the transmembrane domain may be synthetic and may contain mainly hydrophobic residues such as leucine and valine. In a specific example the transmembrane domain is the CD8α transmembrane polypeptide.

[0301] The hinge region refers to an oligo or polypeptide that functions to link the transmembrane domain to the extracellular ligand-binding domain. For example, the hinge region may contain up to 300 amino acids, preferably 10 to 100 amino acids, most preferably 25 to 50 amino acids. The hinge region may be derived from all or part of the extracellular region of CD8, CD4 or CD28, or all or part of a naturally occurring molecule such as all or part of the antibody constant region. Alternatively, the hinge region may be a synthetic sequence corresponding to the natural hinge sequence, or it may be a completely synthetic hinge sequence. In a specific example, the hinge domain may contain a part of the human C D8 alpha chain, the FcyRllla receptor or IgGl. The intracellular signaling domain of the CAR is responsible for activating at least one of the normal effector functions of the cell in which the CAR is placed and / or activating the pathways of proliferation and cell survival. The term "effector function" refers to the special functions of the cell. The effector functions of T cells may be

[0302] e.g., cytolytic activity, or helper activity including the secretion of cytokines. Intracellular signaling domains such as CD3ζ activate the cell in response to the binding of the extracellular domain and proliferation and cell survival. The term "effector function" refers to the special functions of the cell. The effector functions of T cells may be e.g., cytolytic activity, or helper activity including the secretion of cytokines. A personalized signal can be provided. As contemplated, the activation signal can induce effector functions of cells such as, for example, cell lysis activity or cytokine secretion. The intracellular domain of the CAR can include one or more intracellular co-stimulatory domains that transmit co-stimulatory signals to promote cell proliferation, cell survival, and / or cytokine secretion after binding of the extracellular domain.

[0303] Such intracellular co-stimulatory domains include, but are not limited to, CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, C D7, LIGHT, NKG2C, B7-H3, and CD83, N1, or ligands that specifically bind to N6 and the like known in the art. CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, C D7, LIGHT, NKG2C, B7-H3, and CD83, N1, or ligands that specifically bind to N6 and the like known in the art.

[0304] The CAR can be specific for any type of cancer cell. Such cancers include, but are not limited to, carcinomas, lymphomas, sarcomas, blastomas, leukemias, cancers of B-cell origin, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, melanoma, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, Hodgkin lymphoma. In certain embodiments, cancers of B-cell origin include, but are not limited to, B-lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, B-cell non-Hodgkin lymphoma, and multiple myeloma. In certain embodiments, cancers of B-cell origin include, but are not limited to, B-lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, B-cell non-Hodgkin lymphoma, and multiple myeloma.

[0305] The target sequence can further encode an exogenous T cell receptor (TCR). Such an exogenous T cell receptor may include an alpha chain and a beta chain, or alternatively, a gamma chain and beta chain, or alternatively, a gamma chain and ​​​​It may also contain a beta chain or a delta chain. Exogenous TCRs useful in the present invention may have specificity for any antigen or epitope of interest.

[0306] In other embodiments, the sequence of interest can encode a wild-type or modified version of an endogenous gene of interest.

[0307] The sequence of interest can include elements or peptides known in the art that enable the translation of two additional genes from the same mRNA molecule, including but not limited to IRES elements and 2A elements such as T2A elements, P2A elements, E2A elements, and F2 A elements. In a specific embodiment, such an element in the exogenous sequence of interest can be placed upstream of the 5' of the nucleic acid sequence encoding the protein of interest (e.g., CAR). The exogenous sequences of interest described herein can further include additional regulatory sequences. For example, the sequence of interest can include a homologous recombination enhancer sequence, a Kozak sequence, a polyadenylation sequence, a transcription termination sequence, a selection marker sequence (e.g., an antibiotic resistance gene), an origin of replication, etc.

[0308] In addition, the sequences of interest described herein may include at least one nuclear localization signal. Examples of nuclear localization signals are known in the art (see, for example Lange et al., J. Biol. Chem., 2007, 282:5101 - 5105).

[0309] In a specific embodiment, the exogenous sequence of interest includes a polyadenylation sequence or a polyA signal. Thus, the sequence of interest encodes the protein of interest (e.g., CAR). ​It can include a polyA signal located downstream of the 3' of the array. In this way, T cells The transcription of the receptor alpha gene, particularly the coding sequence of the TRAC locus, is interfered with by the polyA signal, and the expression of the T cell receptor alpha subunit is

[0310] In some examples of the present invention, the exogenous sequence of interest is, from 5' to 3', an exogenous splice a ceptor site, a 2A element or an IRES element, the coding sequence of the protein of interest, and poly A signal. In certain examples, the exogenous sequence of the subject includes, from 5' to 3', an exogenous s plice acceptor site, a 2A element or an IRES element, the coding sequence of a CAR or an exogenous T cell receptor , and a polyA signal. In some examples, the exogenous sequence of interest may further include an exogenous branch site located upstream of the 5' of the exogenous splice acceptor site . In various examples of the present invention, the 2A element may be, but is not limited to, a T2A element, a P2A element, an E2A element, or an F2A element.

[0311] The engineered nuclease of the present invention can be delivered to cells in the form of a protein or, preferably, as a nucleic acid encoding the engineered nuclease. Such nucleic acids can be DNA (e.g., circular or linearized plasmid DNA or PCR products) or RNA (e.g., m RNA). In embodiments where the engineered nuclease coding sequence is delivered in DNA form, it must be controllably linked to a promoter to promote the transcription of the nuclease gene . Mammalian promoters suitable for the present invention include the cytomegalovirus early (CMV) promoter (Thomsen et al. (1984), Proc Natl Acad Sci USA 81:659-663), the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter (Gorman et al. (1982), Proc Natl Acad Sci USA 79:6777-6781), the SV40 early promoter (Subramani et al. (1981), Mol Cell Biol 1:854-864), and the human elongation factor 1 alpha (EF1α) promoter (Kim et al. (1996), Gene 179:195-202). Acad Sci USA 81:659-663), the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter (Gorman et al. (1982), Proc Natl Acad Sci USA 79:6777-6781), the SV40 early promoter (Subramani et al. (1981), Mol Cell Biol 1:854-864), and the human elongation factor 1 alpha (EF1α) promoter (Kim et al. (1996), Gene 179:195-202). Proc. Natl. Acad. Sci. USA 81(3):659-63) or the SV40 early promoter Benoist and Chambon (1981), Nature 290(580 4):304-10), etc., along with inducible promoters such as the tetracycline-inducible promoter Dingermann et al. (1992), Mol. Cell Biol. 12(9): 4038-45), etc.

[0312] In some embodiments, since the gene encoding the engineered nuclease is less likely to integrate into the genome of the cell, the mRNA encoding the engineered nuclease is delivered to the cell. Such mRNA encoding the engineered nuclease can be produced using methods known in the art such as in vitro transcription. In some embodiments the mRNA is capped using 7-methyl-guanosine. In some embodiments the mRNA may be polyadenylated. In certain embodiments, the mRNA encoding the engineered nuclease of the invention may be a polycistronic mRNA encoding two or more nucleases that are co-expressed within the cell. The polycistronic mRNA may encode two or more nucleases of the invention that target different recognition sequences within the same target gene. Alternatively, the polycistronic mRNA may encode at least one nuclease described herein and at least one additional nuclease that targets a separate recognition sequence located in the same gene or a second recognition sequence located in a second gene such that cleavage sites are generated in both genes In some embodiments the mRNA is polyadenylated.

[0313] In certain embodiments, the mRNA encoding the engineered nuclease of the invention is a polycistronic mRNA encoding two or more nucleases that are co-expressed within the cell and target different recognition sequences within the same target gene The polycistronic mRNA may encode two or more nucleases of the invention that target different recognition sequences within the same target gene. Alternatively, the polycistronic mRNA may encode at least one nuclease described herein and at least one additional nuclease that targets a separate recognition sequence located in the same gene or a second recognition sequence located in a second gene such that cleavage sites are generated in both genes or targets a second recognition sequence located in a second gene such that cleavage sites are generated in both genes can encode an enzyme. A polycistronic mRNA can be from the same mRNA molecule including, but not limited to, IRES elements, T2A elements, P2A elements, E2A elements, and F2A elements, and can include any element known in the art that enables the translation of two or more genes (i.e., cistrons) of the same.

[0314] It is possible to deliver a purified nuclease protein to a cell to cleave genomic DNA, thereby enabling homologous recombination or non-homologous end joining at the cleavage site with the target sequence by various different mechanisms known in the art.

[0315] In some embodiments, the engineered nuclease protein, or DNA / mRNA encoding the engineered nuclease, is linked to a cell-penetrating peptide or a target ligand to facilitate uptake into the cell. Examples of cell-penetrating peptides known in the art include polyarginine (Jearawiriyapaisarn et al. (2008) Mol Ther. 16:1624-9), the TAT peptide derived from the HIV virus (Hudecz et al. (2005), Med. Res. Rev. 25:679-736), MPG (Simeoni et al. (2003) Nucleic Acids Res. 31:2717-2724), Pep-1 (Deshayes et al. (2004) Biochemistry 43:7698-7706), and HSV-1 VP-22 (Deshayes et al. (2005) Cell Mol Life Sci. 62:1839-49). In another embodiment, the engineered nuclease, or DNA / encoding the engineered nuclease, The mRNA is bound to the nuclease protein / DNA / mRNA by covalent or non-covalent bonds. A is expressed on the target cell so that it binds to and is internalized by the target cell. It is linked to an antibody that recognizes a specific cell surface receptor. The protein / DNA / mRNA is the natural ligand (or natural ligands) of such cell surface receptors. The nucleotide sequence can be covalently or non-covalently linked to the nucleotide sequence (part of the nucleotide sequence) (McCall et al. (2014) Tissue Barriers.2(4):e944449;Dinda et al. (2013 )Curr Pharm Biotechnol.14:1264-74;Kang et al. 2014)Curr Pharm Biotechnol.15(3):220-30; Qian et al. (2014) Expert Opin Drug Metab Toxico l.10(11):1491-508).

[0316] In some embodiments, an engineered nuclease protein or an engineered nuclease DNA / mRNA encoding the enzyme may be isolated using methods known in the art. The nanoparticles are attached by covalent or preferably non-covalent bonds to the nanoparticles, or such nanoparticles are Encapsulated within the particles (Sharma et al. (2014) Biomed Res Int .2014). Nanoparticles are nanoparticles with length scales less than 1 μm, preferably less than 100 nm. Nanoscale delivery systems. Such nanoparticles can be metal, lipid, polymer, or biocompatible. The recombinant meganuclease protein can be designed with a core composed of a polymer. Attach or encapsulate multiple copies of proteins, mRNA, or DNA into the nanoparticle core. This allows for the delivery of copies of the protein / mRNA / DNA to each cell. As the number increases, the intracellular expression of each engineered nuclease increases, maximizing the likelihood that the target recognition sequences will be cleaved. The surface of such nanoparticles may be further modified with a polymer or lipid (e.g., chitosan, a cationic polymer, or a cationic lipid ), conferring additional functionality that enhances the cellular delivery and uptake of the payload (Jian et al. (2012) Biomaterials. 33(30): 7621-30). Nanoparticles can also be advantageously conjugated to targeting molecules to direct the nanoparticles to appropriate cell types and / or increase the likelihood of cellular uptake. Examples of such targeting molecules include antibodies specific for cell surface receptors and natural ligands (or portions of natural ligands) of cell surface receptors.

[0317] In some embodiments, the engineered nuclease or DNA / mRNA encoding the engineered nuclease is encapsulated within liposomes or complexed using cationic lipids (see, e.g., Lipofectamine, Life Technologies Corp., Carlsbad, CA; Zuris et al. (2015) Nat Biotechnol. 33:73-80; Mishra et al. (2011) J Drug Deliv. 2011:863734). Liposome and lipoplex formulations can protect the payload from degradation and promote cellular uptake and delivery efficiency through fusion and / or disruption of the cell membrane of the cell.

[0318] In some embodiments, the engineered nuclease protein, or engineered nuclease ​​​​​​​​​​​DNA / mRNA encoding an enzyme is encapsulated within a polymeric scaffold (e.g., PLGA) or complexed using a cationic polymer (e.g., PEI, PLL) (Tamboli et al. (2011) Ther Deliv. 2(4):523-536). In some embodiments, engineered nuclease protein, or DNA / mRNA encoding an engineered nuclease is combined with an amphiphilic molecule that self-assembles into micelles (Tong et al. (2007) J Gene Med. 9(11):956-66). High molecular micelles may include a micelle shell formed of a hydrophilic polymer (e.g., polyethylene glycol) that can prevent aggregation, mask charge interactions, and reduce extracellular non-specific interactions.

[0319] In some embodiments, engineered nuclease protein, or DNA / mRNA encoding an engineered nuclease is formulated into an emulsion or nanoemulsion (i.e., having an average particle diameter of less than 1 nm) for delivery to cells. The term "emulsion" can refer to, but is not limited to, any of an oil-in-water, water-in-oil, water-in-oil-in-water, or oil-in-water-in-oil dispersion or droplet of lipid structures that can be formed as a result of hydrophobic forces that keep non-polar residues (e.g., long hydrocarbon chains) away from water and orient polar head groups towards water when an immiscible phase is mixed with an aqueous phase. These other lipid structures include, but are not limited to, monolayer, paucilamellar, and multilamellar lipid vesicles, micelles, and lamellar phases. Emulsions include an aqueous phase and a lipophilic phase.

[0320] In some embodiments, engineered nuclease protein, or DNA / mRNA encoding an engineered nuclease is formulated into an emulsion or nanoemulsion (i.e., having an average particle diameter of less than 1 nm) for delivery to cells. The term "emulsion" can refer to, but is not limited to, any of an oil-in-water, water-in-oil, water-in-oil-in-water, or oil-in-water-in-oil dispersion or droplet of lipid structures that can be formed as a result of hydrophobic forces that keep non-polar residues (e.g., long hydrocarbon chains) away from water and orient polar head groups towards water when an immiscible phase is mixed with an aqueous phase. These other lipid structures include, but are not limited to, monolayer, paucilamellar, and multilamellar lipid vesicles, micelles, and lamellar phases. Emulsions include an aqueous phase and a lipophilic phase. ​​​​​​​composed of a phase (typically including an oil and an organic solvent). The emulsion also often includes one or more surfactants. For example, each of U.S. Patent Application Publication Nos. 2002 / 0045667 and 2004 / 00 43041, as well as U.S. Patents Nos. 6,015,832, 6,506,803, 6,635,676, and 6,559,189, which are incorporated herein by reference in their entireties, nanoemulsion formulations are well known.

[0321] In some embodiments, the engineered nuclease protein, or DNA / mRNA encoding the engineered nuclease, can be covalently associated or non-covalently attached to a multifunctional polymer conjugate, a DNA dendrimer, and a polymeric dendrimer ( Mastorakos et al. (2015) Nanoscale. 7(9):3845-56; Cheng et al. (2008) J Pharm Sci. 97(1):123-43). The generation of dendrimers can control the payload capacity and size, and can provide a high payload capacity. Furthermore, the presentation of multiple surface groups can be utilized to improve stability and reduce non-specific interactions. In some embodiments, the engineered nuclease and / or the gene encoding the target sequence is introduced into cells using a viral vector. Such vectors are known in the art and include retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated virus (AAV) vectors (reviewed in Vannucci et al. (2 013) New Microbiol. 36:1-22). In the present invention

[0322] 013) New Microbiol. 36:1-22). ​ A useful recombinant AAV vector can have any serotype that allows for transduction of the virus into cells and insertion of the nuclease gene into the cellular genome. In certain embodiments, the recombinant AAV vector has a serotype of AAV2 or AAV6. The recombinant AAV vector may also be self-complementary so as not to require double-stranded DNA synthesis in the host cell (McCarty et al. (2001) Gene Ther. 8:1248-54). When engineered nuclease genes are delivered via a DNA form (e.g., plasmid) and / or a viral vector (e.g., AAV), they must be controllably linked to a promoter. In some embodiments, this is an endogenous

[0323] promoter from a viral vector (e.g., the LTR of a lentiviral vector), or a viral promoter such as the well-known cytomegalovirus or SV40 virus early promoter. In preferred embodiments, the nuclease gene is controllably linked to a promoter that preferentially drives gene expression in target cells (e.g., T cells ).

[0324]

[0324] The present invention further provides for the introduction of an exogenous sequence of interest into the T cell receptor alpha gene, particularly into the recognition sequence within the target 5' intron. In some embodiments, the exogenous sequence of interest includes a 5' homology arm and a 3' homology arm adjacent to an insert element (i.e., an exogenous splice acceptor site, an IRES or 2A element, the coding sequence of the protein of interest, and / or a polyA signal). Such homology arms have sequence homology with the corresponding sequences 5' upstream and 3' downstream of the nuclease recognition sequence of the target 5' intron at which the cleavage site is generated. have. Generally, the homology arms can have a length of at least 50 base pairs, preferably at least 100 base pairs, and up to 2000 base pairs or more, and have at least 90%, preferably at least 95%, or more sequence homology with their corresponding sequences in the genome.

[0325] The exogenous sequences of the object of the present invention can be introduced into cells by any of the above-mentioned means. In particular In certain embodiments, the exogenous sequences of interest are introduced by viral vectors such as lentiviruses, retroviruses, adenoviruses or preferably by recombinant AAV vectors. Recombinant AAV vectors useful for the introduction of exogenous nucleic acids can have any serotype that allows transduction of cells by the virus and insertion of exogenous nucleic acid sequences into the cellular genome. In certain embodiments the recombinant AAV vector has a serotype of AAV2 or AAV6. The recombinant AAV vector may also be self-complementary so as not to require double-stranded DNA synthesis in the host cell.

[0326] In another specific embodiment, a single-stranded DNA template can be used to introduce the exogenous sequences of interest into cells. The single-stranded DNA can contain the exogenous sequences of interest, and in preferred embodiments, it may include 5' and 3' homology arms that facilitate the insertion of nucleic acid sequences into nuclease cleavage sites by homologous recombination. The single-stranded DNA may further include a 5' AAV inverted terminal repeat (ITR) sequence upstream of the 5' end of the 5' homology arm and a 3' AAV ITR sequence downstream of the 3' end of the 3' homology arm.

[0327] ​​​​​​​In another specific embodiment, the engineered nuclease of the present invention and / or the gene encoding the exogenous sequence of interest of the present invention may be introduced into cells more efficiently using a linearized DNA template. In some examples, the plasmid DNA may be digested with one or more restriction enzymes so that the circular plasmid DNA is linearized prior to transfection into the cells. The gene encoding the exogenous sequence may be introduced into cells more efficiently using a linearized DNA template. In some examples, the plasmid DNA may be digested with one or more restriction enzymes so that the circular plasmid DNA is linearized prior to transfection into the cells. In some examples, the plasmid DNA may be digested with one or more restriction enzymes so that the circular plasmid DNA is linearized prior to transfection into the cells. The engineered T cells of the present invention may require activation prior to the introduction of the nuclease and / or the exogenous sequence of interest. For example, the T cells may be contacted with anti-CD3 and anti-CD28 antibodies that are soluble or bound to a support (i.e., beads) for a period sufficient to activate the cells.

[0328] The engineered T cells of the present invention may require activation prior to the introduction of the nuclease and / or the exogenous sequence of interest. For example, the T cells may be contacted with anti-CD3 and anti-CD28 antibodies that are soluble or bound to a support (i.e., beads) for a period sufficient to activate the cells. The engineered T cells of the present invention may require activation prior to the introduction of the nuclease and / or the exogenous sequence of interest. For example, the T cells may be contacted with anti-CD3 and anti-CD28 antibodies that are soluble or bound to a support (i.e., beads) for a period sufficient to activate the cells. The engineered T cells of the present invention may require activation prior to the introduction of the nuclease and / or the exogenous sequence of interest. For example, the T cells may be contacted with anti-CD3 and anti-CD28 antibodies that are soluble or bound to a support (i.e., beads) for a period sufficient to activate the cells. The engineered T cells of the present invention may require activation prior to the introduction of the nuclease and / or the exogenous sequence of interest. For example, the T cells may be contacted with anti-CD3 and anti-CD28 antibodies that are soluble or bound to a support (i.e., beads) for a period sufficient to activate the cells.

[0329] The genetically modified cells of the present invention can be further modified to express one or more inducible suicide genes, the induction of which causes cell death and enables the selective destruction of the cells in vitro or in vivo. In some examples, the suicide gene may encode a cytotoxic polypeptide, a polypeptide having the ability to convert a non-toxic prodrug into a cytotoxic drug, and / or a polypeptide that activates an intracellular cytotoxic gene pathway. That is, the suicide gene is a nucleic acid that encodes a product that causes cell death either by itself or in the presence of another compound. Representative examples of such suicide genes include those encoding herpes simplex virus thymidine kinase. Additional examples include genes encoding varicella-zoster virus thymidine kinase and the bacterial gene cytosine deaminase, which can convert 5-fluorocytosine into the highly toxic compound 5-fluorouracil. Non-limiting examples of suicide genes include those encoding herpes simplex virus thymidine kinase. The genetically modified cells of the present invention can be further modified to express one or more inducible suicide genes, the induction of which causes cell death and enables the selective destruction of the cells in vitro or in vivo. In some examples, the suicide gene may encode a cytotoxic polypeptide, a polypeptide having the ability to convert a non-toxic prodrug into a cytotoxic drug, and / or a polypeptide that activates an intracellular cytotoxic gene pathway. That is, the suicide gene is a nucleic acid that encodes a product that causes cell death either by itself or in the presence of another compound. Representative examples of such suicide genes include those encoding herpes simplex virus thymidine kinase. Additional examples include genes encoding varicella-zoster virus thymidine kinase and the bacterial gene cytosine deaminase, which can convert 5-fluorocytosine into the highly toxic compound 5-fluorouracil. Non-limiting examples of suicide genes include those encoding herpes simplex virus thymidine kinase. The genetically modified cells of the present invention can be further modified to express one or more inducible suicide genes, the induction of which causes cell death and enables the selective destruction of the cells in vitro or in vivo. In some examples, the suicide gene may encode a cytotoxic polypeptide, a polypeptide having the ability to convert a non-toxic prodrug into a cytotoxic drug, and / or a polypeptide that activates an intracellular cytotoxic gene pathway. That is, the suicide gene is a nucleic acid that encodes a product that causes cell death either by itself or in the presence of another compound. Representative examples of such suicide genes include those encoding herpes simplex virus thymidine kinase. Additional examples include genes encoding varicella-zoster virus thymidine kinase and the bacterial gene cytosine deaminase, which can convert 5-fluorocytosine into the highly toxic compound 5-fluorouracil. Non-limiting examples of suicide genes include those encoding herpes simplex virus thymidine kinase. The genetically modified cells of the present invention can be further modified to express one or more inducible suicide genes, the induction of which causes cell death and enables the selective destruction of the cells in vitro or in vivo. In some examples, the suicide gene may encode a cytotoxic polypeptide, a polypeptide having the ability to convert a non-toxic prodrug into a cytotoxic drug, and / or a polypeptide that activates an intracellular cytotoxic gene pathway. That is, the suicide gene is a nucleic acid that encodes a product that causes cell death either by itself or in the presence of another compound. Representative examples of such suicide genes include those encoding herpes simplex virus thymidine kinase. Additional examples include genes encoding varicella-zoster virus thymidine kinase and the bacterial gene cytosine deaminase, which can convert 5-fluorocytosine into the highly toxic compound 5-fluorouracil. Non-limiting examples of suicide genes include those encoding herpes simplex virus thymidine kinase. The genetically modified cells of the present invention can be further modified to express one or more inducible suicide genes, the induction of which causes cell death and enables the selective destruction of the cells in vitro or in vivo. In some examples, the suicide gene may encode a cytotoxic polypeptide, a polypeptide having the ability to convert a non-toxic prodrug into a cytotoxic drug, and / or a polypeptide that activates an intracellular cytotoxic gene pathway. That is, the suicide gene is a nucleic acid that encodes a product that causes cell death either by itself or in the presence of another compound. Representative examples of such suicide genes include those encoding herpes simplex virus thymidine kinase. Additional examples include genes encoding varicella-zoster virus thymidine kinase and the bacterial gene cytosine deaminase, which can convert 5-fluorocytosine into the highly toxic compound 5-fluorouracil. Non-limiting examples of suicide genes include those encoding herpes simplex virus thymidine kinase. The genetically modified cells of the present invention can be further modified to express one or more inducible suicide genes, the induction of which causes cell death and enables the selective destruction of the cells in vitro or in vivo. In some examples, the suicide gene may encode a cytotoxic polypeptide, a polypeptide having the ability to convert a non-toxic prodrug into a cytotoxic drug, and / or a polypeptide that activates an intracellular cytotoxic gene pathway. That is, the suicide gene is a nucleic acid that encodes a product that causes cell death either by itself or in the presence of another compound. Representative examples of such suicide genes include those encoding herpes simplex virus thymidine kinase. Additional examples include genes encoding varicella-zoster virus thymidine kinase and the bacterial gene cytosine deaminase, which can convert 5-fluorocytosine into the highly toxic compound 5-fluorouracil. Non-limiting examples of suicide genes include those encoding herpes simplex virus thymidine kinase. The genetically modified cells of the present invention can be further modified to express one or more inducible suicide genes, the induction of which causes cell death and enables the selective destruction of the cells in vitro or in vivo. In some examples, the suicide gene may encode a cytotoxic polypeptide, a polypeptide having the ability to convert a non-toxic prodrug into a cytotoxic drug, and / or a polypeptide that activates an intracellular cytotoxic gene pathway. That is, the suicide gene is a nucleic acid that encodes a product that causes cell death either by itself or in the presence of another compound. Representative examples of such suicide genes include those encoding herpes simplex virus thymidine kinase. Additional examples include genes encoding varicella-zoster virus thymidine kinase and the bacterial gene cytosine deaminase, which can convert 5-fluorocytosine into the highly toxic compound 5-fluorouracil. Non-limiting examples of suicide genes include those encoding herpes simplex virus thymidine kinase. The genetically modified cells of the present invention can be further modified to express one or more inducible suicide genes, the induction of which causes cell death and enables the selective destruction of the cells in vitro or in vivo. In some examples, the suicide gene may encode a cytotoxic polypeptide, a polypeptide having the ability to convert a non-toxic prodrug into a cytotoxic drug, and / or a polypeptide that activates an intracellular cytotoxic gene pathway. That is, the suicide gene is a nucleic acid that encodes a product that causes cell death either by itself or in the presence of another compound. Representative examples of such suicide genes include those encoding herpes simplex virus thymidine kinase. Additional examples include genes encoding varicella-zoster virus thymidine kinase and the bacterial gene cytosine deaminase, which can convert 5-fluorocytosine into the highly toxic compound 5-fluorouracil. Non-limiting examples of suicide genes include those encoding herpes simplex virus thymidine kinase. The genetically modified cells of the present invention can be further modified to express one or more inducible suicide genes, the induction of which causes cell death and enables the selective destruction of the cells in vitro or in vivo. In some examples, the suicide gene may encode a cytotoxic polypeptide, a polypeptide having the ability to convert a non-toxic prodrug into a cytotoxic drug, and / or a polypeptide that activates an intracellular cytotoxic gene pathway. That is, the suicide gene is a nucleic acid that encodes a product that causes cell death either by itself or in the presence of another compound. Representative examples of such suicide genes include those encoding herpes simplex virus thymidine kinase. Additional examples include genes encoding varicella-zoster virus thymidine kinase and the bacterial gene cytosine deaminase, which can convert 5-fluorocytosine into the highly toxic compound 5-fluorouracil. Non-limiting examples of suicide genes include those encoding herpes simplex virus thymidine kinase. The genetically modified cells of the present invention can be further modified to express one or more inducible suicide genes, the induction of which causes cell death and enables the selective destruction of the cells in vitro or in vivo. In some examples, the suicide gene may encode a cytotoxic polypeptide, a polypeptide having the ability to convert a non-toxic prodrug into a cytotoxic drug, and / or a polypeptide that activates an intracellular cytotoxic gene pathway. That is, the suicide gene is a nucleic acid that encodes a product that causes cell death either by itself or in the presence of another compound. Representative examples of such suicide genes include those encoding herpes simplex virus thymidine kinase. Additional examples include genes encoding varicella-zoster virus thymidine kinase and the bacterial gene cytosine deaminase, which can convert 5-fluorocytosine into the highly toxic compound 5-fluorouracil. Non-limiting examples of suicide genes include those encoding herpes simplex virus thymidine kinase. and a gene encoding caspase-9, caspase-8, or cytosine deaminase is also included. In some examples, caspase-9 can be activated using a specific chemical inducer of dimerization (CID). The suicide gene may encode a polypeptide that is expressed on the surface of cells that renders the cells sensitive to treatment and / or cytotoxic monoclonal antibodies. In a further example, the suicide gene may encode a recombinant antigen polypeptide that includes an antigen motif recognized by the anti-CD20 mAb rituximab and an epitope that allows for the selection of cells expressing the suicide gene. See, for example, the RQR8 polypeptide that includes two rituximab-binding epitopes and one QBEnd10-binding epitope described in International Publication No. WO 2013 / 153391. In the case of such a gene, rituximab can be administered to a subject, if desired, to induce cell depletion. In a further example, the suicide gene may include a QBEnd10-binding epitope that is expressed in combination with a truncated EGFR polypeptide.

[0330] The T cells modified by the methods and compositions described herein can downregulate the expression of the endogenous T cell receptor and, optionally, can further express a protein of interest (e.g., a CAR). Accordingly, the invention further provides a population of T cells that express a protein of interest and do not express an endogenous T cell receptor. For example, the population can include a plurality of genetically modified T cells of the invention that express a CAR (i.e., are CAR+) or an exogenous T cell receptor (i.e., are exoTCR+) and have reduced expression of the endogenous T cell receptor. ​​​​​​​​​​​​​That is, TCR-). In various embodiments of the present invention, at least 10%, at least 15%, at least 20%, at least 25%, at least 30% , at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75 %, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 10 0% of the cells in the population are the genetically modified T cells described herein. In certain examples, the population is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% , at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% of cells that are both TCR- and CAR+.

[0331] 2.5 Methods for Administering Genetically Modified Cells In some aspects, the present invention provides a pharmaceutical composition comprising the genetically modified T cells of the present invention, or a population of the genetically modified T cells of the present invention, and a pharmaceutically acceptable carrier. Such a pharmaceutical composition can be prepared according to known techniques. See, for example, Remington, The Sci ence And Practice of Pharmacy (21 st ed.200 5). In the manufacture of pharmaceutical formulations according to the present invention, the cells are typically pharmaceutically It is mixed with an acceptable carrier and the resulting composition is administered to a subject. The carrier, of course, must be acceptable in the sense of being compatible with the other ingredients in the formulation and not harmful to the subject. In some embodiments, the pharmaceutical composition of the invention may further comprise one or more additional agents useful for treating the disease of the subject. In further embodiments, the pharmaceutical composition of the invention may further comprise cytokines (e.g., IL-2, IL-7, IL-15, and / or IL-21, etc.) and other biomolecules that promote in vivo cell proliferation and engraftment of the genetically modified T cells. The pharmaceutical composition comprising the genetically modified T cells of the invention may be administered in the same composition as the additional agent or the biomolecule, or alternatively may be co-administered in a separate

[0332] composition. The present disclosure also provides the genetically modified cells or populations described herein for use as a medicament. The present disclosure further provides the use of the genetically modified cells or populations described herein in the manufacture of a medicament for treating a disease of a subject in need thereof. In such an aspect, the medicament is useful for cancer immunotherapy of a subject in need thereof.

[0333] The pharmaceutical composition of the invention can be useful for treating any condition that can be targeted by adoptive T cell immunotherapy. Non-limiting examples of cancers that can be treated with the pharmaceutical composition and medicaments of the present disclosure include cancers of B cell origin, neuroblastoma, osteosarcoma, prostate cancer, renal cell carcinoma, or malignant melanoma within the eye, renal cancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, non-Hodgkin lymphoma, esophageal cancer, small intestine cancer, endocrine cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, pediatric solid tumor, lymphocytic lymphoma, bladder cancer, cancer of the kidney or ureter, renal pelvic carcinoma, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor , brainstem glioma, pituitary adenoma, Kaposi sarcoma, epidermoid carcinoma, squamous cell carcinoma, environmentally induced cancers including those induced by asbestos, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, acute myeloid lymphoma, chronic myeloid leukemia, chronic lymphocytic leukemia, immunoblastic large cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides, undifferentiated large cell lymphoma, T cell lymphoma, and any combination of these cancers, including but not limited to carcinomas, lymphomas, sarcomas, melanomas, blastomas, leukemias, and germ cell tumors. In certain embodiments, cancers of B cell origin include, but are not limited to, B cell lineage acute lymphoblastic leukemia, B cell chronic lymphocytic leukemia, B cell lymphoma, diffuse large B cell lymphoma, pre-B ALL (pediatric indication), mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, Burkitt lymphoma, multiple myeloma, and B cell non-Hodgkin lymphoma. .

[0334] In some of these embodiments of treating cancer with the genetically modified cells of the present disclosure, additional treatments such as radiation, surgery, or chemotherapeutic agents are further applied to the subject to whom the genetically modified cells have been administered. .

[0335] The present invention further provides a population of genetically modified cells comprising a plurality of genetically modified cells described herein, which comprise an exogenous nucleic acid molecule encoding a target sequence in the genome, wherein the exogenous nucleic acid molecule is inserted into the target 5' intron of the T cell receptor alpha gene, and the cell surface expression of the endogenous TCR is reduced. Thus, in various embodiments of the present invention, a population of genetically modified cells is provided, wherein at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40 %, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 8 5%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% of the cells in the population are the genetically modified cells described herein. In a further embodiment of the present invention, a population of genetically modified cells is provided, wherein at least 10%, at least 15%, at least 20%, at least 25 %, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 7 0%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% of the cells in the population are the genetically modified cells described herein that further express a chimeric antigen receptor.

[0336] 2.6 Methods for Producing Recombinant Virus Vectors ​​​ Another aspect disclosed herein is the administration of the genetically modified T cells of the present disclosure to a subject in need thereof. In certain embodiments, the pharmaceutical composition described herein is administered to a subject in need thereof. For example, an effective amount of the cell population can be administered to a subject having a disease. In certain embodiments, the disease can be cancer, and the administration of the genetically modified T cells of the present invention results in immunotherapy. The administered cells can suppress proliferation and reduce the number in the recipient, and kill target cells. Unlike antibody therapy, the genetically modified T cells of the present disclosure can replicate and expand in vivo, resulting in long-term persistence that may lead to sustained control of the disease. Examples of possible routes of administration include parenteral (e.g., intravenous (IV), intramuscular (IM), intradermal, subcutaneous (SC), or infusion) administration. Further, the administration can be by continuous infusion, or by single

[0337] or multiple bolus administrations. In specific embodiments, one or both of the agents are infused over a period of about 12 hours, 6 hours, 4 hours, 3 hours, 2 hours, or less than 1 hour. In yet other embodiments, the infusion starts slowly and then increases over time. In some embodiments, the genetically modified T cells of the present disclosure target tumor antigens for the purpose of treating cancer. Such cancers include, but are not limited to, carcinomas, lymphomas, sarcomas, blastomas, leukemias, cancers of B cell origin, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, melanoma, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, Hodgkin lymphoma.

[0338] ​​​​​​. In certain embodiments, cancers and disorders include, but are not limited to, pre-B ALL (pediatric indication), adult ALL, mantle cell lymphoma, diffuse large B cell lymphoma, salvage after allogeneic bone marrow transplantation, etc. These cancers can be treated, for example, using combinations of CARs targeting CD19, CD20, CD22, and / or ROR1. In some non-limiting examples, the genetically modified eukaryotic cells or populations thereof of the present disclosure target cancers of B cell origin, neuroblastoma, osteosarcoma, prostate cancer, renal cell carcinoma, rhabdomyosarcoma, liver cancer, gastric cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, breast cancer, lung cancer, malignant melanoma of the skin or intraocular, renal cancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, anal canal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar cancer, non-Hodgkin lymphoma, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, pediatric solid tumor, lymphoblastic lymphoma, bladder cancer, cancer of the kidney or ureter, renal pelvis carcinoma, central nervous system (CNS) neoplasm, primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brainstem glioma, pituitary adenoma, Kaposi sarcoma, epidermoid carcinoma, squamous cell carcinoma, environmentally induced cancers including those induced by asbestos, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, acute myeloid lymphoma, chronic myeloid leukemia, chronic lymphocytic leukemia, immunoblastic large cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides, anaplastic large cell lymphoma, T cell lymphoma, and any combination of these cancers, including but not limited to carcinomas, lymphomas, sarcomas, melanomas, blastomas, leukemias, and germ cell tumors. In certain specific embodiments Adult ALL, mantle cell lymphoma, diffuse large B cell lymphoma, salvage after allogeneic bone marrow transplantation, etc. are included, but not limited to these. These cancers can be treated, for example, using combinations of CARs targeting CD1 9, CD20, CD22, and / or ROR1. In some non-limiting examples, the genetically modified eukaryotic cells or populations thereof of the present disclosure target cancers of B cell origin, neuroblastoma, osteosarcoma, prostate cancer, renal cell carcinoma, rhabdomyosarcoma, liver cancer, gastric cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, breast cancer, lung cancer , malignant melanoma of the skin or intraocular, renal cancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, anal canal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube carcinoma, endometrial carcinoma , cervical carcinoma, vaginal carcinoma, vulvar cancer, non-Hodgkin lymphoma, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, pediatric solid tumor, lymphoblastic lymphoma, bladder cancer, cancer of the kidney or ureter, renal pelvis carcinoma, central nervous system (CNS) neoplasm, primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brainstem glioma, pituitary adenoma, Kaposi sarcoma, epidermoid carcinoma, squamous cell carcinoma, environmentally induced cancers including those induced by asbestos, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, acute myeloid lymphoma, chronic myeloid leukemia, chronic lymphocytic leukemia, immunoblastic large cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides, anaplastic large cell lymphoma, T cell lymphoma, and any combination of these cancers, including but not limited to carcinomas, lymphomas, sarcomas, melanomas, blastomas, leukemias, and germ cell tumors. In certain embodiments, cancers and disorders include, but are not limited to, pre-B ALL (pediatric indication), adult ALL, mantle cell lymphoma, diffuse large B cell lymphoma, salvage after allogeneic bone marrow transplantation, etc. These cancers can be treated, for example, using combinations of CARs targeting CD1 9, CD20, CD22, and / or ROR1. In some non-limiting examples, the genetically modified eukaryotic cells or populations thereof of the present disclosure target cancers of B cell origin, neuroblastoma, osteosarcoma, prostate cancer, renal cell carcinoma, rhabdomyosarcoma, liver cancer, gastric cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, breast cancer, lung cancer , malignant melanoma of the skin or intraocular, renal cancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, anal canal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube carcinoma, endometrial carcinoma , cervical carcinoma, vaginal carcinoma, vulvar cancer, non-Hodgkin lymphoma, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, pediatric solid tumor, lymphoblastic lymphoma, bladder cancer, cancer of the kidney or ureter, renal pelvis carcinoma, central nervous system (CNS) neoplasm, primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brainstem glioma, pituitary adenoma, Kaposi sarcoma, epidermoid carcinoma, squamous cell carcinoma, environmentally induced cancers including those induced by asbestos, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, acute myeloid lymphoma, chronic myeloid leukemia, chronic lymphocytic leukemia, immunoblastic large cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides, anaplastic large cell lymphoma, T cell lymphoma, and any combination of these cancers, including but not limited to carcinomas, lymphomas, sarcomas, melanomas, blastomas, leukemias, and germ cell tumors. In terms of morphology, B-cell origin cancers include, but are not limited to, B-cell lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, B-cell lymphoma, diffuse large B-cell lymphoma, pre-B ALL (pediatric indication), mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, Burkitt lymphoma, multiple myeloma, and B-cell non-Hodgkin lymphoma. When an "effective amount" or "therapeutic amount" is indicated, the exact amount administered can be determined by a physician considering individual differences in age, weight, tumor size (if present), degree of infection or metastasis, and the condition of the patient (subject). In some embodiments, the pharmaceutical composition containing the genetically modified cells described herein is administered at a dosage of 10 cells / kg body weight to 10 cells / kg body weight, including all integer values within those ranges. In further embodiments, the dosage is 10

[0339] cells / kg body weight to 10 cells / kg body weight, including all integer values within those ranges. In some embodiments, the cell composition is administered multiple times at these dosages. The cells can be administered using infusion techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988 for reference). The optimal dosage and treatment plan for a particular patient can be readily determined by one of ordinary skill in the medical art by monitoring the patient for signs of the disease and adjusting the treatment accordingly. In some embodiments, the administration of the genetically modified T cells of the present disclosure is at least for the target disease or condition including all integer values within those ranges of 10 4 cells / kg body weight to 10 9 cells / kg body weight. In further embodiments, the dosage is including all integer values within those ranges of 10 5 cells / kg body weight to 10 6 cells / kg body weight. In some embodiments, the cell composition is administered multiple times at these dosages. The cells can be administered using infusion techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988 for reference). The optimal dosage and treatment plan for a particular patient can be readily determined by one of ordinary skill in the medical art by monitoring the patient for signs of the disease and adjusting the treatment accordingly. The cells can be administered using infusion techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988 for reference). See, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988. The optimal dosage and treatment plan for a particular patient can be readily determined by one of ordinary skill in the medical art by monitoring the patient for signs of the disease and adjusting the treatment accordingly. The optimal dosage and treatment plan for a particular patient can be readily determined by one of ordinary skill in the medical art by monitoring the patient for signs of the disease and adjusting the treatment accordingly. The optimal dosage and treatment plan for a particular patient can be readily determined by one of ordinary skill in the medical art by monitoring the patient for signs of the disease and adjusting the treatment accordingly.

[0340] In some embodiments, the administration of the genetically modified T cells of the present disclosure is at least for the target disease or condition Reduce at least one symptom. For example, administration of the genetically modified T cells of the present disclosure reduces at least one symptom of cancer of cancer. Symptoms of cancer are well known in the art and can be determined by known techniques.

[0341] 2.7 Engineered Nuclease Variants In some embodiments, the present invention provides a recombinant AAV vector for use in the methods of the present invention. Recombinant AAV vectors are typically produced in mammalian cell lines such as HEK-293. The viral cap and rep genes are removed from the vector to prevent self-replication and create room for delivery of a therapeutic gene (s) (e.g., an endonuclease gene). Therefore, they need to be provided in trans to a packaging cell line. In addition, "helper" (e.g., adenovirus) components necessary to support replication need to be provided (Cots D, Bosch A, Chillon M ( 2013) Curr. Gene Ther. 13(5):370-81). In many cases, recombinant AAV vectors are produced using triple transfection, where a cell line is transfected with a first plasmid encoding "helper" components, a second plasmid containing the cap and rep genes, and a third plasmid containing viral ITRs containing intervening DNA sequences to be packaged into the virus. The virus particles containing the genome (the ITRs of interest and intervening gene(s)) encapsulated in the capsid are then isolated from the cells by freeze-thaw cycles, sonication, detergents, or other means known in the art. The particles are then subjected to cesium chloride density gradient centrifugation or other means known in the art. Other means known in the art. Other means known in the art. Purify using affinity chromatography and then deliver the gene(s) of interest to an organism such as a cell, tissue, or human patient.

[0342] Since recombinant AAV particles are usually produced (manufactured) intracellularly, care must be taken when practicing the present invention to ensure that site-specific endonucleases are not expressed in the packaging cells. Since the viral genome of the present invention contains recognition sequences for endonucleases, the endonucleases expressed in the packaging cell line can cleave the viral genome before it is packaged into viral particles. This results in reduced packaging efficiency and / or packaging of fragmented genomes. To prevent endonuclease expression in packaging cells, several approaches can be used:

[0343] 1. The endonuclease may be placed under the control of a tissue-specific promoter that is not activated in the packaging cells. For example, if a viral vector is developed for delivery of an endonuclease gene(s) to muscle tissue, a muscle-specific promoter can be used. Examples of muscle-specific promoters include C5-12 (Liu et al. (2004) Hum Gene Ther. 15:783-92), muscle-specific creatine kinase (MCK) promoter (Yuasa, et al. (2002) Gene Ther. 9:1576-88), or smooth muscle 22 (SM22) promoter (Haase, et al. (20 13) BMC Biotechnol. 13:49-54). Examples of CNS (neuron)-specific promoters include NSE, synapsin, and MeCP2 promoter ​ include (Lentz et al. (2012) Neurobiol Dis. 48:179 -88). Examples of liver-specific promoters include albumin promoter (Palb, etc.), human α1-antitrypsin (Pa1AT, etc.), and hemopexin (Phpx, etc.) (Kr amer, MG et al., (2003) Mol. Therapy 7:375-85). Examples of eye-specific promoters include opsin and corneal epithelial-specific K12 promoter (Martin KRG, Klein RL, and Quigley HA (2002) Methods (28):267-75) (Tong Y et al. (2007 ) J Gene Med, 9:956-66). These promoters, or other tissue-specific promoters known in the art are not highly active in HEK-293 cells, so when incorporated into the viral vectors of the present invention, it is not expected to result in significant levels of endonuclease gene expression in packaging cells. Similarly, the viral vectors of the present invention are contemplated for use with non-compatible tissue-specific promoters (i.e., using well-known HeLa cell line (human epithelial cells) and liver-specific hemopexin promoter ) and other cell lines. Other examples of tissue-specific promoters include : synovial sarcoma PDZD4 (cerebellum), C6 (liver), ASB5 (muscle), PPP1R12B( heart), SLC5A12 (kidney), cholesterol-regulating APOM (liver), ADPRHL 1 (heart), and single-gene malformation syndrome TP73L (muscle) (Jacox E et al. (2010) PLoS One v.5(8):e12274).

[0344] 2. Alternatively, the vector can be packaged into cells of different species in which the endonuclease is unlikely to be expressed. For example, virus particles can be produced in microbial, insect, or plant cells using mammalian promoters such as the well-known cytomegalovirus or SV40 virus early promoter that are not active in non-mammalian packaging cells. In a preferred embodiment, the virus particles are produced in insect cells using the baculovirus system described by Gao et al. (Gao, H. et al. (2007) J. Biotechnol. 131(2):138-43). The endonuclease under the control of a mammalian promoter is unlikely to be expressed in these cells (Airenne, KJ et al. (2013) Mol. Ther. 21(4):739-49). Furthermore, insect cells utilize mRNA splicing motifs different from those of mammalian cells. Thus, it is possible to incorporate mammalian introns such as the human growth hormone (hGH) intron or the SV40 large T antigen intron into the coding sequence of the endonuclease. Since these introns are not efficiently spliced from the pre-mRNA transcripts of insect cells, the insect cells do not express functional endonuclease and package the full-length genome. In contrast, mammalian cells to which the obtained recombinant AAV particles are delivered properly splice the pre-mRNA and express functional endonuclease protein. Haifeng Chen reported using the hGH and SV40 large T antigen introns to attenuate the expression of the toxic proteins barnase and diphtheria toxin fragment A in insect packaging cells, enabling the production of recombinant AAV vectors carrying these toxin genes (Chen, H (2 Inactive in non-mammalian packaging cells, virus particles can be produced in microbial, insect, or plant cells using mammalian promoters such as the well-known cytomegalovirus or SV40 virus early promoter. In a preferred embodiment, the virus particles are produced in insect cells using the baculovirus system described by Gao et al. (Gao, H. et al. (2007) J. Biotechnol. 131(2):138-43). The endonuclease under the control of a mammalian promoter is unlikely to be expressed in these cells (Airenne, KJ et al. (2013) Mol. Ther. 21(4):739-49). Furthermore, insect cells utilize mRNA splicing motifs different from those of mammalian cells. Thus, it is possible to incorporate mammalian introns such as the human growth hormone (hGH) intron or the SV40 large T antigen intron into the coding sequence of the endonuclease. Since these introns are not efficiently spliced from the pre-mRNA transcripts of insect cells, the insect cells do not express functional endonuclease and package the full-length genome. In contrast, mammalian cells to which the obtained recombinant AAV particles are delivered properly splice the pre-mRNA and express functional endonuclease protein. Haifeng Chen reported using the hGH and SV40 large T antigen introns to attenuate the expression of the toxic proteins barnase and diphtheria toxin fragment A in insect packaging cells, enabling the production of recombinant AAV vectors carrying these toxin genes (Chen, H (2 Inactive in non-mammalian packaging cells, virus particles can be produced in microbial, insect, or plant cells using mammalian promoters such as the well-known cytomegalovirus or SV40 virus early promoter. In a preferred embodiment, the virus particles are produced in insect cells using the baculovirus system described by Gao et al. (Gao, H. et al. (2007) J. Biotechnol. 131(2):138-43). The endonuclease under the control of a mammalian promoter is unlikely to be expressed in these cells (Airenne, KJ et al. (2013) Mol. Ther. 21(4):739-49). Furthermore, insect cells utilize mRNA splicing motifs different from those of mammalian cells. Thus, it is possible to incorporate mammalian introns such as the human growth hormone (hGH) intron or the SV40 large T antigen intron into the coding sequence of the endonuclease. Since these introns are not efficiently spliced from the pre-mRNA transcripts of insect cells, the insect cells do not express functional endonuclease and package the full-length genome. In contrast, mammalian cells to which the obtained recombinant AAV particles are delivered properly splice the pre-mRNA and express functional endonuclease protein. Haifeng Chen reported using the hGH and SV40 large T antigen introns to attenuate the expression of the toxic proteins barnase and diphtheria toxin fragment A in insect packaging cells, enabling the production of recombinant AAV vectors carrying these toxin genes (Chen, H (2 Inactive in non-mammalian packaging cells, virus particles can be produced in microbial, insect, or plant cells using mammalian promoters such as the well-known cytomegalovirus or SV40 virus early promoter. In a preferred embodiment, the virus particles are produced in insect cells using the baculovirus system described by Gao et al. (Gao, H. et al. (2007) J. Biotechnol. 131(2):138-43). 012) Mol Ther Nucleic Acids.1(11):e57).

[0345] 3. The endonuclease gene can be controllably linked to an inducible promoter such that a small molecule inducer is required for endonuclease expression. Examples of inducible promoters include the Tet-On system (Clontech; Chen H et al., (2015) BMC Biotechnol.15(1):4)) and the RheoSwitch system ( Intrexon; Sowa G et al. (2011) Spine,36(10):E623- 8). Along with both systems, similar systems known in the art activate transcription in response to small molecule activators (doxycycline or ecdysone, respectively), depending on ligand-inducible transcription factors (mutants of Tet repressor and ecdysone receptor, respectively). Implementing the present invention using such ligand-inducible transcriptional activators involves: 1) a step of placing the endonuclease gene under the control of a promoter responsive to the corresponding transcription factor, wherein the endonuclease gene has a binding site (which may be plural) for the transcription factor; and 2) a step of including a gene encoding the transcription factor in the packaged viral genome. When the transcriptional activator is not provided in the same cells, the latter step is necessary because the endonuclease will not be expressed in the target cells or tissues after recombinant AAV delivery. Then, the transcriptional activator induces endonuclease gene expression only in cells or tissues treated with the cognate small molecule activator. This approach allows for the selection of when and to which tissue to deliver the small molecule inducer, thereby enabling endonuclease It is advantageous because it can spatiotemporally regulate Crease gene expression. However, since it is necessary to include an inducer in the viral genome, the carrying capacity is significantly limited, and this application has a drawback in this regard.

[0346] 4. In another preferred embodiment, the recombinant AAV particles are produced in a mammalian cell line that expresses a transcriptional repressor that inhibits the expression of the endonuclease. Transcriptional repressors are known in the art and include Tet repressor, Lac repressor, Cro repressor, and lambda repressor. Many nuclear hormone receptors, such as the ecdysone receptor, also act as transcriptional repressors in the absence of their cognate hormone ligand. To practice the present invention, the packaging cells are transfected / transduced with a vector encoding the transcriptional repressor, and the endonuclease gene (packaging vector) within the viral genome is controllably linked to a promoter modified to include a binding site for the repressor such that the repressor silences the promoter. The gene encoding the transcriptional repressor can be placed at various positions. The gene encoding the transcriptional repressor may be encoded on a separate vector; incorporated into the packaging vector outside the ITR sequence; incorporated into the cap / rep vector or the adenovirus helper vector, or most preferably, stably incorporated into the genome of the packaging cells such that it is constitutively expressed. Methods for modifying common mammalian promoters to incorporate transcriptional repression sites are known in the art. For example, Chang and Roninson modified strong constitutive CMV and RSV promoters to include the operator of the Lac repressor ​​​​​​​​​​​In cells that are modified to express a repressor, gene expression from the modified promoter is significantly attenuated (Chang BD, and Roninson IB (1 996) Gene 183:137-42). By using a non-human transcriptional repressor, transcription of the endonuclease gene is suppressed only in packaging cells that express the repressor, ensuring that it is not suppressed in target cells or tissues transduced with the resulting recombinant AAV vector.

[0347] In some embodiments, gene delivery is achieved via a lentiviral vector. In contrast to other retroviruses, lentiviruses can, in some cases, be used to transduce certain non-dividing cells. Non-limiting examples of lentiviral vectors include those derived from lentiviruses such as human immunodeficiency virus 1 (HIV-1), HIV-2, simian immunodeficiency virus (SIV), human T-lymphotropic virus 1 (HTLV-1), HTLV-2, or equine infectious anemia virus (EIAV). For example, for example, the genes env, vif, vpr, vpu, and nef are deleted to attenuate the HIV pathogenicity genes to generate a lentiviral vector that is safer for therapeutic purposes. Lentiviral vectors are known in the art; see Naldini et al., (1996 and 1998); Zufferey et al., (1997); Dull et al., 199 8, U.S. Patent Nos. 6,013,516; and 5,994,136). In some embodiments, these viral vectors are plasmid-based or viral-based and contain sequences essential for the incorporation, selection, and introduction of foreign nucleic acids into host cells. configured to maintain. Known lentiviruses are American Type C ulture Collection (“ATCC”; 10801 Universit y Blvd., Manassas, Va. 20110 - 2209) or other depository institutions or can be easily obtained from collections, or can be isolated from known sources of supply using generally available techniques.

[0348] In a specific embodiment, the lentiviral vector is prepared using a plasmid encoding the gag, pol, tat, and rev genes cloned from the human immunodeficiency virus (HIV) and a second plasmid encoding an envelope protein derived from vesicular stomatitis virus (VSV - G) used for pseudotyped virus particles. Transfer vectors such as the pCDH - EF1 - MCS vector can be used with appropriate promoters and coding sequences as needed. Then, all three plasmids are transfected into lentiviral cells (such as Lenti - X - 293T cells), and then the lentivirus can be collected, concentrated, and screened after an appropriate incubation time. Thus, a method for producing a recombinant lentiviral vector containing an exogenous sequence for the purposes described herein or an engineered nuclease of the present invention is provided herein.

[0349] Characterization of Meganucleases that Recognize and Cleave Recognition Sequences of the Target 5' Intron of the T Cell Receptor Alpha Gene Embodiments of the present invention include the engineered nucleases described herein, particularly engineered meganucleases, and variants thereof. Further embodiments of the present invention include an isolated polynucleotide containing a nucleic acid sequence encoding the engineered meganucleases described herein. ​​​​​​​​It includes nucleotides and variants of such polynucleotides.

[0350] As used herein, "variant" is intended to mean a substantially similar sequence. A "variant" polypeptide is intended to mean a polypeptide derived from a "native" polypeptide by deletion or addition of one or more amino acids at one or more internal sites of the native protein, and / or substitution of one or more amino acids at one or more sites on one or more native polypeptides. As used herein, a "native" polynucleotide or polypeptide includes the parental sequence from which the variant is derived. Variant polypeptides included in embodiments are biologically active. That is, they continue to retain the ability to recognize and cleave recognition sequences found in the target 5' intron of the human T cell receptor alpha gene, including, for example, the TRC11-12 recognition sequence (SEQ ID NO: 4), the TRC15-16 recognition sequence (SEQ ID NO: 6), the TRC17-18 recognition sequence (SEQ ID NO: 8), and the TRC19-20 recognition sequence (SEQ ID NO: 10). Such variants may be due to, for example, human manipulation. Biologically active variants of the native polypeptides of embodiments (e.g., SEQ ID NOs: 12-27), or biologically active variants of the recognition half-site binding subunits described herein (e.g., SEQ ID NOs: 28-59), are at least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% identical to the amino acid sequence of the native polypeptide or native subunit. As used herein, "variant" is intended to mean a substantially similar sequence. A "variant" polypeptide is intended to mean a polypeptide derived from a "native" polypeptide by deletion or addition of one or more amino acids at one or more internal sites of the native protein, and / or substitution of one or more amino acids at one or more sites on one or more native polypeptides. As used herein, a "native" polynucleotide or polypeptide includes the parental sequence from which the variant is derived. Variant polypeptides included in embodiments are biologically active. That is, they continue to retain the ability to recognize and cleave recognition sequences found in the target 5' intron of the human T cell receptor alpha gene, including, for example, the TRC11-12 recognition sequence (SEQ ID NO: 4), the TRC15-16 recognition sequence (SEQ ID NO: 6), the TRC17-18 recognition sequence (SEQ ID NO: 8), and the TRC19-20 recognition sequence (SEQ ID NO: 10). Such variants may be due to, for example, human manipulation. Biologically active variants of the native polypeptides of embodiments (e.g., SEQ ID NOs: 12-27), or biologically active variants of the recognition half-site binding subunits described herein (e.g., SEQ ID NOs: 28-59), are at least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% identical to the amino acid sequence of the native polypeptide or native subunit. As used herein, "variant" is intended to mean a substantially similar sequence. A "variant" polypeptide is intended to mean a polypeptide derived from a "native" polypeptide by deletion or addition of one or more amino acids at one or more internal sites of the native protein, and / or substitution of one or more amino acids at one or more sites on one or more native polypeptides. As used herein, a "native" polynucleotide or polypeptide includes the parental sequence from which the variant is derived. Variant polypeptides included in embodiments are biologically active. That is, they continue to retain the ability to recognize and cleave recognition sequences found in the target 5' intron of the human T cell receptor alpha gene, including, for example, the TRC11-12 recognition sequence (SEQ ID NO: 4), the TRC15-16 recognition sequence (SEQ ID NO: 6), the TRC17-18 recognition sequence (SEQ ID NO: 8), and the TRC19-20 recognition sequence (SEQ ID NO: 10). Such variants may be due to, for example, human manipulation. Biologically active variants of the native polypeptides of embodiments (e.g., SEQ ID NOs: 12-27), or biologically active variants of the recognition half-site binding subunits described herein (e.g., SEQ ID NOs: 28-59), are at least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% identical to the amino acid sequence of the native polypeptide or native subunit. As used herein, "variant" is intended to mean a substantially similar sequence. A "variant" polypeptide is intended to mean a polypeptide derived from a "native" polypeptide by deletion or addition of one or more amino acids at one or more internal sites of the native protein, and / or substitution of one or more amino acids at one or more sites on one or more native polypeptides. As used herein, a "native" polynucleotide or polypeptide includes the parental sequence from which the variant is derived. Variant polypeptides included in embodiments are biologically active. That is, they continue to retain the ability to recognize and cleave recognition sequences found in the target 5' intron of the human T cell receptor alpha gene, including, for example, the TRC11-12 recognition sequence (SEQ ID NO: 4), the TRC15-16 recognition sequence (SEQ ID NO: 6), the TRC17-18 recognition sequence (SEQ ID NO: 8), and the TRC19-20 recognition sequence (SEQ ID NO: 10). Such variants may be due to, for example, human manipulation. Biologically active variants of the native polypeptides of embodiments (e.g., SEQ ID NOs: 12-27), or biologically active variants of the recognition half-site binding subunits described herein (e.g., SEQ ID NOs: 28-59), are at least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% identical to the amino acid sequence of the native polypeptide or native subunit. As used herein, "variant" is intended to mean a substantially similar sequence. A "variant" polypeptide is intended to mean a polypeptide derived from a "native" polypeptide by deletion or addition of one or more amino acids at one or more internal sites of the native protein, and / or substitution of one or more amino acids at one or more sites on one or more native polypeptides. As used herein, a "native" polynucleotide or polypeptide includes the parental sequence from which the variant is derived. Variant polypeptides included in embodiments are biologically active. That is, they continue to retain the ability to recognize and cleave recognition sequences found in the target 5' intron of the human T cell receptor alpha gene, including, for example, the TRC11-12 recognition sequence (SEQ ID NO: 4), the TRC15-16 recognition sequence (SEQ ID NO: 6), the TRC17-18 recognition sequence (SEQ ID NO: 8), and the TRC19-20 recognition sequence (SEQ ID NO: 10). Such variants may be due to, for example, human manipulation. Biologically active variants of the native polypeptides of embodiments (e.g., SEQ ID NOs: 12-27), or biologically active variants of the recognition half-site binding subunits described herein (e.g., SEQ ID NOs: 28-59), are at least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% identical to the amino acid sequence of the native polypeptide or native subunit. As used herein, "variant" is intended to mean a substantially similar sequence. A "variant" polypeptide is intended to mean a polypeptide derived from a "native" polypeptide by deletion or addition of one or more amino acids at one or more internal sites of the native protein, and / or substitution of one or more amino acids at one or more sites on one or more native polypeptides. As used herein, a "native" polynucleotide or polypeptide includes the parental sequence from which the variant is derived. Variant polypeptides included in embodiments are biologically active. That is, they continue to retain the ability to recognize and cleave recognition sequences found in the target 5' intron of the human T cell receptor alpha gene, including, for example, the TRC11-12 recognition sequence (SEQ ID NO: 4), the TRC15-16 recognition sequence (SEQ ID NO: 6), the TRC17-18 recognition sequence (SEQ ID NO: 8), and the TRC19-20 recognition sequence (SEQ ID NO: 10). Such variants may be due to, for example, human manipulation. Biologically active variants of the native polypeptides of embodiments (e.g., SEQ ID NOs: 12-27), or biologically active variants of the recognition half-site binding subunits described herein (e.g., SEQ ID NOs: 28-59), are at least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% identical to the amino acid sequence of the native polypeptide or native subunit. As used herein, "variant" is intended to mean a substantially similar sequence. A "variant" polypeptide is intended to mean a polypeptide derived from a "native" polypeptide by deletion or addition of one or more amino acids at one or more internal sites of the native protein, and / or substitution of one or more amino acids at one or more sites on one or more native polypeptides. As used herein, a "native" polynucleotide or polypeptide includes the parental sequence from which the variant is derived. Variant polypeptides included in embodiments are biologically active. That is, they continue to retain the ability to recognize and cleave recognition sequences found in the target 5' intron of the human T cell receptor alpha gene, including, for example, the TRC11-12 recognition sequence (SEQ ID NO: 4), the TRC15-16 recognition sequence (SEQ ID NO: 6), the TRC17-18 recognition sequence (SEQ ID NO: 8), and the TRC19-20 recognition sequence (SEQ ID NO: 10). Such variants may be due to, for example, human manipulation. Biologically active variants of the native polypeptides of embodiments (e.g., SEQ ID NOs: 12-27), or biologically active variants of the recognition half-site binding subunits described herein (e.g., SEQ ID NOs: 28-59), are at least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% identical to the amino acid sequence of the native polypeptide or native subunit. As used herein, "variant" is intended to mean a substantially similar sequence. A "variant" polypeptide is intended to mean a polypeptide derived from a "native" polypeptide by deletion or addition of one or more amino acids at one or more internal sites of the native protein, and / or substitution of one or more amino acids at one or more sites on one or more native polypeptides. As used herein, a "native" polynucleotide or polypeptide includes the parental sequence from which the variant is derived. Variant polypeptides included in embodiments are biologically active. That is, they continue to retain the ability to recognize and cleave recognition sequences found in the target 5' intron of the human T cell receptor alpha gene, including, for example, the TRC11-12 recognition sequence (SEQ ID NO: 4), the TRC15-16 recognition sequence (SEQ ID NO: 6), the TRC17-18 recognition sequence (SEQ ID NO: 8), and the TRC19-20 recognition sequence (SEQ ID NO: 10). Such variants may be due to, for example, human manipulation. Biologically active variants of the native polypeptides of embodiments (e.g., SEQ ID NOs: 12-27), or biologically active variants of the recognition half-site binding subunits described herein (e.g., SEQ ID NOs: 28-59), are at least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% identical to the amino acid sequence of the native polypeptide or native subunit. As used herein, "variant" is intended to mean a substantially similar sequence. have sequence identity determined by a 7%, about 98%, or about 99% sequence alignment program and the parameters described elsewhere in this specification. The biologically active variants of the polypeptides or subunits of the embodiments may differ from the polypeptide or subunit by only about 1 to 40 amino acid residues, only about 1 to 20, only about 1 to 10, only about 5, only 4, 3, 2, or 1 amino acid residue. .

[0351] The polypeptides of the embodiments can be modified by various methods including amino acid substitutions, deletions, truncations, and insertions. Methods of such manipulation are generally known in the art. For example, variants of amino acid sequences can be prepared by DNA mutagenesis. Methods of mutagenesis and polynucleotide modification are well known in the art. For example, Kunkel (1 985) Proc. Natl. Acad. Sci. USA 82:488-492; Ku nkel et al. (1987) Methods in Enzymol. 154:367-38 2; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds . (1983) Techniques in Molecular Biology (M acMillan Publishing Company, New York), and are cited. Guidance regarding appropriate amino acid substitutions that do not affect the biological activity of the protein of interest is incorporated herein by reference in its entirety from Dayho ff et al. (1978) Atlas of Protein Sequence and S tructure (Natl. Biomed. Res. Found., Washingt on, D.C.). on, D.C.). may be found in models of (Washington, D.C.). Conservative substitutions such as replacing one amino acid with another amino acid having similar properties may be optimal.

[0352] A number of amino acid modifications to the DNA recognition domain of wild-type I-CreI meganuclease have been previously identified (e.g., U.S. Patent No. 8,021,867), and individually or in combination, the resulting rationally designed meganucleases have different half-site specificities from the wild-type enzyme, resulting in engineered meganucleases with altered specificities at individual bases within the half-sites of the DNA recognition sequence. Table 5 shows potential substitutions made in recombinant meganuclease monomers or subunits to enhance specificity based on the bases present at each half-site position (-1 to -9) of the recognition half-site.

[0353] [Table 5]

[0354] Entries in bold are wild-type contact residues and do not constitute "modifications" as used herein. An asterisk indicates that the residue contacts a base on the antisense strand.

[0355] In the case of polynucleotides, "variant" includes deletions and / or additions of one or more nucleotides at one or more sites within a native polynucleotide. One of ordinary skill in the art will recognize that variants of the nucleic acids of the embodiments are constructed such that the open reading frame is maintained. In the case of polynucleotides, conservative variants include sequences that encode one amino acid sequence of the polypeptide of the embodiment due to the degeneracy of the genetic code. Variant polynucleotides ​​​​​​​​​​Nucleotides include, for example, those generated by using site-directed mutagenesis, but are induced by synthesis such as those still encoding the recombinant meganucleases of the embodiments including polynucleotides. Generally, variants of a particular polynucleotide of an embodiment have sequence identity determined by an alignment program and other parameters described elsewhere herein to that particular polynucleotide of at least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more. Variants of a particular polynucleotide of an embodiment (i.e., the reference polynucleotide) can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by the variant polynucleotide and the polypeptide encoded by the reference polynucleotide. Deletions, insertions, and substitutions of the protein sequences encompassed herein are not expected to result in a fundamental change in the properties of the polypeptide. However, when it is difficult to predict the exact effect before making a substitution, deletion, or insertion, one of ordinary skill in the art will understand that the effect can be evaluated by screening the polypeptide for its ability to preferentially recognize and cleave recognition sequences found within the target 5' intron of the human T cell receptor alpha gene.

[0356] EXAMPLES

[0357] The present invention is further illustrated by the following examples, which are not to be construed as limiting. ​​​​​​​​​​​should not. One of ordinary skill in the art can recognize or confirm numerous equivalents to the specific substances and procedures described herein using only routine experimentation. Such equivalents are intended to be included in the claims that follow the examples below. The engineered meganucleases (SEQ ID NOs: 12 - 15), collectively referred to herein as "TRC11 - 12 meganucleases", were engineered to recognize and cleave the TRC11 - 12 recognition sequence (SEQ ID NO: 4) present in the target 5' intron of the human T - cell receptor alpha gene. Each TRC11 - 12 recombinant meganuclease comprises an N - terminal nuclear localization signal derived from SV40, a first meganuclease subunit, a linker sequence, and a second meganuclease subunit. The first subunit of each TRC11 - 12 meganuclease binds to the TRC12 recognition half - site of SEQ ID NO: 4, and the second subunit binds to the TRC11 recognition half - site (see Figure 2). The TRC12 - binding subunit and the TRC11 - binding subunit each contain a 56 - base pair hypervariable region designated as HVR1 and HVR2, respectively. The TRC12 - binding subunit is highly conserved outside the HVR1 region. Similarly, the TRC11 - binding subunit is highly conserved outside the HVR2 region. The TRC11 - binding regions of SEQ ID NOs: 12 - 15 are provided as SEQ ID NOs: 28 - 31, respectively. SEQ ID NOs: 28 - 31 are each meganuclease

[0358] Example 1 1. Meganucleases that Recognize and Cleave the TRC11-12 Recognition Sequence 2. Meganucleases that Recognize and Cleave the TRC15-16 Recognition Sequence 3. Meganucleases that Recognize and Cleave the TRC17-18 Recognition Sequence The engineered meganucleases (SEQ ID NOs: 12 - 15), collectively referred to herein as "TRC11 - 12 meganucleases", were engineered to recognize and cleave the TRC11 - 12 recognition sequence (SEQ ID NO: 4) present in the target 5' intron of the human T - cell receptor alpha gene. Each TRC11 - 12 recombinant meganuclease comprises an N - terminal nuclear localization signal derived from SV40, a first meganuclease subunit, a linker sequence, and a second meganuclease subunit. The first subunit of each TRC11 - 12 meganuclease binds to the TRC12 recognition half - site of SEQ ID NO: 4, and the second subunit binds to the TRC11 recognition half - site (see Figure 2). The engineered meganucleases (SEQ ID NOs: 12 - 15), collectively referred to herein as "TRC11 - 12 meganucleases", were engineered to recognize and cleave the TRC11 - 12 recognition sequence (SEQ ID NO: 4) present in the target 5' intron of the human T - cell receptor alpha gene. Each TRC11 - 12 recombinant meganuclease comprises an N - terminal nuclear localization signal derived from SV40, a first meganuclease subunit, a linker sequence, and a second meganuclease subunit. The first subunit of each TRC11 - 12 meganuclease binds to the TRC12 recognition half - site of SEQ ID NO: 4, and the second subunit binds to the TRC11 recognition half - site (see Figure 2). The engineered meganucleases (SEQ ID NOs: 12 - 15), collectively referred to herein as "TRC11 - 12 meganucleases", were engineered to recognize and cleave the TRC11 - 12 recognition sequence (SEQ ID NO: 4) present in the target 5' intron of the human T - cell receptor alpha gene. Each TRC11 - 12 recombinant meganuclease comprises an N - terminal nuclear localization signal derived from SV40, a first meganuclease subunit, a linker sequence, and a second meganuclease subunit. The first subunit of each TRC11 - 12 meganuclease binds to the TRC12 recognition half - site of SEQ ID NO: 4, and the second subunit binds to the TRC11 recognition half - site (see Figure 2). The engineered meganucleases (SEQ ID NOs: 12 - 15), collectively referred to herein as "TRC11 - 12 meganucleases", were engineered to recognize and cleave the TRC11 - 12 recognition sequence (SEQ ID NO: 4) present in the target 5' intron of the human T - cell receptor alpha gene. Each TRC11 - 12 recombinant meganuclease comprises an N - terminal nuclear localization signal derived from SV40, a first meganuclease subunit, a linker sequence, and a second meganuclease subunit. The first subunit of each TRC11 - 12 meganuclease binds to the TRC12 recognition half - site of SEQ ID NO: 4, and the second subunit binds to the TRC11 recognition half - site (see Figure 2). The engineered meganucleases (SEQ ID NOs: 12 - 15), collectively referred to herein as "TRC11 - 12 meganucleases", were engineered to recognize and cleave the TRC11 - 12 recognition sequence (SEQ ID NO: 4) present in the target 5' intron of the human T - cell receptor alpha gene. Each TRC11 - 12 recombinant meganuclease comprises an N - terminal nuclear localization signal derived from SV40, a first meganuclease subunit, a linker sequence, and a second meganuclease subunit. The first subunit of each TRC11 - 12 meganuclease binds to the TRC12 recognition half - site of SEQ ID NO: 4, and the second subunit binds to the TRC11 recognition half - site (see Figure 2). The engineered meganucleases (SEQ ID NOs: 12 - 15), collectively referred to herein as "TRC11 - 12 meganucleases", were engineered to recognize and cleave the TRC11 - 12 recognition sequence (SEQ ID NO: 4) present in the target 5' intron of the human T - cell receptor alpha gene. Each TRC11 - 12 recombinant meganuclease comprises an N - terminal nuclear localization signal derived from SV40, a first meganuclease subunit, a linker sequence, and a second meganuclease subunit. The first subunit of each TRC11 - 12 meganuclease binds to the TRC12 recognition half - site of SEQ ID NO: 4, and the second subunit binds to the TRC11 recognition half - site (see Figure 2). The engineered meganucleases (SEQ ID NOs: 12 - 15), collectively referred to herein as "TRC11 - 12 meganucleases", were engineered to recognize and cleave the TRC11 - 12 recognition sequence (SEQ ID NO: 4) present in the target 5' intron of the human T - cell receptor alpha gene. Each TRC11 - 12 recombinant meganuclease comprises an N - terminal nuclear localization signal derived from SV40, a first meganuclease subunit, a linker sequence, and a second meganuclease subunit. The first subunit of each TRC11 - 12 meganuclease binds to the TRC12 recognition half - site of SEQ ID NO: 4, and the second subunit binds to the TRC11 recognition half - site (see Figure 2). The engineered meganucleases (SEQ ID NOs: 12 - 15), collectively referred to herein as "TRC11 - 12 meganucleases", were engineered to recognize and cleave the TRC11 - 12 recognition sequence (SEQ ID NO: 4) present in the target 5' intron of the human T - cell receptor alpha gene. Each TRC11 - 12 recombinant meganuclease comprises an N - terminal nuclear localization signal derived from SV40, a first meganuclease subunit, a linker sequence, and a second meganuclease subunit. The first subunit of each TRC11 - 12 meganuclease binds to the TRC12 recognition half - site of SEQ ID NO: 4, and the second subunit binds to the TRC11 recognition half - site (see Figure 2).

[0359] The TRC12 - binding subunit and the TRC11 - binding subunit each contain a 56 - base pair hypervariable region designated as HVR1 and HVR2, respectively. The TRC12 - binding subunit is highly conserved outside the HVR1 region. Similarly, the TRC11 - binding subunit is highly conserved outside the HVR2 region. The TRC11 - binding regions of SEQ ID NOs: 12 - 15 are provided as SEQ ID NOs: 28 - 31, respectively. SEQ ID NOs: 28 - 31 are each meganuclease The TRC12 - binding subunit and the TRC11 - binding subunit each contain a 56 - base pair hypervariable region designated as HVR1 and HVR2, respectively. The TRC12 - binding subunit is highly conserved outside the HVR1 region. Similarly, the TRC11 - binding subunit is highly conserved outside the HVR2 region. The TRC11 - binding regions of SEQ ID NOs: 12 - 15 are provided as SEQ ID NOs: 28 - 31, respectively. SEQ ID NOs: 28 - 31 are each meganuclease The TRC12 - binding subunit and the TRC11 - binding subunit each contain a 56 - base pair hypervariable region designated as HVR1 and HVR2, respectively. The TRC12 - binding subunit is highly conserved outside the HVR1 region. Similarly, the TRC11 - binding subunit is highly conserved outside the HVR2 region. The TRC11 - binding regions of SEQ ID NOs: 12 - 15 are provided as SEQ ID NOs: 28 - 31, respectively. SEQ ID NOs: 28 - 31 are each meganuclease The TRC12 - binding subunit and the TRC11 - binding subunit each contain a 56 - base pair hypervariable region designated as HVR1 and HVR2, respectively. The TRC12 - binding subunit is highly conserved outside the HVR1 region. Similarly, the TRC11 - binding subunit is highly conserved outside the HVR2 region. The TRC11 - binding regions of SEQ ID NOs: 12 - 15 are provided as SEQ ID NOs: 28 - 31, respectively. SEQ ID NOs: 28 - 31 are each meganuclease The TRC12 - binding subunit and the TRC11 - binding subunit each contain a 56 - base pair hypervariable region designated as HVR1 and HVR2, respectively. The TRC12 - binding subunit is highly conserved outside the HVR1 region. Similarly, the TRC11 - binding subunit is highly conserved outside the HVR2 region. The TRC11 - binding regions of SEQ ID NOs: 12 - 15 are provided as SEQ ID NOs: 28 - 31, respectively. SEQ ID NOs: 28 - 31 are each meganuclease SEQ ID NO: 28, which is the TRC11 binding region of meganuclease TRC11-12x.4 (SEQ ID NO: 12) shares at least 90% sequence identity with the TRC12 binding regions of SEQ ID NOs: 12-15. are provided as SEQ ID NOs: 32-35, respectively. SEQ ID NOs: 32-35 are each the SEQ ID NO: 32, which is the TRC12 binding region of meganuclease TRC11-12x.4 (SEQ ID NO: 12), and shares at least 90% sequence identity with it.

[0360] 4. Meganucleases that Recognize and Cleave the TRC19-20 Recognition Sequence The engineered meganucleases collectively referred to herein as "TRC15-16 meganucleases" (SEQ ID NOs: 16-19) are engineered to recognize and cleave the TRC15-16 recognition sequence (SEQ ID NO: 6) present in the target 5' intron of the human T cell receptor alpha gene. Each TRC15-16 recombinant meganuclease comp...

Claims

Claim 1 A meganuclease engineered to recognize and cleave a recognition sequence within an intron of the human T cell receptor alpha gene located 5' upstream of TRAC exon 1, wherein the engineered meganuclease comprises a first subunit and a second subunit, the first subunit binds to a first recognition half-site of the recognition sequence and comprises a first hypervariable (HVR1) region, and the second subunit binds to a second recognition half-site of the recognition sequence and comprises a second hypervariable (HVR2) region. A meganuclease according to claim 1, wherein the intron comprises SEQ ID NO: 3, and the engineered meganuclease has no recognition sequence within an endogenous splice donor site or an endogenous splice acceptor site adjacent to the intron. A meganuclease according to claim 1 or 2, wherein the recognition sequence comprises SEQ ID NO:

4. A meganuclease according to claim 3, wherein the HVR1 region comprises an amino acid sequence having at least 80% sequence identity with an amino acid sequence corresponding to residues 215-270 of any one of SEQ ID NOs: 12-15. A meganuclease according to claim 3 or 4, wherein the HVR1 region comprises residues corresponding to residues 215, 217, 211, 266, and 268 of any one of SEQ ID NOs: 12-15. A meganuclease according to claim 3-5, wherein the HVR1 region comprises residues 215-270 of any one of SEQ ID NOs: 12-15. A meganuclease according to any one of claims 3-6, wherein the HVR2 region comprises an amino acid sequence having at least 80% sequence identity with an amino acid sequence corresponding to residues 24-79 of any one of SEQ ID NOs: 12-15. A meganuclease according to any one of claims 3-7, wherein the HVR2 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of any one of SEQ ID NOs: 12-15. A meganuclease according to any one of claims 3-8, wherein the HVR2 region comprises residues 24-79 of any one of SEQ ID NOs: 12-15. ９、２２１、２２３、２２４、２２９、２３１、２３３、２３５、２３７、２５９、２６ Claim 10 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ The first subunit comprises an amino acid sequence having at least 80% sequence identity to any one of residues 198 to 344 of SEQ ID NOs: 12 to 15, and the second sub unit comprises an amino acid sequence having at least 80% sequence identity to any one of residues 7 to 153 of SEQ ID NOs: 12 to 15, the engineered meganuclease according to any one of claims 3 to 9 %.

11. The engineered meganuclease according to any one of claims 3 to 10, wherein the first subunit comprises any one of residues 198 to 344 of SEQ ID NOs: 12 to 15 .

12. The engineered meganuclease according to any one of claims 3 to 11, wherein the second subunit comprises any one of residues 7 to 153 of SEQ ID NOs: 12 to 15 .

13. The engineered meganuclease according to any one of claims 3 to 12, wherein the engineered meganuclease comprises a linker that covalently links the first sub unit and the second subunit .

14. The engineered meganuclease according to any one of claims 3 to 13, wherein the engineered meganuclease comprises an amino acid sequence of any one of SEQ ID NOs: 12 to 15 .

15. The engineered meganuclease according to claim 1 or 2, wherein the recognition sequence comprises SEQ ID NO: 6 .

16. The engineered meganuclease according to claim 15, wherein the HVR1 region comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence corresponding to any one of residues 24 to 79 of SEQ ID NOs: 16 to 19 .

17. The engineered meganuclease according to claim 15 or 16, wherein the HVR1 region comprises residues corresponding to any one of residues 24, 26, 28, 3 0, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NOs: 16 to 19 .

18. The engineered meganuclease according to any one of claims 15 to 17, wherein the HVR1 region comprises a residue corresponding to residue 64 of any one of SEQ ID NOs: 16 to 19 .

19. The engineered meganuclease according to any one of claims 15 to 18, wherein the HVR1 region comprises any one of residues 24 to 79 of SEQ ID NOs: 16 to 19 .

20. The engineered meganuclease according to any one of claims 15 to 18, wherein the HVR2 region corresponds to any one of residues 215 to 270 of SEQ ID NOs: 16 to 19 ​ ​ ​ An amino acid sequence having at least 80% sequence identity with the amino acid sequence to be used, comprising the engineered meganuclease according to any one of claims 15 to 19. **Claim 21** The engineered meganuclease according to any one of claims 15 to 20, wherein the HVR2 region comprises residues corresponding to residues 215, 217, 21 ９、２２１、２２３、２２４、２２９、２３１、２３３、２３５、２３７、２５９、２６ 1, 266, and 268 of any one of SEQ ID NOs: 16 to 19. The engineered meganuclease described above. **Claim 22** The engineered meganuclease according to any one of claims 15 to 21, wherein the HVR2 region comprises residues 215 to 270 of any one of SEQ ID NOs: 16 to 19. The engineered meganuclease described above. **Claim 23** The engineered meganuclease according to any one of claims 15 to 22, wherein the first subunit comprises an amino acid sequence having at least 80% sequence identity with residues 7 to 153 of any one of SEQ ID NOs: 16 to 19, and the second subunit The engineered meganuclease according to any one of claims 15 to 22, wherein the first subunit comprises an amino acid sequence having at least 80% sequence identity with residues 7 to 153 of any one of SEQ ID NOs: 16 to 19, and the second subunit The engineered meganuclease according to any one of claims 15 to 22, wherein the first subunit comprises an amino acid sequence having at least 80% sequence identity with residues 7 to 153 of any one of SEQ ID NOs: 16 to 19, and the second subunit The engineered meganuclease according to any one of claims 15 to 22, wherein the first subunit comprises an amino acid sequence having at least 80% sequence identity with residues 7 to 153 of any one of SEQ ID NOs: 16 to 19, and the second subunit The engineered meganuclease described above. **Claim 24** The engineered meganuclease according to any one of claims 15 to 23, wherein the first subunit comprises residues 7 to 153 of any one of SEQ ID NOs: 16 to 19. The engineered meganuclease described above. **Claim 25** The engineered meganuclease according to any one of claims 15 to 24, wherein the second subunit comprises residues 198 to 344 of any one of SEQ ID NOs: 16 to 19. The engineered meganuclease described above. **Claim 26** The engineered meganuclease according to any one of claims 15 to 25, wherein the engineered meganuclease comprises a linker that covalently links the first subunit The engineered meganuclease according to any one of claims 15 to 25, wherein the engineered meganuclease comprises a linker that covalently links the first subunit The engineered meganuclease described above. **Claim 27** The engineered meganuclease according to any one of claims 15 to 26, wherein the engineered meganuclease comprises an amino acid sequence of any one of SEQ ID NOs: 16 to 19. The engineered meganuclease described above. **Claim 28** The engineered meganuclease according to claim 1 or 2, wherein the recognition sequence comprises SEQ ID NO:

8. The engineered meganuclease described above. **Claim 29** The engineered meganuclease according to claim 28, wherein the HVR1 region comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence corresponding to residues 24 to 79 of any one of SEQ ID NOs: 20 to 23. The engineered meganuclease described above. The engineered meganuclease described above. **Claim 30** The engineered meganuclease according to claim 28, wherein the HVR1 region comprises residues 24, 26, 28, 3 of any one of SEQ ID NOs: 20 to 23. The engineered meganuclease according to claim 28 or 29, comprising residues corresponding to 0, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77. The engineered meganuclease according to claim 28 or 29, comprising residues corresponding to 0, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77. **Claim 31** The engineered meganuclease according to any one of claims 28 to 30, wherein the HVR1 region comprises a residue corresponding to residue 66 of any one of SEQ ID NOs: 20 to 23. The engineered meganuclease according to any one of claims 28 to 30, wherein the HVR1 region comprises a residue corresponding to residue 66 of any one of SEQ ID NOs: 20 to 23. **Claim 32** The engineered meganuclease according to any one of claims 28 to 31, wherein the HVR1 region comprises residues 24 to 79 of any one of SEQ ID NOs: 20 to 23. The engineered meganuclease according to any one of claims 28 to 31, wherein the HVR1 region comprises residues 24 to 79 of any one of SEQ ID NOs: 20 to 23. **Claim 33** The engineered meganuclease according to any one of claims 28 to 32, wherein the HVR2 region comprises an amino acid sequence having at least 80% sequence identity with an amino acid sequence corresponding to residues 215 to 270 of any one of SEQ ID NOs: 20 to 23. The engineered meganuclease according to any one of claims 28 to 32, wherein the HVR2 region comprises an amino acid sequence having at least 80% sequence identity with an amino acid sequence corresponding to residues 215 to 270 of any one of SEQ ID NOs: 20 to 23. The engineered meganuclease according to any one of claims 28 to 32, wherein the HVR2 region comprises an amino acid sequence having at least 80% sequence identity with an amino acid sequence corresponding to residues 215 to 270 of any one of SEQ ID NOs: 20 to 23. **Claim 34** The engineered meganuclease according to any one of claims 28 to 33, wherein the HVR2 region comprises residues corresponding to residues 215, 217, 211, 266, and 268 of any one of SEQ ID NOs: 20 to 23. ９、２２１、２２３、２２４、２２９、２３１、２３３、２３５、２３７、２５９、２６ The engineered meganuclease according to any one of claims 28 to 33, wherein the HVR2 region comprises residues corresponding to residues 215, 217, 211, 266, and 268 of any one of SEQ ID NOs: 20 to 23. The engineered meganuclease according to any one of claims 28 to 33, wherein the HVR2 region comprises residues corresponding to residues 215, 217, 211, 266, and 268 of any one of SEQ ID NOs: 20 to 23. **Claim 35** The engineered meganuclease according to any one of claims 28 to 34, wherein the HVR2 region comprises residues 215 to 270 of any one of SEQ ID NOs: 20 to 23. The engineered meganuclease according to any one of claims 28 to 34, wherein the HVR2 region comprises residues 215 to 270 of any one of SEQ ID NOs: 20 to 23. **Claim 36** The engineered meganuclease according to any one of claims 28 to 35, wherein the first subunit comprises an amino acid sequence having at least 80% sequence identity with respect to residues 7 to 153 of any one of SEQ ID NOs: 20 to 23, and the second subunit comprises an amino acid sequence having at least 80% sequence identity with respect to residues 198 to 344 of any one of SEQ ID NOs: 20 to 23. The engineered meganuclease according to any one of claims 28 to 35, wherein the first subunit comprises an amino acid sequence having at least 80% sequence identity with respect to residues 7 to 153 of any one of SEQ ID NOs: 20 to 23, and the second subunit comprises an amino acid sequence having at least 80% sequence identity with respect to residues 198 to 344 of any one of SEQ ID NOs: 20 to 23. The engineered meganuclease according to any one of claims 28 to 35, wherein the first subunit comprises an amino acid sequence having at least 80% sequence identity with respect to residues 7 to 153 of any one of SEQ ID NOs: 20 to 23, and the second subunit comprises an amino acid sequence having at least 80% sequence identity with respect to residues 198 to 344 of any one of SEQ ID NOs: 20 to 23. The engineered meganuclease according to any one of claims 28 to 35, wherein the first subunit comprises an amino acid sequence having at least 80% sequence identity with respect to residues 7 to 153 of any one of SEQ ID NOs: 20 to 23, and the second subunit comprises an amino acid sequence having at least 80% sequence identity with respect to residues 198 to 344 of any one of SEQ ID NOs: 20 to 23. The engineered meganuclease according to any one of claims 28 to 35, wherein the first subunit comprises an amino acid sequence having at least 80% sequence identity with respect to residues 7 to 153 of any one of SEQ ID NOs: 20 to 23, and the second subunit comprises an amino acid sequence having at least 80% sequence identity with respect to residues 198 to 344 of any one of SEQ ID NOs: 20 to 23. **Claim 37** The engineered meganuclease according to any one of claims 28 to 36, wherein the first subunit comprises residues 7 to 153 of any one of SEQ ID NOs: 20 to 23. The engineered meganuclease according to any one of claims 28 to 36, wherein the first subunit comprises residues 7 to 153 of any one of SEQ ID NOs: 20 to 23. **Claim 38** The engineered meganuclease according to any one of claims 28 to 37, wherein the second subunit comprises residues 198 to 344 of any one of SEQ ID NOs: 20 to 23. The engineered meganuclease according to any one of claims 28 to 37, wherein the second subunit comprises residues 198 to 344 of any one of SEQ ID NOs: 20 to 23. **Claim 39** The engineered meganuclease according to any one of claims 28 to 38, wherein the engineered meganuclease comprises a linker, and the linker covalently links the first subunit and the second subunit. The engineered meganuclease according to any one of claims 28 to 38, wherein the engineered meganuclease comprises a linker, and the linker covalently links the first subunit and the second subunit. The engineered meganuclease according to any one of claims 28 to 38, wherein the engineered meganuclease comprises a linker, and the linker covalently links the first subunit and the second subunit. **Claim 40** The engineered meganuclease contains an amino acid sequence of any one of SEQ ID NOs: 20 to 23 The engineered meganuclease according to any one of claims 28 to 39. **Claim 41** The engineered meganuclease according to claim 1 or 2, wherein the recognition sequence contains SEQ ID NO:

10. **Claim 42** The engineered meganuclease according to claim 41, wherein the HVR1 region contains an amino acid sequence having at least 80% sequence identity with the amino acid sequence corresponding to residues 24 to 79 of any one of SEQ ID NOs: 24 to 27. **Claim 43** The engineered meganuclease according to claim 41 or 42, wherein the HVR1 region contains residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of any one of SEQ ID NOs: 24 to 27. **Claim 44** The engineered meganuclease according to any one of claims 41 to 43, wherein the HVR1 region contains residues 24 to 79 of any one of SEQ ID NOs: 24 to 27. **Claim 45** The engineered meganuclease according to any one of claims 41 to 44, wherein the HVR2 region contains an amino acid sequence having at least 80% sequence identity with the amino acid sequence corresponding to residues 215 to 270 of any one of SEQ ID NOs: 24 to 27. **Claim 46** The engineered meganuclease according to any one of claims 41 to 45, wherein the HVR2 region contains residues corresponding to residues 215, 217, 211, 266, and 268 of any one of SEQ ID NOs: 24 to 27. **Claim 47** The engineered meganuclease according to any one of claims 41 to 46, wherein the HVR2 region contains residues 215 to 270 of any one of SEQ ID NOs: 24 to 27. **Claim 48** The engineered meganuclease according to any one of claims 41 to 47, wherein the first subunit contains an amino acid sequence having at least 80% sequence identity with respect to residues 7 to 153 of any one of SEQ ID NOs: 24 to 27, and the second subunit contains an amino acid sequence having at least 80% sequence identity with respect to residues 198 to 344 of any one of SEQ ID NOs: 24 to 27. **Claim 49** The engineered meganuclease according to any one of claims 41 to 48, wherein the first subunit contains residues 7 to 153 of any one of SEQ ID NOs: 24 to 27. **Claim 50** ​ ９、２２１、２２３、２２４、２２９、２３１、２３３、２３５、２３７、２５９、２６ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ The second subunit contains any one of residues 198 to 344 of SEQ ID NOs: 24 to 27 The engineered meganuclease according to any one of claims 41 to 49. **Claim 51** The engineered meganuclease contains a linker, and the linker covalently bonds the first subunit and the second subunit. The engineered meganuclease according to any one of claims 41 to 50 . **Claim 52** The engineered meganuclease contains an amino acid sequence of any one of SEQ ID NOs: 24 to 27 The engineered meganuclease according to any one of claims 41 to 51. **Claim 53** A polynucleotide comprising a nucleic acid sequence encoding the engineered meganuclease according to any one of claims 1 to 52 . **Claim 54** The polynucleotide according to claim 53, wherein the polynucleotide is mRNA. **Claim 55** The mRNA is a polycistronic mRNA encoding the engineered meganuclease according to any one of claims 1 to 52 and at least one additional polypeptide or nucleic acid. The polynucleotide according to claim 54 . **Claim 56** A recombinant DNA construct comprising the polynucleotide according to claim 53. **Claim 57** The recombinant DNA construct according to claim 56, wherein the recombinant DNA construct encodes a viral vector . **Claim 58** The viral vector is an adenoviral vector, a lentiviral vector, a retroviral vector , or an adeno-associated virus (AAV) vector. The recombinant DNA construct according to claim 57 . **Claim 59** The viral vector is a recombinant AAV vector. The recombinant DNA construct according to claim 57 or claim 58 . **Claim 60** A viral vector comprising the polynucleotide according to claim 53. **Claim 61** The viral vector is an adenoviral vector, a lentiviral vector, a retroviral vector , or an AAV vector. The viral vector according to claim 60 . **Claim 62** The viral vector is a recombinant AAV vector. The viral vector according to claim 60 or claim 61 . **Claim 63** A method for producing a genetically modified T cell containing an exogenous sequence of interest inserted into the chromosome of the T cell, the method comprising The first nucleic acid sequence encoding the engineered meganuclease according to any one of claims 1 to 52, wherein the engineered meganuclease is expressed in the T cell A first nucleic acid sequence; and And (b) introducing one or more nucleic acids of a second nucleic acid sequence containing the target sequence; The engineered meganuclease generates a cleavage site on the chromosome at a recognition sequence within an intron of the human T cell receptor alpha gene located upstream of the 5' of the TRAC exon 1; 、 The target sequence is inserted into the chromosome at the cleavage site; The target sequence contains an exogenous splice acceptor site and / or a poly A signal; The endogenous splice donor site and the endogenous splice acceptor site adjacent to the intron are not modified and / or remain functional, the method.

64. The method according to claim 63, wherein the cell surface expression of the endogenous T cell receptor is reduced when compared with non-modified control cells.

65. The method according to claim 63 or 64, wherein the intron contains SEQ ID NO:

3.

66. The method according to any one of claims 63 to 65, wherein (a) the recognition sequence contains SEQ ID NO: 4, and the engineered meganuclease is the engineered meganuclease according to any one of claims 3 To 14; (b) the recognition sequence contains SEQ ID NO: 6, and the engineered meganuclease is the engineered meganuclease according to any one of claims 1 5 to 27; (c) the recognition sequence contains SEQ ID NO: 8, and the engineered meganuclease is the engineered meganuclease according to any one of claims 2 8 to 40; or (d) the recognition sequence contains SEQ ID NO: 10, and the engineered meganuclease is the engineered meganuclease according to any one of claims 41 to 52, the method.

67. The method according to any one of claims 63 to 66, wherein the second nucleic acid sequence further contains a sequence homologous to the sequence adjacent to the cleavage site, and the target sequence is inserted into the cleavage site by homologous recombination.

68. The method according to any one of claims 63 to 67, wherein the T cell is a human T cell or a cell derived therefrom.

69. The target sequence, from 5' to 3', an exogenous splice acceptor site, a 2A element or ​ ​ ​ ​ ​ ​ ​ ​ Claim 6, which comprises an IRES element, a coding sequence of a target protein, and a polyA signal The method according to any one of claims 3 to 68

70. The method according to claim 69, wherein the 2A element is a T2A element, a P2A element, an E2A element, or an F2A element The method according to claim 69

71. The method according to claim 69 or 70, wherein the 2A element is a T2A element

72. The method according to any one of claims 63 to 71, wherein the target sequence comprises a coding sequence of a chimeric antigen receptor or an exogenous T cell receptor The method according to any one of claims 63 to 71

73. The method according to claim 72, wherein the chimeric antigen receptor or the exogenous T cell receptor comprises an extracellular ligand-binding domain having specificity for a tumor-specific antigen The method according to claim 72

74. The method according to any one of claims 63 to 73, wherein at least the first nucleic acid sequence is introduced into the T cell by mRNA The method according to any one of claims 63 to 73

75. The method according to any one of claims 63 to 74, wherein at least the second nucleic acid sequence is introduced into the T cell by a viral vector The method according to any one of claims 63 to 74

76. The method according to claim 75, wherein the viral vector is an adenovirus vector, a lentivirus vector, a retrovirus vector, or an AAV vector The method according to claim 75

77. The method according to claim 75 or 76, wherein the viral vector is a recombinant AAV vector The method according to claim 75 or 76

78. A method for producing a genetically modified T cell comprising an exogenous sequence of interest inserted into the chromosome of the T cell, the method comprising (a) introducing the engineered meganuclease according to any one of claims 1 to 52 into the T cell; and (b) transfecting the T cell with a nucleic acid comprising the sequence of interest, wherein The engineered meganuclease generates a cleavage site on the chromosome at a recognition sequence within an intron of the human T cell receptor alpha gene located upstream of the 5' of the TRAC exon 1; The sequence of interest is inserted into the chromosome at the cleavage site; The sequence of interest comprises an exogenous splice acceptor site and / or a polyA signal; The endogenous splice donor site and the endogenous splice acceptor site adjacent to the intron remain unmodified and / or functional, the method The sequence of interest is inserted into the chromosome at the cleavage site; The sequence of interest comprises an exogenous splice acceptor site and / or a polyA signal; The endogenous splice donor site and the endogenous splice acceptor site adjacent to the intron remain unmodified and / or functional, the method The endogenous splice donor site and the endogenous splice acceptor site adjacent to the intron remain unmodified and / or functional, the method The endogenous splice donor site and the endogenous splice acceptor site adjacent to the intron remain unmodified and / or functional, the method

79. The method according to claim 78, wherein the cell surface expression of the endogenous T cell receptor is reduced when compared to unmodified control cells The method according to claim 78

80. The method according to claim 78 or 79, wherein the intron contains SEQ ID NO:

3.

81. The method according to any one of claims 78 to 80, wherein (a) the recognition sequence contains SEQ ID NO: 4, and the engineered meganuclease is the engineered meganuclease according to any one of claims 3 to 14; (b) the recognition sequence contains SEQ ID NO: 6, and the engineered meganuclease is the engineered meganuclease according to any one of claims 1 5 to 27; (c) the recognition sequence contains SEQ ID NO: 8, and the engineered meganuclease is the engineered meganuclease according to any one of claims 2 8 to 40; or (d) the recognition sequence contains SEQ ID NO: 10, and the engineered meganuclease is the engineered meganuclease according to any one of claims 41 to 52.

82. The method according to claim 81, wherein the nucleic acid further contains a sequence homologous to the sequence adjacent to the cleavage site, and the target sequence is inserted into the cleavage site by homologous recombination.

83. The method according to claim 81 or 82, wherein the T cell is a human T cell or a cell derived therefrom.

84. The method according to any one of claims 78 to 83, wherein the target sequence contains, from 5' to 3', an exogenous splice acceptor site, a 2A element or an IRES element, a coding sequence of the target protein, and a polyA signal.

85. The method according to claim 84, wherein the 2A element is a T2A element, a P2A element, an E2A element, or an F2A element.

86. The method according to claim 84 or 85, wherein the 2A element is a T2A element.

87. The method according to any one of claims 78 to 86, wherein the target sequence contains a coding sequence of a chimeric antigen receptor or an exogenous T cell receptor.

88. The method according to claim 87, wherein the chimeric antigen receptor or the exogenous T cell receptor contains an extracellular ligand-binding domain specific for a tumor-specific antigen.

89. The method according to any one of claims 78 to 88, wherein the nucleic acid is introduced into the T cell by a viral vector.

90. The method according to claim 89, wherein the viral vector is an adenovirus vector, a lentivirus vector, a retrovirus vector, or an AAV vector.

91. The method according to claim 89 or 90, wherein the viral vector is a recombinant AAV vector. Method. **Claim 92** A method for producing a genetically modified T cell comprising a modified human T cell receptor alpha gene, wherein the method comprises: (a) introducing into the T cell: (i) a first nucleic acid sequence encoding an engineered nuclease, the engineered nuclease being a first nucleic acid sequence expressed in the T cell, or (ii) an engineered nuclease protein; and (b) introducing into the cell a second nucleic acid sequence comprising the exogenous sequence of interest; wherein the engineered nuclease generates a cleavage site at a recognition sequence within an intron of the human T cell receptor alpha gene located upstream of the 5' of the TRAC exon 1; the sequence of interest is inserted into the human T cell receptor alpha gene at the cleavage site; the sequence of interest comprises an exogenous splice acceptor site and / or a poly A signal; and the endogenous splice donor site and endogenous splice acceptor site adjacent to the intron remain unmodified and / or functional. **Claim 93** The method according to claim 92, wherein the cell surface expression of the endogenous T cell receptor is reduced when compared to non-modified control cells. **Claim 94** The method according to claim 93 or 94, wherein the intron comprises SEQ ID NO:

3. **Claim 95** The method according to any one of claims 92 to 94, wherein the second nucleic acid sequence comprises, from 5' to 3': (a) a 5' homology arm homologous to a 5' upstream sequence adjacent to the cleavage site; (b) the exogenous sequence of interest; and (c) a 3' homology arm homologous to a 3' downstream sequence adjacent to the cleavage site; wherein the exogenous sequence of interest is inserted into the human T cell receptor alpha gene at the cleavage site by homologous recombination. **Claim 96** The method according to any one of claims 92 to 95, wherein the genetically modified T cell is a genetically modified human T cell or a cell derived therefrom. **Claim 97** The method according to any one of claims 92 to 96, wherein the exogenous sequence of interest comprises, from 5' to 3', an exogenous splice acceptor site, a 2A element or an IRES element, a coding sequence of a protein of interest, and a poly A signal. **Claim 98** The method according to claim 97, wherein the 2A element is a T2A element, a P2A element, an E2A element, or an F2A element. **Claim 99** ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ The method according to claim 97 or 98, wherein the 2A element is a T2A element.

100. The method according to any one of claims 92 to 99, wherein the target sequence contains a coding sequence of a chimeric antigen receptor or an exogenous T cell receptor.

101. The method according to claim 100, wherein the chimeric antigen receptor or the exogenous T cell receptor contains an extracellular ligand-binding domain having specificity for a tumor-specific antigen.

102. The method according to any one of claims 92 to 101, wherein at least the first nucleic acid sequence is introduced into the T cell by mRNA.

103. The method according to any one of claims 92 to 102, wherein at least the second nucleic acid sequence is introduced into the T cell by a viral vector.

104. The method according to claim 103, wherein the viral vector is an adenovirus vector, a lentivirus vector, a retrovirus vector, or an adeno-associated virus (AAV) vector.

105. The method according to claim 103 or 104, wherein the viral vector is a recombinant AAV vector.

106. The method according to any one of claims 92 to 105, wherein the engineered nuclease is an engineered meganuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a compact TALEN, a CRISPR nuclease, or a megaTAL.

107. The method according to any one of claims 92 to 106, wherein the engineered nuclease is an engineered meganuclease.

108. The method according to claim 107, wherein the engineered meganuclease has specificity for a recognition sequence containing SEQ ID NO:

4.

109. The method according to claim 107 or 108, wherein the engineered meganuclease is the engineered meganuclease according to any one of claims 3 to 14.

110. The method according to claim 107, wherein the engineered meganuclease has specificity for a recognition sequence containing SEQ ID NO:

6.

111. The method according to claim 107 or 110, wherein the engineered meganuclease is the engineered meganuclease according to any one of claims 15 to 27.

112. The method according to claim 107, wherein the engineered meganuclease has specificity for a recognition sequence containing SEQ ID NO:

8. ​ ​ ​ The method according to claim 107.

113. The method according to claim 107 or 112, wherein the engineered meganuclease is the engineered meganuclease according to any one of claims 28 to 40.

114. The method according to claim 107, wherein the engineered meganuclease is specific for a recognition sequence comprising SEQ ID NO:

10.

115. The method according to claim 107 or 114, wherein the engineered meganuclease is the engineered meganuclease according to any one of claims 41 to 52.

116. A genetically modified T cell prepared by the method according to any one of claims 63 to 115.

117. A genetically modified T cell comprising a modified human T cell receptor alpha gene in its genome, wherein the modified human T cell receptor alpha gene comprises an exogenous sequence of interest inserted into an intron within the T cell receptor alpha gene located upstream of the 5' of TRAC exon 1, the exogenous sequence of interest comprises an exogenous splice acceptor site and / or a poly A signal, the endogenous splice donor site and the endogenous splice acceptor site adjacent to the intron are not modified and / or remain functional, and the cell surface expression of the endogenous T cell receptor is reduced when compared to unmodified control cells.

118. The genetically modified T cell according to claim 117, wherein the intron comprises SEQ ID NO:

3.

119. The genetically modified T cell according to claim 117 or 118, wherein the genetically modified T cell is a genetically modified human T cell or a cell derived therefrom.

120. The genetically modified T cell according to any one of claims 117 to 119, wherein the exogenous sequence of interest comprises, from 5' to 3', an exogenous splice acceptor site, a 2A element or an IRES element, a coding sequence of a protein of interest, and a poly A signal.

121. The genetically modified T cell according to claim 120, wherein the 2A element is a T2A element, a P2A element, an E2A element, or an F2A element.

122. The genetically modified T cell according to claim 120 or 121, wherein the 2A element is a T2A element. 。

123. The genetically modified T cell according to any one of claims 117 to 122, wherein the sequence of interest comprises a coding sequence of a chimeric antigen receptor or an exogenous T cell receptor.

124. The chimeric antigen receptor or the exogenous T cell receptor has specificity for a tumor-specific antigen The genetically modified T cell according to claim 123, comprising an extracellular ligand-binding domain having specificity for a tumor-specific antigen.

125. The exogenous sequence of interest is inserted into the intron at an engineered meganuclease recognition site, a TALEN recognition site , a zinc finger nuclease recognition site, a CRISPR recognition site, or a megaTAL recognition site, the genetically modified T cell according to any one of claims 117 to 124.

126. The exogenous sequence of interest is inserted into the intron at an engineered meganuclease recognition site The genetically modified T cell according to any one of claims 117 to 125, wherein the insertion is made.

127. The exogenous sequence of interest is inserted into the intron within SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10 The genetically modified T cell according to any one of claims 117 to 126, wherein the insertion is made.

128. A population of genetically modified T cells comprising a plurality of the genetically modified T cells according to any one of claims 116 to 127 .

129. At least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45 %, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 9 0%, at least 95%, or up to 100% of the cells in the population are those of the genetically modified T cells according to any one of claims 116 to 127 The population according to claim 128.

130. The genetically modified T cell is a genetically modified human T cell or a cell derived therefrom The population according to claim 128 or claim 129.

131. The sequence of interest comprises a coding sequence of a chimeric antigen receptor or an exogenous T cell receptor, the claim The population according to any one of 128 to 130.

132. The chimeric antigen receptor or the exogenous T cell receptor has specificity for a tumor-specific antigen The population according to claim 131, comprising an extracellular ligand-binding domain having the same.

133. The cell surface expression of the endogenous T cell receptor is reduced on the genetically modified T cell when compared to unmodified control cells The population according to any one of claims 128 to 132.

134. A pharmaceutical composition useful for treating a disease in a subject in need thereof, said pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of the genetically modified T cells according to any one of claims 116 to 127. A pharmaceutical composition.

135. The pharmaceutical composition according to claim 134, wherein the genetically modified T cells are genetically modified human T cells or cells derived therefrom. A pharmaceutical composition according to claim 134.

136. The pharmaceutical composition according to claim 134 or 135, wherein the target sequence comprises a coding sequence of a chimeric antigen receptor or an exogenous T cell receptor. A pharmaceutical composition according to claim 134 or 135.

137. The pharmaceutical composition according to claim 136, wherein the chimeric antigen receptor or the exogenous T cell receptor comprises an extracellular ligand-binding domain specific for a tumor-specific antigen. A pharmaceutical composition according to claim 136.

138. The pharmaceutical composition according to any one of claims 134 to 137, wherein the cell surface expression of the endogenous T cell receptor is reduced on the genetically modified T cells when compared to unmodified control cells. A pharmaceutical composition according to any one of claims 134 to 137. 。

139. A method of treating a disease in a subject in need thereof, comprising administering to the subject the genetically modified T cells according to any one of claims 116 to 127. A method of treating a disease in a subject in need thereof.

140. The method according to claim 139, wherein the method comprises administering to the subject the pharmaceutical composition according to any one of claims 134 to 138. A method according to claim 139.

141. The method is an immunotherapy for treating cancer in a subject in need thereof, the genetically modified T cells are genetically modified human T cells or cells derived therefrom, and the target sequence comprises a coding sequence of a chimeric antigen receptor or an extracellular ligand-binding domain specific for a tumor-specific antigen of an exogenous T cell receptor, and the cell surface expression of the endogenous T cell receptor is reduced on the genetically modified T cells when compared to unmodified control cells. The method according to claim 139 or 140. The method according to claim 139 or 140, wherein the target sequence comprises a coding sequence of a chimeric antigen receptor or an extracellular ligand-binding domain specific for a tumor-specific antigen of an exogenous T cell receptor, and the cell surface expression of the endogenous T cell receptor is reduced on the genetically modified T cells when compared to unmodified control cells. The method according to claim 139 or 140, wherein the target sequence comprises a coding sequence of a chimeric antigen receptor or an extracellular ligand-binding domain specific for a tumor-specific antigen of an exogenous T cell receptor, and the cell surface expression of the endogenous T cell receptor is reduced on the genetically modified T cells when compared to unmodified control cells. The method according to claim 139 or 140, wherein the target sequence comprises a coding sequence of a chimeric antigen receptor or an extracellular ligand-binding domain specific for a tumor-specific antigen of an exogenous T cell receptor, and the cell surface expression of the endogenous T cell receptor is reduced on the genetically modified T cells when compared to unmodified control cells. A method according to claim 139 or 140.

142. The method according to claim 141, wherein the cancer is selected from the group consisting of carcinomas, lymphomas, sarcomas, blastomas, and leukemias. A method according to claim 141.

143. The method according to claim 141 or 142, wherein the cancer is selected from the group consisting of cancers of B-cell origin, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, melanoma, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, and Hodgkin lymphoma. The method according to claim 141 or 142, wherein the cancer is selected from the group consisting of cancers of B-cell origin, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, melanoma, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, and Hodgkin lymphoma. A method according to claim 141 or 142.

144. The cancer of B-cell origin is B-lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia , the method according to claim 14, selected from the group consisting of B-cell non-Hodgkin lymphoma and multiple myeloma. 3.

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