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

Abstract

To provide engineered meganucleases.SOLUTION: Provided is an engineered meganuclease that recognizes and cleaves a recognition sequence within an intron of the human T cell receptor alpha gene located 5 ' upstream of TRAC exon 1, wherein said engineered meganuclease comprises a first subunit and a second subunit, wherein said first subunit binds to a first recognition half-site of said recognition sequence and comprises a first hypervariable region, and wherein said second subunit binds to a second recognition half-site of said recognition sequence and comprises a second hypervariable region.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 provides 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. Regarding 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. Created October 31, 2017 The resulting ASCII copy is named P109070024US01-SEQ-HJD. It is 124,035 bytes in size. [Background technology]

[0003] T cell adoptive immunotherapy is a promising approach to cancer treatment. This strategy targets specific tumor-associated Using genetically modified isolated human T cells to enhance specificity for related antigens Genetic modifications include the use of chimeric antigen receptors or exogenous 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) target major histocompatibility complexes in a non-restrictive manner. Induce tumor immune reactivity in combination. T cell adoptive immunotherapy is a promising treatment for B cell malignancies (e.g., Acute lymphoblastic leukemia (ALL), B-cell non-Hodgkin's lymphoma (NHL), and chronic lymphocytic leukemia (CRL) leukemia), multiple myeloma, neuroblastoma, glioblastoma, progressive glioma, ovarian cancer, mesothelioma It has been used as a clinical therapy for many cancers, including ovarian cancer, melanoma, and pancreatic cancer.

[0004] Despite their potential utility as cancer treatments, adoptive immunotherapy using CAR T cells The expression of endogenous T cell receptors on the cell surface is limited in part. CAR T cells expressing cellular receptors have been shown to mediate major and minor histocompatibility responses after administration to allogeneic patients. This can lead to the development of graft-versus-host disease (GVHD). Clinical trials have primarily focused on the use of autologous CAR T cells, in which patients' T cells are isolated and genetically engineered. After modification to incorporate a chimeric antigen receptor, the cells are reinfused into the same patient. Although providing immune tolerance to the administered CAR T cells, this approach does not guarantee that the patient's cancer will be cured until the diagnosis is made. The study is limited by both the time and expense required to generate patient-specific CAR T cells after they are acquired. Receive an agreement.

[0005] Therefore, the expression of endogenous T cell receptors is reduced and it does not initiate GVHD upon administration. Developing "off-the-shelf" CAR T cells prepared using T cells from healthy donors It would be advantageous to generate and validate such a product prior to diagnosis and to provide it immediately to the patient, if necessary. Therefore, endogenous T cells can be made available to prevent the development of GVHD. There is a need to develop allogeneic CAR T cells that lack cell receptors.

[0006] Genetic modifications of genomic DNA are engineered to recognize the DNA sequence of a desired locus. It can also be performed using site-specific, infrequently cutting endonucleases. Methods for producing such site-specific endonucleases are known in the art. For example, zinc finger nucleases (ZFNs) can be used to recognize and cut specific sites in the genome. ZFNs can be engineered to cleave the nuclease domain of the FokI restriction enzyme. It is a chimeric protein containing a zinc finger DNA binding domain fused to a zinc finger. The finger domain was redesigned by rational or experimental means to have a length of approximately 18 base pairs. It is possible to generate proteins that bind to a specific DNA sequence. By fusing the protein domain to the FokI nuclease, we were able to achieve genome-wide specificity for D ZFNs can target cleavage of genes in a wide range of eukaryotes. It has been widely used to target additions, deletions, and substitutions (reviewed in Non-Patent Document 1). Similarly, TAL effector nucleases (TALENs) r nuclease) that can cut specific sites in genomic DNA. Similar to ZFNs, TALENs are engineered nucleotide sequences fused to a FokI nuclease domain. It contains a site-specific DNA binding domain (reviewed in Non-Patent Document 2). In the case of , the DNA-binding domains are TAs, each of which specifically recognizes a single DNA base pair. The ZFNs and TAs in the practice of the present invention comprise a tandem array of L effector domains. The limitation of LEN is that they are heterodimers, and therefore, they are unable to function in cells. The production of a monofunctional nuclease requires the co-expression of two protein monomers.

[0007] Compact TALENs utilize an alternative endonuclease structure that avoids the need for dimerization Compact TALENs have an I-TevI ​​homing endonuclease (Non-Patent Document 3). Engineered site-specific TALF fused to a nuclease domain derived from ribosomal enzyme Unlike FokI, I-TevI ​​consists of two vector DNA-binding domains. Compact TALENs are useful for generating double-stranded DNA breaks because they do not need to dimerize. It functions as a nomadic

[0008] Engineered endonucleases based on the CRISPR system are also known in the art. CRISPR endonucleases are characterized by two Components: (1) Caspase effector nucleases, typically microbial Ca s9, Cpf1, or another suitable nuclease; and (2) a nuclease that A short "guide R" containing a targeting sequence of approximately 20 nucleotides that guides the target to the desired location. Multiple guide RNAs, each with a different targeting sequence, are inserted into the same cell. By expressing the gene in cells, DNA cleavage can be targeted simultaneously at multiple sites in the genome. Therefore, CRISPR nucleases are suitable for the present invention. A major drawback of the RISPR system is the reported high frequency of off-target DNA breaks, which can lead to serious side effects in human patients. This may limit the usefulness of the system for treating patients (Non-Patent Document 6).

[0009] Homing endonucleases are 15-base pair endonucleases commonly found in plant and fungal genomes. A group of naturally occurring nucleases that recognize cleavage sites of ~40 base pairs. Guendonuclease is a group 1 enzyme that breaks down self-splicing introns and inteins. Homing endonucleases are frequently associated with parasitic DNA elements in the chromosome. by homologous recombination or gene insertion at a specific location in the host genome by generating a double-strand break in the Homing end naturally promotes cell entry and mobilizes cellular DNA repair mechanisms (Non-Patent Document 7). Nucleases generally fall into four families: the LAGLIDADG (SEQ ID NO: 2) family -, GIY-YIG family, His-Cys box family and HNH family These families are classified into structural motifs that influence catalytic activity and recognition sequences. For example, members of the LAGLIDADG (SEQ ID NO: 2) family are , either one or two of the conserved LAGLIDADG (SEQ ID NO: 2) motifs. It is characterized by having a peak (see Non-Patent Document 8). LAGLIDADG (SEQ ID NO: 2) homing enzyme with a single copy of the motif The endonuclease forms a homodimer, but the LAGLIDADG (SEQ ID NO: 2) motif Members with two copies of the nucleotide are found as monomers.

[0010] I-CreI (SEQ ID NO: 1) is a gene encoding the alga Chlamydomonas reinhardtii A 22-base pair recognition sequence was identified in the chloroplast chromosome of Cydomonas reinhardtii. The LAGLIDADG (SEQ ID NO: 2) family of homing endonucleases recognizes and cleaves Genetic selection techniques were used to select for the wild-type I-CreI cleavage site. Recently, I-CreI and other homing Endonucleases are selected from a wide variety of genomes, including mammalian, yeast, plant, bacterial, and viral genomes. MonoLAs can be comprehensively redesigned to target a wide variety of DNA sites. A method for rationally designing the GLIDADG (SEQ ID NO: 2) homing endonuclease is disclosed. It was described in Patent Document 1.

[0011] As first described in US Pat. No. 5,629,999, I-CreI and its engineered derivatives Usually a dimer, with the C-terminus of the first subunit linked to the N-terminus of the second subunit These can be fused to a single polypeptide using a short peptide linker (Non-Patent Document 1 3 and 14). Thus, functional "single-chain" meganucleases can be expressed from a single transcript. It can be done.

[0012] Engineered meganucleotides for DNA target cleavage in the human T-cell receptor alpha gene The use of ATPases has been previously disclosed. For example, in US Pat. Nos. 5,999,223 and 5,999,238, the applicant T cell receptor alpha constant region (TRAC) An engineered gene specific for the recognition sequence of exon 1 of the constant region (Kanagawa constant region) gene. Patent Documents 3 and 4 disclose a meganuclease that is capable of binding to a meganuclease cleavage site. Also disclosed is a method for targeted insertion of a CAR coding sequence. It is engineered to target the recognition sequence within RAC exon 1 (SEQ ID NO: 3 in Patent Document 5). Patent document 5 discloses a chimeric antigen I-OnuI meganuclease mutant. Although the receptor can be expressed in TCR knockout cells, the authors did not disclose inserting a code sequence into the meganuclease cleavage site.

[0013] Small hairpin RNA, zinc finger nuclease (ZFN), transcriptional activator Like effector nuclease (TALEN), megaTAL, and CRISPR systems The use and internalization of other nucleases, including nucleases (e.g., Non-Patent Document 15; Patent Documents 6-8), Mechanisms for preventing expression of the intrinsic TCR have also been disclosed.

[0014] Furthermore, Non-Patent Document 16 reports that the 5' end of TRAC exon 1 and the region immediately adjacent to TRAC exon 1 CAR to a site spanning both endogenous splice acceptor sites located 5' upstream We disclose the use of the CRISPR / Cas9 system to target the insertion of coding sequences. showed that the predicted double-strand break site of Cas9 nuclease is the splice acceptor site. (Eyquem, see Supplementary Figure 1A). The acceptor site is a site where Cas9 disruption occurs in the absence of a donor template. This is evidenced by the fact that it causes TCR knockout in 70% of T cells. and is required for TCR expression (Eyquem, see Supplementary Figure 1C, second panel). stomach).

[0015] In particular, the nucleases and CRISPR systems disclosed in the prior art each Expression of genes and functional T cell receptors; e.g., TRAC exons, or endogenous splices of T cell receptor genes at sites or loci important for the formation of the receptor site Insertion of the CAR coding sequence into these cleavage sites results in CAR-positive Although T cell receptor negative (TCR-) cells that are CAR+ may be generated, this A significant drawback of this approach is that TCR expression is abrogated if the donor template is not inserted. Non-homologous end joining (NHEJ) is an error-prone process at the break site. s end-joining).

[0016] As a result, previous methods for producing CAR T cells have not been fully developed for preclinical and clinical use. A mixed population of TCR- / CAR+ and TCR- / CAR- cells requiring enrichment For example, as mentioned above, Supplementary Figure 1C of Eyquem shows that the donor template is present. If Cas9 does not insert the splice acceptor site 5' upstream of TRAC exon 1, Upon disruption, approximately 70% of the cells were TCR- / CAR-. However, even in the presence of the CAR donor template (1e6 AAV6), TCR- / CAR A mixed population of TCR- and TCR- / CAR+ cells was generated. When DNA was delivered with an AAV6 MOI of 1e6, 45.6% of T cells were TC Although it was R- / CAR+, it was prone to errors caused by Cas9 and subsequent NHEJ. A significant proportion of cells (30.7%) had cleavage within the splice acceptor site due to repair. %) were TCR- / CAR-. [Prior art documents] [Patent documents]

[0017] [Patent Document 1] International Publication No. 2007 / 047859 [Patent Document 2] International Publication No. 2009 / 059195 [Patent Document 3] International Publication No. 2017 / 062439 [Patent Document 4] International Publication No. 2017 / 062451 [Patent Document 5] International Publication No. 2014 / 191527 [Patent Document 6] U.S. Patent No. 8,956,828 [Patent Document 7] U.S. Patent Application Publication No. 2014 / 0301990 [Patent Document 8] U.S. Patent Application Publication No. 2012 / 0321667 [Non-patent literature]

[0018] [Non-Patent Document 1] Durai et al. (2005), Nucleic Acids Res 33,5978 [Non-patent document 2] Mak et al. (2013), Curr Opin Struct Biol. 23:93-9 [Non-patent document 3] Beurdeley et al. (2013), Nat Commun. 4:1762) [Non-patent document 4] Ran et al. (2013), Nat Protoc. 8:2281-2308 [Non-Patent Document 5] Mali et al. (2013), Nat Methods 10:957-63 [Non-patent document 6] Fu et al. (2013), Nat Biotechnol. 31:822-6 [Non-Patent Document 7] Stoddard(2006),Q.Rev.Biophys.38:49-95 [Non-patent document 8] Chevalier et al. (2001), Nucleic Acids Res. 29(18):3757-3774 [Non-Patent Document 9] Sussman et al. (2004), J.Mol.Biol.342:31-41 [Non-Patent Document 10] Chames et al. (2005), Nucleic Acids Res.33:e178 [Non-Patent Document 11] Seligman et al. (2002), Nucleic Acids Res.30:3870-9 [Non-Patent Document 12] Arnould et al. (2006), J. Mol. Biol. 355:443-58 [Non-Patent Document 13] Li et al. (2009), Nucleic Acids Res.37:1650-62 [Non-Patent Document 14] Grizot et al. (2009), Nucleic Acids Res.37:5405-19 [Non-Patent Document 15] Osborn et al. (2016), Molecular Therapy 24(3):570-581 [Non-Patent Document 16] Eyquem et al. (2017), Nature 543:113-117 Summary of the Invention [Problem to be solved by the invention]

[0019] [Means for solving the problem]

[0020] In contrast, the present invention modifies the T gene to insert a sequence of interest, such as a CAR coding sequence. We take a counterintuitive approach to targeting elements of the TCR gene that are essential for TCR expression. Rather than targeting the TCR alpha gene, the present invention targets the TCR alpha gene located 5' upstream of TRAC exon 1. Targets gene introns. Endogenous splice donor sites and Nucleotides within this non-coding intron are not expected unless the endogenous splice acceptor site is altered. Double-strand breaks caused by NHEJ are not amenable to T cell division, even though NHEJ generates indels at the break site. It has no substantial effect on CR expression.

[0021] Contrary to convention, targeting recognition sequences in introns allows for at least the exogenous sequence to be targeted. The sequence of interest containing the price acceptor site and / or poly A signal is then isolated, e.g., from a homologous Recombination disrupts TCR expression only if it is inserted at the cleavage site. The TCR cells produced in accordance with the invention contain a sequence of interest inserted into the intron cleavage site. By extension, if the inserted sequence of interest further contains a CAR coding sequence, the resulting Most or all of the TCR- cells in the resulting cell population will be TCR- / CAR+, which is more than before. In stark contrast to the law, the resulting population includes a significant proportion of TRC- / CAR- Thus, the present invention provides a method for enriching CAR+ cells from a mixed population of TRC- cells. This significantly advances the field by eliminating the cumbersome need to compress the

[0022] Furthermore, in some embodiments of the present invention, the sequence of interest inserted into the intron is Contains a 2A element 5' upstream of the sequence (e.g., CAR coding sequence) (see Figure 1) The inclusion of this 2A element allows the endogenous T cell receptor antigen to be expressed, rather than the exogenous promoter. This allows expression of the coding sequence driven by the promoter of the ruffa gene. Thus, expression of polypeptides such as CARs may mediate T cell feedback normally associated with TCR expression. It is controlled by a lock mechanism.

[0023] The present invention relates to a genetically modified human T cell receptor alpha gene in its genome. The modified human T cell receptor alpha gene is in an intron within the T cell receptor alpha gene located 5' upstream of TRAC exon 1 The exogenous sequence of interest inserted into the intron may be and a T cell that can contain an exogenous splice acceptor site and / or poly(A) signal. In some embodiments, the sequence of interest disrupts expression of the receptor alpha subunit. It can also include a coding sequence for a polypeptide (e.g., a CAR coding sequence). Endogenous splice donor and endogenous splice acceptor sites adjacent to the intron The site remains unaltered and / or functional within the cell. Cell surface expression of the receptor is reduced when compared to unmodified control cells.

[0024] The present invention also provides compositions and methods for producing populations of T cells, as well as genetically modified T cells. The present invention further provides a method for treating cancer by administering genetically modified T cells. The present invention provides a method of immunotherapy for tumor remission, wherein T cells are directed against a tumor-specific antigen (e.g., a CAR). They express receptors that

[0025] Thus, in one aspect of the present invention, a human T cell receptor agonist located 5' upstream of TRAC exon 1 is An engineered meganucleotide that recognizes and cleaves a recognition sequence within an intron of the receptor alpha gene. a meganuclease, wherein the engineered meganuclease comprises a first subunit and a second subunit. a first subunit that binds to a first recognition half-site of the recognition sequence; , a first hypervariable (HVR1) region, and the second subunit comprises a second hypervariable (HVR2) region of the recognition sequence. The engineered meganucleotides bind to the recognition half-sites of the nucleotides and contain a second hypervariable (HVR2) region. In some embodiments, the intron comprises SEQ ID NO: 3 and is engineered The meganucleases selected are those that target endogenous splice donor sites or endogenous splice donor sites adjacent to introns. It does not have a recognition sequence within the splice acceptor site.

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

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

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

[0029] In some such embodiments, the HVR1 region comprises any one of SEQ ID NOs: 12-15. Contains residues 215-270.

[0030] In some such embodiments, the HVR2 region comprises any one of SEQ ID NOs: 12-15. Identical to the amino acid sequence corresponding to residues 24-79 by at least 80%, at least 85%, or at least and amino acid sequences that share 90%, at least 95%, or more sequence identity with each other. .

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

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

[0033] In some such embodiments, the first subunit is any of SEQ ID NOs: 12-15. At least 80%, at least 85%, at least 9 for residues 198-344 0%, at least 95%, or more sequence identity, The subunits of SEQ ID NOs: 12 to 15 are selected from residues 7 to 153 of any one of SEQ ID NOs: 12 to 15 and at least 8 0%, at least 85%, at least 90%, at least 95% or more sequence identity It contains an amino acid sequence with identical identity.

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

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

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

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

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

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

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

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

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

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

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

[0045] In some such embodiments, the first subunit is any of SEQ ID NOs: 16-19. At least 80%, at least 85%, at least 90% for one residue 7-153 , comprising an amino acid sequence having at least 95% or more sequence identity, and a second subunit The subunit is a combination of residues 198 to 344 of any one of SEQ ID NOs: 16 to 19 and at least 80 %, at least 85%, at least 90%, at least 95% or more sequence identity Contains the amino acid sequence.

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

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

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

[0049] In some such embodiments, the engineered meganuclease has the sequence set forth in SEQ ID NOs: 16-19. It contains any one of the amino acid sequences.

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

[0051] In some such embodiments, the HVR1 region comprises any one of SEQ ID NOs: 20-23. Identical to the amino acid sequence corresponding to residues 24-79 by at least 80%, at least 85%, or at least and amino acid sequences that share 90%, at least 95%, or more sequence identity with each other. nothing.

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

[0053] In some such embodiments, the HVR1 region comprises any one of SEQ ID NOs: 20-23. Contains the residue corresponding to residue 66.

[0054] In some such embodiments, the HVR1 region comprises any one of SEQ ID NOs: 20-23. Contains residues 24-79.

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

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

[0057] In some such embodiments, the HVR2 region comprises any one of SEQ ID NOs: 20-23. Contains residues 215-270.

[0058] In some such embodiments, the first subunit is any of SEQ ID NOs: 20-23. At least 80%, at least 85%, at least 90% for one residue 7-153 , comprising an amino acid sequence having at least 95% or more sequence identity, and a second subunit The subunit is a combination of residues 198 to 344 of any one of SEQ ID NOs: 20 to 23 and at least 80 %, at least 85%, at least 90%, at least 95%, or more sequence identity It contains an amino acid sequence having the following properties:

[0059] In some such embodiments, the first subunit is any of SEQ ID NOs: 20-23. One of the residues contains residues 7 to 153.

[0060] In some such embodiments, the second subunit is any of SEQ ID NOs: 20-23. One contains residues 198-344.

[0061] In some such embodiments, the engineered meganuclease comprises a linker, The CAR covalently links the first and second subunits.

[0062] In some such embodiments, the engineered meganuclease has the sequence of SEQ ID NOs: 20-23. It contains any one of the amino acid sequences.

[0063] In certain embodiments, the recognition sequence is SEQ ID NO: 10 (i.e., the TRC19-20 recognition sequence). column).

[0064] In some such embodiments, the HVR1 region comprises any one of SEQ ID NOs: 24-27. Identical to the amino acid sequence corresponding to residues 24-79 by at least 80%, at least 85%, or at least and amino acid sequences that share 90%, at least 95%, or more sequence identity with each other. .

[0065] In some such embodiments, the HVR1 region comprises 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 comprises any one of SEQ ID NOs: 24-27. Contains residues 24-79.

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

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

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

[0070] In some such embodiments, the first subunit is any of SEQ ID NOs: 24-27. At least 80%, at least 85%, at least 90% for one residue 7-153 , comprising an amino acid sequence having at least 95% or more sequence identity, and a second subunit The subunit is a combination of residues 198 to 344 of any one of SEQ ID NOs: 24 to 27 and at least 80 %, at least 85%, at least 90%, at least 95%, or more sequence identity It contains an amino acid sequence having the following properties:

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

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

[0073] In some such embodiments, the engineered meganuclease comprises a linker, The CAR covalently links the first and second subunits.

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

[0075] In another aspect, the present invention provides a method for producing a nucleic acid encoding an engineered meganuclease as described herein. A polynucleotide comprising a nucleic acid sequence that

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

[0077] In a further aspect, the mRNA is a nucleotide sequence encoding the engineered meganucleases described herein and Polycistronic mRNA encoding at least one additional polypeptide or nucleic acid. be.

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

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

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

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

[0082] In another aspect, the present invention provides a gene comprising an exogenous sequence of interest inserted into the chromosome of a T cell. A method for producing modified T cells is provided, the method comprising: (a) administering to a T cell a gene encoding a modified T cell as described herein; a first nucleic acid sequence encoding an engineered meganuclease, (b) a first nucleic acid sequence from which the meganuclease is expressed in T cells; and (b) a sequence of interest. a second nucleic acid sequence comprising: The enzyme is located 5' upstream of TRAC exon 1 of the human T cell receptor alpha gene. a chromosomal cleavage site at a recognition sequence within the intron; the sequence of interest is inserted into the chromosome at the exogenous splice acceptor site and / or contains a poly A signal; and the endogenous splice donor site and The primary splice acceptor site remains unaltered and / or functional.

[0083] In some embodiments of the above methods, the T cells are V and J segment rearranged. These are precursor T cells that have not undergone this process.

[0084] In certain embodiments of the above methods, the cell surface expression of the endogenous T cell receptor is It is decreased when compared with the control cells.

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

[0086] 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 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. In some embodiments of the above method, the recognition sequence is SEQ ID NO:8. The engineered meganuclease comprises the nucleotide sequence described herein that recognizes and cleaves SEQ ID NO:8. In some embodiments of the above method, the meganuclease is an engineered meganuclease. The string contains SEQ ID NO: 10, and the engineered meganuclease recognizes and cleaves SEQ ID NO: 10. The engineered meganucleases described herein are

[0087] In certain embodiments of the above method, the second nucleic acid sequence is complementary to the sequence adjacent to the cleavage site. The desired sequence is inserted into the cleavage site by homologous recombination.

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

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

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

[0091] In some embodiments of the above methods, the sequence of interest is expressed by a chimeric antigen receptor or an exogenous T cell. In certain embodiments of the above method, the coding sequence for a chimeric antigen receptor or an exogenous antigen receptor is T-cell receptors contain extracellular ligand-binding domains with specificity for tumor-specific antigens. Includes:

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

[0093] In certain embodiments of the above method, the at least second nucleic acid sequence is a viral vector. In certain embodiments of the above methods, the viral vector is introduced into the T cell by Denoviral vector, lentiviral vector, retroviral vector, or AAV In a specific embodiment of the above method, the viral vector is a recombinant AAV vector. He is a ctor.

[0094] In another aspect, the present invention provides a gene comprising an exogenous sequence of interest inserted into the chromosome of a T cell. Methods for producing modified T cells are provided, the methods comprising: (a) administering to a T cell a gene encoding a nucleotide sequence described herein; (b) introducing an engineered meganuclease containing a nucleic acid sequence of interest; and and transfecting the T cells with the engineered meganuclease. The intron of the human T cell receptor alpha gene located 5' upstream of TRAC exon 1 A recognition sequence within the chromosome is used to generate a cleavage site in the chromosome; the sequence of interest is then stained at the cleavage site. the sequence of interest is inserted into the host; the sequence of interest may be inserted into the exogenous splice acceptor site and / or polyA signal; and the endogenous splice donor site and 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 rearranged. These are precursor T cells that have not undergone this process.

[0096] In some embodiments of the above methods, the endogenous T cell receptors are increased when compared to unmodified control cells. 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 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. In some embodiments of the above method, the recognition sequence is SEQ ID NO:8. The engineered meganuclease comprises the nucleotide sequence described herein that recognizes and cleaves SEQ ID NO:8. In some embodiments of the above method, the meganuclease is an engineered meganuclease. The string contains SEQ ID NO: 10, and the engineered meganuclease recognizes and cleaves SEQ ID NO: 10. The engineered meganucleases described herein are

[0099] In certain embodiments of the above method, the nucleic acid contains a sequence homologous to a sequence adjacent to the cleavage site. Furthermore, the sequence of interest is inserted into the cleavage site by homologous recombination.

[0100] In some embodiments of the above methods, the T cells are human T cells or cells derived therefrom. do.

[0101] In certain embodiments of the above method, the sequence of interest is 5' to 3' separated from the exogenous splice. an acceptor site, a 2A element or an IRES element, the coding sequence for the protein of interest, and In certain embodiments of the above method, the 2A element comprises a T2A element, a P2A element, an E2A element, or an F2A element. In a specific embodiment of the above method, is a T2A element.

[0102] In some embodiments of the above method, the sequence of interest is an exogenous splice acceptor site. and an exogenous branch site located 5' upstream of

[0103] In some embodiments of the above methods, the sequence of interest is expressed by a chimeric antigen receptor or an exogenous T cell. In certain embodiments of the above method, the coding sequence for a chimeric antigen receptor or an exogenous antigen receptor is T-cell receptors contain extracellular ligand-binding domains with specificity for tumor-specific antigens. Includes:

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

[0105] In another aspect, the present invention provides a method for the production of genetically modified T cells comprising a modified human T cell receptor alpha gene. The present invention provides a method for producing T cells, the method comprising: (a) administering to a T cell: (i) an engineered nucleic acid; a first nucleic acid sequence encoding a nuclease, wherein the engineered nuclease is (ii) a first nucleic acid sequence expressed in a manner consistent with the invention; or (iii) an engineered nuclease protein. and (b) introducing into said cell a second nucleic acid sequence comprising an exogenous sequence of interest. and wherein the engineered nuclease is located 5' upstream of TRAC exon 1. A cleavage site is generated in the recognition sequence within the intron of the human T cell receptor alpha gene located above. wherein said sequence of interest is inserted into the human T cell receptor alpha gene at said cleavage site; The sequence of interest may include an exogenous splice acceptor site and / or a polyA signal. the endogenous splice donor site and endogenous splice acceptor site adjacent to the intron; The target site remains unaltered and / or functional.

[0106] In some embodiments of the above methods, the T cells are V and J segment rearranged. These are precursor T cells that have not undergone this process.

[0107] In some embodiments of the above methods, the endogenous T cell receptors are increased when compared to unmodified control cells. The body has reduced cell surface expression.

[0108] In certain embodiments of the above methods, 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 cleavage site (b) a 5' homology arm homologous to the 5' upstream sequence adjacent to the position; (c) the exogenous sequence of interest; a 3' homology arm homologous to the 3' downstream sequence adjacent to the cleavage site, wherein the exogenous gene of interest The sex sequence 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 is an exogenous splice acceptor site. and an exogenous branch site located 5' upstream of

[0111] In certain embodiments of the above methods, the genetically modified T cells are genetically modified human T cells, or are cells derived from it.

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

[0113] In some embodiments of the above methods, the sequence of interest is expressed by a chimeric antigen receptor or an exogenous T cell. In certain embodiments of the above method, the coding sequence for a chimeric antigen receptor or Exogenous T cell receptors contain extracellular ligand-binding domains with specificity for tumor-specific antigens. Including Inn.

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

[0115] In certain embodiments of the above methods, the at least second nucleic acid sequence is a viral vector In certain embodiments of the above methods, the viral vector is introduced into the T cell by Denovirus vector, lentivirus vector, retrovirus vector, or adenovirus vector In a specific embodiment of the above method, the viral vector is an AAV vector. The vector is a recombinant AAV vector.

[0116] In some embodiments of the above methods, the engineered nuclease is an engineered meganuclease. ases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases TALEN, Compact TALEN, CRISPR nuclease, or me In certain embodiments of the above method, the engineered nuclease is an engineered nuclease. It is a meganuclease.

[0117] In some embodiments of the above methods, the engineered meganuclease comprises SEQ ID NO:4. In some such embodiments of the above method, the engineered The meganuclease is an engineered meganuclease described herein that recognizes and cleaves SEQ ID NO:4. It is a ganucleases.

[0118] In some embodiments of the above methods, the engineered meganuclease comprises SEQ ID NO:6. In some such embodiments of the above method, the engineered The meganuclease is an engineered meganuclease described herein that recognizes and cleaves SEQ ID NO:6. It is a ganucleases.

[0119] In some embodiments of the above methods, the engineered meganuclease comprises SEQ ID NO:8. In some such embodiments of the above method, the engineered The engineered meganuclease described herein recognizes and cleaves SEQ ID NO:8. It is a meganuclease.

[0120] In some embodiments of the above methods, the engineered meganuclease comprises SEQ ID NO: 10. In some such embodiments of the above method, the engineered The resulting meganuclease recognizes and cleaves SEQ ID NO: 10. It is a meganuclease.

[0121] In another aspect, the present invention provides a method for producing genetically modified T cells, comprising: The present invention provides genetically modified T cells prepared by any of the methods.

[0122] In another aspect, the present invention provides a method for detecting a human T cell receptor alpha gene comprising: Genetically modified T cells are provided, wherein the modified human T cell receptor alpha gene is TRAC. It is inserted into an intron within the T-cell receptor alpha gene, located 5' upstream of exon 1. the exogenous sequence of interest comprises an exogenous splice acceptor site and and / or poly(A) signals, and endogenous splice donor sites adjacent to the introns and The primary splice acceptor site remains unaltered and / or functional. , cell surface expression of endogenous T cell receptors is reduced when compared to unmodified control cells .

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

[0124] In certain embodiments, the genetically modified T cells are genetically modified human T cells, or cells derived therefrom. These are the cells that come from the nucleus.

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

[0126] In some embodiments, the exogenous sequence of interest is located within the 5′ end of the exogenous splice acceptor site. 'Further comprising an exogenous branch site located upstream.

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

[0128] In some embodiments, the exogenous sequence of interest comprises an engineered meganuclease recognition site, TALEN recognition site, zinc finger nuclease recognition site, CRISPR recognition site, or inserted into an intron at a megaTAL recognition site. The target sequence is inserted into an intron with an engineered meganuclease recognition site. In embodiments, the exogenous sequence of interest is inserted into an intron within SEQ ID NO: 4. In one embodiment, the exogenous sequence of interest is inserted into an intron within SEQ ID NO:6. In another embodiment, the exogenous sequence of interest is inserted into an intron within SEQ ID NO: 8. In this case, the exogenous sequence of interest is inserted into an intron within SEQ ID NO:10.

[0129] In another aspect, the present invention provides a method for the production of a genetically modified T cell comprising administering to a subject ... A population of modified T cells is provided.

[0130] In some embodiments, at least 10%, at least 15%, at least a few of the cells in the population 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 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 genes described herein These are genetically modified T cells.

[0131] In certain embodiments, the genetically modified T cells are genetically modified human T cells, or cells derived therefrom. These are cells that

[0132] In some embodiments, the exogenous sequence of interest present in the genetically modified T cell is a chimeric antigen In certain embodiments, the coding sequence for a chimeric antigen receptor or an exogenous T cell receptor is The receptor or exogenous T cell receptor binds to extracellular ligands with specificity for tumor-specific antigens. Contains a binding domain.

[0133] In some embodiments, cell surface expression of endogenous T cell receptors is increased compared to unmodified control cells. When the expression level of the gene-modified T cells was increased, the expression level was reduced.

[0134] In another aspect, the present invention provides a pharmaceutical composition useful for treating a disease in a subject in need thereof. The present invention provides a pharmaceutical composition useful for treating a disease, the pharmaceutical composition comprising a pharmaceutically acceptable carrier and The 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. These are the cells that come from the nucleus.

[0136] In some embodiments, the exogenous sequence of interest present in the genetically modified T cell is a chimeric antigen In certain embodiments, the coding sequence for a chimeric antigen receptor or an exogenous T cell receptor is The receptor or exogenous T cell receptor binds to extracellular ligands with specificity for tumor-specific antigens. Contains a binding domain.

[0137] In some embodiments, cell surface expression of endogenous T cell receptors is increased compared to unmodified control cells. When the expression level of the gene-modified T cells was increased, the expression level was reduced.

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

[0139] In some embodiments, the methods include administering to a subject a pharmaceutical composition described herein. This includes:

[0140] In certain embodiments, the method includes treating 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 above-mentioned exogenous factor present in genetically modified T cells The functional sequence is a chimeric antigen receptor or an extracellular ligand-binding domain having specificity for a tumor-specific antigen. It contains the coding sequence of an exogenous T cell receptor, including the domain, and inhibits cell surface expression of an endogenous T cell receptor. Expression is reduced on genetically modified T cells when compared to unmodified control cells.

[0141] In some embodiments of the above methods, the cancer is selected from the group consisting of carcinoma, lymphoma, sarcoma, blastoma, and leukemia. Cancer of the present invention is selected from the group consisting of:

[0142] In certain embodiments of the above methods, 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's lymphoma.

[0143] In certain embodiments of the above methods, the cancer of B-cell origin is B-lineage acute lymphoblastic leukemia. , consisting of B-cell chronic lymphocytic leukemia, B-cell non-Hodgkin's lymphoma, and multiple myeloma is selected from the group.

[0144] In another aspect, the present invention provides a method for producing a recombinant human ovarian cancer cell line comprising administering to a mammalian animal the genetic modification described herein for use as a pharmaceutical. The present disclosure further provides methods for using the genetically modified cells described herein. a compound according to any one of claims 1 to 4, wherein the compound is a compound selected from the group consisting of benzodiazepines, ... Uses of the genetically modified cells are provided. In one such aspect, the medicament is useful for treating cancer.

[0145] In another aspect, the present invention provides a compound according to the invention, comprising a compound of the formula (I) or (II) as described herein for use in the treatment of a disease, preferably in the treatment of cancer. The present invention provides genetically modified cells as described in the document. [Brief explanation of the drawings]

[0146] [Figure 1] FIG. 1 is a diagram of a sample strategy for inserting and expressing an exogenous sequence of interest into an intron of the T cell receptor alpha gene, rearranged to encode a functional T cell receptor alpha subunit. As shown, the exogenous sequence of interest is inserted into an intron of the T cell receptor alpha gene 5' upstream of TRAC exon 1. The endogenous splice acceptor site and endogenous splice acceptor site flanking the target 5' intron remain intact. Following nuclease cleavage, the exogenous sequence of interest described herein is inserted into the intron. As shown, the sequence of interest contains at least the exogenous splice acceptor site and / or a polyA signal that, when inserted into the intron, disrupts expression of the T cell receptor alpha subunit. The inserted sequence of interest can optionally include a T2A element, represented by a T2A element. The inserted sequence of interest can also optionally include a coding sequence for a polypeptide of interest, represented by a chimeric antigen receptor coding sequence. Optionally, the sequence of interest can further comprise an exogenous branch site located 5' upstream of the exogenous splice acceptor site. [Figure 2]1 shows the TRC recognition sequences of the targeted 5' intron of the human T-cell receptor alpha gene. Each recognition sequence targeted by the engineered meganucleases of the present invention contains two recognition half-sites. Each recognition half-site consists of 9 base pairs separated by a 4-base pair central sequence. The TRC11-12 recognition sequence (SEQ ID NO: 4) contains two recognition half-sites designated TRC11 and TRC12. The TRC15-16 recognition sequence (SEQ ID NO: 6) contains two recognition half-sites designated TRC15 and TRC16. The TRC17-18 recognition sequence (SEQ ID NO: 8) contains two recognition half-sites designated TRC17 and TRC18. The TRC19-20 recognition sequence (SEQ ID NO: 10) contains two recognition half-sites designated TRC19 and TRC20. [Figure 3] The engineered meganucleases of the present invention comprise two subunits: a first subunit comprising an HVR1 region that binds to a first recognition half-site (e.g., TRC11, TRC15, TRC17, or TRC19), and a second subunit comprising an HVR2 region that binds to a second recognition half-site (e.g., TRC12, TRC16, TRC18, or TRC20). In embodiments where the engineered meganuclease is a single-chain meganuclease, the first subunit comprising the HVR1 region can be positioned as either the N- or C-terminal subunit. Similarly, the second subunit comprising the HVR2 region can be positioned as either the N- or C-terminal subunit. [Figure 4]Figure 1 shows a schematic of a reporter assay in CHO cells evaluating engineered meganucleases targeting recognition sequences found in the target 5' intron of the T-cell receptor alpha gene. For the engineered meganucleases described herein, CHO cell lines were generated in which a reporter cassette was stably integrated into the cell's genome. The reporter cassette is composed, in 5' to 3' order: the SV40 early promoter; the 5' 2 / 3 of the GFP gene; the recognition sequence for the engineered meganuclease of the present invention (e.g., the TRC11-12 recognition sequence); the recognition sequence for the CHO-23 / 24 meganuclease (WO 2012 / 167192); and the 3' 2 / 3 of the GFP gene. Cells stably transfected with this cassette did not express GFP in the absence of a DNA cleavage inducer. Meganucleases were introduced by transfection of DNA or mRNA from a plasmid encoding each meganuclease. When DNA cleavage was induced at either of the meganuclease recognition sequences, the overlapping regions of the GFP gene recombined with each other, resulting in a functional GFP gene. The percentage of cells expressing GFP can be determined by flow cytometry as an indirect measure of the frequency of genome cleavage by the engineered meganuclease. [Figure 5A-D]Figure 1 shows the efficiency of engineered meganucleases to recognize and cleave recognition sequences found in the target 5' intron of the T cell receptor alpha gene in a CHO cell reporter assay. Engineered meganucleases set forth in SEQ ID NOS: 12-15 were engineered to target the TRC11-12 recognition sequence (SEQ ID NO: 4). Engineered meganucleases set forth in SEQ ID NOS: 16-19 were engineered to target the TRC15-16 recognition sequence (SEQ ID NO: 6) and screened for efficacy in a CHO cell reporter assay. Engineered meganucleases set forth in SEQ ID NOS: 20-23 were engineered to target the TRC17-18 recognition sequence (SEQ ID NO: 8). Engineered meganucleases set forth in SEQ ID NOS: 24-27 were engineered to target the TRC19-20 recognition sequence (SEQ ID NO: 10). The results shown indicate the percentage of GFP-expressing cells observed in each assay, indicating the effectiveness of each meganuclease in cleaving the target recognition sequence or the CHO-23 / 24 recognition sequence. Additionally, a negative control (bs) was included in each assay. Figure 5A shows meganucleases targeting the TRC11-12 recognition sequence. Figure 5B shows meganucleases targeting the TRC15-16 recognition sequence. Figure 5C shows meganucleases targeting the TRC17-18 recognition sequence. Figure 5D shows meganucleases targeting the TRC19-20 recognition sequence. [Figure 6A-D]Figure 1 shows the efficiency of engineered meganucleases to recognize and cleave recognition sequences in the intron of the human T-cell receptor alpha gene 5' upstream of TRAC exon 1 in a CHO cell reporter assay. Engineered meganucleases set forth in SEQ ID NOS: 12-15 were engineered to target the TRC11-12 recognition sequence (SEQ ID NO: 4). Engineered meganucleases set forth in SEQ ID NOS: 16-19 were engineered to target the TRC15-16 recognition sequence (SEQ ID NO: 6) and screened for efficacy in a CHO cell reporter assay. Engineered meganucleases set forth in SEQ ID NOS: 20-23 were engineered to target the TRC17-18 recognition sequence (SEQ ID NO: 8). Engineered meganucleases set forth in SEQ ID NOS: 24-27 were engineered to target the TRC19-20 recognition sequence (SEQ ID NO: 10). Engineered meganucleases were screened for efficacy in a CHO cell reporter assay at multiple time points over a 7-day period post-nucleofection. The results shown show the percentage of GFP-expressing cells observed in each assay over the 7-day period of analysis, indicating the effectiveness of each meganuclease at cleaving the target recognition sequence or the CHO-23 / 24 recognition sequence as a function of time. Figure 6A shows meganucleases targeting the TRC11-12 recognition sequence. Figure 6B shows meganucleases targeting the TRC15-16 recognition sequence. Figure 6C shows meganucleases targeting the TRC17-18 recognition sequence. Figure 6D shows meganucleases targeting the TRC19-20 recognition sequence. [Figure 7] T7E assay of T cell lysates. Human CD3+ T cells were isolated from PBMCs by magnetic separation and activated for 72 hours. Activated human T cells were electroporated with TRC11-12 or TRC15-16 meganuclease mRNA, and genomic DNA (gDNA) was harvested from the cells 72 hours post-transfection. A T7 endonuclease I (T7E) assay was performed to estimate genetic modifications at the endogenous TRC11-12 or TRC15-16 recognition sequences. [Figure 8]Cleavage at the recognition sequence of the targeted 5' intron does not affect T cell receptor expression. Human T cells were enriched from apheresis samples obtained from human donors and stimulated for 3 days using anti-CD3 / anti-CD28 beads in the presence of IL-2. After 3 days, T cells were harvested, the beads were removed, and 1 μg of the indicated meganuclease RNA was introduced into the T cell sample. Nucleofected cells were cultured for 6 days before flow cytometry analysis. CD3 surface expression, representing endogenous T cell receptor expression, was measured by labeling T cell samples with anti-CD3-BrilliantViolet711 and GhostDye-510. T cells were nucleofected with either TRC1-2x.87EE (an engineered nuclease targeting TRAC exon 1) or no RNA (mock), serving as positive and negative controls for TRAC locus editing, respectively, as shown in Figure 8A and Figure 8B. Four additional samples were also nucleofected with RNA encoding one different nuclease variant from the TRC15-16 family, all of which target the TRC15-16 recognition sequence in the 5' intron. Gene disruption by editing the TRAC locus results in the failure of TCRα synthesis and the failure of the TCR complex (including CD3) to be displayed on the surface of edited cells. Over half of the T cells edited with TRC1-2x.87EE were TCR-negative due to truncation of exon 1 and error-prone repair of the cleavage site by NHEJ (Figure 8B). In comparison, the frequency of TCR-negative cells after editing with TRC15-16x.3l, TRC15-16x.63, TRC15-16x.87, and TRC15-16x.89 was only 4%–8% (Figure 8C, Figure 8D, Figure 8E, and Figure 8F, respectively). [Figure 9A-C]9A-9C show donor templates for exogenous sequences of interest. Donor templates are provided that include homology arms, an exogenous splice acceptor site, a CAR coding sequence, and a polyA signal. Figure 9A provides an exemplary donor template (SEQ ID NO: 60) suitable for insertion into the TRC11-12 recognition sequence. Figure 9B provides an exemplary donor template (SEQ ID NO: 61) suitable for insertion into the TRC15-16 recognition sequence. Figure 9C provides an exemplary donor template (SEQ ID NO: 62) suitable for insertion into the TRC17-18 recognition sequence. [Figure 10A-D] Figure 10 shows the insertion of the GFP coding sequence into the targeted 5' intron. T cells were nucleofected with mRNA encoding the TRC11-12x.82 nuclease and transduced with an AAV6 vector containing the 7227 construct encoding a T2A sequence followed by a promoterless GFP coding sequence. Additional T cells were nucleofected with mRNA encoding the TRC15-16.x31 nuclease and transduced with an AAV6 vector containing the 7228 construct encoding a T2A sequence followed by a promoterless GFP coding sequence. TCR knockout and GFP expression were measured by flow cytometry 5 days after transfection / transduction. Figure 10A shows the expression of CD3 (x-axis) and GFP (y-axis) after donor template insertion with the TRC11-12 recognition sequence. Figure 10B shows the GFP expression (x-axis) and cell number (y-axis) after donor template insertion with the TRC11-12 recognition sequence. Figure 10C shows CD3 (x-axis) and GFP (y-axis) expression after donor template insertion with the TRC15-16 recognition sequence. Figure 10D shows GFP expression (x-axis) and cell number (y-axis) after donor template insertion with the TRC15-16 recognition sequence. [Figure 11A-C]Insertion of anti-CD19 CAR coding sequences into the targeted 5' intron. T cells were nucleofected with mRNA encoding the TRC11-12x.82 nuclease and transduced with an AAV6 vector containing the 7225 construct encoding the T2A sequence followed by a promoterless anti-CD19 CAR coding sequence. Additional T cells were nucleofected with mRNA encoding the TRC15-16.x31 nuclease and transduced with an AAV6 vector containing the 7226 construct encoding the T2A sequence followed by a promoterless anti-CD19 CAR coding sequence. TCR knockout and CAR expression were measured by flow cytometry 5 days after transfection / transduction. Figure 11A shows CAR expression in CD3 cells from the negative control group (TRC enzyme only). Figure 11B shows CAR expression in CD3 cells after donor template insertion with the TRC11-12 recognition sequence. Figure 11C shows CAR expression in CD3 cells after donor template insertion with the TRC15-16 recognition sequence.

[0147] A brief description of arrays 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 a 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 the TRC11-12x.4 meganuclease.

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

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

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

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

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

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

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

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

[0167] SEQ ID NO: 21 shows the amino acid sequence of the TRC17-18x.82 meganuclease.

[0168] SEQ ID NO: 22 shows the amino acid sequence of the TRC17-18x.18 meganuclease.

[0169] SEQ ID NO: 23 shows the amino acid sequence of the TRC17-18x.71 meganuclease.

[0170] SEQ ID NO: 24 shows the amino acid sequence of the TRC19-20x.85 meganuclease.

[0171] SEQ ID NO: 25 shows the amino acid sequence of the TRC19-20x.74 meganuclease.

[0172] SEQ ID NO: 26 shows the amino acid sequence of the TRC19-20x.71 meganuclease.

[0173] SEQ ID NO: 27 shows the amino acid sequence of the TRC19-20x.87 meganuclease.

[0174] SEQ ID NO: 28 is the TRC11-12x.4 meganuclease TRC11 binding subunit The amino acid sequence of the

[0175] SEQ ID NO: 29 is the TRC11-12x.82 meganuclease TRC11 binding subunit The amino acid sequence of TT is shown.

[0176] SEQ ID NO: 30 is the TRC11-12x.60 meganuclease TRC11 binding subunit The amino acid sequence of TT is shown.

[0177] SEQ ID NO: 31 is the TRC11-12x.63 meganuclease TRC11 binding subunit The amino acid sequence of TT is shown.

[0178] SEQ ID NO: 32 is the TRC11-12x.4 meganuclease TRC12 binding subunit The amino acid sequence of the

[0179] SEQ ID NO: 33 is the TRC11-12x.82 meganuclease TRC12 binding subunit The amino acid sequence of TT is shown.

[0180] SEQ ID NO: 34 is the TRC11-12x.60 meganuclease TRC12 binding subunit The amino acid sequence of TT is shown.

[0181] SEQ ID NO: 35 is the TRC11-12x.63 meganuclease TRC12 binding subunit The amino acid sequence of TT is shown.

[0182] SEQ ID NO: 36 is the TRC15-16x.31 meganuclease TRC15 binding subunit The amino acid sequence of TT is shown.

[0183] SEQ ID NO: 37 is the TRC15-16x.87 meganuclease TRC15 binding subunit The amino acid sequence of TT is shown.

[0184] SEQ ID NO: 38 is the TRC15-16x.63 meganuclease TRC15 binding subunit The amino acid sequence of TT is shown.

[0185] SEQ ID NO: 39 is the TRC15-16x.89 meganuclease TRC15 binding subunit The amino acid sequence of TT is shown.

[0186] SEQ ID NO: 40 is the TRC15-16x.31 meganuclease TRC16 binding subunit The amino acid sequence of TT is shown.

[0187] SEQ ID NO: 41 is the TRC15-16x.87 meganuclease TRC16 binding subunit The amino acid sequence of TT is shown.

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

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

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

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

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

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

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

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

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

[0197] SEQ ID NO: 51 is the TRC17-18x.71 meganuclease TRC18 binding subunit The amino acid sequence of TT is shown.

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

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

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

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

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

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

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

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

[0206] SEQ ID NO: 60 is a DNA fragment containing an anti-CD19 CAR that can be inserted into the TRC11-12 recognition sequence. The nucleic acid sequence of the parent template is shown.

[0207] SEQ ID NO: 61 is a DNA fragment containing an anti-CD19 CAR that can be inserted into the TRC15-16 recognition sequence. The nucleic acid sequence of the parent template is shown.

[0208] SEQ ID NO: 62 is a DNA fragment containing an anti-CD19 CAR that can be inserted into the TRC17-18 recognition sequence. The nucleic acid sequence of the parent template is shown.

[0209] SEQ ID NO: 63 encodes a GFP protein that can be inserted into the TRC11-12 recognition sequence. 1 shows the nucleic acid sequence of the 7227 donor template.

[0210] SEQ ID NO: 64 encodes an anti-CD19 CAR that can be inserted into the TRC11-12 recognition sequence. 1 shows the nucleic acid sequence of the 7225 donor template.

[0211] SEQ ID NO: 65 encodes a GFP protein that can be inserted into the TRC15-16 recognition sequence. 1 shows the nucleic acid sequence of the 7228 donor template.

[0212] SEQ ID NO: 66 encodes an anti-CD19 CAR that can be inserted into the TRC15-16 recognition sequence. 7 shows the nucleic acid sequence of the 7226 donor template.

[0213] (definition) 1.1 References and Definitions The patent and scientific literature referenced herein establishes knowledge that is available to those skilled in the art. Issued U.S. patents, allowed applications, published foreign applications, and Ge Each reference containing an nBank database sequence is specifically and individually indicated by reference. The present invention is incorporated by reference to the same extent as if it were incorporated herein. .

[0214] This invention may be embodied in many different forms and is not limited to the embodiments set forth herein. Rather, these embodiments should not be construed as constituting an entire or part of the invention unless such disclosure is deemed to be thorough and complete. These and other related drawings are provided so that this invention will be fully understood by those skilled in the art. Features illustrated with respect to a particular embodiment may be incorporated into other embodiments and may be used interchangeably with other embodiments. Features shown may be omitted from the embodiment. Numerous variations and additions to the embodiments proposed herein will occur to those skilled in the art without departing from the invention. It will be clear to

[0215] Unless otherwise defined, all technical and scientific terms used herein are within the meaning of the present invention. The term "complex" has the same meaning as commonly understood by one of ordinary skill in the art to which it pertains. The terminology used in the description is for the purpose of describing particular embodiments only. It is not intended to limit the invention.

[0216] All publications, patent applications, patents, and other references mentioned herein are hereby incorporated by reference. and is incorporated herein in its entirety.

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

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

[0219] As used herein, the term "meganuclease" refers to a nucleic acid having more than 12 base pairs. It refers to an endonuclease that binds to double-stranded DNA at a recognition sequence. The recognition sequence of the meganuclease is 22 base pairs. The enzyme may be an endonuclease having, for example, DNA binding specificity, DNA cleavage activity, modified compared to native I-CreI with respect to DNA binding affinity or dimerization properties, It may refer to engineered variants of I-CreI. Methods for producing variants are well known in the art (see, e.g., International Publication No. 2007 As used herein, meganucleases, as heterodimers, include: or a pair of DNA-binding domains linked into a single polypeptide using a peptide linker It binds to double-stranded DNA as a "single-stranded meganuclease" that is used for "homing end The term "nuclease" is synonymous with the term "meganuclease." The meganucleases, when expressed in cells, particularly human T cells, are capable of carrying out the methods described herein. No adverse effects on cell viability or meganuclease cleavage activity as measured using the Substantially allowing cells to be transfected and maintained at 37°C without observing significant degradation. It is non-toxic.

[0220] As used herein, the term "single-chain meganuclease" refers to a single-chain meganuclease that is linked by a linker. refers to a polypeptide containing a pair of nuclease subunits linked by a single chain. Ganuclease has the following structure: N-terminal subunit-linker-C-terminal subunit The two meganuclease subunits generally do not have identical amino acid sequences and are non-identical. Therefore, single-stranded meganucleases usually recognize pseudopalindromic or cleaves non-palindromic recognition sequences. Single-chain meganucleases are actually dimers. Although not a single-chain heterodimer, it is referred to as a "single-chain heterodimer meganuclease." For clarity, unless otherwise specified, the term "meganuclease" is used. The term may refer to a dimeric or single-chain meganuclease.

[0221] As used herein, the term "linker" refers to a linker that connects two meganuclease subunits. Linker refers to an exogenous peptide sequence used to join multiple peptides into a single polypeptide. The polypeptide may have a sequence found in a naturally occurring protein or may have a sequence not found in any naturally occurring protein. The linker may be an artificial sequence that cannot be synthesized. The linker may be flexible and lack secondary structure. The linker may be a hydroxyl group or may have a tendency to form a particular three-dimensional structure under physiological conditions. Examples include U.S. Patent No. 8,445,251 and U.S. Patent No. 9,434,931. In some embodiments, the linker includes, but is not limited to, and having an amino acid sequence containing residues 154 to 195 of any one of SEQ ID NOs: 12 to 27. Good too.

[0222] As used herein, the term "zinc finger nuclease" or "ZFN" refers to a , restriction endonucleases, homing endonucleases, S1 nucleases, Mang Mung bean nuclease, pancreatic DNAse I, Micrococcal nuclease, and yeast HO endonuclease, fused to a nuclease domain derived from an endonuclease or exonuclease that does not It refers to a chimeric protein containing a zinc finger DNA-binding domain combined with a zinc finger. Nuclease domains useful for designing nucleases include FokI, FoM, Type II restriction endonucleases, including but not limited to the restriction enzyme StsI Additional Type II restriction endonucleases include those derived from WO 2006 / 024444. No. 7 / 014275, which is incorporated herein by reference in its entirety. The structure of the zinc finger domain is stabilized by the coordination of zinc ions. DNA-binding proteins containing zinc finger domains bind to DNA in a sequence-specific manner The zinc finger domain may be a naturally occurring sequence or may be derived by rational or experimental means. The protein was then redesigned to produce a protein that binds to a predetermined DNA sequence approximately 18 base pairs in length. For example, see U.S. Pat. No. 6,629,493, each of which is incorporated herein by reference in its entirety. Patent Nos. 5,789,538, 5,925,523, and 6,007,988 Nos. 6,013,453, 6,200,759, and WO 95 / 194 No. 31, No. 96 / 06166, No. 98 / 53057, No. 98 / 54311, Nos. 00 / 27878, 01 / 60970, 01 / 88197, and See, for example, US Pat. No. 02 / 099084. This engineered protein domain was cloned into the FokI gene. By fusing it to a nuclease domain such as nuclease, it can be expressed with genome-level specificity. NA cleavage can be targeted. Target sites, zinc finger proteins, and The choice of method for designing and constructing zinc finger nucleases is well known to those skilled in the art. and U.S. Patent Application Publication Nos. 20030232410 and 20050208489. , No. 2005064474, No. 20050026157, No. 20060188 987 and WO 07 / 014275, each of which is incorporated in its entirety. The present specification is incorporated by reference.

[0223] As used herein, the term "TALEN" refers to a restriction endonuclease, homogenous endonuclease (HMO) endonuclease, S1 nuclease, mung bean nuclease, pancreatic DNAs e I, micrococcal nuclease, and yeast HO endonuclease, but these Nuclease domains from endonucleases or exonucleases, including but not limited to or a DNA binding domain containing multiple TAL domain repeats fused to its active portion. For example, the entire contents of which are incorporated herein by reference. See Christian et al. (2010) Genetics 186:757-761. Nuclease domains useful for designing TALENs include FokI, Fo M, StsI, HhaI, HindIII, Nod, BbvCI, EcoRI, BglI and AlwI, derived from type II restriction endonucleases including, but not limited to, Additional type II restriction endonucleases include those described in WO 2007 / 01 4275. In some embodiments, the nuclease domain of the TALEN is The FokI nuclease domain or an active portion thereof. Used in the infection process by plant pathogens of the genus Xanthomonas These may be derived from the TALE (transcription activator-like effector) family of proteins. The AL domain repeats consist of 33–34 repeats with variable 12th and 13th amino acids. The amino acid sequence. Repeat variable dipeptide (RVD) These two positions, called dipeptides, are highly variable and specific nucleic acids Each base pair in the DNA target sequence exhibits a unique RVD-associated recognition. In some embodiments, the TALEN contacts a single TAL repeat. Contains 22 TAL domain repeats. TALENs induce nonspecific DNA cleavage. Two DNA recognition regions flanking a central region (i.e., a "spacer") are required. The term "spacer" in relation to EN is recognized by each monomer that constitutes a TALEN. and a nucleic acid sequence separating two nucleic acid sequences that are linked together. A TAL domain repeat is It may be the native sequence of a naturally occurring TALE protein, or may be a rational or experimentally determined sequence. They can also be artificially redesigned to produce proteins that bind to a given DNA sequence ( See, for example, Boch et al. (2009), each of which is incorporated herein by reference in its entirety. )Science326(5959):1509-1512and Moscow an d See Bogdanove (2009) Science 326 (5959): 1501 Examples of specific sequences and RVDs and their corresponding target nucleotides For information on how to engineer TALENs, see U.S. Patent Application Publication No. 2011014594 See also WO 2010 / 079430 and WO 2010 / 079430. In some embodiments, Each nuclease (e.g., FokI) monomer is a TAL enzyme that recognizes a different DNA sequence. It may be fused to an effector sequence and is inactive only when the two recognition sites are in close proximity. The monomers come together to create a functional enzyme.

[0224] As used herein, the term "compact TALEN" refers to a I-TevI ​​homing I-endonuclease, or MmeI, EndA, End1, I-BasI, I-Tev II, I-TevIII, I-TwoI, MspI, MvaI, NucA, NucM etc. Examples of compounds included in Table 2 of U.S. Patent Application No. 20130117869 include, but are not limited to, Any of the endonucleases listed in the literature (which are incorporated herein by reference in their entirety) 16-22 TAL domain repeats fused in any orientation to any part of the Compact TALENs are endonucleases that contain a DNA-binding domain. A dual target with an intervening DNA spacer that does not require dimerization for processing activity In some embodiments, compact TALENs can be used in a variety of applications, including 16-22 gene fragments. It contains TAL domain repeats.

[0225] As used herein, the term "CRISPR" refers to Cas9, Cpf1 or other suitable By hybridizing with caspases, such as nucleases, to their recognition sites in genomic DNA, Caspase-based endonucleases containing guide RNAs that direct caspase DNA cleavage The caspase component of CRISPR is responsible for RNA-guided DNA endonucleases. 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 another embodiment, the caspase is a class II, type V enzyme, such as Cpf1. The guide RNA consists of a direct repeat and a sequence complementary to the target recognition site. and a suitable guide sequence (often referred to as a spacer in the context of endogenous CRISPR systems). In certain embodiments, CRISPR is a method for targeting a directed repeat sequence present on a guide RNA. Direct repeat sequence (sometimes called tracr mate sequence) tracrRNA (transactivating CRNA) that is (fully or partially) complementary to In certain embodiments, the caspase further comprises a RISPR RNA. It cleaves one strand of oligonucleotide and acts as a nickase, leaving only one strand of target DNA. It can be mutated with respect to the corresponding wild-type enzyme so as to lack the ability to cleave. Non-limiting examples of caspase enzymes that function as caspases include the RuvC I catalytic domain and the have the D10A mutation in the genotype, or the H840A, N854A, or N863A mutation Cas9 enzyme is an example.

[0226] As used herein, the term "megaTAL" refers to an engineered sequence-specific homeostasis vector. Transcription activator-like effector (TALE) DNA binding with a transcription endonuclease It refers to a single-stranded nuclease containing a domain.

[0227] The terms "recombinant" or "engineered" as used herein with respect to proteins The nucleic acid encoding the protein and the genetic information for the cell or organism that expresses the protein. It means having an altered amino acid sequence as a result of the application of molecular engineering techniques. In this context, the terms "recombinant" or "engineered" refer to any substance that has been produced as a result of the application of genetic engineering techniques. Genetic engineering techniques include PCR and DNA sequencing. NA cloning techniques; transfection, transformation, and other gene transfer techniques; homologous recombination; These include, but are not limited to, site-directed mutagenesis; and gene fusion. By definition, a protein has the same amino acid sequence as a naturally occurring protein, but is clonal in a heterologous host. Proteins produced by scanning and expression are not considered recombinant or engineered. It will not be done.

[0228] As used herein, the term "wild type" refers to a population of alleles of the same type of gene. refers to the most common naturally occurring alleles (i.e., polynucleotide sequences) in the wild A polypeptide encoded by a wild-type allele has its original function. The term also refers to a polypeptide encoded by a wild-type allele. Genes (i.e., polynucleotides) and polypeptides are sequences that are identical to the wild-type sequence(s). a mutant or variant pair containing one or more mutations and / or substitutions compared to A wild-type allele or polypeptide is produced. can confer a normal phenotype to an organism, whereas a mutant or variant allele or polypeptide The peptides can, in some cases, confer a phenotypic change. can be distinguished from recombinant, engineered, or non-naturally occurring nucleases.

[0229] The term "modified" as used herein in reference to recombinant or engineered proteins , the amino acid residues in the recombinant sequence relative to a reference sequence (e.g., a wild-type or native sequence) It means an insertion, deletion, or substitution.

[0230] As used herein, the term "recognition sequence" refers to a sequence that is amplified by an endonuclease. Refers to the DNA sequence that is bound and cleaved. In the case of meganucleases, the recognition sequence is a four base pair sequence. Contains a pair of 9-base pair "half sites" in opposite orientation separated by a single stranded meganuclear In the case of enzymes, the N-terminal domain of the protein contacts the first half-site and the C The terminal domain contacts the second half-site. Cleavage by the meganuclease results in 4 This results in a 3' "overhang" of base pairs. "Overhang" or "sticky end" is a short single strand that can be generated by endonuclease cleavage of a double-stranded DNA sequence. The single-stranded DNA segment is a single-stranded meganuclease derived from I-CreI. In the case of clease, the overhang contains 10 to 13 bases of the 22 base pair recognition sequence. In the case of compact TALENs, the recognition sequence is the first sequence recognized by the I-TevI ​​domain. 1 CNNNGN sequence followed by a nonspecific spacer of 4 to 16 base pairs in length, followed by A second sequence of 16-22 bp in length recognized by the TAL effector domain is A compact TAL may contain a sequence of 5′ T bases (this sequence typically has a 5′ T base). Cleavage by EN results in a two-base pair 3' overhang. The sequence is the sequence for the guide RNA to bind and direct Cas9 cleavage, typically 1 6 to 24 base pairs. Perfect complementarity between the guide sequence and the recognition sequence is essential for cleavage. CRISPR cleavage is not necessarily required for blunting in response to caspases. end (class II, type II caspases, etc.) or overhanging end (class II, type V caspases, etc.) In those embodiments in which CpfI caspase is utilized, In this state, cleavage by the CRISPR complex containing it generates a 5' overhang, which In certain embodiments, a 5-nucleotide 5' overhang is generated. In addition, a PAM (protospacer adjacent motif) is located near the recognition sequence complementary to the guide RNA. It also requires sequence recognition. The exact sequence, length requirements for PAM, and the distance from the target sequence The distance varies depending on the caspase enzyme, but the PAM is usually located close to the target / recognition sequence. PAM sequences of specific caspase enzymes are known in the art. (See, for example, U.S. Pat. No. 8,233,999, each of which is incorporated herein by reference in its entirety. ,697,359 and U.S. Patent Application Publication No. 20160208243) The PAM sequence of a novel or engineered caspase enzyme can be determined by PAM depletion assays (e.g., Karvelis et al. (2017) Methods 12, the entire contents of which are incorporated herein by reference. 1-122:3-8) or other methods known in the art. In the case of zinc fingers, the DNA binding domain is typically 2 base pairs to 1 It recognizes an 18-bp recognition sequence containing a pair of 9-base pair "half sites" separated by 0 base pairs. Cleavage by the cleavage enzyme produces blunt ends or 5' overhangs of variable length (often 4 base pairs). Make a ring.

[0231] As used herein, the term "target site" or "target sequence" refers to a recognition site for a nuclease. A region of a cell's chromosomal DNA that contains a recognition sequence.

[0232] As used herein, the term "DNA binding affinity" or "binding affinity" refers to a The tendency of the cleavage enzyme to bind non-covalently to the reference DNA molecule (e.g., the recognition sequence or any sequence). The binding affinity is expressed as the dissociation constant K d As used herein, As shown, the K d statistically significantly higher than the reference nuclease A nuclease is "altered" if the ATP content increases or decreases by a significant percent change. It has binding affinity.

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

[0234] As used herein, the term "non-homologous end joining" or "NHEJ" refers to the joining of two non-homologous end Natural cells repair double-strand DNA breaks by directly joining identical DNA segments This refers to the process (e.g., Cahill et al. (2006), Front. Biosci. 1 1:1958-1976). DNA repair by non-homologous end joining corrects errors. Repair sites are prone to non-templated addition or deletion of DNA sequences. In some cases, cleavage at the target recognition sequence leads to NHEJ at the target recognition site. DNA produced by NHEJ after nuclease-induced cleavage of a target site in the coding sequence of a gene. A repair can introduce mutations into coding sequences, such as frameshift mutations, that disrupt gene function. Therefore, engineered nucleases can be used to effectively clone genes in cell populations. You can check out.

[0235] As used herein, a "chimeric antigen receptor" or "CAR" refers to a chimeric antigen receptor (CAR) that has specificity for an antigen. engineered receptors that are grafted onto or grafted onto immune effector cells (e.g., human T cells) Chimeric antigen receptors typically comprise at least an extracellular ligand-binding domain. or portion thereof and an intracellular domain containing one or more signaling domains and / or costimulatory domains. Includes the main.

[0236] In some embodiments, the extracellular ligand binding domain or portion is a monoclonal antibody. It is a form of single-chain variable fragment (scFV) derived from provides protection against cancer cells or other disease-causing cells or particles In some embodiments, the scFv is a surface-predominant epitope or antigen. In various embodiments, the extracellular ligand-binding domain is attached via a Kerr sequence. is specific for any antigen or epitope of interest. In some embodiments, the scFv The may be murine, humanized, or fully human.

[0237] The extracellular domain of the chimeric antigen receptor binds to the autoantigen-specific B cell receptor on B lymphocytes. These may contain autoantigens that can be recognized by the immune system (Payne et al. (2016), Science 3 53(6295):179-184), autoreactions in antibody-mediated autoimmune diseases Such CARs instruct T cells to specifically target and kill B lymphocytes. These may be referred to as chimeric autoantibody receptors (CAARs), and their use is encompassed by the present invention. do.

[0238] The extracellular domain of a chimeric antigen receptor may also bind to a naturally occurring ligand for an antigen of interest. The antigen-binding fragments may include fragments of naturally occurring ligands that retain the ability to bind to the antigen of interest. good.

[0239] The intracellular stimulatory domain transmits activation signals to immune effector cells after antigen binding. Such cytoplasmic signaling domains may include one or more cytoplasmic signaling domains. Domains include, but are not limited to, CD3ζ.

[0240] The intracellular stimulatory domain also transmits proliferation and / or cell survival signals after ligand binding. As used herein, "co-stimulatory" may include one or more intracellular costimulatory domains. The "domain" is a polypeptide that transmits intracellular proliferation and / or cell survival signals upon activation. Costimulatory domain activation occurs when two costimulatory domain polypeptides interact with each other. Activation can occur following mono-dimerization of a co-stimulatory domain containing a co-stimulatory domain. It also occurs after activation of a tract (e.g., a chimeric antigen receptor or an inducible regulatory construct). Generally, the costimulatory domain is a transmembrane costimulatory receptor, particularly a costimulatory receptor cellular domain. Such intracellular costimulatory domains may be derived from the intracellular portion. It may be any of the following, including but not limited to, CD27, CD28, CD8, 4-1B B(CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function Related antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83, N1, N6, or any combination thereof. may include:

[0241] Chimeric antigen receptors are designed to bind to 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 (α chain), p75 (β chain or γ chain), Fc receptor subunit chains of receptors (e.g., Fcγ receptor III), or CD8 alpha chains Alternatively, the transmembrane domain may be synthetic and may contain leucine. and valine.

[0242] The hinge region functions to connect 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 composed of all or part of the extracellular region of CD8, CD4, or CD28. or may be derived from all or part of a naturally occurring molecule, such as all or part of an antibody constant region. Alternatively, the hinge region may be a synthetic sequence that 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 identical to that of a T Immune effector cells (e.g., human T cells) that may or may not endogenously express CR The expression of exogenous TCRs on immune effector cells is Specific epitopes or antigens (e.g., cancer cells, or other disease-causing cells or particles) It can confer specificity for epitopes or antigens that are preferentially present on the surface of the molecule. Such exogenous T cell receptors may comprise an alpha chain and a beta chain, or The exogenous TCR useful in the present invention may comprise any TCR of interest. The antibody may have specificity for any antigen or epitope.

[0244] As used herein, the term "decreased expression" refers to the decrease in expression of a gene relative to a control cell. The term "reduced" refers to a reduction in the expression of endogenous T cell receptors on the cell surface of modified T cells. also show that endogenous polypeptides (i.e., endogenous polypeptides) are present at the cell surface when compared to a population of control cells. It can refer to a decrease in the proportion of cells in a cell population that express the T cell receptor (TCR). The degradation can be up to 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% %, 90%, 95%, 96%, 97%, 98%, 99%, or up to 100% Therefore, the term "reduced" refers to both partial and complete knockdown of endogenous T cell receptors. This includes both complete knockdown and complete knockdown.

[0245] As used herein with respect to both amino acid and nucleic acid sequences, the term "percent "Sequence identity", "sequence identity", "percentage similarity", "sequence similarity", etc. The similarity between the amino acid residues or nucleotides selected is maximized, and the same or similar residues or nucleotides are not included. The number of octides, the total number of residues or nucleotides, and the presence of gaps in the sequence alignment It refers to a measure of similarity between two sequences based on the alignment of the sequences, which is a function of length and Various algorithms and computers are available to determine sequence similarity using standard parameters. A computer program can be used. As used herein, sequence similarity is defined as For amino acid sequences, use the BLASTp program; for nucleic acid sequences, use the BLASTn program. Both were measured using a rhamnogram and were analyzed by the National Center for Biotechnology Information. technology information(www.ncbi.nlm.nih.g ov / ) and are described, for example, in Altschul et al. (1990), J. Mol. Biol.215:403-410;Gish and States(1993),N ature Genet.3:266-272;Madden et al. (1996), Meth Enzymol.266:131-141; Altschul et al. (1997), Nuc Leic Acids Res.25:33 89-3402; Zhang et al. (200 0), J. Comput. Biol. 7(1-2): 203-14. As used herein, the percent similarity of two amino acid sequences is determined by the BLASTp algorithm. The score is based on the following parameters for the algorithm: word size = 3; gap opening = 1; Start penalty = -11; Gap extension penalty = -1; Scoring matrix = BL As used herein, the percent similarity of two nucleic acid sequences is determined by the BLAS The score is based on the following parameters of the Tn algorithm: word size = 11; Gap Open Penalty = -5; Gap Widening Penalty = -2; Match Reward = 1; Miss Match penalty = -3.

[0246] As used herein with respect to modifications of two proteins or amino acid sequences, "to" means "to" a protein or amino acid sequence. The term "responsive to" means that a particular modification of a first protein is the same as a modification of a second protein. Used to indicate substitution of amino acid residues, so that two proteins have the same standard sequence When subjected to alignment (for example, using the BLASTp program), The amino acid position of the modification of one protein corresponds to the amino acid position of the modification of the second protein. Thus, amino acid "A" of residue "X" of the first protein The modification to X and Y is performed when residue X and residue Y match each other in a sequence alignment. The residues "Y" in the second protein may be different in number, despite the fact that " corresponds to a modification to the amino acid "A".

[0247] As used herein, the term "recognition half-site," "recognition sequence half-site," or simply " The term "half site" refers to the moiety of a homodimeric or heterodimeric meganuclease. recognized by a nucleomer or by one subunit of a single-chain meganuclease It refers to a nucleic acid sequence within a double-stranded DNA molecule.

[0248] As used herein, the term "hypervariable region" refers to amino acids having relatively high variability. It refers to a localized sequence within a meganuclease monomer or subunit that contains an acid. The variable region may be about 50-60 contiguous residues, about 53-57 contiguous residues, or preferably In some embodiments, the hypervariable region residues can comprise about 56 residues. It may correspond to positions 24 to 79 or positions 215 to 270 of any one of Nos. 12 to 27. The variable region may contain one or more residues that contact the DNA bases in the recognition sequence. The hypervariable region can be modified to alter the base preference of the monomer or subunit. The region also binds to the DNA backbone when the meganuclease associates with the double-stranded DNA recognition sequence. Such residues may be modified to enhance the DNA backbone and target recognition. The binding affinity of the meganuclease to the recognition sequence can be altered. In embodiments, the hypervariable region can include 1 to 20 residues that exhibit variability, and the bases They may be modified to affect selectivity and / or DNA binding affinity. In this embodiment, the hypervariable region contains about 15 to 18 residues that exhibit variability, and is responsible for base preference and / or or may be modified to affect DNA binding affinity. The variable residues within the hypervariable region are at positions 24, 26, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 5 , 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 In another embodiment, the variable residues within the hypervariable region correspond to SEQ ID NOs: 12-2. 7 any one of positions 215, 217, 219, 221, 223, 224, 229, 2 One or more of 31, 233, 235, 237, 259, 261, 266, and 268 corresponds to the number.

[0249] As used herein, "T cell receptor alpha gene" or "TCR alpha gene" The terms are used interchangeably and refer to T cell receptor alpha subunits. T cell receptor alpha refers to the locus within the cell. After rearrangement, the T cell receptor alpha gene is expressed as an endogenous promoter, rearranged V and J segments, endogenous splice donor region positions, introns, endogenous splice acceptor sites, and subunit coding sequences. The TRAC locus includes the chromosome. See, e.g., Figure 1.

[0250] As used herein, "intron within the T cell receptor alpha gene" and "target 5 The term 'intron' refers to the rearranged T cell receptor alpha gene, as shown in Figure 1. 5' upstream of TRAC exon 1, 3' downstream of the V and J segments in the gene refers to an intron located in the nucleus, and is divided into an endogenous splice donor site and an endogenous splice acceptor site. The target 5' intron is a sequence comprising SEQ ID NO: 3 and the It is possible to have functional variants thereof that retain the nuclease recognition sequence involved.

[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. Wild-type sequence identified by NCBI Gen ID NO. 28755 and its function Includes sexual variants.

[0252] As used herein, the term "endogenous splice donor site" refers to an endogenous TCR antigen. 3' downstream of the lufa gene promoter and rearranged V and J segments, The term refers to a naturally occurring splice donor site located 5' upstream of the target intron. The "endogenous splice acceptor site" refers to the site 3' downstream of the target intron and the TRA C refers to the naturally occurring splice acceptor site immediately 5' upstream of exon 1. The endogenous splice donor site and endogenous splice acceptor site were determined as described by Desmet et al. Nucleic Acid Research (2009) 37(9):e67) can be identified within the gene by methods known in the art, such as those described in "Functionality" of endogenous splice donor and endogenous splice acceptor sites The term "pairing" refers to the ability to perform splicing of the intervening intron sequence. Point.

[0253] As used herein, the term "exogenous splice acceptor site" refers to an exogenous splice acceptor site that is an exogenous splice acceptor site of interest. The splice acceptor site included in the target sequence is introduced into the target 5' intron. The exogenous splice acceptor site is naturally present in the human T-cell receptor alpha gene. or a splice acceptor sequence that does not naturally occur in the gene. (e.g., consensus sequences or heterologous sequences). The scepter site is required to facilitate intron splicing, and is used to identify exogenous branch sites. Such branching sites may further comprise a branching site that is naturally present in the T cell receptor alpha gene. or branch site sequences that do not naturally occur in the gene (e.g., The nucleic acid sequence may contain a heterologous sequence (sus sequence or heterologous sequence).

[0254] "Recombinant DNA construct", "recombinant construct", "expression cassette", "Expression construct," "chimeric construct," "construct," and "recombinant The terms "DNA fragment" and "DNA fragment" are used interchangeably herein and may refer to single-stranded or double-stranded polynucleotides. Recombinant constructs contain regulatory sequences and sequences not found together in nature. Artificial single- or double-stranded polynucleotides, including but not limited to, sequences and coding sequences. For example, a recombinant DNA construct may contain multiple copies of a gene from different sources. regulatory and coding sequences that are derived from the same source and in a manner different from that found in nature Such a construct may contain regulatory and coding sequences arranged in a manner similar to that described above. It may be used or may be used in combination with a vector.

[0255] As used herein, a "vector" or "recombinant DNA vector" refers to a vector that is capable of expressing a given host cell. A replication system and sequence capable of transcribing and translating a sequence encoding a polypeptide in If a vector is used, the choice of vector may be The choice will depend 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 vectors of the present invention. There are known methods in the art that are suitable for delivering genes encoding meganucleases of the present invention to target cells. These include, but are not limited to, other vectors known in the art. Successfully transform and select host cells containing either the isolated nucleotide or nucleic acid sequence. and are familiar with the genetic elements that must be present on a vector in order for it to propagate.

[0256] As used herein, the term "vector" may also refer to a viral vector. The vectors include retroviral vectors, lentiviral vectors, and adenoviral vectors. vectors, and adeno-associated viral vectors (AAV). Not determined.

[0257] As used herein, a "polycistronic" mRNA is one that contains two or more coding sequences (i.e., A single messenger R contains a cistron (i.e., a cistron) that encodes two or more proteins. Polycistronic mRNAs contain IRES elements, T2A elements, P2A elements, E Two or more sequences from the same mRNA molecule, including but not limited to, F2A elements, and F2A elements. The gene may contain any element known in the art that allows for translation of the gene. Cut.

[0258] As used herein, "human T cells" or "T cells" refer to T cells derived from a donor, particularly a human donor. It refers to isolated T cells. T cells and cells derived from them have not been passaged in culture. isolated T cells, T cells passaged and maintained under cell culture conditions without immortalization, and Examples of such T cells include T cells that can be immortalized and maintained indefinitely under cell culture conditions.

[0259] As used herein, "human natural killer cells" or "human NK cells" or "Natural Killer Cells" "Natural killer cells" or "NK cells" are cytotoxic lymphocytes important to the innate immune system. NK cells are a type of lymphocyte that play a key role in the adaptive immune response of vertebrates. NK cells have a similar role to that of cytotoxic T cells. They respond quickly to virus-infected cells. , responds to tumor formation and acts approximately 3 days after infection.

[0260] As used herein, a "control" or "control cell" refers to a control cell that is a genetically modified cell of a genotype or A control cell refers to a cell that provides a reference point for measuring phenotypic changes. (a) wild-type cells, i.e., the same as the starting material for the genetic changes that resulted in the genetically modified cells; (b) cells of the same genotype as the genetically modified cells, but transformed with a construct (i.e., with a construct that has no known effect on the property of interest) or (c) a cell that is genetically identical to the genetically modified cell but has an altered genotype. or exposed to conditions or stimuli that induce the expression of a phenotype, or to further genetic modifications. The cells may include cells that are not present in the body.

[0261] As used herein, the term "treatment" or "treatment of a subject" refers to the treatment of a subject with a disease. For example, the subject may have a disease such as cancer. The treatment has the potential to be an immunotherapy for the treatment of disease. These include preventing the onset or recurrence of disease, alleviating symptoms, and directly or indirectly reducing the pathology of disease. reduction in outcomes, slowing of disease progression, improvement or palliation of disease state, and remission or improved prognosis In some embodiments, the genetic material described herein includes, but is not limited to, The modified cells are administered during treatment in the form of a pharmaceutical composition of the present invention.

[0262] The terms "effective amount" or "therapeutically effective amount" refer to a beneficial or desirable biological and / or A therapeutically effective amount refers to an amount sufficient to produce a clinical result. Depending on the disease and its severity, and the age, weight, physical condition and responsiveness of the subject to be treated, It changes depending on the situation.

[0263] As used herein, the term "cancer" refers to an abnormality that causes a malignant growth or tumor. Any neoplastic disease (whether invasive or metastatic) characterized by uncontrolled cell division. It should be understood to include.

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

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

[0266] As used herein, the term "sarcoma" refers to a tumor composed of a substance like embryonic connective tissue, A tumor generally composed of closely packed cells embedded in a fibrous, heterogeneous, or homogeneous substance Refers to...

[0267] As used herein, the term "melanoma" refers to a tumor of the melanocytic system of the skin and other organs. This refers to a tumor that arises from

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

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

[0270] As used herein, the recitation of a numerical range for a variable includes any one of the values ​​within that range. The present invention is intended to convey that the present invention may be practiced with various variables. For a continuous variable, the variable may be equal to any integer value within the numeric range, including the endpoints of the range. Similarly, for variables that are inherently continuous, the variable is a numeric range that includes the endpoints of that range. It may be equal to any real value within the range. For example, without limitation, it may have a value between 0 and 2. A variable described as "one" may take on the values ​​0, 1, or 2 if the variable is discrete in nature. and if the variable is continuous in nature, the values ​​0.0, 0.1, 0.01, 0.00 It can take the value 1 or any other real value between 0 and 2 inclusive.

[0271] 2.1 Principles of the invention The present invention relates, in part, to the use of a sequence of interest (e.g., an exogenous splice acceptor site and / or polynucleotide). A signal) in the target 5' intron of the T cell receptor alpha gene Insertion into the cleavage site allows the production of TCR- cells only if the insert is present In the absence of an insert, the nuclease-modified insert The CAR promoter is simply removed and the endogenous gene is expressed. In examples where the TCR- / CAR sequence is included, the invention provides a method for identifying TCR- / CAR- The present invention provides a method for producing a population of CAR+ cells. The desired sequence can be expressed in the same manner.

[0272] In contrast, conventional nuclease-based approaches to generate modified T cells of the endogenous splice acceptor site 5' upstream of RAC and / or TRAC exon 1 Targets coding sequences, resulting in the creation of indels that disrupt protein expression. NHEJ at the cleavage site of the cleavage enzyme results in a significant proportion of TCR- / CAR- cells. Highly mixed populations of TCR-cells can be generated.

[0273] Thus, by reducing the need to purify a mixed population of TCR-cells, the present invention Allogeneic CAR T cells expressing antigen-specific CARs and with reduced expression of endogenous TCRs This provides a simplified method for producing a population of cells that can be administered to an allogeneic subject. When administered, these drugs showed reduced or no induction of graft-versus-host disease (GVHD). Furthermore, the inclusion of a 2A element in the exogenous sequence of interest may prevent exogenous promoter activity. The promoter of the endogenous T cell receptor alpha gene is not the promoter of the α-terminal T cell receptor. In this way, the expression of a polypeptide such as a CAR is controlled by the TCR It is regulated by a T cell feedback mechanism normally associated with its expression.

[0274] 2.2 Recognizes and cleaves the recognition sequence within the target 5' intron of the T cell receptor alpha gene Nuclease for In the art, site-specific nucleases are used to isolate DNA fragments in the genome of living cells. These DNA breaks can lead to mutagenic NHEJ repair or transcription. resulting in permanent alteration of the genome via homologous recombination with transgenic DNA sequences. NHEJ is known to induce mutagenesis at the cleavage site, resulting in the formation of allelic residues. NHEJ-associated mutagenesis can result in premature stop codons, aberrant non- Inactivating the allele through the generation of a frameshift mutation that produces a functional protein or may induce mechanisms such as nonsense-mediated mRNA decay. The use of nucleases to induce selective mutagenesis was used to identify specific mutations present in the wild-type allele. Can be used to target mutations or sequences, and to induce double-strand breaks at the target locus The use of nucleases to induce genomic DNA fragments is particularly useful in transgenic mice that are flanked by sequences homologous to the genomic target. It is known to stimulate homologous recombination of nicked DNA sequences. The nucleic acid sequence can be inserted into the target locus. Such exogenous nucleic acids can be, for example, chimeric It may encode an antigen receptor, an exogenous TCR, or any sequence or polypeptide of interest. good.

[0275] In different embodiments, a variety of different types of nucleases are useful in practicing the present invention. In one embodiment, the present invention can be practiced using engineered meganucleases. In another embodiment, the present invention can be used to detect CRISPR nucleases or CRISPR nicksers. This can be done using CRISPR and CRISPR enzymes that recognize specific DNA sites. Methods for generating RISPR nickases are described in the art, e.g., Ran et al. (2013) N at Protoc. 8:2281-308. In another embodiment, The present invention can be practiced using TALENs or compact TALENs. Methods for generating TALE domains that bind to DNA sites in a gene encoding a TALE are known in the art, e.g., Re Known in yon et al. (2012) Nat Biotechnol. 30:460-5 In another embodiment, the present invention relates to a method for producing a protein comprising a zinc finger nuclease (ZFN). In a further embodiment, the present invention can be implemented using megaTAL. can be implemented.

[0276] In a preferred embodiment, the nuclease used to practice the present invention is a single-stranded megakaryon The single-chain meganuclease is a nuclease consisting of a pair of nucleotides linked by a linker peptide. It contains an N-terminal subunit and a C-terminal subunit. It recognizes one half of the sequence (i.e., the recognition half-site) and the site of DNA cleavage is the site of two subunits. The DNA strand break occurs in the center of the recognition sequence near the boundary of the DNA fragment. The cleavage is correlated by four base pair pairs to generate a four base pair 3' single-stranded overhang. Be killed.

[0277] In some instances, the engineered meganucleases of the invention comprise a TRC11-12 recognition sequence (SEQ ID NO: 4). The enzymes 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 meganuclease of the invention may comprise the TRC15-16 recognition sequence ( The engineered meganuclei are engineered to recognize and cleave the nucleotide sequence (SEQ ID NO: 6). The enzymes 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 meganuclease of the invention may comprise the TRC17-18 recognition sequence ( The engineered meganuclei are engineered to recognize and cleave the nucleotide sequence (SEQ ID NO: 8). The enzymes 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 meganuclease of the invention may comprise the TRC19-20 recognition sequence ( The engineered meganucleoside is engineered to recognize and cleave the nucleotide sequence (SEQ ID NO: 10). The enzymes are collectively referred to herein as "TRC19-20 meganucleases." Exemplary TRC19-20 meganucleases are provided in SEQ ID NOs: 24-27.

[0281] The engineered meganuclease of the present invention comprises a first hypervariable (HVR1) region. and a second subunit comprising a second hypervariable (HVR2) region. The first subunit binds to the first recognition half-site (TRC11, TRC15) of the recognition sequence. The second subunit binds to the recognition sequence (e.g., TRC17, or TRC19 half site), and the third subunit binds to the recognition sequence (e.g., TRC18, TRC19 half site). The second recognition half site of the row (e.g., TRC12, TRC16, TRC18, or TRC19) The recombinant meganuclease binds to the single-chain meganuclease In some embodiments, the first and second subunits comprise an HVR1 region, and the first HVR2 region The first subunit that binds to the fusiform cells is located as the N-terminal subunit, HVR2 The second subunit contains the C-terminal region and binds to the second half-site. In another embodiment, the first and second sub-units are oriented so as to be disposed in a The first subunit contains the HVR1 region and binds to the first half-site at the C-terminus. The second half-site is arranged as a subunit, contains the HVR2 region, and binds to the second half-site. The present invention is oriented such that the subunit of Exemplary TRC11-12 meganucleases of the present invention are provided in Table 1. C15-16 meganucleases are provided in Table 2. Exemplary TRC17-18 meganucleases of the present invention Ganucleases are provided in Table 3. Exemplary TRC19-20 Meganucleases of the Invention are provided in Table 4.

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

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

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

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

[0286] 2.3 Methods for producing genetically modified cells After rearrangement, the human T-cell receptor alpha gene contains many elements. Without being bound by theory, these elements include, from 5' to 3', the endogenous promoter, The synthesized V and J segments, endogenous splice donor sites, introns (all i.e., target 5' intron), endogenous splice acceptor site, and exon and TRAC gene containing alpha subunit encoding and interspace introns See, for example, Figure 1.

[0287] The invention disclosed herein provides a method for producing 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. , obtained from many sources including thymus tissue, tissue from the site of infection, ascites, pleural effusion, 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, T cells can be any T cell line known to those of skill in the art. It is obtained from a unit of blood collected from a subject using any number of techniques known in the art. 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 The 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 target acceptor site. The nucleic acid sequence is a sequence set forth in SEQ ID NO: 3 or a sequence having at least 7 sequences identical to the nucleic acid sequence set forth in SEQ ID NO: 3. 5%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity and includes a sequence that contains a recognition sequence for an engineered nuclease described herein.

[0289] In some embodiments, the exogenous sequence of interest is an engineered meganuclease, a zinc finger Finger nuclease, TALEN, compact TALEN, CRISPR nuclease or at double-strand 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' site of the endogenous TCR alpha gene promoter. The 5' upstream of the target intron is located in the rearranged V and J segments. 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 Figure 1.

[0291] In specific embodiments, the engineered nucleases disclosed herein inhibit endogenous splices. It does not modify either the splice donor site or the endogenous splice acceptor site, but This is because both sites must retain functionality for practicing the present invention. In some embodiments, the endogenous splice donor site and / or the endogenous splice acceptor site The -sites retain the ability of each site to pair with the other and splice the intron (i.e., Therefore, as used herein, 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 the endogenous splice acceptor site. It has the ability to remove Ron.

[0292] In certain embodiments, the sequence of interest can include an exogenous splice acceptor site. As used herein, the terms "exogenous" or "heterologous" with respect to a nucleotide sequence The term "naturally occurring" refers to a plant that is purely synthetic, derived from an exogenous species, or, if derived from the same species, is intentionally Substantially altered from the naturally occurring form of the composition and / or genomic locus by significant human intervention Therefore, an exogenous splice acceptor site is a purely synthetic sequence. splice acceptor sites or human genomes with altered sequences or genomic loci It may also be a splice acceptor site from the

[0293] In a specific embodiment, the exogenous splice acceptor site is a splice acceptor site that occupies an intervening intron sequence. It can partner with an endogenous splice donor site to splice out. In this way, the exogenous splice acceptor site is aligned with the endogenous splice donor site. By competing with the endogenous splice acceptor site for target 5' endonucleases It may interfere with the natural splicing of RON.

[0294] In various embodiments, the exogenous sequence of interest comprises a coding sequence for a protein of interest. It is contemplated that the coding sequence may be for any protein of interest. can be.

[0295] In certain embodiments, the exogenous sequence of interest comprises a nucleic acid sequence encoding a CAR. Generally, a CAR of the present disclosure comprises at least an extracellular domain and an intracellular domain. In some embodiments, the extracellular domain is also referred to as a ligand-binding domain or moiety. In some embodiments, the target-specific binding element comprises an intracellular domain or a cytoplasmic domain. The domains comprise at least one costimulatory domain and one or more signaling domains, e.g., CD3ζ. In other embodiments, the CAR comprises a signaling domain such as CD3ζ. The cells may contain only one or more constructs on separate constructs that are expressed within the cells. The antibody may contain a number of costimulatory domains.

[0296] In some embodiments, a CAR useful in the present invention comprises a ligand binding domain or The ligand-binding domain contains an extracellular target-specific binding element, also called the ligand-binding moiety. The choice of ligand depends on the type and number of ligands that define the surface of the target cell. The dopamine-binding domain acts as a cell surface marker on target cells associated with specific disease states. The CAR may be selected to recognize a ligand that binds to the CAR. Examples of cell surface markers that can act as ligands for the domain include viral, bacterial and These include those associated with bacterial and parasitic infections, autoimmune diseases, and cancer cells. In some embodiments, the CAR is a desired ligand that specifically binds to an antigen on a tumor cell. The binding moiety is engineered to target tumor-specific antigens of interest. For purposes of this disclosure, "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 binds any antigen or are specific for an epitope, particularly any tumor antigen or epitope of interest. In some embodiments, the target antigen is ErbB2 (HER2 / neu), cancer tumor infantile 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, ductal epithelial mucin, gp36, TAG-72, glycosphingolipid, 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, Prostase-specific antigen (PSA), PAP, NY-ESO-1, LAGA-la, p5 3. Prostein, PSMA, surviving and telomerase, Prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, Ephrin B2, insulin growth factor (IGFl)-1, IGF-II, IGF-1 receptor, Mesothelin, a major histocompatibility complex (MHC) molecule that presents tumor-specific peptide epitopes Addition of 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigen, and fibronectin Additional domain A (EDA) and additional domain B (EDB) of tenascin-C and the Al domain of tenascin-C. tumor-associated surface antigens such as fibroblast-associated protein (fap); CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD38, CD1 23, CD133, CD138, CTLA-4, B7-1(CD80), B7-2(CD 86), endoglin, major histocompatibility complex (MHC ) molecule, BCMA (CD269, TNFRSF17), CS1, or HIV-specific antigen (H Virus-specific surface antigens such as EBV-specific antigens, CMV-specific antigens, E HPV-specific antigens such as E6 or E7 oncoproteins, Russ virus-specific antigens, influenza As well as influenza virus-specific antigens, 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 binds to an autoantibody on a B lymphocyte. These include autoantigens that can be recognized by autospecific B cell receptors (Payne et al., 2011). 6)See Science, Vol.353(6295):179-184 , T cells to specifically target and kill autoreactive B lymphocytes in antibody-mediated autoimmune diseases. Such CARs are sometimes called chimeric autoantibody receptors (CAARs). be.

[0299] In some embodiments, the extracellular domain of the chimeric antigen receptor is a chimeric antigen receptor for an antigen of interest. A naturally occurring ligand, or a naturally occurring ligand that retains the ability to bind to an antigen of interest. It may also contain fragments of

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

[0301] The hinge region functions to connect 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 composed of all or part of the extracellular region of CD8, CD4, or CD28. or may be derived from all or part of a naturally occurring molecule, such as all or part of an antibody constant region. Alternatively, the hinge region may be a synthetic sequence that 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.

[0302] The intracellular signaling domain of CAR is responsible for the normal effector functions of the cell in which it is placed. Responsible for the activation of at least one of the functions and / or pathways of proliferation and cell survival. The term "effector function" refers to a specialized function of a cell. The effector function of T cells is The activity may be, for example, cytolytic activity or helper activity including secretion of cytokines. Intracellular signaling domains such as CD3ζ activate cells in response to binding of the extracellular domain. As discussed, an activation signal can provide, for example, a cell It is possible to induce cellular effector functions such as cytolytic activity or cytokine secretion.

[0303] The intracellular domain of CAR, after binding to the extracellular domain, regulates cell proliferation, cell survival, and and / or one or more intracellular co-stimulatory signals that promote cytokine secretion. Such intracellular costimulatory domains can include, but are not limited to, However, 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 N6 specifically Examples of suitable ligands include those known in the art that bind to such ligands.

[0304] CARs can be specific for any type of cancer cell. Such cancers include carcinomas, lymphomas, and tumors, sarcomas, blastomas, leukemia, 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's lymphoma In certain embodiments, cancers of B-cell origin and These include B-lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, and B-cell non-homologous leukemia. These include, but are not limited to, lymphoma, and multiple myeloma.

[0305] The sequence of interest may further encode an exogenous T cell receptor (TCR). The exogenous T cell receptor may comprise an alpha chain and a beta chain, or alternatively, a gamma chain and The exogenous TCR useful in the present invention may comprise any antigen or may have specificity for an epitope.

[0306] In other embodiments, the sequence of interest is a wild-type or modified version of an endogenous gene of interest. Can be coded.

[0307] The sequence of interest may include an IRES element, as well as T2A, P2A, E2A, and F2A elements. two more A elements from the same mRNA molecule, including but not limited to two A elements, It may contain elements or peptides known in the art that allow for translation of the gene. In specific embodiments, such elements in the exogenous sequence of interest are It can be placed 5' upstream of a nucleic acid sequence encoding a protein (e.g., a CAR).

[0308] The exogenous sequences of interest described herein may further comprise additional regulatory sequences. For example, the sequence of interest may be a homologous recombination enhancer sequence, a Kozak sequence, a polyadenylation sequence, or a nucleotide sequence. sequence, transcription termination sequence, selection marker sequence (e.g., antibiotic resistance gene), origin of replication, etc. The sequences of interest described herein may also contain at least one nuclear localization Examples of nuclear localization signals are known in the art (e.g., , Lange et al., J. Biol. Chem., 2007, 282:5101-5105 See the reference.

[0309] In specific embodiments, the exogenous sequence of interest is a polyadenylation sequence or polyA signal. Thus, the sequence of interest encodes a protein of interest (e.g., a CAR). A poly(A) signal located 3' downstream of the sequence can be included. In this way, T cells Transcription of the receptor alpha gene, specifically the coding sequence of the TRAC locus, is initiated by a poly(A) signal. This interferes with the expression of the T cell receptor alpha subunit.

[0310] In some examples of the present invention, the exogenous sequence of interest may be 5' to 3' exogenous splice amino acid sequence. acceptor site, 2A element or IRES element, coding sequence for the protein of interest, and polynucleotides In certain instances, the subject exogenous sequence includes, from 5' to 3', an exogenous sequence. Price acceptor site, 2A element or IRES element, CAR or exogenous T cell receptor In some instances, the exogenous sequence of interest comprises an exogenous The fragment may further comprise an exogenous branch site located 5' upstream of the exogenous splice acceptor site. In various embodiments of the present invention, the 2A element may be a T2A element, a P2A element, an E2A element, or It may be, but is not limited to, an F2A element.

[0311] The engineered nucleases of the present invention may be used in the form of proteins or, preferably, engineered Such nucleic acids can be delivered to cells as DNA (e.g., For example, circular or linearized plasmid DNA or PCR products) or RNA (for example, m The engineered nuclease coding sequence may be delivered in the form of DNA. In embodiments, the nuclease gene is operably linked to a promoter to promote transcription of the nuclease gene. Mammalian promoters suitable for the present invention include promoters from cytomegalovirus (Cytomegalovirus) and cytomegalovirus (Cytomegalovirus). The CMV early promoter (Thomsen et al. (1984), Proc Natl Acad Sci USA.81(3):659-63) or SV40 early promoter ( Benoist and Chambon(1981),Nature.290(580 4):304-10), as well as tetracycline-inducible promoters. Dingermann et al. (1992), Mol Cell Biol. 12(9): 4038-45).

[0312] In some embodiments, the gene encoding the engineered nuclease is located in the genome of the cell. The engineered nuclease-encoding mRNA is then transferred to the cells, reducing the likelihood of integration. Such mRNA encoding the engineered nuclease is delivered to the cells in vitro. The vector may be produced using methods known in the art, such as rotranscription. In some embodiments, the mRNA is capped using 7-methyl-guanosine. The mRNA may be polyadenylated.

[0313] In certain embodiments, mRNA encoding an engineered nuclease of the invention is expressed in cells Polycistronic mRNAs encoding two or more nucleases simultaneously expressed within a single cell. Polycistronic mRNAs may target different recognition sequences within the same target gene. Alternatively, a polycistronic mRNA may encode two or more nucleases of the invention. NA is a nuclease that contains at least one nuclease described herein and a nuclease that is located in the same gene. target distinct recognition sequences or to generate cleavage sites in both genes. At least one additional nuclease targeting a second recognition sequence located in a second gene. The polycistronic mRNA may contain an IRES element, a T2A element, a P2A element, E2A element, and F2A element from the same mRNA molecule, The term "cistron" is used to refer to a gene encoding a cistron, which is known in the art to allow translation of two or more genes (i.e., cistrons) of the same gene. It can contain any element that is

[0314] Purified nuclease proteins can be delivered to cells to cleave genomic DNA. This allows the desired sequence to be isolated by a variety of different mechanisms known in the art. This allows homologous recombination or non-homologous end joining at the cleavage site.

[0315] In some embodiments, an engineered nuclease protein or an engineered nuclease The DNA / mRNA encoding the enzyme is cell-permeable to facilitate its uptake into cells. The peptide or targeting ligand is linked to a cell-penetrating peptide known in the art. Examples include polyarginine (Jearawiriyapaisarn et al. (2008) M ol Ther.16:1624-9), TAT peptide derived from HIV virus (Hud ecz et al. (2005), Med. Res. Rev. 25:679-736), MPG(S Imeoni et al. (2003) Nucleic Acids Res. 31:2717-2 724), Pep-1 (Deshayes et al. (2004) Biochemistry 4 3:7698-7706, and HSV-1 VP-22 (Deshayes et al. (2005 ) Cell Mol Life Sci. 62:1839-49. In the form of an engineered nuclease or DNA / encoding an engineered nuclease mRNA is bound to nuclease proteins / DNA / mRNA by covalent or non-covalent bonds. A is expressed on target cells so that it binds to and is internalized by the target cells. 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. (part of the molecule) can be covalently or non-covalently linked (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 Opinion 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 can be isolated using methods known in the art. Covalently or preferably non-covalently linked to nanoparticles or to such nanoparticles. Encapsulated within particles (Sharma et al. (2014) Biomed Res Int 2014). Nanoparticles are particles 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. Attaching or encapsulating 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, cleaving the target recognition sequence. The surface of such nanoparticles forms a core-shell nanoparticle. Therefore, polymers or lipids (e.g., chitosan, cationic polymers, or cationic lipids) ), the surface of which may be further modified with additional compounds that enhance cellular delivery and uptake of the payload. Adding functionalities (Jian et al. (2012) Biomaterials. 33(30): 7621-30). The nanoparticles may further be used to target the nanoparticles to the appropriate cell type and / or to induce cellular responses. To increase the likelihood of cellular uptake, the compound may be advantageously conjugated to a targeting molecule. Examples include antibodies specific for cell surface receptors and natural ligands (or natural ligands) for cell surface receptors. Some of the natural ligands are also mentioned.

[0317] In some embodiments, the engineered nuclease or a nucleic acid encoding the engineered nuclease is The DNA / mRNA is encapsulated in liposomes or transported using cationic lipids. (e.g., Lipofectamine, Life Technologies) Logies Corp., Carlsbad, CA; Zuris et al. (2015) Nat Biotechnol.33:73-80;Mishra et al. (2011) J Drug Deliv. 2011:863734). Liposomes and Lipoplexes The drug formulation protects the payload from degradation and delivers it to the cells through fusion with and / or disruption of the cell membrane. Cellular uptake and delivery efficiency can be enhanced.

[0318] In some embodiments, an engineered nuclease protein or an engineered nuclease The DNA / mRNA encoding the enzyme is encapsulated within a polymer scaffold (e.g., PLGA). or complexed using cationic polymers (e.g., PEI, PLL) (Ta mboli et al. (2011) Ther Deliv.2(4):523-536).

[0319] In some embodiments, an engineered nuclease protein or an engineered nuclease The DNA / mRNA encoding the enzyme is combined with amphiphilic molecules that self-assemble into micelles. (Tong et al. (2007) J Gene Med. 9(11):956-66). Molecular micelles prevent aggregation, mask charge interactions, and reduce extracellular nonspecific interactions. A micellar shell formed from a hydrophilic polymer (e.g., polyethylene glycol) that can be reduced May include:

[0320] In some embodiments, an engineered nuclease protein or an engineered nuclease The DNA / mRNA encoding the enzyme is packaged in an emulsion or nanoemulsion for delivery to cells. The emulsion is formulated into an emulsion (i.e., with an average particle diameter of less than 1 nm). The term "aqueous phase" refers to the formation of a water-immiscible phase when mixed with an aqueous phase, with non-polar residues (e.g., They form as a result of hydrophobic forces that direct the long hydrocarbon chains away from water and the polar head groups towards water. and oil-in-water, water-in-oil, water-in-oil-in-water, or oil-in-water-in-oil, including lipid structures that can These other lipid structures and These include unilamellar, paucilamellar, and multilamellar lipid vesicles, micelles, and Emulsions include, but are not limited to, aqueous and lamellar phases. Emulsions are often also called emulsions. For example, each of the surfactants is incorporated herein by reference in its entirety. Nos. 2002 / 0045667 and 2004 / 0045667, which form part of the present application. 43041, as well as U.S. Patent Nos. 6,015,832, 6,506,803, and As described in US Pat. Nos. 6,635,676 and 6,559,189, nanoemulsifiers The leuconazole formulation is well known.

[0321] In some embodiments, an engineered nuclease protein or an engineered nuclease The DNA / mRNA encoding the enzyme is then synthesized into a multifunctional polymer conjugate, a DNA dendrimer. and polymer dendrimers, and can be covalently associated or non-covalently attached to the polymer dendrimers ( Mastorakos et al. (2015) Nanoscale.7(9):3845-56; Cheng et al. (2008) J Pharm Sci. 97(1):123-43). The creation of drimers allows for control of payload volume and size, providing high payload capacity. Furthermore, the presentation of multiple surface groups can be utilized to improve stability and reduce non-specific binding. Interactions can be reduced.

[0322] In some embodiments, the gene encoding the engineered nuclease and / or sequence of interest is The gene is introduced into the cell using a viral vector. Such vectors are known in the art. It is known that retroviral vectors, lentiviral vectors, and adenoviral vectors vectors, and adeno-associated virus (AAV) vectors (Vannucci et al. (2012) (Reviewed in New Microbiol. 36:1-22). Useful recombinant AAV vectors are useful for viral transduction of cells and nucleation into the cellular genome. The recombinant vector may have any serotype that allows for the insertion of a recombinant ATPase gene. AAV vectors have the serotypes AAV2 or AAV6. Recombinant AAV vectors are also It 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).

[0323] The engineered nuclease gene may be in the form of DNA (e.g., a plasmid) and / or a virus. When delivered via a vector (e.g., AAV), they are operably linked to a promoter. In some embodiments, this is due to the endogenous DNA fragments derived from the viral vector. promoter (e.g., LTR in lentiviral vectors), or a well-known site Viral promoters such as the tomegalovirus or SV40 virus early promoter. In a preferred embodiment, the nuclease gene is expressed in a target cell (e.g., a T cell). ) is operably linked to a promoter that drives gene expression preferentially in

[0324] The present invention further provides a method for targeting a recognition sequence within the T cell receptor alpha gene, particularly within the target 5' intron. In some embodiments, the exogenous sequence of interest is Insert elements (i.e., exogenous splice acceptor sites, IRES or 2A elements, 5' homology arms adjacent to the coding sequence of the target protein and / or poly(A) signal and 3' homology arms. Such homology arms are located at the target 5' end where the cleavage site is generated. The sequence homology is determined by the corresponding sequences 5' upstream and 3' downstream of the nuclease recognition sequence of the nucleotide. Generally, the homology arms have at least 50 base pairs, preferably at least 100 base pairs, and their counterparts in the genome, which can be up to 2000 base pairs or more in length. A sequence that is at least 90%, preferably at least 95%, or more similar to the sequence The sequences may have sequence homology.

[0325] The exogenous sequences of interest of the present invention can be introduced into cells by any of the aforementioned means. In certain embodiments, the exogenous sequence of interest is a lentivirus, retrovirus, adenovirus, or The vector is introduced by a viral vector such as fluviral vector, or preferably a recombinant AAV vector. Recombinant AAV vectors useful for the introduction of exogenous nucleic acids are useful for viral transduction of cells and for the production of cellular The vector may have any serotype that allows for the insertion of an exogenous nucleic acid sequence into the cell genome. In some embodiments, the recombinant AAV vector has an AAV2 or AAV6 serotype. V vectors are also self-complementary so as not to require double-stranded DNA synthesis in the host cell. may be.

[0326] In another specific embodiment, a single-stranded DNA template is used to encode the exogenous sequence of interest. The single-stranded DNA can contain an exogenous sequence of interest and is preferably In a preferred embodiment, the method further comprises facilitating insertion of a nucleic acid sequence into a nuclease cleavage site by homologous recombination. The single-stranded DNA may comprise a 5' homology arm and a 3' homology arm. 5' AAV inverted terminal repeat (ITR) sequence 5' upstream of the homologous arm, and the 3' homologous arm It may further comprise a 3' AAV ITR sequence 3' downstream of the

[0327] In another particular embodiment, the engineered nucleases of the invention and / or the target compounds of the invention are The gene encoding the exogenous sequence is transfected using a linearized DNA template. In some instances, the circular plasmid DNA can be introduced into cells prior to transfection. The plasmid DNA may be digested with one or more restriction enzymes such that A is linearized.

[0328] T cells modified according to the present invention may be modified by the addition of a nuclease and / or an exogenous sequence of interest prior to the introduction of the nuclease and / or the exogenous sequence of interest. For example, T cells may require activation for a period of time sufficient to activate the cells. anti-CD3 and anti-CD28 antibodies, either soluble or bound to a support (i.e., beads) can be brought into contact with.

[0329] The genetically modified cells of the present invention are further modified to express one or more inducible suicide genes. Its induction can lead to cell death and can be used in vitro or in vivo In some instances, the suicide gene is a cytotoxic polypeptide. , a polypeptide capable of converting a non-toxic prodrug into a cytotoxic drug, and / or It may encode a polypeptide that activates a cytotoxic gene pathway within the cell, i.e., a suicide gene. Genes encode products that, by themselves or in the presence of other compounds, cause cell death A typical example of such a suicide gene is the thymidine kinase gene of herpes simplex virus. An additional example is a gene encoding the thymidine kinase of the varicella-zoster virus. and the gene that converts 5-fluorocytosine to the highly toxic compound 5-fluorouracil. The bacterial gene cytosine deaminase can be converted into a suicide gene. Non-limiting examples of suicide genes include and genes encoding caspase-9, caspase-8, or cytosine deaminase. In some instances, specific chemical dimerization inducers (CIDs) are also included. caspase-9 was activated using a dimerization inducer. The suicide gene can activate the cells by inducing therapeutic and / or cytotoxic monoclonal antibodies. It may also encode a polypeptide expressed on the surface of a cell that renders it sensitive to an antibody. In one example, the suicide gene is an antigen molecule recognized by the anti-CD20 mAb rituximab. Recombinant antigen polypeptides containing epitopes that allow selection of cells expressing the chief and suicide genes. For example, the polypeptides described in WO2013153391 may be encoded. It contains two rituximab-binding epitopes and one QBEnd10-binding epitope. See RQR8 polypeptide. For such genes, rituximab is optionally In a further example, the suicide gene can be administered to a subject to induce cell depletion. A polypeptide comprising a QBEnd10-binding epitope expressed in combination with a truncated EGFR polypeptide. It can be seen.

[0330] T cells modified by the methods and compositions described herein are capable of expressing endogenous T cell receptors. The expression of the target protein (e.g., CAR) can be reduced, and optionally, the target protein (e.g., CAR) can be further expressed. Thus, the present invention further provides a method for expressing a protein of interest and targeting endogenous T cells. For example, the population may be a population of T cells that do not express a CAR. Multiple genetically modified T cells of the present invention or exogenous T cell receptors (i.e., CAR+) These cells can contain T cell receptors (i.e., exoTCR+) and have reduced expression of endogenous T cell receptors ( In various embodiments of the present invention, at least one of the cells in the population 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%, less 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 at least 96%, at least 97%, at least 98%, at least 99%, or up to 10% In certain instances, the population is comprised of at least 100% of the T cells in the population. 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 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 It may contain up to 100% cells that are both TCR- and CAR+.

[0331] 2.4 Pharmaceutical Compositions In some aspects, the present invention provides a method for producing a genetically modified T cell of the present invention, or a genetically modified T A pharmaceutical composition is provided that includes a population of cells and a pharmaceutically acceptable carrier. The product can be prepared according to known techniques. See, e.g., Remington, The Sci ence And Practice of Pharmacy(21 st ed.200 In the manufacture of pharmaceutical formulations according to the invention, the cells are typically pharmaceutically Upon mixing with an acceptable carrier, the resulting composition is administered to a subject. It 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 compositions of the present invention are used to treat a disease in a subject. In a further embodiment, the pharmaceutical composition of the present invention may further comprise one or more additional agents useful in treating The composition contains cytokines that promote in vivo cell proliferation and engraftment of genetically modified T cells. Biomolecules such as IL-2, IL-7, IL-15, and / or IL-21 The pharmaceutical composition comprising the genetically modified T cells of the present invention may further comprise an additional drug or may be administered in the same composition as the biomolecule, or alternatively in a separate composition. It may be administered at the same time.

[0332] The present disclosure also provides a method for producing a pharmaceutical composition comprising the genetically modified cells or The present disclosure further provides a method for producing a genetically modified cell or population thereof as described herein. In the manufacture of a medicament for treating a disease in a subject in need thereof, In one aspect, the invention provides a method for the treatment of a patient in need thereof, comprising administering to the patient a genetically modified cell or a population thereof. The present invention is useful for cancer immunotherapy of subjects with the above-mentioned conditions.

[0333] The pharmaceutical compositions of the present invention are useful for the treatment of any condition that can be targeted by T cell adoptive immunotherapy. Non-limiting examples of cancers that may be treated with the pharmaceutical compositions and medicaments of the present disclosure include B Cancer of cell origin, neuroblastoma, osteosarcoma, prostate cancer, renal cell carcinoma, rhabdomyosarcoma, liver cancer, gastric cancer ( gastric cancer), bone cancer, pancreatic cancer, skin cancer, head and neck cancer, breast cancer, lung cancer, skin cancer or intraocular malignant melanoma, 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, child Cervical carcinoma, vaginal carcinoma, vulvar carcinoma, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer Adenocarcinoma, parathyroid carcinoma, adrenal carcinoma, soft tissue sarcoma, urethral cancer, penile cancer, pediatric solid tumors, lymphocytic Lymphoma, bladder cancer, kidney or ureter cancer, renal pelvic carcinoma, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor , brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, asbestos Environmentally induced cancers, including those induced by multiple myeloma, Hodgkin's lymphoma, and non-Hodgkin's lymphoma acute myeloid lymphoma, chronic myeloid leukemia, chronic lymphocytic leukemia, immunoblastic large cell lymphoma lymphoma, acute lymphoblastic leukemia, mycosis fungoides, anaplastic large cell lymphoma, T-cell lymphoma carcinoma, lymphoma, including but not limited to, leukemia ... , sarcoma, melanoma, blastoma, leukemia, and germ cell tumors. Cancers of B-cell origin include B-cell acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, and Hematologic malignancies, B-cell lymphoma, diffuse large B-cell lymphoma, pre-B ALL (pediatric use) Symptoms), mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, Burkitt lymphoma , multiple myeloma, and B-cell non-Hodgkin's lymphoma. .

[0334] In some of these embodiments of treating cancer with the genetically modified cells of the present disclosure, the genetically modified cells The subject to whom the cells have been administered may then be administered additional treatments such as radiation, surgery, or chemotherapy. .

[0335] The present invention further includes exogenous nucleic acid molecules encoding sequences of interest in the genome, as described herein. A population of genetically modified cells is provided, comprising a plurality of the genetically modified cells described, wherein the exogenous The nucleic acid molecule is inserted into the targeted 5' intron of the T cell receptor alpha gene, and the endogenous TCR Thus, in various embodiments of the present invention, the cell surface expression of A population of cells is provided, wherein at least 10%, at least 15%, at least 18%, or at least 20% of the cells in the population are 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 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 genes described herein In a further embodiment of the present invention, a population of genetically modified cells is provided, the population At least 10%, at least 15%, at least 20%, at least 25% of the cells in the population %, at least 30%, at least 35%, at least 40%, at least 45%, at least 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 genes described herein further express a chimeric antigen receptor. It is a genetically modified cell.

[0336] 2.5 Methods of administration of genetically modified cells Another aspect disclosed herein is the administration of a genetically modified T cell of the present disclosure to a subject in need thereof. In certain embodiments, the pharmaceutical compositions described herein are For example, an effective amount of the cell population is administered to a subject having a disease. In certain embodiments, the disease may be cancer, and the genetically modified T cells of the present invention may The administration of these cells is immunotherapy. The administered cells suppress proliferation and reduce the number of cells in the recipient. Unlike antibody therapy, the genetically modified T cells of the present disclosure The cells are able to replicate and expand in vivo, resulting in sustained disease control. This provides long-term sustainability that may continue.

[0337] Examples of possible routes of administration include parenteral (e.g., intravenous (IV), intramuscular (IM), dermal) In addition, administration can be by continuous infusion or by a single bolus. or by multiple bolus administration. One or both of the above is less than about 12 hours, 6 hours, 4 hours, 3 hours, 2 hours, or 1 hour. In yet other embodiments, the infusion occurs slowly at first, then Then it increases over time.

[0338] In some embodiments, the genetically modified T cells of the present disclosure target tumor antigens for the purpose of treating cancer. Such cancers include carcinomas, lymphomas, sarcomas, blastomas, leukemias, and tumors of B-cell origin. cancer, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, melanoma, prostate cancer, colon cancer, renal cell carcinoma, including, but not limited to, ovarian cancer, rhabdomyosarcoma, leukemia, and Hodgkin's lymphoma In specific embodiments, cancers and disorders include pre-B ALL (pediatric indications), adult Human ALL, mantle cell lymphoma, diffuse large B-cell lymphoma, and post-allogeneic bone marrow transplantation These cancers include, but are not limited to, CD1 9. Using a combination of CARs targeting CD20, CD22, and / or ROR1 In some non-limiting examples, the genetically modified eukaryotic cells or populations thereof of the present disclosure can be used to treat 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 in the skin or eyes, kidney cancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, anal cancer. hilar cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube carcinoma, endometrial cancer Cancer, cervical carcinoma, vaginal carcinoma, vulvar cancer, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, Thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, pediatric solid tumors, lymphoma lymphoma, bladder cancer, kidney or ureter cancer, renal pelvic carcinoma, neoplasms of the central nervous system (CNS), Primary CNS lymphoma, tumor angiogenesis, spinal axis tumor r), brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, asbestos-induced Environmentally induced cancers, including those induced by acute myeloid lymphoma, chronic myeloid leukemia, chronic lymphocytic leukemia, immunoblastic myeloid leukemia Acute lymphoblastic leukemia, mycosis fungoides, anaplastic large cell lymphoma, T-cell lymphoma Cancers, including but not limited to, lymphomas, pancreatic cancers, and any combination of these cancers. Targets include leukemia, sarcoma, melanoma, blastoma, leukemia, and germ cell tumors. In terms of morphology, cancers of B-cell origin include B-cell acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, and Lymphoid leukemia, B-cell lymphoma, diffuse large B-cell lymphoma, pre-B AL L (pediatric indications), mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, Burkitt's lymphoma These include, but are not limited to, lymphoma, multiple myeloma, and B-cell non-Hodgkin's lymphoma. Not determined.

[0339] When an "effective amount" or "therapeutic amount" is referred to, the exact amount to be administered will vary depending on age, weight, tumor size, and other factors. Considering the size (if present), the degree of infection or metastasis, and individual differences in the condition of the patient (subject). In some embodiments, the genetic information described herein can be used to determine the risk of developing a cancer. Pharmaceutical compositions containing the modified cells may be prepared in any of the following ranges: 10 to 12, including all integer values ​​within those ranges. 4 cells / kg Weight ~10 9 In a further embodiment, the dosage is: 10, including all integer values ​​within those ranges 5 cells / kg body weight ~10 6 cells / kg body weight In some embodiments, the cell composition is administered multiple times at these dosages. can be administered using injection techniques commonly known in immunotherapy (e.g., Ros See Berg et al., New Eng. J. of Med. 319:1676, 1988. The optimal dosage and treatment regimen for a particular patient will depend on the patient's disease symptoms. The present invention can be readily achieved by those skilled in the art of medicine by monitoring the patient and adjusting treatment accordingly. can be determined.

[0340] In some embodiments, administration of the genetically modified T cells of the present disclosure results in at least one treatment for the target disease or condition. For example, administration of the genetically modified T cells of the present disclosure reduces at least one symptom of cancer. Symptoms of cancer are well known in the art and may be alleviated by administering a steroid or other anti-cancer drug to at least one patient. , can be determined by known techniques.

[0341] 2.6 Methods for producing recombinant viral vectors In some embodiments, the present invention provides recombinant AAV vectors 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, allowing it to self-replicate. prevent the production of HIV-1 and deliver the therapeutic gene(s) (e.g., an endonuclease gene) These must be provided in trans to the packaging cell line to allow for access to the In addition, "helpers" (e.g., adenovirus) are required to support replication. Components must be provided (Cots D, Bosch A, Chillon M 2013) Curr. Gene Ther. 13(5):370-81). In many cases, The recombinant AAV vector is transfected into the cell line by transfecting it with a first plasmid encoding the "helper" components. , a second plasmid containing the cap gene and rep gene, and the A third plasmid containing the viral ITRs containing the intervening DNA sequence for The capsids are then produced using triple transfection. a virus containing the genome (ITRs and intervening gene(s) of interest) enclosed in a The particles may be dehydrated by freeze-thaw cycling, sonication, surfactants, or other methods known in the art. The particles are then isolated from the cells by cesium chloride density gradient centrifugation or other means. Purification using affinity chromatography followed by incorporation into cells, tissues, or human patients The gene of interest is delivered to an organism such as a human.

[0342] Recombinant AAV particles are typically produced intracellularly, ensuring site-specific endonuclease activity. Care must be taken when practicing this invention to ensure that nucleases are not expressed in the packaging cells. Since the viral genome of the present invention contains an endonuclease recognition sequence, The endonuclease expressed in the packaging cell line is packaged into viral particles. This allows the viral genome to be cleaved before it is packaged, thereby reducing the packaging efficiency. This results in the packaging of under-expressed and / or fragmented genomes. To prevent endonuclease expression in the You can:

[0343] 1. The endonuclease is a tissue-specific protein that is not activated in the packaging cells. For example, the expression of an endonuclease gene (or genes) in muscle tissue may be placed under the control of a motor. If viral vectors are developed for the delivery of muscle-specific promoters, Examples of muscle-specific promoters include C5-12 (Liu (2004) Hum Gene Ther. 15:783-92), muscle-specific Cleave MCK promoter (Yuasa, et al. (2002) Gene Ther. 9:1576-88), or the smooth muscle 22 (SM22) promoter (Haase, et al. (20 13) BMC Biotechnol.13:49-54) Examples of gene-specific promoters include the NSE, synapsin, and MeCP2 promoters. (Lentz et al. (2012) Neurobiol Dis. 48:179 Examples of liver-specific promoters include the 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 the corneal epithelium-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 those known in the art, Other known tissue-specific promoters are not highly active in HEK-293 cells. Therefore, when incorporated into the viral vector of the present invention, significant would not be expected to result in significant levels of endonuclease gene expression. In addition, the viral vectors of the present invention may be modified to incorporate incompatible tissue-specific promoters (i.e., Using the well-known HeLa cell line (human epithelial cells) and the liver-specific hemopexin promoter, ) are contemplated for use in 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 the monogenic 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 a different species in which the endonuclease is unlikely to be expressed. For example, viral particles can be caged in non-mammalian packaging cells. It is not a sexually transmitted virus, but is a well-known cytomegalovirus or SV40 virus early promoter. Mammalian promoters such as α, β ... In a preferred embodiment, the viral particles are prepared as described by Gao et al. .Biotechnol.131(2):138-43) It is produced in insect cells using a viral system. Creases are unlikely to be expressed in these cells (Airenne, KJ et al. (201 3) Mol. Ther. 21(4):739-49). Furthermore, insect cells are more efficient than mammalian cells. Therefore, human growth hormone (HGH) utilizes a different mRNA splicing motif. GH) intron or mammalian intron such as SV40 large T antigen intron These introns can be incorporated into the coding sequence of the clease. Insect cells lack functional endogenous mRNA because it is not efficiently spliced ​​from the pre-mRNA transcript. In contrast, the resulting recombinant Mammalian cells into which AAV particles are delivered properly splice and produce functional pre-mRNA. Haifeng Chen is a researcher in the field of HGH and Using the SV40 large T antigen intron to express virulence proteins in insect packaging cells It attenuates the expression of the protein barnase and diphtheria toxin fragment A, and inhibits the expression of these toxin genes. Chen, H (2 012) Mol Ther Nucleic Acids.1(11):e57).

[0345] 3. The endonuclease gene requires a small molecule inducer for endonuclease expression. If desired, it can be operably linked to an inducible promoter. Examples of inducible promoters For example, the Tet-On system (Clontech; Chen H et al., (2015) B MC Biotechnol.15(1):4)) and the RheoSwitch system ( Intrexon;Sowa G et al. (2011) Spine,36(10):E623- 8) In addition to these systems, similar systems known in the art include , which activate transcription in response to small molecule activators (doxycycline or ecdysone, respectively). Ligand-inducible transcription factors (Tet repressor and ecdysone receptor, respectively) that activate the The present invention is practiced using such ligand-inducible transcriptional activators. These are: 1) the endonuclease gene is under the control of a promoter that responds to the corresponding transcription factor. The step of placing the endonuclease gene under a target gene, wherein the endonuclease gene has a binding site (or multiple sites) for a transcription factor. and 2) incorporating transcription factors into the packaged viral genome. If a transcriptional activator is not also provided to the cell, the gene encoding the transcriptional activator may be included in the cell. In this case, the endonuclease is not expressed in target cells or tissues after recombinant AAV delivery. The latter step is necessary. The transcriptional activator is then treated with its cognate small molecule activator. This approach induces endonuclease gene expression only in the treated cells or tissues. By choosing when and to which tissues to deliver small molecule inducers, This is advantageous because it allows spatiotemporal regulation of gene expression. The need to include an inducer in the genome severely limits carrying capacity and makes this approach Roach has its drawbacks.

[0346] 4. In another preferred embodiment, the recombinant AAV particles prevent expression of the endonuclease. The transcriptional repressor is produced in a mammalian cell line that expresses a transcriptional repressor. known as the Tet repressor, Lac repressor, Cro repressor, and Many nuclear hormone receptors, such as the ecdysone receptor, have homologous In the absence of a hormone ligand, it also acts as a transcriptional repressor. To achieve this, packaging cells are transfected / transduced with a vector encoding a transcriptional repressor. The endonuclease gene (packaging vector) in the viral genome is inserted Contains a binding site for a repressor so that the repressor can silence the promoter The gene encoding the transcriptional repressor is operably linked to a promoter modified so as to The gene encoding the transcriptional repressor can be placed in various locations. may be integrated into a packaging vector outside the ITR sequences; It may be incorporated into a cap / rep vector or an adenovirus helper vector. or, most preferably, stably integrated into the genome of the packaging cell so as to be constitutively expressed. A general mammalian promoter may be modified to incorporate a transcription repression site. Methods for this purpose are known in the art. For example, Chang and Roninson Strong constitutive CMV and RSV promoters to contain the Lac repressor operator In cells expressing the repressor, gene expression from the modified promoter is suppressed. Chang BD, and Roninson IB (1 996) Gene 183:137-42). The use of non-human transcriptional repressors Transcription of the endonuclease gene is repressed only in packaging cells expressing the repressor and is not suppressed in target cells or tissues transduced with the resulting recombinant AAV vector. Ensure that

[0347] In some embodiments, gene transfer is achieved via a lentiviral vector. In contrast to other retroviruses, lentiviruses can, in some cases, translocate certain non-dividing cells. Non-limiting examples of lentiviral vectors include human immunodeficiency virus (HIV) vectors, which can be used to transduce cells. Human immunodeficiency virus type 1 (HIV-1), HIV-2, simian immunodeficiency virus (SIV), human T-lymphotropic virus 1 (HTLV-1), HTLV-2, or equine infectious anemia virus Examples include those derived from lentiviruses such as E1AV. Deletion of env, vif, vpr, vpu, and nef genes attenuates HIV pathogenicity genes This allows the production of lentiviral vectors, making the vectors safer for therapeutic purposes. Lentiviral vectors are known in the art and are described in 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. It contains sequences essential for the uptake, selection, and transfer of foreign nucleic acid into the host cell. Known lentiviruses are American Type C ulture Collection(“ATCC”;10801 University 20110-2209) or other depository institutions or readily available from collections or known sources using commonly available techniques It can be isolated from a source.

[0348] In a specific embodiment, the lentiviral vector is a human immunodeficiency virus (HIV) vector. A plasmid encoding the gag, pol, tat, and rev genes cloned from smids and vesicular stomatitis virus (VSV-G) pseudotyped against It is prepared using a second plasmid encoding the derived envelope protein. The transfer vector, such as the pCDH-EF1-MCS vector, contains an appropriate promoter, All three plasmids were then transformed into The virus is transfected into cells (such as Lenti-X-293T cells) and then the appropriate After the incubation period, lentivirus can be collected, concentrated, and screened. Therefore, the exogenous sequences of interest described herein or the engineered nucleic acids of the invention Provided herein are methods for producing recombinant lentiviral vectors containing the enzyme.

[0349] 2.7 Engineered nuclease mutants Embodiments of the present invention relate to the engineered nucleases described herein, particularly engineered Further embodiments of the present invention include the meganucleases described herein. An isolated polynucleotide comprising a nucleic acid sequence encoding the engineered meganuclease described. The present invention encompasses polynucleotides, polypeptides, and variants of such polynucleotides.

[0350] As used herein, "variant" is intended to mean a substantially similar sequence. A "variant" polypeptide is one that has one or more alterations at one or more internal sites of the native protein. is a deletion or addition of multiple amino acids and / or a modification of one or more naturally occurring polypeptides. Derived from a "native" polypeptide by substitution of one or more amino acids at sites As used herein, "natural" polynucleotides are intended to mean polypeptides. The term "mutant" includes the parent sequence from which the variant is derived. Polypeptides are biologically active, that is, they induce the desired biological activity of the native protein. biological activity; i.e., for example, TRC11-12 recognition sequence (SEQ ID NO: 4), TRC15- 16 recognition sequence (SEQ ID NO: 6), TRC17-18 recognition sequence (SEQ ID NO: 8), and TRC1 Targets 5' of the human T cell receptor alpha gene, containing the 9-20 recognition sequence (SEQ ID NO: 10) They retain the ability to recognize and cleave the recognition sequence found in introns. The biological activity of the native polypeptides of the embodiments may be affected by, for example, human manipulation. a functional variant (e.g., SEQ ID NOS: 12-27), or a recognition half-sequence described herein. Biologically active variants of the ATP-binding subunit (e.g., SEQ ID NOS: 28-59) are naturally occurring At least about 40%, about 4%, or 5%, approx. 50%, approx. 55%, approx. 60%, approx. 65%, approx. 70%, approx. 75%, approx. 80%, approx. 8 5%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 9 7%, about 98%, or about 99% sequence alignment programs and elsewhere herein. The polypeptides of the embodiment have sequence identity as determined by the parameters set forth in A biologically active variant of a polypeptide or subunit is a variant of that polypeptide or subunit. , only about 1 to 40 amino acid residues, only about 1 to 20, only about 1 to 10 , may differ by as few as 5, as few as 4, 3, 2, or 1 amino acid residue .

[0351] Polypeptides of the embodiments may be modified in a variety of ways, including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. Variants of the amino acid sequence can be prepared by mutation of the DNA. Methods for modifying thiol are well known in the art. See, for example, Kunkel (1999). 985)Proc.Natl.Acad.Sci.USA 82:488-492;Ku nkel et al. (1987) Methods in Enzymol. 154:367-38 2; U.S. Patent No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology(M (acMillan Publishing Company, New York), and See those cited references. Guidance regarding amino acid substitutions can be found in Dayho, M.D., incorporated herein by reference. ff et al. (1978) Atlas of Protein Sequence and S structure(Natl.Biomed.Res.Found.,Washingt on, DC) model. Conservative substitutions, such as exchanging a given amino acid for another amino acid, may be optimal.

[0352] Substantial amino acid modifications to the DNA recognition domain of wild-type I-CreI meganuclease have been previously identified (e.g., U.S. Pat. No. 8,021,867) and are known to be useful either alone or in combination. In addition, the resulting rationally designed meganucleases have half-length differences that are distinct from the wild-type enzymes. The specificity is determined by the individual bases within the half-sites of the DNA recognition sequence, so that the recognition sequence has site specificity. Table 5 shows the structure of each half of the recognition half site, resulting in a modified engineered meganuclease. To enhance specificity based on the bases present at the site positions (-1 to -9), recombinant DNA technology was used. Potential substitutions made in the ganucleases monomers or subunits are shown.

[0353] [Table 5]

[0354] Entries in bold are wild-type contact residues and constitute "modifications" as used herein. An asterisk indicates that the residue is in contact with a base in the antisense strand.

[0355] In the case of polynucleotides, a "variant" is one or more sites within a naturally occurring polynucleotide. Those skilled in the art will recognize that the nucleic acid of the embodiment may be modified by one or more deletions and / or additions of one or more nucleotides in the sequence. It is recognized that mutants of the In the case of polynucleotides, conservative variants are practically A variant polypeptide includes a sequence encoding one of the amino acid sequences of the variant polypeptide. The nucleotides may be generated, for example, by using site-directed mutagenesis, but Derived synthetically, such as those that still encode the recombinant meganuclease of the embodiment. In general, variants of particular polynucleotides of the embodiments include , at least about 40%, about 45%, about 50%, about 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99% or more of the sequence alignment programs and those described elsewhere herein. The specific polynucleotides of the present invention have sequence identity as determined by the parameters set forth below. A variant of a polynucleotide (i.e., a reference polynucleotide) is a polynucleotide that is amplified by a variant polynucleotide. Polypeptides encoded by the reference polynucleotides It can also be assessed by comparing the percent sequence identity between

[0356] Deletions, insertions, and substitutions in the protein sequences encompassed herein may be made without affecting the properties of the polypeptide. However, substitutions, deletions, or insertions are not expected to result in fundamental changes to the In cases where it is difficult to predict the exact effect before administration, those skilled in the art may The ability to preferentially recognize and cleave recognition sequences found within the target 5' intron of a gene It will be understood that the effectiveness of the polypeptides will be assessed by screening them. cormorant. [Example]

[0357] This invention is further illustrated by the following examples, which should not be construed as limiting. Those skilled in the art will be able to identify and implement the specific compounds described herein using no more than routine experimentation. Numerous equivalents to quality and procedure can be recognized or identified. Such equivalents include: It is intended that the invention be encompassed within the scope of the claims that follow the examples below.

[0358] Example 1 Megagenes that recognize and cleave target 5' intron recognition sequences of the T cell receptor alpha gene Characterization of nuclease 1. Meganuclease that recognizes and cleaves the TRC11-12 recognition sequence The engineered meganucleases, collectively referred to herein as "TRC11-12 meganucleases," The nuclease (SEQ ID NOs: 12-15) targets the 5' end of the human T cell receptor alpha gene. It is designed to recognize and cleave the TRC11-12 recognition sequence (SEQ ID NO: 4) present in the nucleotide. Each TRC11-12 recombinant meganuclease was engineered to contain an N-terminal SV40-derived A nuclear localization signal, a first meganuclease subunit, a linker sequence, and a second meganuclease subunit. The first subunit of each TRC11-12 meganuclease The first subunit binds to the TRC12 recognition half-site of SEQ ID NO: 4, and the second subunit binds to the TR It binds to the C11 recognition half-site (see Figure 2).

[0359] The TRC12-binding subunit and the TRC11-binding subunit are HVR1 and HVR2, respectively. Each contains a 56 base pair hypervariable region called VR2. TRC12 binding subunit is highly conserved outside the HVR1 region. Similarly, the TRC11 binding subunit is also The TRC11 binding regions of SEQ ID NOs: 12 to 15 are highly conserved outside the VR2 region. SEQ ID NOs: 28 to 31 are provided as SEQ ID NOs: 28 to 31, respectively. SEQ ID NO: 28, which is the TRC11-binding region of enzyme TRC11-12x.4 (SEQ ID NO: 12) Shares at least 90% sequence identity with the TRC12-binding regions of SEQ ID NOs: 12 to 15 are provided as SEQ ID NOs: 32 to 35, respectively. SEQ ID NOs: 32 to 35 each represent the sequence SEQ ID NO: 12, which is the TRC12-binding region of the cleavage enzyme TRC11-12x.4 (SEQ ID NO: 12) It shares at least 90% sequence identity with no. 32.

[0360] 2. Meganuclease that recognizes and cleaves the TRC15-16 recognition sequence The engineered meganucleases, collectively referred to herein as "TRC15-16 meganucleases," The nuclease (SEQ ID NOs: 16-19) targets the 5' end of the human T cell receptor alpha gene. It is designed to recognize and cleave the TRC15-16 recognition sequence (SEQ ID NO: 6) present in the nucleotide. Each TRC15-16 recombinant meganuclease was engineered to contain an N-terminal fragment derived from SV40. a nucleotidic localization signal, a first meganuclease subunit, a linker sequence, and a second meganuclease subunit; Contains meganuclease subunits. The first subunit of each TRC15-16 meganuclease The first subunit binds to the TRC15 recognition half-site of SEQ ID NO: 6, and the second subunit binds to the TRC15 recognition half-site of SEQ ID NO: 6. Binds to the TRC16 recognition half-site (see Figure 2).

[0361] The TRC15-binding subunit and the TRC16-binding subunit are HVR1 and HVR2, respectively. Each contains a 56 base pair hypervariable region called VR2. TRC15 binding subunit is highly conserved outside the HVR1 region. Similarly, the TRC16 binding subunit is also The TRC15 binding regions of SEQ ID NOs: 16 to 19 are highly conserved outside the VR2 region. SEQ ID NOs: 36 to 39 are provided as SEQ ID NOs: 36 to 39, respectively. SEQ ID NO:3, which is the TRC15-binding region of the enzyme TRC15-16x.31 (SEQ ID NO:16) 6. The TRC16 binding domains of SEQ ID NOs: 16 to 19 share at least 90% sequence identity with The regions are provided as SEQ ID NOS: 40-43, respectively. The sequence of the TRC16-binding domain of the nuclease TRC15-16x.31 (SEQ ID NO: 16) It shares at least 90% sequence identity with sequence no. 40.

[0362] 3. Meganuclease that recognizes and cleaves the TRC17-18 recognition sequence The engineered TRC17-18 meganucleases, collectively referred to herein as "TRC17-18 meganucleases," The meganucleases (SEQ ID NOs: 20-23) target the human T cell receptor alpha gene. It recognizes and cleaves the TRC17-18 recognition sequence (SEQ ID NO: 8) present in the 5' intron. The TRC17-18 recombinant meganucleases were each engineered to an N-terminal nuclear localization signal corresponding to the first meganuclease subunit, a linker sequence, and and a second meganuclease subunit. The first subunit binds to the TRC17 recognition half-site of SEQ ID NO: 8, and the second subunit binds to the TRC17 recognition half-site of SEQ ID NO: 8. Nit binds to the TRC18 recognition half-site (see Figure 2).

[0363] The TRC17-binding subunit and the TRC18-binding subunit are HVR1 and HVR2, respectively. Each contains a 56 base pair hypervariable region called VR2. TRC17 binding subunit is highly conserved outside the HVR1 region. Similarly, the TRC18 binding subunit is also The TRC17 binding regions of SEQ ID NOs: 20 to 23 are highly conserved outside the VR2 region. SEQ ID NOs: 44 to 47 are provided as meganucleases 44 to 47, respectively. SEQ ID NO: 20, which is the TRC17-binding domain of the enzyme TRC17-18x.15 SEQ ID NO: 20-23 share at least 90% sequence identity with TRC18. The combined regions are provided as SEQ ID NOs: 48 to 51, respectively. and the TRC18-binding region of meganuclease TRC17-18x.15 (SEQ ID NO: 20). It shares at least 90% sequence identity with SEQ ID NO: 48, which is

[0364] 4. Meganuclease that recognizes and cleaves the TRC19-20 recognition sequence The engineered meganucleases, collectively referred to herein as "TRC19-20 meganucleases," The meganucleases (SEQ ID NOs: 24-27) target the human T cell receptor alpha gene. Recognizes and cleaves the TRC19-20 recognition sequence (SEQ ID NO: 10) present in the 5' intron The TRC19-20 recombinant meganucleases were each engineered to the resulting N-terminal nuclear localization signal, the first meganuclease subunit, a linker sequence, and a second meganuclease subunit. The first subunit binds to the TRC19 recognition half-site of SEQ ID NO: 10, and the second subunit binds to the TRC19 recognition half-site of SEQ ID NO: 10. The subunit binds to the TRC20 recognition half-site (see Figure 2).

[0365] The TRC19-binding subunit and the TRC20-binding subunit are HVR1 and HVR2, respectively. Each contains a 56 base pair hypervariable region called VR2. TRC19 binding subunit is highly conserved outside the HVR1 region. Similarly, the TRC20 binding subunit is also The TRC19 binding regions of SEQ ID NOs: 24 to 27 are highly conserved outside the VR2 region. SEQ ID NOs: 52 to 55 are provided as SEQ ID NOs: 52 to 55, respectively. SEQ ID NO:5, which is the TRC19-binding region of the enzyme TRC19-20x.85 (SEQ ID NO:24) 2. The TRC20 binding domains of SEQ ID NOs: 24 to 27 share at least 90% sequence identity with The regions are provided as SEQ ID NOS: 56-59, respectively. The sequence of the TRC20-binding domain of the nuclease TRC19-20x.85 (SEQ ID NO: 24) Shares at least 90% sequence identity with sequence no. 56.

[0366] 5. Cleavage of the TRC recognition sequence in CHO cell reporter assays TRC11-12, TRC15-16, TRC17-18, and TRC19-20 Mega Nucleases are identified by their respective recognition sequences (SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and To determine whether each recombinant meganuclear fragment can recognize and cleave the nucleotide sequence (SEQ ID NO: 10), The enzyme activity was evaluated using a CHO cell reporter assay as previously described (International Publication No. 2012 / 003064). (See 167192 and Figure 4.) To perform the assay, CHO cells with an embedded non-functional green fluorescent protein (GFP) gene expression cassette We created a cell reporter line. The GFP gene in each cell line was transduced by meganuclease. Intracellular cleavage of either recognition sequence stimulates a homologous recombination event resulting in a functional GFP gene As shown, it is interrupted by a pair of recognition sequences.

[0367] In the CHO reporter cell line developed for this study, a One recognition sequence is the TRC11-12 recognition sequence (SEQ ID NO: 4), the other is the TRC15-16 recognition sequence (SEQ ID NO: 6), TRC17-18 recognition sequence (SEQ ID NO: 8), or TRC19-20 recognition sequence The second recognition sequence inserted into the GFP gene was CHO-2 3 / 24 recognition sequence, and was identified by a control meganuclease called "CHO-23 / 24." TRC11-21 recognition sequence and CHO-23 / 24 recognition sequence CHO reporter cells containing the TR are referred to herein as "TRC11-12 cells." CHO reporter cells containing the C15-16 recognition sequence and the CHO-23 / 24 recognition sequence were These are referred to herein as "TRC15-16 cells." CHO reporter cells containing the -23 / 24 recognition sequence are referred to herein as "TRC17-18 The TRC19-20 recognition sequence and the CHO-23 / 24 recognition sequence are referred to as "cells." The reporter cells are referred to herein as "TRC19-20 cells."

[0368] CHO reporter cells were transfected with the corresponding engineered meganuclease encoding (e.g. , TRC11-12 cells were transfected with a plasmid encoding the TRC11-12 meganuclease. DNA) or a plasmid encoding the CHO-23 / 34 meganuclease. Each assay was transfected with 4 e 5 CHO reporter cells were transfected with Lipofectamine. 96 using Ctaamine 2000 (ThermoFisher) according to the manufacturer's instructions. Transfection was performed with 50 ng of plasmid DNA in a well plate. 48 hours after transfection Cells were evaluated by flow cytometry during the transfection period and compared with untransfected negative controls (bs). The percentage of GFP-positive cells was determined. As shown in Figures 5A, 5B, 5C, and 5D, In total, all TRC meganucleases expressed their corresponding recognition sequences at frequencies significantly higher than the negative control. It was found that GFP-positive cells were produced in cell lines containing

[0369] TRC11-12, TRC15-16, TRC17-18, and TRC19-20 The efficacy of the constructed meganucleases was also assessed by transfecting the meganucleases into the corresponding reporter cell lines. The time-dependent evaluation was performed 2, 5, and 7 days after the introduction of the Follow the instructions to use the BioRad Gene Pulser Xcell to generate each reporter -Cell line (1.0×10 6 cells), 1 × 10 per cell 6 Copy the corresponding meganuclei At specific time intervals, the cells were analyzed by flow cytometry. The percentage of GFP-positive cells was determined by immunofluorescence microscopy. As shown in Figure 6D, % GFP expression was significantly higher in TRC11-12, TRC15-16, and TRC1 The results varied between meganucleases TRC19-20 and TRC7-18, and were approximately the same throughout the study. Some maintained the same %GFP, while others showed a decrease in %GFP expression after 5 or 7 days. It showed a small amount.

[0370] 6. Conclusion These studies have led to the development of the TRC11-12 meganuclease, TRC15- 16 meganuclease, TRC17-18 meganuclease, and TRC19-20 meganuclease Demonstration that the nucleases can efficiently target and cleave their respective recognition sequences within cells did.

[0371] Example 2 Generation of indels at TRC recognition sequences in human T cells 1. Background This study demonstrated that the engineered nucleases included in the present invention inhibit the recognition of each of the T cells in human T cells. We demonstrated that the recognition sequence can be cleaved. Human CD3+ T cells were isolated from PBMCs by magnetic separation. They were isolated and activated for 72 hours using antibodies against CD3 and CD28. 6 activation Human T cells were transfected using the Lonza 4D-Nucleofector according to the manufacturer's instructions. 2e per cell 6 Copy the given TRC11-12 or TRC15-16 At 72 hours after transfection, the cells were transfected with ribosomal RNA containing ribosomal RNA. Genomic DNA (gDNA) was collected from the nuclease and subjected to a T7 endonuclease I (T7E) assay. Genetic modifications were performed in the recognition sequences of endogenous TRC11-12 or TRC15-16. The T7E assay revealed that the genes of TRC11-12 or TRC15-6 were The locus is amplified by PCR using primers flanking two recognition sequences. If there is an indel (random insertion or deletion) within the target locus, the resulting PCR product will be The PCR product will consist of a mix of wild-type and mutant alleles. Slow reannealing allowed the wild-type and This allows the formation of heteroduplexes consisting of the mutant allele and the base and / or bulge. The T7E1 enzyme cleaves at the mismatch site, which can be visualized by gel electrophoresis. This gives rise to possible cleavage products.

[0372] 2.Results Mock-electroporated Cell and control gDNA (lanes 1 and 2, respectively) show total chromatin without any T7E digestion bands. Long PCR showed a single band corresponding to TRC11-12 or This indicates that the recognition sequence of TRC15-16 is not present (Figure 7). , TRC11-12x.63, and TRC11-12x.82, respectively. Lanes 3, 5, and 6, which correspond to irradiated cells, show indels within the recognition site. The full-length PCR band is shown along with a shorter T7E digestion band. Lane 4, corresponding to cells electroporated at 0.60, shows only the full-length PCR band. This indicates that there are no indels in the recognition site. Electroporated in TRC15-16x.87 and TRC15-16x.89, respectively. Lanes 7, 8, and 10, corresponding to cells with IgG1A, show indels within the recognition site. , showing the full-length PCR band along with the shorter T7E digestion band. TRC15-16x.63 Lane 9, corresponding to cells electroporated with , shows only the full-length PCR band. , indicating that there were no indels in the recognition site.

[0373] 3. Conclusion These data suggest that some TRC11-12 and TRC15-16 nucleases Demonstrate that it can cleave each recognition sequence of human T cells. TRC11-12x.4 , TRC11-12x.63, TRC11-12x.82, TRC15-16x.31, Electroporation was performed using either TRC15-16x.87 or TRC15-16x.89. All irradiated cells expressed T7E- Digestion bands were shown.

[0374] Example 3 Effect of TRC recognition sequence cleavage on T cell receptor expression 1. Background The purpose of these experiments was to identify TRCs within the target 5' intron of the T cell receptor alpha gene. Cleavage of the recognition sequence and subsequent repair by NHEJ affect the expression of endogenous T cell receptors. The purpose of this study was to demonstrate whether this would have an effect.

[0375] CD3 positive selection kit and Robo-Sep automated magnetic separator (both Stem Cell Human T cells were magnetically enriched using a magnetic field analyzer (II Technologies). Cells were enriched from apheresis samples obtained from compensated healthy human volunteers. T cell activators (anti-CD3 / anti-CD28) Dynabeads (LifeT 1:1 in the presence of 10 ng / ml IL-2 using T cells were stimulated for 3 days at a cell:bead ratio of 1000. After 3 days, T cells were harvested and analyzed by DynaMag. Remove the Dynabeads using a magnet (Life Technologies) Using a Lonza 4-D nucleofector, 1 μg of the indicated Meganuclease R Nucleofected cells were analyzed by flow cytometry. The T cell samples were cultured for 6 days before analysis. CD3 surface display was 2.0x 10 5 1 μl anti-CD3-BrilliantViolet711 per cell sample (BioLegend product 300464) and 0.3 μl of GhostDye-510 ( The measurements were performed by labeling with a fluorophore (Tombo Biosciences). Data were acquired using a n-Coulter CytoFLEX-S cytometer .

[0376] 2.Results T cells were treated with an engineered nucleoside targeting TRC1-2x.87EE (TRAC exon 1). Nucleofection was performed with either RNA (pre-cleavage) or RNA (mock), and the TRAC gene was then transfected. These served as positive and negative controls for gene editing and are shown in Figures 8A and 8B. An additional sample also contained one different nuclease variant from the TRC15-16 family. Nucleofection with RNA encoding all members of the 5' intron. The RC15-16 recognition sequence is targeted. Editing the TRAC locus results in gene disruption. When the TCRα chain is generated, the TCR complex (including CD3) is not synthesized on the surface of the edited cell. More than half of the TRC1-2x.87EE-edited T cells contain exon 1 TRC-negative due to cleavage of the cleavage site and error-prone repair by NHEJ In comparison, TRC15-16x.31 and TRC15-16 x.63, TRC15-16x.87, and TRC15-16x.89 after editing The frequency of CR-negative cells was only 4% to 8% (Figures 8C, 8D, 8E, and 8F, respectively). and Figure 8F).

[0377] 3. Conclusion These experiments demonstrated that the targeting recognition sequence upstream of the 5' intron of TRAC exon 1 is intrinsic. Although they can generate T7E receptors (as observed in the T7E assay), they lack cell surface expression of endogenous T cell receptors. The present inventors demonstrate that the exogenous splice acceptor has no substantial effect on the A construct containing the cleavage site, the CAR coding sequence, and the polyA signal Insertion of this gene is expected to knock out TCR expression in cells.

[0378] Example 4 Insertion of a sequence of interest into the targeted 5' intron The goal of these experiments was to generate a double-strand break in the target 5' intron and induce homologous recombination. (i) the exogenous splice acceptor is inserted into the cleavage site by inserting an exogenous sequence of interest into the cleavage site; The presence of a target site and / or polyA signal disrupts the expression of endogenous T cell receptors, ii) allow expression of the protein of interest encoded by the insert;

[0379] In these examples, the exogenous sequence of interest contains many of the elements shown in the construct in Figure 9. Each construct is flanked by 5' and 3' homology arms. These arms are located 5' upstream and 3' downstream of the respective TRC recognition sequences in the target 5' intron. 'There is homology to the downstream sequence. The size of each homology and the corresponding The percent homology of the arms to the sequence determines the likelihood of homologous recombination of the construct into the cleavage site. The 5' homology arm is flanked by: Both an exogenous branch site for splicing and an exogenous splice acceptor site Next, a signal peptide, anti-CD19 scFv, CD 8 hinge and transmembrane domains, N6 costimulatory domain, and CD3ζ signaling domain The CAR coding sequence contains a T2A element 5' of the anti-CD19 CAR sequence. The string is followed by a bi-polyA signal and finally a 3' homology arm. The specific constructs provided in 9C are TRC11-12, TRC15-1, and TRC16-1, respectively. 6, and TRC17-18, and are provided in SEQ ID NOs: 60 to 62. do.

[0380] Donor human T cells were obtained, activated, and transfected with TRC megakaryons as described in the previous examples. The donor template containing the exogenous sequence of interest can be transfected with the cleavage mRNA. The rates (e.g., SEQ ID NOS: 60-62, Figures 9A-9C) were calculated using methods known in the art. The donor template can be introduced by any number of means, preferably by a combination of donor templates. Transduction can be achieved by transduction of recombinant AAV. Compared to transfection with RNA, transduction and transfection can be performed at any time. It is preferable to carry out the above-mentioned treatment. The level of T cell receptor expression and the level of CAR expression are , by flow cytometry (as described above and by methods known in the art), Any cell that does not have an insert at the cleavage site can be determined in the cell at any time point. Because TCR- cells continue to express their endogenous TCR, the majority of TCR- cells obtained by this method also express CAR It is expected to be +.

[0381] In certain studies, 5' introns targeting promoterless GFP or CAR coding sequences When the endogenous TCR promoter is introduced using the construct of the present invention, In this study, we demonstrate that apheresis can drive the expression of these proteins. Samples were collected from healthy, informed, and compensated donors and were CD3 positive selection kit according to the instructions (Stem Cell Technologies) T cells were enriched using 5% fetal bovine serum and 10 ng / ml I Immunotherapy was performed in X-VIVO 15 medium (Lonza) supplemented with L-2 (Gibco). Cult T cell stimulator (anti-CD2 / CD3 / CD28-Stem Cell Tec After 3 days of stimulation, cells were harvested and activated using 4-DNase. Two cells were isolated by electroporation using a nucleofector (Lonza). T cells were transfected with RNA encoding one of the TRC nucleases. received RNA encoding either TRC15-16x.31 or TRC12x.82. It was.

[0382] Cells that received TRC11-12x.82 RNA expressed TRC11-12x.82. Two sequences containing regions of homology with the genomic sequence adjacent to the recognition sequence (i.e., the TRC11-12 recognition sequence) The construct was transduced with one of the AAV6 vectors: Construct 7227 (SEQ ID NO: 63) One vector containing the T2A sequence followed by a promoterless GFP gene. The other vector contains a T2A sequence followed by a promoterless CAR gene. The construct contains construct 7225 (SEQ ID NO: 64) encoding the child.

[0383] Cells that received TRC15-16.x31 RNA were designated as TRC15-16x.31-recognized cells. It contains regions of homology with genomic sequences adjacent to the recognition sequence (i.e., the TRC15-16 recognition sequence). The mice were transduced with one of two AAV6 vectors: construct 7228 (SEQ ID NO: 6) 5) One vector contains a T2A sequence followed by a promoterless GFP gene. and the other vector encodes the T2A sequence followed by a promoterless CA Construct 7226 (SEQ ID NO: 66) encodes the R gene.

[0384] All transductions were performed at a multiplicity of infection (MOI) of 50,000 (viral genomes / cell). The cells were cultured in X-VIVO15 medium supplemented with 5% FBS and 30 ng / ml IL-2. The cell culture was maintained for an additional 5 days. On day 5, CD3 (anti-CD3-APC / 750 or anti-BV 711, BioLegend) and CAR (anti-FMC63-biotin + streptavidin Cells were stained for PEG-PE (prepared in-house) and Beckman-Coulter Cysteine TRC knockout was measured by measuring the signal on a toFLEX-S flow cytometer. Analysis of knock-out, and knock-in of GFP or CAR was performed.

[0385] 2.Results The frequency of TCR knockout cells (CD3+ vs. CD3-) and GFP knockin cells The frequency of cysts is shown in Figure 10. After administration of TRC11-12x.82 and AAV6-7227, 18% of all T cells in culture were CD3-GFP+ (Figure 10A). When gating on the CD3- population only, 85% of the cells were GFP+ (Figure 10 B) Administration of TRC15-16x.31 and AAV6-7228 increased CD3- / GF The frequency of P+ cells was slightly lower (12.6%, Fig. 10C), but the GF of the CD3- population The frequency of P+ cells was still above 80% (Fig. 10D).

[0386] CAR knock-in into the target 5' intron (FMC63+) was performed using the CAR vector shown in Figure 11. Compared with edited cells that were not transduced with TATA (Figure 11A), anti-FMC63 and anti-CD Staining the samples with 3 revealed a knockout population alongside the vector-transduced samples. The knock-in population is identified (the histogram in Figure 11 is gated on CD3-negative events). CAR constructs without promoters in the recognition sequences of TRC11-12 and TCR15-16. When the tract was inserted (Figure 11B and Figure 11C, respectively), CD3- / CAR+ An event occurs, and the endogenous TCR promoter encodes the CAR upon insertion into the target 5' intron. It was shown that the expression of the sequence was enhanced.

[0387] 3. Conclusion TCR intron-specific meganucleases together with corresponding homologous T2A transgenesis Observation of CD3- / GFP+ or CD3- / CAR+ events after administration of the constructs , demonstrating that endogenous TCR transcriptional control elements can be used to drive expression of a protein of interest. .

Claims

1. The introductory portion of the human T-cell receptor alpha gene is located 5' upstream of TRAC exon 1. an engineered meganuclease that recognizes and cleaves a recognition sequence within a nucleic acid sequence, 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 has a first hypervariable (HVR1) region, and the second subunit comprises a second recognition half-sequence of the recognition sequence. An engineered meganuclease that binds to a nucleotide and contains a second hypervariable (HVR2) region.

2. the intron comprises SEQ ID NO:3, and the engineered meganuclease comprises a Within the endogenous splice donor site or endogenous splice acceptor site adjacent to the 2. The engineered meganuclease of claim 1, which has no recognition sequence.

3. 3. The engineered meganuclear of claim 1 or 2, wherein the recognition sequence comprises SEQ ID NO:

4. See.

4. the HVR1 region corresponds to residues 215 to 270 of any one of SEQ ID NOs: 12 to 15; and wherein the amino acid sequence has at least 80% sequence identity with an amino acid sequence corresponding to the amino acid sequence of claim 1.

3. The engineered meganuclease according to claim 3.

5. the HVR1 region is selected from residues 215, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 2 ９、２２１、２２３、２２４、２２９、２３１、２３３、２３５、２３７、２５９、２６ 5. The engineered megaprotein of claim 3 or 4, comprising residues corresponding to 1, 266, and 268. Nuclease.

6. the HVR1 region comprises residues 215-270 of any one of SEQ ID NOs: 12-15; The engineered meganuclease of any one of claims 3 to 5.

7. the HVR2 region corresponds to residues 24 to 79 of any one of SEQ ID NOs: 12 to 15 The amino acid sequence of claim 3 to claim 5 has at least 80% sequence identity with the amino acid sequence of claim 1.

7. The engineered meganuclease of any one of claims 6.

8. the HVR2 region is selected from residues 24, 26, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, Corresponding to 0, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 The engineered meganuclease of any one of claims 3 to 7, comprising a residue selected from the group consisting of:

9. the HVR2 region comprises residues 24-79 of any one of SEQ ID NOs: 12-15. Item 9. The engineered meganuclease of any one of items 3 to 8.

10. the first subunit is selected from residues 198 to 344 of any one of SEQ ID NOs: 12 to 15 and wherein said second subunit comprises an amino acid sequence having at least 80% sequence identity to said The unit has at least 80% affinity to residues 7-153 of any one of SEQ ID NOs: 12-15.

10. The method of claim 3, wherein the amino acid sequence has a sequence identity of at least 100%. Meganucleases.

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

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

13. the engineered meganuclease comprises a linker, the linker being linked to the first subunit The method according to any one of claims 3 to 12, wherein the first subunit is covalently linked to the second subunit. Engineered meganucleases.

14. the engineered meganuclease has the amino acid sequence of any one of SEQ ID NOs: 12-15; The engineered meganuclease of any one of claims 3 to 13, comprising an array.

15. 3. The engineered meganuclear of claim 1 or 2, wherein the recognition sequence comprises SEQ ID NO:

6. See.

16. the HVR1 region corresponds to residues 24 to 79 of any one of SEQ ID NOs: 16 to 19 15. The amino acid sequence of claim 15, which has at least 80% sequence identity with the amino acid sequence of 2. An engineered meganuclease according to claim 1.

17. the HVR1 region is selected from residues 24, 26, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, Corresponding to 0, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 17. The engineered meganuclease of claim 15 or 16, comprising a residue comprising:

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

19. the HVR1 region comprises residues 24-79 of any one of SEQ ID NOs: 16-19.

19. The engineered meganuclease of any one of paragraphs 15 to 18.

20. the HVR2 region corresponds to residues 215 to 270 of any one of SEQ ID NOs: 16 to 19; and wherein the amino acid sequence has at least 80% sequence identity with an amino acid sequence corresponding to the amino acid sequence of claim 1.

20. The engineered meganuclease of any one of 15 to 19.

21. the HVR2 region is selected from residues 215, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 2 ９、２２１、２２３、２２４、２２９、２３１、２３３、２３５、２３７、２５９、２６ 21. The method of claim 15, comprising residues corresponding to 1, 266, and 268. Engineered meganucleases.

22. the HVR2 region comprises residues 215-270 of any one of SEQ ID NOs: 16-19; The engineered meganuclease of any one of claims 15 to 21.

23. The first subunit corresponds to residues 7 to 153 of any one of SEQ ID NOs: 16 to 19. and the second subunit comprises an amino acid sequence having at least 80% sequence identity with the first subunit. The fragment has at least 80% identity to residues 198-344 of any one of SEQ ID NOs: 16-19. % sequence identity of the amino acid sequence of any one of claims 15 to 22 Engineered meganucleases.

24. the first subunit comprises residues 7 to 153 of any one of SEQ ID NOs: 16 to 19.

24. The engineered meganuclease of any one of claims 15 to 23.

25. the second subunit comprises residues 198 to 344 of any one of SEQ ID NOs: 16 to 19; 25. The engineered meganuclease of any one of claims 15 to 24, comprising:

26. the engineered meganuclease comprises a linker, the linker being linked to the first subunit 26. The method according to claim 15, wherein the polymerizable monomer unit and the second subunit are covalently bonded to each other.

1. The engineered meganucleases described.

27. the engineered meganuclease has the amino acid sequence of any one of SEQ ID NOs: 16-19; 27. The engineered meganuclease of any one of claims 15 to 26, comprising an array.

28. 3. The engineered meganuclear of claim 1 or 2, wherein the recognition sequence comprises SEQ ID NO:

8. See.

29. the HVR1 region corresponds to residues 24 to 79 of any one of SEQ ID NOs: 20 to 23 28. The amino acid sequence of claim 27, wherein the amino acid sequence has at least 80% sequence identity with the amino acid sequence of claim 27.

2. An engineered meganuclease according to claim 1.

30. the HVR1 region is selected from residues 24, 26, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, Corresponding to 0, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 30. The engineered meganuclease of claim 28 or 29, comprising a residue comprising:

31. the HVR1 region comprises a residue corresponding to residue 66 of any one of SEQ ID NOs: 20-23; 31. The engineered meganuclease of any one of claims 28 to 30, comprising:

32. the HVR1 region comprises residues 24-79 of any one of SEQ ID NOs: 20-23.

32. The engineered meganuclease of any one of paragraphs 28 to 31.

33. the HVR2 region corresponds to residues 215 to 270 of any one of SEQ ID NOs: 20 to 23 and wherein the amino acid sequence has at least 80% sequence identity with an amino acid sequence corresponding to the amino acid sequence of claim 1.

33. The engineered meganuclease of any one of claims 28 to 32.

34. the HVR2 region is selected from residues 215, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 2 ９、２２１、２２３、２２４、２２９、２３１、２３３、２３５、２３７、２５９、２６ 34. The method of claim 28, comprising residues corresponding to 1, 266, and 268. Engineered meganucleases.

35. the HVR2 region comprises residues 215-270 of any one of SEQ ID NOs: 20-23; 35. The engineered meganuclease of any one of claims 28 to 34.

36. The first subunit corresponds 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 the first subunit. nucleotide sequence of at least 80 to residues 198-344 of any one of SEQ ID NOs: 20-23 % sequence identity of the amino acid sequence of any one of claims 28 to 35 Engineered meganucleases.

37. the first subunit comprises residues 7 to 153 of any one of SEQ ID NOs: 20 to 23; 37. The engineered meganuclease of any one of claims 28 to 36.

38. the second subunit comprises residues 198 to 344 of any one of SEQ ID NOs: 20 to 23; 38. The engineered meganuclease of any one of claims 28 to 37, comprising:

39. the engineered meganuclease comprises a linker, the linker being linked to the first subunit 39. The method according to claim 28, wherein the polymerizable monomer is a polymerizable compound, and the polymerizable monomer is a polymerizable compound.

1. The engineered meganucleases described.

40. the engineered meganuclease has the amino acid sequence of any one of SEQ ID NOs: 20-23; 40. The engineered meganuclease of any one of claims 28 to 39, comprising an array.

41. 3. The engineered meganuclea of ​​claim 1 or 2, wherein the recognition sequence comprises SEQ ID NO:

10. -ze.

42. the HVR1 region corresponds to residues 24 to 79 of any one of SEQ ID NOs: 24 to 27 42. The amino acid sequence of claim 41, which comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of 2. An engineered meganuclease according to claim 1.

43. the HVR1 region is selected from residues 24, 26, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, Corresponding to 0, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 43. The engineered meganuclease of claim 41 or 42, comprising a residue comprising:

44. the HVR1 region comprises residues 24-79 of any one of SEQ ID NOs:24-27.

44. The engineered meganuclease of any one of paragraphs 41 to 43.

45. the HVR2 region corresponds to residues 215 to 270 of any one of SEQ ID NOs: 24 to 27 and wherein the amino acid sequence has at least 80% sequence identity with an amino acid sequence corresponding to the amino acid sequence of claim 1.

45. The engineered meganuclease of any one of items 41 to 44.

46. the HVR2 region is selected from residues 215, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 2 ９、２２１、２２３、２２４、２２９、２３１、２３３、２３５、２３７、２５９、２６ 46. ​​The method of claim 41, comprising residues corresponding to 1, 266, and 268. Engineered meganucleases.

47. the HVR2 region comprises residues 215 to 270 of any one of SEQ ID NOs: 24 to 27 47. The engineered meganuclease of any one of claims 41 to 46.

48. The first subunit corresponds to residues 7 to 153 of any one of SEQ ID NOs: 24 to 27. and the second subunit comprises an amino acid sequence having at least 80% sequence identity with the first subunit. The fragment has at least 80% identity to residues 198-344 of any one of SEQ ID NOs: 24-27. % sequence identity of the amino acid sequence of any one of claims 41 to 47 Engineered meganucleases.

49. the first subunit comprises residues 7 to 153 of any one of SEQ ID NOs: 24 to 27; 49. The engineered meganuclease of any one of claims 41 to 48.

50. the second subunit is selected from residues 198 to 344 of any one of SEQ ID NOs: 24 to 27 50. The engineered meganuclease of any one of claims 41 to 49, comprising:

51. the engineered meganuclease comprises a linker, the linker being linked to the first subunit 51. The method according to claim 41, wherein the polymerizable monomer is a polymerizable compound, and the polymerizable monomer is a polymerizable compound.

1. The engineered meganucleases described.

52. the engineered meganuclease has the amino acid sequence of any one of SEQ ID NOs: 24-27; 52. The engineered meganuclease of any one of claims 41 to 51, comprising an array.

53. A nucleic acid encoding the engineered meganuclease of any one of claims 1 to 52. A polynucleotide comprising a nucleic acid sequence.

54. 54. The polynucleotide of claim 53, wherein the polynucleotide is mRNA.

55. The mRNA is the engineered meganuclear vector of any one of claims 1 to 52. a polycistronic nucleic acid encoding a polypeptide and at least one additional polypeptide or nucleic acid; 55. The polynucleotide of claim 54, which is mRNA.

56. 54. A recombinant DNA construct comprising the polynucleotide of claim 53.

57. 57. The method of claim 56, wherein the recombinant DNA construct encodes a viral vector. Recombinant DNA constructs of

58. The viral vector is an adenoviral vector, a lentiviral vector, a retroviral vector, or a 58. The vector of claim 57, which is a viral vector or an adeno-associated viral (AAV) vector. The recombinant DNA constructs described above.

59. 59. The method of claim 57 or claim 58, wherein the viral vector is a recombinant AAV vector. The recombinant DNA constructs described above.

60. 54. A viral vector comprising the polynucleotide of claim 53.

61. The viral vector is an adenoviral vector, a lentiviral vector, a retroviral vector, or a 61. The viral vector of claim 60, which is a viral vector or an AAV vector.

62. 62. The method of claim 60 or claim 61, wherein the viral vector is a recombinant AAV vector. The viral vectors described above.

63. Methods for producing genetically modified T cells containing an exogenous sequence of interest inserted into the chromosome of the T cell The method comprises administering to a T cell: (a) encoding the engineered meganuclease of any one of claims 1 to 52; a first nucleic acid sequence encoding the engineered meganuclease, wherein the engineered meganuclease is expressed in said T cell; a first nucleic acid sequence expressed as a (b) a second nucleic acid sequence comprising a sequence of interest; 、 The engineered meganuclease comprises a human T cell line located 5' upstream of TRAC exon 1. generating a breakage site in said chromosome at a recognition sequence within an intron of a cellular receptor alpha gene; the sequence of interest is inserted into the chromosome at the cleavage site; The sequence of interest may contain an exogenous splice acceptor site and / or a polyA signal. fruit; The endogenous splice donor site and endogenous splice acceptor site flanking the intron The method wherein the target site remains unaltered and / or functional.

64. reduced cell surface expression of endogenous T cell receptors compared to unmodified control cells; 64. The method of claim 63.

65. 65. The method of claim 63 or 64, wherein the intron comprises SEQ ID NO:

3.

66. 66. The method according to any one of claims 63 to 65, (a) the recognition sequence comprises SEQ ID NO: 4 and the engineered meganuclease comprises the sequence of claim 3 15. The engineered meganuclease of any one of claims 1 to 14; (b) the recognition sequence comprises SEQ ID NO:6 and the engineered meganuclease comprises the sequence of claim 1 28. The engineered meganuclease of any one of claims 5 to 27; (c) the recognition sequence comprises SEQ ID NO: 8 and the engineered meganuclease comprises the sequence of claim 2 or the engineered meganuclease of any one of claims 8 to 40; (d) the recognition sequence comprises SEQ ID NO: 10 and the engineered meganuclease comprises 53. A method according to any one of claims 41 to 52, wherein the engineered meganuclease is

67. the second nucleic acid sequence further comprises a sequence homologous to a sequence adjacent to the cleavage site, 67. Any one of claims 63 to 66, wherein a target sequence is inserted into the cleavage site by homologous recombination. The method described in paragraph .

68. Any of claims 63 to 67, wherein the T cells are human T cells or cells derived therefrom.

1. The method according to claim 1.

69. The sequence of interest may contain, from 5' to 3', an exogenous splice acceptor site, a 2A element or comprises an IRES element, a coding sequence for a protein of interest, and a polyA signal.

69. The method of any one of claims 3 to 68.

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

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

72. wherein the sequence of interest comprises a coding sequence for a chimeric antigen receptor or an exogenous T cell receptor.

72. The method according to any one of claims 63 to 71.

73. The chimeric antigen receptor or exogenous T cell receptor has specificity for a tumor-specific antigen.

73. The method of claim 72, comprising an extracellular ligand-binding domain.

74. 63. The method of claim 63, wherein at least the first nucleic acid sequence is introduced into the T cell by mRNA.

74. The method according to any one of claims 1 to 73.

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

76. The viral vector is an adenoviral vector, a lentiviral vector, a retroviral vector, or a 76. The method of claim 75, wherein the vector is a viral vector or an AAV vector.

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

78. Methods for producing genetically modified T cells containing an exogenous sequence of interest inserted into the chromosome of the T cell The method comprises: (a) injecting the engineered meganuclease of any one of claims 1 to 52 into a T cell introducing the compound into the cell; and (b) transfecting the T cells with a nucleic acid comprising the sequence of interest; The engineered meganuclease comprises a human T cell line located 5' upstream of TRAC exon 1. generating a breakage site in said chromosome at a recognition sequence within an intron of a cellular receptor alpha gene; the sequence of interest is inserted into the chromosome at the cleavage site; The sequence of interest may contain an exogenous splice acceptor site and / or a polyA signal. fruit; The endogenous splice donor site and endogenous splice acceptor site flanking the intron The method wherein the target site remains unaltered and / or functional.

79. reduced cell surface expression of endogenous T cell receptors compared to unmodified control cells; 79. The method of claim 78.

80. 80. The method of claim 78 or 79, wherein the intron comprises SEQ ID NO:

3.

81. 81. The method according to any one of claims 78 to 80, (a) the recognition sequence comprises SEQ ID NO: 4 and the engineered meganuclease comprises the sequence of claim 3 15. The engineered meganuclease of any one of claims 1 to 14; (b) the recognition sequence comprises SEQ ID NO:6 and the engineered meganuclease comprises the sequence of claim 1 28. The engineered meganuclease of any one of claims 5 to 27; (c) the recognition sequence comprises SEQ ID NO: 8 and the engineered meganuclease comprises the sequence of claim 2 41. The engineered meganuclease of any one of claims 8 to 40; or (d) the recognition sequence comprises SEQ ID NO: 10 and the engineered meganuclease comprises 53. The method of claim 41, wherein the engineered meganuclease is any one of items 41 to 52.

82. the nucleic acid further comprises a sequence homologous to a sequence adjacent to the cleavage site, and the sequence of interest is 82. The method of claim 81, wherein the insertion occurs at the cleavage site by homologous recombination.

83. 83. The method of claim 81 or 82, wherein the T cell is a human T cell or a cell derived therefrom. How to do it.

84. The sequence of interest may contain, from 5' to 3', an exogenous splice acceptor site, a 2A element or comprises an IRES element, a coding sequence for a protein of interest, and a polyA signal.

84. The method according to any one of claims 8 to 83.

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

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

87. wherein the sequence of interest comprises a coding sequence for a chimeric antigen receptor or an exogenous T cell receptor.

87. The method of any one of claims 78 to 86.

88. The chimeric antigen receptor or exogenous T cell receptor has specificity for a tumor-specific antigen.

88. The method of claim 87, comprising an extracellular ligand-binding domain.

89. 89. The method of claim 78, wherein the nucleic acid is introduced into the T cells by a viral vector.

10. The method according to any one of claims 1 to 9.

90. The viral vector is an adenoviral vector, a lentiviral vector, a retroviral vector, or a 90. The method of claim 89, wherein the vector is a viral vector or an AAV vector.

91. The method of claim 89 or 90, wherein the viral vector is a recombinant AAV vector. Law.

92. Method for producing genetically modified T cells containing a modified human T cell receptor alpha gene The method comprises: (a) To T cells: (i) a first nucleic acid sequence encoding an engineered nuclease, the nuclease is a first nucleic acid sequence expressed in the T cell; or (ii) engineered nuclease proteins; to introduce; and (b) introducing into said cell a second nucleic acid sequence comprising an exogenous sequence of interest; Including, The engineered nuclease is located 5' upstream of TRAC exon 1 of the human TRAC gene. generating a cleavage site at a recognition sequence within an intron of the cell receptor alpha gene; said sequence of interest is inserted into said human T-cell receptor alpha gene at said cleavage site; The sequence of interest may contain an exogenous splice acceptor site and / or a polyA signal. fruit; The endogenous splice donor site and endogenous splice acceptor site flanking the intron The method wherein the target site remains unaltered and / or functional.

93. reduced cell surface expression of endogenous T cell receptors compared to unmodified control cells; 93. The method of claim 92.

94. 95. The method of claim 93 or 94, wherein the intron comprises SEQ ID NO:

3.

95. 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) the exogenous sequence of interest; and (c) a 3' homology arm that is homologous to a 3' downstream sequence adjacent to the cleavage site; The exogenous sequence of interest is inserted into the human T cell receptor alpha at the cleavage site by homologous recombination.

95. The method of any one of claims 92 to 94, wherein the gene is inserted into the fa gene.

96. The genetically modified T cells are genetically modified human T cells or cells derived therefrom. The method of any one of claims 92 to 95.

97. The exogenous sequence of interest comprises, 5' to 3', an exogenous splice acceptor site, 2A a coding sequence for a protein of interest, and a poly(A) signal. The method of any one of claims 92 to 96.

98. the 2A element is a T2A element, a P2A element, an E2A element, or an F2A element. Item 98. The method according to item 97.

99. 99. The method of claim 97 or 98, wherein the 2A element is a T2A element.

100. wherein the sequence of interest comprises a coding sequence for a chimeric antigen receptor or an exogenous T cell receptor. The method of any one of claims 92 to 99.

101. The chimeric antigen receptor or exogenous T cell receptor has specificity for a tumor-specific antigen. The method of claim 100, comprising an extracellular ligand-binding domain.

102. 93. Claim 92, wherein at least the first nucleic acid sequence is introduced into the T cell by mRNA.

102. The method according to any one of claims 1 to 101.

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

104. The viral vector is an adenoviral vector, a lentiviral vector, a retroviral vector, or a 104. The method of claim 103, which is a viral vector or an adeno-associated virus (AAV) vector. The method described.

105. 105. The method of claim 103 or 104, wherein the viral vector is a recombinant AAV vector. How to do it.

106. The engineered nuclease may be an engineered meganuclease, a zinc finger nuclease, or ZFNs, transcription activator-like effector nucleases (TALENs), and 92. A pactoTALEN, a CRISPR nuclease, or a megaTAL.

106. The method according to any one of claims 1 to 105.

107. 92-1, wherein the engineered nuclease is an engineered meganuclease.

06. The method according to any one of claims 06 to 06.

108. The engineered meganuclease has specificity for a recognition sequence comprising SEQ ID NO:

4. The method of claim 107.

109. The engineered meganuclease according to any one of claims 3 to 14.

109. The method of claim 107 or 108, wherein the meganuclease is a

110. The engineered meganuclease has specificity for a recognition sequence comprising SEQ ID NO:

6. The method of claim 107.

111. The engineered meganuclease of any one of claims 15 to 27.

111. The method of claim 107 or 110, wherein the meganuclease is a recombinant meganuclease.

112. The engineered meganuclease has specificity for a recognition sequence comprising SEQ ID NO:

8. The method of claim 107.

113. The engineered meganuclease of any one of claims 28 to 40.

113. The method of claim 107 or 112, wherein the meganuclease is a recombinant meganuclease.

114. The engineered meganuclease has specificity for a recognition sequence comprising SEQ ID NO:

10. The method of claim 107.

115. The engineered meganuclease of any one of claims 41 to 52.

115. The method of claim 107 or 114, wherein the meganuclease is a modified meganuclease.

116. A genetically modified T cell prepared by the method of 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. The modified human T cell receptor alpha gene is located 5' upstream of TRAC exon 1 comprising an exogenous sequence of interest inserted into an intron within the T cell receptor alpha gene, The exogenous sequence of interest may include an exogenous splice acceptor site and / or a polyA signal. and an endogenous splice donor site and an endogenous splice access site adjacent to the intron. The peptide site remains unaltered and / or functional, and is an endogenous T cell receptor. wherein cell surface expression of is reduced as compared to unmodified control cells.

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

3.

119. The genetically modified T cells are genetically modified human T cells or cells derived therefrom. Item 119. The genetically modified T cell of item 117 or 118.

120. The exogenous sequence of interest comprises, 5' to 3', an exogenous splice acceptor site, 2A a coding sequence for a protein of interest, and a poly(A) signal.

120. The genetically modified T cell of any one of claims 117 to 119.

121. 2A element is a T2A element, a P2A element, an E2A element, or an F2A element.

120. A genetically modified T cell according to 120.

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

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

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

125. The exogenous sequence of interest may comprise an engineered meganuclease recognition site, a TALEN recognition site, , zinc finger nuclease recognition site, CRISPR recognition site, or megaTAL 125. The method of claim 117, wherein the nucleic acid sequence of claim 117 is inserted into the intron at a recognition site. Genetically modified T cells.

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

127. The exogenous sequence of interest is selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO:

10. The gene modification according to any one of claims 117 to 126, wherein the gene modification is inserted into the intron at Mutant T cells.

128. A genetically modified T cell comprising a plurality of the genetically modified T cells of any one of claims 116 to 127. Population of child modified T cells.

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 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 composition is in accordance with any one of claims 116 to 127.

129. The population of claim 128, wherein the genetically modified T cells are

130. The genetically modified T cells are genetically modified human T cells or cells derived therefrom. , the population described in claim 128 or claim 129.

131. wherein the sequence of interest comprises a coding sequence for a chimeric antigen receptor or an exogenous T cell receptor. The population described in any one of claims 128 to 130.

132. The chimeric antigen receptor or the exogenous T cell receptor has specificity for a tumor-specific antigen.

132. The population of claim 131 , comprising an extracellular ligand-binding domain having the following structure:

133. The cell surface expression of endogenous T cell receptors is increased by the gene modification compared to unmodified control cells.

133. The population of any one of claims 128 to 132, which is reduced on transformed T cells.

134. 1. A pharmaceutical composition useful for treating a disease in a subject in need thereof, comprising: A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of any of claims 116 to 127. A pharmaceutical composition comprising the genetically modified T cell of any one of claims 1 to 14.

135. The genetically modified T cells are genetically modified human T cells or cells derived therefrom. Item 135. The pharmaceutical composition according to Item 134.

136. wherein the sequence of interest comprises a coding sequence for a chimeric antigen receptor or an exogenous T cell receptor.

136. The pharmaceutical composition of claim 134 or 135.

137. The chimeric antigen receptor or the exogenous T cell receptor is specific for a tumor-specific antigen.

137. The pharmaceutical composition of claim 136, comprising an extracellular ligand-binding domain having the following structure:

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

139. Administering the genetically modified T cells of any one of claims 116 to 127 to a subject and (c) administering to a subject in need thereof a therapeutically effective amount of a compound selected from the group consisting of benzodiazepine, ...

140. The method comprises administering the pharmaceutical composition of any one of claims 134 to 138 to the subject.

140. The method of claim 139, comprising administering to the body.

141. The method is an immunotherapy for the treatment of cancer in a subject in need thereof, The genetically modified T cells are genetically modified human T cells or cells derived therefrom, The sequence is an extracellular ligand-binding domain with specificity for a chimeric antigen receptor or tumor-specific antigen. a coding sequence for an exogenous T cell receptor containing an endogenous T cell receptor, and is reduced on the genetically modified T cells compared to unmodified control cells. 139 or 140.

142. the cancer is selected from the group consisting of carcinoma, lymphoma, sarcoma, blastoma, and leukemia cancers; The method of claim 141.

143. The cancer is a cancer of B-cell origin, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, melanoma, or prostate cancer. , colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, and Hodgkin's lymphoma 143. The method of claim 141 or claim 142, wherein the method is selected from:

144. The cancer of B-cell origin is B-lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, 15. The method of claim 14, wherein the cancer is selected from the group consisting of B-cell non-Hodgkin's lymphoma, B-cell non-Hodgkin's lymphoma, and multiple myeloma.

3. The method according to claim 3.

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