Engineered meganucleases with recognition sequences found in the human t cell receptor alpha constant region gene

Abstract

To provide means for creating T cells from a third party donor by reducing the expression of the endogenous T cell receptor and not initiating GVHD upon administration.SOLUTION: Disclosed herein are recombinant meganucleases engineered to recognize and cleave a recognition sequence present in the human T cell receptor alpha constant region gene. The present disclosure further relates to the use of such recombinant meganucleases in methods for producing genetically modified eukaryotic cells.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application relates to the "Human T-cell receptor alpha constant region gene" filed on February 19, 2016. U.S. Provisional Patent Issued: "Meganucleases Engineered for Recognition Sequences Found in Genes" Application No. 62 / 297,445 and the "Human T Cell Receptor" patent application filed on October 5, 2015 "Meganucleases engineered against recognition sequences found in human alpha constant region genes" This application claims priority to U.S. Provisional Patent Application No. 62 / 237,382, entitled , the disclosures of which are incorporated herein by reference in their entireties.

[0002] The present invention relates to the fields of oncology, cancer immunotherapy, molecular biology, and recombinant nucleic acid technology. The present invention relates to a method for detecting and regulating a recognition sequence found in the human T cell receptor alpha constant region gene. The present invention further relates to recombinant meganucleases engineered to cleave and / or amplify the nucleotide sequences of a target gene. The use of such recombinant meganucleases in methods for producing transformed eukaryotic cells is also disclosed. Regarding.

[0003] Reference to sequence listing submitted as a text file via EFS-WEB This application contains a sequence listing that was submitted in ASCII format via EFS-Web. and is incorporated herein by reference in its entirety. The name of the resulting ASCII copy is 2000706_00179WO1.txt. It is 264,046 bytes in size. [Background technology]

[0004] T cell adoptive immunotherapy is a promising approach for cancer treatment. This strategy targets specific Isolated human T cells genetically modified to enhance their specificity for tumor-associated antigens Genetic modification utilizes chimeric antigen receptors (CARs) to transfer antigen specificity to T cells. This may involve the expression of exogenous T cell receptors (ARs) or exogenous T cell receptors. Antigen receptors derive their specificity from the variable domains of monoclonal antibodies. Therefore, T cells expressing chimeric antigen receptors (CAR T cells) bind to major histocompatibility complexes (MCCs). To date, T cell adoptive immunotherapy has not been limited to B cells. Alveolar malignancies (e.g., acute lymphoblastic leukemia (ALL), B-cell non-Hodgkin's lymphoma ( NHL, and chronic lymphocytic leukemia), multiple myeloma, neuroblastoma, glioblastoma, advanced Clinical treatment of multiple cancers, including glioma, ovarian cancer, mesothelioma, melanoma, and pancreatic cancer It has been used as a

[0005] Despite their potential utility as cancer treatments, adoptive immunotherapy with CAR T cells has To some extent, this has been limited by the expression of endogenous T cell receptors on the cell surface. CAR T cells expressing histocompatibility receptors have been shown to be major and minor histocompatible following administration to allogeneic patients. These antigens can lead to the development of graft-versus-host disease (GVHD). The experiment involves isolating a patient's T cells, genetically modifying them to incorporate a chimeric antigen receptor, and then The current approach is largely focused on the use of autologous CAR T cells, which are then reinfused into the same patient. Previous approaches provide immune tolerance to the administered CAR T cells, but this approach The study explores the time and cost required to generate patient-specific CAR T cells after a patient's cancer is diagnosed. The constraints are both on the

[0006] Therefore, it reduces the expression of endogenous T cell receptors and prevents the development of GVHD upon administration. Developing "off-the-shelf" CAR T cells prepared using T cells from three donors Such products would be advantageous because they would be generated and validated prior to diagnosis and, if necessary, Therefore, it is possible to provide the patient with endogenous factors to prevent the occurrence of GVHD. There is a need to develop allogeneic CAR T cells that lack the human T cell receptor.

[0007] Genetic modification of genomic DNA allows the gene to recognize specific DNA sequences at a desired locus. engineered site-specific, rare-cutting homing endonucleases (" This can be done using homing endonucleases (also called meganucleases). The enzymes cleave the 15-40 base pair cleavage sites commonly found in plant and fungal genomes. They are a group of naturally occurring nucleases that recognize the parasite DNA element , frequently associated with, for example, group 1 self-splicing introns and inteins They create double-strand breaks in chromosomes that mobilize the cell's DNA repair machinery. By allowing the host genome to undergo homologous recombination or gene insertion at specific locations, Homing endonucleases are generally LAGLIDADG (sequence Row number 7) Family, GIY-YIG family, His-Cys box family These families are classified into four families: the HNH family and the ATP family. They are characterized by structural motifs that influence their affinity and recognition sequence. For example, LAGLIDADG (SEQ ID NO: 7) family members contain the conserved LAGLIDADG (SEQ ID NO: 7) They are characterized by having either one or two copies of the motif (Non-Patent Document 2). LAGLIDADG with a single copy of the LAGLIDADG (SEQ ID NO: 7) motif (SEQ ID NO: 7) Homing endonuclease forms a homodimer, whereas LAGLID Members with two copies of the ADG (SEQ ID NO: 7) motif are found as monomers. do.

[0008] Methods for generating engineered site-specific recombinant meganucleases are known in the art. I-CreI (SEQ ID NO: 6) is a gene encoding the enzyme I-CreI from the alga Chlamydomonas reinhardtii (C 22 base pairs in the chloroplast chromosome of Hlamydomonas reinhardtii The homing endonuclease LAGLIDADG (sequence number LAGLIDADG) recognizes and cleaves the recognition sequence. (Column number 7) is a member of the family. To achieve this, genetic selection techniques have been used (Non-Patent Documents 3-6). CreI and other homing endonucleases are expressed in mammalian, yeast, plant, bacterial, and Comprehensive targeting of a wide range of DNA sites, including sites in the viral genome Redesignable mono-LAGLIDADG (SEQ ID NO: 7) homing endonuclease A rational design method is described (Patent Document 1).

[0009] As first described in US Pat. No. 5,629,999, I-CreI and its engineered derivatives It is usually a dimer, but contains a short peptide that connects the C-terminus of the first subunit to the N-terminus of the second. Using a linker, it is possible to fuse the two proteins into a single polypeptide (Non-Patent Documents 7-8 ) Therefore, it is possible to express a functional "single-chain" meganuclease from a single transcript. Such engineered meganucleases have extremely low off-target cleavage frequency. By delivering a gene encoding a single-chain meganuclease into cells, TC Capable of specifically and preferentially targeting, cleaving, and disrupting the R alpha constant region gene is.

[0010] Engineered meganucleotides for cleaving DNA targets in the human T cell receptor alpha constant region The use of cleavage has been previously disclosed in US Pat. No. 5,629,493, which discloses a TCR alpha- It targets the recognition sequence in exon 1 of the human constant region gene (SEQ ID NO: 3 in Patent Document 3). Patent Document 3 discloses a mutant of I-OnuI meganuclease engineered as follows: discuss that chimeric antigen receptors can be expressed in TCR knockout cells. In the study, the authors described a chimeric nucleotide sequence encoding a meganuclease cleavage site in the TCR alpha constant region gene. There is no disclosure of the insertion of antigen receptor coding sequences.

[0011] The use of other nucleases and mechanisms to disrupt endogenous TCR expression has also been disclosed. For example, zinc finger nucleotides for disrupting TCR genes in human T cells have been used. The use of zinc finger ATPases is described in US Pat. No. 5,623,297 and US Pat. No. 5,623,297. Nucleases and transcription activator-like effector nucleases (TALENs), as well as single It has a single guide RNA engineered to target TCR genes in isolated T cells. Patent document 7 describes the use of the CRISPR / Cas system to induce specific leukemia in T cells. Use of small hairpin RNAs to target nucleic acids encoding target TCR and / or CD3 chains has disclosed. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] International Publication No. 2007 / 047859 Pamphlet [Patent Document 2] International Publication No. 2009 / 059195 Brochure [Patent Document 3] International Publication No. 2014 / 191527 Brochure [Patent Document 4] U.S. Patent No. 8,955,828 [Patent Document 5] US Patent Application Publication No. 2014 / 034902 [Patent Document 6] US Patent Application Publication No. 2014 / 0301990 [Patent Document 7] US Patent Application Publication No. 2012 / 0321667 [Non-patent literature]

[0013] [Non-Patent Document 1] Q.Rev.Biophys.38(2006):49-95 [Non-patent document 2] Nucleic Acids Res.29(18)(2001):3757-3774 [Non-patent document 3] J. Mol. Biol. 342(2004):31-41 [Non-patent document 4] Nucleic Acids Res.33(2005):e178 [Non-Patent Document 5] Nucleic Acids Res.30(2002):3870-9 [Non-patent document 6] J. Mol. Biol. 355(2006):443-58 [Non-Patent Document 7] Nucleic Acids Res.37(2009):1650-62 [Non-patent document 8] Nucleic Acids Res.37(2009):5405-19 Summary of the Invention [Problem to be solved by the invention]

[0014] However, the present invention improves upon the teachings of the prior art. The present inventors have developed a method for determining the human TCR alpha constant. exogenous polynucleotide sequences inserted into common region genes (e.g., chimeric antigen receptors or exogenous TCR coding sequence) while simultaneously suppressing the expression of endogenous T cell receptors on the cell surface. Furthermore, the prior art is the first to teach the destruction of genetically modified cells. A ganucleases or recognition sequences, or their use in generating such genetically modified cells. The document does not teach the use of these. [Means for solving the problem]

[0015] The present invention relates to a method for identifying the residues of the human T cell receptor (TCR) alpha constant region gene (SEQ ID NO: 1). A recombinant megagene engineered to recognize and cleave the recognition sequence found within 93-208 Such meganucleases can be used to target the TCR alpha constant region gene. and thus are useful for disrupting cell surface TCR expression and / or function. Therefore, the meganucleases of the present invention have utility in immunotherapy, including cancer immunotherapy. Meganuclease cleavage may result in the mutagenic effects of non-homologous end joining or homologous recombination. disrupting gene function by either facilitating the introduction of exogenous polynucleotides into genes via In some embodiments, the introduced exogenous polynucleotide demonstrates that meganucleases are useful for generating allogeneic CART cells lacking endogenous TCRs. Thus, the present invention includes nucleic acid sequences encoding chimeric antigen receptors.

[0016] The present invention further provides a method for producing a genetically modified eukaryotic cell by using a recombinant meganuclease protein. a method comprising delivering a gene encoding a protein or a recombinant meganuclease into a eukaryotic cell; Further provide the law.

[0017] Thus, in one aspect, the present invention provides a method for the production of a human T cell receptor alpha constant region gene (sequence A recombinant meganuclease that recognizes and cleaves the recognition sequence within residues 93 to 208 of Such a recombinant meganuclease comprises a first subunit and a second subunit. a first subunit that binds to a first recognition half-site of the recognition sequence and a first superunit that binds to a first recognition half-site of the recognition sequence; The second subunit comprises a variable (HVR1) region and binds to a second recognition half-site of the recognition sequence. and contains the second hypervariable (HVR2) region.

[0018] In one embodiment, the recognition sequence comprises SEQ ID NO:3 (ie, the TRC1-2 recognition sequence).

[0019] In one such embodiment, the first meganuclease subunit is SEQ ID NO:8 Residues 198 to 344 of any one of SEQ ID NOS: 19 to 27 Groups 7 to 153 and at least 80%, at least 85%, at least 90%, or at least The second meganuclease subunit also contains an amino acid sequence with 95% sequence identity. The target is residues 7 to 153 of any one of SEQ ID NOs: 8 to 18 or any one of SEQ ID NOs: 19 to 27. At least 80%, at least 85%, at least 90% of residues 198-344 %, or at least 95% sequence identity.

[0020] In another such embodiment, the HVR1 region comprises (a) any one of SEQ ID NOs: 8-18. or (b) position 215 of any one of SEQ ID NOs: 19 to 27; or In another such embodiment, the HVR1 region comprises a Y at position (b) position 233 of any one of SEQ ID NOs: 19 to 27; or (c) position 4 of any one of SEQ ID NOs: 19 to 27 In another such embodiment, the HVR1 region comprises a G at the position corresponding to position 2. (a) positions 215 and 233, respectively, of any one of SEQ ID NOs: 8 to 18; or (b) the sequence Y and G at positions corresponding to positions 24 and 42, respectively, of any one of Nos. 19 to 27 Includes one or more of the following.

[0021] In another such embodiment, the HVR2 region comprises (a) any one of SEQ ID NOs: 8-18. (b) position 26 of any one of SEQ ID NOs: 19 to 27; or (b) position 217 of any one of SEQ ID NOs: 19 to 27 In another such embodiment, the HVR2 region comprises (a) a T at position 8 of SEQ ID NO: 8. (b) position 28 of any one of SEQ ID NOs: 19 to 27; or (b) position 219 of any one of SEQ ID NOs: 19 to 27 In another such embodiment, the HVR2 region comprises F or Y at the position corresponding to (a) position 38 of any one of SEQ ID NOs: 8 to 18; or (b) position 38 of any one of SEQ ID NOs: 19 to 27 In another such embodiment, any one of the amino acids includes an F at a position corresponding to position 229. The HVR2 region is (a) position 44 of any one of SEQ ID NOs: 8 to 18; or (b) position 44 of SEQ ID NO: and S at a position corresponding to position 235 of any one of 19 to 27. In this embodiment, the HVR2 region comprises: (a) position 46 of any one of SEQ ID NOs: 8 to 18; or b) containing F or Y at a position corresponding to position 237 of any one of SEQ ID NOs: 19 to 27 In another such embodiment, the HVR2 region comprises (a) any one of SEQ ID NOs: 8-18. or (b) one of SEQ ID NOs: 19-27; Any one of the positions corresponding to positions 217, 219, 229, 235, and 237, respectively includes one or more of T, F or Y, F, S and F or Y and R.

[0022] In another such embodiment, the HVR1 region is selected from the group consisting of any one of SEQ ID NOs: 8-18. or residues 24 to 79 of any one of SEQ ID NOs: 19 to 27. In another such embodiment, the HVR2 region comprises any one of SEQ ID NOs: 8-18. It comprises residues 24 to 79 or residues 215 to 270 of any one of SEQ ID NOs: 19 to 27.

[0023] In another such embodiment, the first meganuclease subunit is SEQ ID NO: Residues 198 to 344 of any one of SEQ ID NOs: 8 to 18 or any one of SEQ ID NOs: 19 to 27 In another such embodiment, the second meganuclease subunit comprises residues 7 to 153. The unit is composed of residues 7 to 153 of any one of SEQ ID NOs: 8 to 18 or residues 19 to 27 and any one of residues 198 to 344.

[0024] In another such embodiment, the recombinant meganuclease comprises a single-chain meganuclease comprising a linker. a nuclease, wherein the linker is shared between the first and second subunits Combine.

[0025] In another such embodiment, the recombinant meganuclease is any of SEQ ID NOs: 8-27. It contains one of the amino acid sequences.

[0026] In a further embodiment, the recognition sequence is SEQ ID NO: 4 (i.e., the TRC3-4 recognition sequence). Includes.

[0027] In one such embodiment, the first meganuclease subunit is SEQ ID NO:2 8 or 29 residues 7 to 153 and at least 80%, at least 85%, at least 90% or a second meganuclear comprising an amino acid sequence having at least 95% sequence identity. The ze subunit is at least 80% identical to residues 198-344 of SEQ ID NO: 28 or 29, amino acids with at least 85%, at least 90%, or at least 95% sequence identity Contains the acid sequence.

[0028] In another such embodiment, the HVR1 region comprises the amino acid sequence at position 24 of SEQ ID NO: 28 or 29. In another such embodiment, the HVR1 region comprises a Y at the corresponding position. In another such embodiment, H The VR1 region contains a Y at a position corresponding to position 46 of SEQ ID NO: 28 or 29. In certain embodiments, the HVR1 region comprises a sequence identical to that at positions 24 and 26 of SEQ ID NO: 28 or 29, respectively. , and one or more of Y, T, and Y at positions corresponding to 46.

[0029] In another such embodiment, the HVR2 region comprises position 215 of SEQ ID NO: 28 or 29. In another such embodiment, the HVR2 region comprises an H at the position corresponding to SEQ ID NO: In another such embodiment, the amino acid sequence of SEQ ID NO: 28 or 29 contains a T at a position corresponding to position 266 of SEQ ID NO: 28 or 29. Alternatively, the HVR2 region contains a C at a position corresponding to position 268 of SEQ ID NO: 28 or 29. In such embodiments, the HVR2 region comprises amino acids 215, 266, 270, 280, 290, 300, 315, 326, 330, 340, 350, 360, 370, 380, 390, 400, 415, 426, 430, 440, 450, 460, 470, and containing one or more of H, T, and C at the position corresponding to position 268.

[0030] In another such embodiment, the HVR1 region comprises residue 24 of SEQ ID NO: 28 or 29. In another such embodiment, the HVR2 region comprises SEQ ID NO: 28 or 29. 9, including residues 215–270.

[0031] In another such embodiment, the first meganuclease subunit is SEQ ID NO: In another such embodiment, the second meganucleotide comprises residues 7 to 153 of 28 or 29. The nuclease subunit comprises residues 198 to 344 of SEQ ID NO:28 or 29.

[0032] In another such embodiment, the recombinant meganuclease comprises a single-chain meganuclease comprising a linker. a nuclease, wherein the linker is shared between the first and second subunits Combine.

[0033] In another such embodiment, the recombinant meganuclease is SEQ ID NO: 28 or 29 It contains the amino acid sequence of

[0034] In a further embodiment, the recognition sequence comprises SEQ ID NO:5 (i.e., the TRC7-8 recognition sequence). include.

[0035] In one such embodiment, the first meganuclease subunit is SEQ ID NO:3 Residues 7 to 153 of SEQ ID NO: 30 or residues 198 to 344 of SEQ ID NO: 31 or 32 and at least 8 0%, at least 85%, at least 90%, or at least 95% sequence identity and the second meganuclease subunit comprises an amino acid sequence comprising residue 1 of SEQ ID NO: 30. 98 to 344 or residues 7 to 153 of SEQ ID NO: 31 or 32, amino acids with at least 85%, at least 90%, or at least 95% sequence identity Contains arrays.

[0036] In another such embodiment, the HVR1 region comprises (a) position 24 of SEQ ID NO: 30; or or (b) contains a Y at a position corresponding to position 215 of SEQ ID NO: 31 or 32.

[0037] In another such embodiment, the HVR2 region comprises: (a) position 215 of SEQ ID NO: 30; or (b) containing Y or W at a position corresponding to position 24 of SEQ ID NO: 31 or 32. In such embodiments, the HVR2 region comprises: (a) position 231 of SEQ ID NO: 30; or (b) containing M, L, or W at a position corresponding to position 40 of SEQ ID NO: 31 or 32; In another such embodiment, the HVR2 region comprises: (a) position 237 of SEQ ID NO: 30; or or (b) contains a Y at a position corresponding to position 46 of SEQ ID NO: 31 or 32. In one embodiment, the HVR2 region comprises: (a) a sequence comprising the amino acid sequence at positions 215 and 231 of SEQ ID NO: 30, respectively; , and 237; or (b) positions 24, 40, and 237 of SEQ ID NO: 31 or 32, respectively. At the position corresponding to position 46, it contains one or more of Y or W, M, L or W and Y.

[0038] In another such embodiment, the HVR1 region comprises residues 24-79 or 25 of SEQ ID NO: 30. In another such embodiment, the nucleotide sequence comprises residues 215-270 of SEQ ID NO: 31 or 32. The HVR2 region is defined as residues 215 to 270 of SEQ ID NO: 30 or residues 31 or 32 of SEQ ID NO: 33. 2, containing residues 24–79.

[0039] In another such embodiment, the first meganuclease subunit is SEQ ID NO: 30 or residues 198 to 344 of SEQ ID NO: 31 or 32. In such an embodiment, the second meganuclease subunit has the sequence of SEQ ID NO: 30 Contains residues 198 to 344 or residues 7 to 153 of SEQ ID NO: 31 or 32.

[0040] In another such embodiment, the recombinant meganuclease comprises a single-chain meganuclease comprising a linker. a nuclease, wherein the linker is shared between the first and second subunits Combine.

[0041] In another such embodiment, the recombinant meganuclease is selected from the group consisting of SEQ ID NOs: 30-32. It contains any one of the amino acid sequences.

[0042] In another aspect, the present invention provides a method for producing a recombinant meganuclease encoding the recombinant meganuclease described herein. An isolated polynucleotide comprising the nucleic acid sequence is provided.

[0043] In various embodiments of the present invention, the isolated polynucleotide can be mRNA. In this embodiment, the mRNA encodes a single-chain recombinant meganuclease as described herein. In other embodiments, the mRNA can comprise at least one of the sequences described herein. The coding sequence for one meganuclease and at least one further protein (e.g., Polycistronic mRNA (e.g., bicistronic mRNA) containing the coding sequence for a second nuclease In certain embodiments, the polycistronic may be a polysymmetric polymer (e.g., polysymmetric, polyisotropic ... Contrasting mRNAs are expressed in the same gene (e.g., T-cell receptor alpha constant region gene). and encoding two or more meganucleases described herein that target different recognition sequences. In other embodiments, the polycistronic mRNA can be The meganuclease described herein and the same gene (e.g., T cell receptor alpha constant region gene) It recognizes and cleaves a different recognition sequence within a gene of interest in the genome. A second nuclease can be encoded that recognizes and cleaves a different recognition sequence. In such embodiments, polycistronic mRNAs produced using such polycistronic mRNAs Genetically modified cells can have multiple genes knocked out simultaneously. Genetically modified cells produced using such polycistronic mRNAs Insertion into the genome at one or more of the cleavage sites generated by the administered nuclease. In a further embodiment, the polycistronic amino acid sequence may be an exogenous sequence. The mRNA contains at least one meganuclease described herein and one or more genes useful for the cell. encoding two additional proteins, improving the efficiency of insertion of an exogenous sequence of interest into the cleavage site; and / or is beneficial in treating a disease.

[0044] In another aspect, the present invention provides a method for producing a recombinant meganuclease encoding the recombinant meganuclease described herein. A recombinant DNA construct is provided, comprising an isolated polynucleotide comprising a nucleic acid sequence. In one embodiment, the recombinant DNA construct encodes a viral vector. Thus, the recombinant DNA construct encodes a recombinant AAV vector.

[0045] In another aspect, the present invention provides a method for producing a recombinant meganuclease encoding the recombinant meganuclease described herein. In another aspect, a viral vector is provided, comprising an isolated polynucleotide comprising a nucleic acid sequence. In accordance with the present invention, a method for producing a recombinant meganuclease comprising the steps of: The present invention provides a recombinant AAV vector comprising an isolated polynucleotide comprising:

[0046] In another aspect, the present invention provides a method for the detection of a gene comprising an exogenous sequence of interest inserted into the chromosome of a eukaryotic cell. A method for producing a genetically modified eukaryotic cell, comprising: (b) a first nucleic acid sequence encoding a recombinant meganuclease; and (b) a second nucleic acid sequence comprising a sequence of interest. 2 nucleic acid sequence, The meganuclease has a recognition sequence comprising SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. A method for creating a cleavage site in a chromosome and inserting a sequence of interest into the chromosome at the cleavage site. to provide.

[0047] In one embodiment of this method, the second nucleic acid contains a sequence homologous to the sequence adjacent to the cleavage site. wherein the sequence of interest is inserted into the cleavage site by homologous recombination.

[0048] In another embodiment of this method, the second nucleic acid lacks substantial homology to the cleavage site; The sequence of interest is inserted into the chromosome by non-homologous end joining.

[0049] In another embodiment of this method, the eukaryotic cell is a human T cell or a cell derived therefrom. do.

[0050] In another embodiment of this method, the sequence of interest encodes a chimeric antigen receptor. In another embodiment of the method, the sequence of interest encodes an exogenous T cell receptor.

[0051] In another embodiment of this method, at least the first nucleic acid sequence is delivered to the cell by mRNA. In some such embodiments, the mRNA is introduced into a At least one recombinant meganuclease and at least one further protein (e.g. For example, it may be a polycistronic mRNA encoding a second nuclease.

[0052] In another embodiment of this method, the at least second nucleic acid sequence is incorporated into a viral vector. In another embodiment of this method, the first nucleic acid sequence and The two nucleic acid sequences can be delivered by the same viral vector or by separate viral vectors. It is introduced into a eukaryotic cell.

[0053] In another embodiment of this method, the at least second nucleic acid sequence is a recombinant AAV vector. In another embodiment of this method, the first nucleic acid sequence and The first and second nucleic acid sequences may be expressed by the same recombinant AAV vector or by separate recombinant AAV vectors. It is introduced into eukaryotic cells by a vector.

[0054] In another embodiment of this method, the at least second nucleic acid sequence is a single-stranded DNA template. It is introduced into eukaryotic cells using

[0055] In a particular embodiment of this method, the recombinant meganuclease described herein is The first nucleic acid sequence is introduced into the cell by mRNA, and the second nucleic acid sequence containing the exogenous sequence of interest is introduced into the cell by mRNA. The nucleic acid sequence is introduced into the cell using a viral vector, preferably a recombinant AAV vector. The cells are human T cells and the sequence of interest encodes a chimeric antigen receptor. In an embodiment, the method comprises a chimeric antigen receptor, and Genetically modified T cells are generated that have reduced cell surface expression of the cytotoxic T cell receptor.

[0056] In another aspect, the present invention provides a method for the detection of a gene comprising an exogenous sequence of interest inserted into the chromosome of a eukaryotic cell. A method for producing a genetically modified eukaryotic cell, comprising: (a) a recombinant megakaryotic cell as described herein; (b) introducing a nuclease into a eukaryotic cell; and (b) introducing into said eukaryotic cell a nuclease containing a sequence of interest. transfecting a recombinant meganuclease comprising the sequence of SEQ ID NO: 3, A cleavage site is generated in the chromosome at a recognition sequence containing SEQ ID NO: 4 or SEQ ID NO: 5, and the target sequence is The method provides a method in which the sequence is inserted into the chromosome at the break site.

[0057] In one embodiment of this method, the nucleic acid further comprises a sequence homologous to a sequence adjacent to the cleavage site. and the sequence of interest is inserted into the cleavage site by homologous recombination.

[0058] In another embodiment of this method, the nucleic acid lacks substantial homology to the cleavage site and is The sequence is inserted into the chromosome by non-homologous end joining.

[0059] In another embodiment of this method, the eukaryotic cell is a human T cell or a cell derived therefrom. do.

[0060] In another embodiment of this method, the sequence of interest encodes a chimeric antigen receptor. In another embodiment of the method, the sequence of interest encodes an exogenous T cell receptor.

[0061] In another embodiment of this method, the nucleic acid sequence is introduced into the eukaryotic cell by a viral vector. It will be introduced.

[0062] In another embodiment of this method, the nucleic acid sequence is delivered to a eukaryotic cell by a recombinant AAV vector. is introduced into the cells.

[0063] In another embodiment of this method, the nucleic acid sequence is isolated from a eukaryotic cell using a single-stranded DNA template. will be introduced.

[0064] In another aspect, the present invention provides a method for the production of a gene encoding ... for a eukaryotic cell by disrupting a target sequence in the chromosome of the eukaryotic cell. A method for producing a genetically modified eukaryotic cell, comprising injecting the eukaryotic cell with a recombinant megagene as described herein. transfecting a nucleic acid encoding a nuclease into the recombinant meganuclease; The enzyme cleaves the chromosome at a recognition sequence comprising SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5. A method for generating a cleavage site and disrupting a target sequence at the cleavage site by non-homologous end joining. to provide.

[0065] In one embodiment of this method, the eukaryotic cell is a human T cell or a cell derived therefrom. do.

[0066] In another embodiment of this method, the nucleic acid encoding the recombinant meganuclease is mRNA. In some such embodiments, the mRNA is at least one of the at least two ... at least one recombinant meganuclease and at least one further protein (e.g., The mRNA may be a polycistronic mRNA encoding a nuclease (a second nuclease).

[0067] In another embodiment, the method further comprises introducing into the eukaryotic cell a second nucleic acid comprising the exogenous sequence of interest. In one such embodiment of this method, the second The nucleic acid further comprises a sequence homologous to a sequence adjacent to the cleavage site, and the sequence of interest is is inserted into the cleavage site by

[0068] In another embodiment of this method, the sequence of interest encodes a chimeric antigen receptor. In another embodiment of the method, the sequence of interest encodes an exogenous T cell receptor.

[0069] In another embodiment of this method, the at least second nucleic acid is delivered by a viral vector. and introduced into eukaryotic cells.

[0070] In another embodiment of this method, the at least second nucleic acid is contained in a recombinant AAV vector. Thus, it is introduced into eukaryotic cells.

[0071] In another embodiment of this method, the at least second nucleic acid sequence is a single-stranded DNA template. It is introduced into eukaryotic cells using

[0072] In another aspect, the present invention provides a method for the production of a gene encoding ... for a eukaryotic cell by disrupting a target sequence in the chromosome of the eukaryotic cell. A method for producing a genetically modified eukaryotic cell, comprising administering to the cell a recombinant meganuclease as described herein. into a eukaryotic cell, wherein the meganuclease is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, Alternatively, a cleavage site is generated in the chromosome at a recognition sequence containing SEQ ID NO: 5, and the target sequence is the cleavage site. The method provides a method for disrupting a region at a site by non-homologous end joining.

[0073] In one embodiment of this method, the eukaryotic cell is a human T cell or a cell derived therefrom. do.

[0074] In another embodiment, the method comprises transfecting a nucleic acid comprising an exogenous sequence of interest into a eukaryotic cell. The method further comprises the step of infecting the cells.

[0075] In one such embodiment of this method, the nucleic acid is homologous to a sequence adjacent to the cleavage site. The cleavage site further comprises a sequence, and the sequence of interest is inserted into the cleavage site by homologous recombination.

[0076] In another such embodiment of this method, the sequence of interest encodes a chimeric antigen receptor. In another such embodiment of this method, the sequence of interest is an exogenous T cell receptor Encode the body.

[0077] In another such embodiment of this method, the nucleic acid is delivered to a eukaryotic cell by a viral vector. It is introduced into cells.

[0078] In another such embodiment of this method, the nucleic acid is delivered by a recombinant AAV vector. It is introduced into a eukaryotic cell.

[0079] In another embodiment of this method, the nucleic acid sequence is isolated from a eukaryotic cell using a single-stranded DNA template. will be introduced.

[0080] In another aspect, the present invention provides a method for producing a genetically modified non-human organism, comprising the steps of: a step of producing a genetically modified non-human eukaryotic cell according to the method described in the document, and and growing the eukaryotic cell to produce the genetically modified non-human organism.

[0081] In one embodiment of this method, the non-human eukaryotic cell is a gamete, a zygote, a blastocyst, an embryonic stem cell, or a mammalian cell. cells, and protoplast cells.

[0082] In another aspect, the present invention provides a method for the treatment of a genetically modified organism as described herein for use as a pharmaceutical. The present invention further provides a method for treating a disease in a subject in need thereof. The present invention also provides the use of a genetically modified eukaryotic cell as described herein in the manufacture of a medicament for the treatment of a cancer. In one such embodiment, the medicament is useful for treating cancer.

[0083] In some embodiments, the CAR comprises an extracellular antigen-binding domain. In embodiments, the extracellular ligand binding domain or portion is derived from a monoclonal antibody. They may be in the form of single chain variable fragments (scFv), which target specific epitopes or antigens ( Predominantly present on the surface of cells, such as cancer cells or other disease-causing cells or particles The scFv provides specificity to the target epitope or antigen. The extracellular ligand-binding domain can bind to any antigen or epitope of interest. In some embodiments, the scFv can be humanized. The extracellular domain of the chimeric antigen receptor also binds to autoantigen-specific B cell receptors on B lymphocytes. can be recognized by the body and thus autoreactive B lymphocytes in antibody-mediated autoimmune diseases These include self-antigens that direct T cells to specifically target and kill leukocytes (Payne et al., (2016), Science 353(6295):179-184. Such CARs are called chimeric autoantibody receptors (CAARs), and their use is described in the present invention. is included in.

[0084] In another aspect, the present invention provides an immunosuppressant for treating cancer in a subject in need thereof. Methods of therapy include the use of genetically modified cells and and a pharmaceutically acceptable carrier. do.

[0085] In some embodiments, the cancer is selected from the group consisting of carcinoma, lymphoma, sarcoma, blastoma, and leukemia. is selected from the group consisting of:

[0086] In some embodiments, the cancer is a cancer 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 In some embodiments, the tumor is of B-cell origin. These cancers include B-lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, and B-cell non-homologous leukemia. lymphoma.

[0087] These and other aspects and embodiments of the invention are described in detail below with reference to the detailed description and claims. For clarity, separate embodiments may be considered. Certain features of the invention which are described in the context of embodiments may also be provided in combination in a single embodiment. All combinations of the embodiments are specifically encompassed by the present invention and may be combined. Each combination is disclosed herein just as if it were individually and explicitly disclosed. Conversely, various features of the invention may be described in the context of a single embodiment, for the sake of brevity. , may be provided separately or in any suitable subcombination. All subcombinations of features are also specifically embraced by the present invention, and exactly such Each such subcombination is expressly incorporated herein by reference as if it were individually and explicitly disclosed herein. Embodiments of each aspect of the invention disclosed herein are intended to be illustrative, mutatis mutandis. The same applies to each other aspect of the present invention. [Brief explanation of the drawings]

[0088] [Figure 1] Figure 1A shows the TRC recognition sequences in the human TRC alpha constant region gene. Each recognition sequence targeted by a recombinant meganuclease of the present invention contains two recognition half-sites. Each recognition half-site contains 9 base pairs separated by a 4-base pair central sequence. The TRC1-2 recognition sequence (SEQ ID NO:3) spans nucleotides 187-208 of the human T-cell alpha constant region (SEQ ID NO:1) and contains two recognition half-sites designated TRC1 and TRC2. The TRC3-4 recognition sequence (SEQ ID NO:4) spans nucleotides 93-114 of the human T-cell alpha constant region (SEQ ID NO:1) and contains two recognition half-sites designated TRC3 and TRC4. The TRC7-8 recognition sequence (SEQ ID NO:5) spans nucleotides 118-139 of the human T-cell alpha constant region (SEQ ID NO:1) and contains two recognition half-sites designated TRC7 and TRC8. B) The recombinant meganuclease of the present invention comprises two subunits, where a first subunit comprising an HVR1 region binds to a first recognition half-site (e.g., TRC1, TRC3, or TRC7) and a second subunit comprising an HVR2 region binds to a second recognition half-site (e.g., TRC2, TRC4, or TRC8). In embodiments where the recombinant 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 2A-2B]Amino acid alignments of TRC1 binding subunits are shown. Figures 2A-2B show that some recombinant meganucleases encompassed by the present invention contain one subunit that binds to the 9-base pair TRC1 recognition half-site of SEQ ID NO:3. Amino acid sequence alignments are provided for the TRC1 binding subunits (SEQ ID NOs:33-52) of recombinant meganucleases shown in SEQ ID NOs:8-27. As shown, the TRC1 binding subunits of SEQ ID NOs:8-18 contain residues 198-344, and the TRC1 binding subunits of SEQ ID NOs:19-27 contain residues 7-153. Each TRC1 binding subunit contains a 56-amino acid hypervariable region as shown. Variable residues within the hypervariable region are shaded, and the most frequently occurring amino acid at each position is further highlighted, with the most common residue in bold and the second most common in bold and italics. Residues outside the hypervariable region are identical in each subunit, except for the Q or E residues at positions 80 or 271 (see U.S. Patent No. 8,021,867). All TRC1-binding subunits provided in Figure 2 share at least 90% sequence identity with the TRC1-binding subunit (residues 198-344) of the TRC1-2x.87EE meganuclease (SEQ ID NO: 33). The residue numbers shown are those of SEQ ID NOs: 8-27. [Figure 3A-3B]Amino acid alignments of TRC2-binding subunits are shown. Figures 3A-3B show that some recombinant meganucleases encompassed by the present invention contain one subunit that binds to the 9-base pair TRC2 recognition half-site of SEQ ID NO:3. Amino acid sequence alignments are provided for the TRC2-binding subunits (SEQ ID NOs:58-77) of recombinant meganucleases shown in SEQ ID NOs:8-27. As shown, the TRC2-binding subunits of SEQ ID NOs:8-18 contain residues 7-153, and the TRC2-binding subunits of SEQ ID NOs:19-27 contain residues 198-344. Each TRC2-binding subunit contains a 56-amino acid hypervariable region as shown. Variable residues within the hypervariable region are shaded, and the most frequently occurring amino acid at each position is further highlighted, with the most common residue in bold and the second most common in bold and italic. Residues outside the hypervariable region are identical in each subunit, except for the Q or E residues at positions 80 or 271 (see U.S. Pat. No. 8,021,867) and the R residue at position 330 (gray shaded and underlined) in meganucleases TRC1-2x.87EE, TRC1-2x.87QE, TRC1-2x.87EQ, TRC1-2x.87, and TRC1-2x.163. All TRC2-binding subunits provided in Figure 3 share at least 90% sequence identity with the TRC2-binding subunit (residues 7-153) of the TRC1-2x.87EE meganuclease (SEQ ID NO: 58). Residue numbers shown are those of SEQ ID NOs: 8-27. [Figure 4]

[0033] Figure 1 shows an amino acid alignment of TRC3-binding subunits. Some recombinant meganucleases encompassed by the present invention contain one subunit that binds to the 9-base pair TRC3 recognition half-site of SEQ ID NO:4. An amino acid sequence alignment is provided for the TRC3-binding subunits (SEQ ID NOs:53 and 54) of recombinant meganucleases shown in SEQ ID NOs:28 and 29. As shown, the TRC3-binding subunits of SEQ ID NOs:28 and 29 contain residues 7-153. Each TRC3-binding subunit contains a 56-amino acid hypervariable region as shown. Variable residues within the hypervariable region are shaded. Residues outside the hypervariable region are identical in each subunit, except for a Q or E residue at position 80 (see U.S. Patent No. 8,021,867). The TRC3-binding subunits of the TRC3-4x.3 and TRC3-4x.19 meganucleases share 97% sequence identity. The residue numbers shown are those of SEQ ID NOs:28 and 29. [Figure 5]

[0033] Figure 1 shows an amino acid alignment of TRC4-binding subunits. Some recombinant meganucleases encompassed by the present invention contain one subunit that binds to the 9-base-pair TRC4 recognition half-site of SEQ ID NO:4. An amino acid sequence alignment is provided for the TRC4-binding subunits (SEQ ID NOs:78 and 79) of recombinant meganucleases shown in SEQ ID NOs:28 and 29. As shown, the TRC4-binding subunits of SEQ ID NOs:28 and 29 contain residues 198-344. Each TRC4-binding subunit contains a 56-amino acid hypervariable region as shown. Variable residues within the hypervariable region are shaded. Residues outside the hypervariable region are identical in each subunit, except for a Q or E residue at position 80 (see U.S. Patent No. 8,021,867). The TRC4-binding subunits of the TRC3-4x.3 and TRC3-4x.19 meganucleases share 97% sequence identity. The residue numbers shown are those of SEQ ID NOs:28 and 29. [Figures 6A-6B]Amino acid alignments of TRC7 binding subunits are shown. Figures 6A-6B show that some recombinant meganucleases encompassed by the present invention contain one subunit that binds to the 9-base pair TRC7 recognition half-site of SEQ ID NO:5. Amino acid sequence alignments are provided for the TRC7 binding subunits (SEQ ID NOs:55-57) of recombinant meganucleases shown in SEQ ID NOs:30-32. As shown, the TRC7 binding subunit of SEQ ID NO:30 contains residues 7-153, and the TRC7 binding subunits of SEQ ID NOs:31 and 32 contain residues 198-344. Each TRC7 binding subunit contains a 56-amino acid hypervariable region as shown. Variable residues within the hypervariable region are shaded, and the most frequently occurring amino acid at each position is further highlighted, with the most common residue in bold and the second most common in bold and italic. Residues outside the hypervariable region are identical in each subunit, except for the Q or E residues at positions 80 or 271 (see U.S. Patent No. 8,021,867). All TRC7 binding subunits provided in Figure 6 share at least 90% sequence identity with the TRC7 binding subunit (residues 7-153) of the TRC7-8x.7 meganuclease (SEQ ID NO: 55). Residue numbers shown are those of SEQ ID NOs: 30-32. [Figures 7A-7B]7A-7B show amino acid alignments of TRC8-binding subunits. Figures 7A-7B show that some recombinant meganucleases encompassed by the present invention contain one subunit that binds to the 9-base pair TRC8 recognition half-site of SEQ ID NO:5. Amino acid sequence alignments are provided for the TRC8-binding subunits (SEQ ID NOs:80-82) of recombinant meganucleases shown in SEQ ID NOs:30-32. As shown, the TRC8-binding subunit of SEQ ID NO:30 contains residues 198-344, and the TRC8-binding subunits of SEQ ID NOs:31 and 32 contain residues 7-153. Each TRC8-binding subunit contains a 56-amino acid hypervariable region, as shown. Variable residues within the hypervariable region are shaded, and the most frequently occurring amino acid at each position is further highlighted, with the most common residue in bold and the second most common in bold and italics. Residues outside the hypervariable region are identical in each subunit, except for the Q or E residues at positions 80 or 271 (see U.S. Patent No. 8,021,867). All TRC8-binding subunits provided in Figure 7 share at least 90% sequence identity with the TRC8-binding subunit (residues 198-344) of the TRC7-8x.7 meganuclease (SEQ ID NO: 80). Residue numbers shown are those of SEQ ID NOs: 30-32. [Figure 8]Figure 1 shows a schematic diagram of a reporter assay in CHO cells to evaluate recombinant meganucleases targeting a recognition sequence found in the T cell receptor alpha constant region (SEQ ID NO: 1). For the recombinant meganucleases described herein, CHO cell lines were generated in which a reporter cassette was stably integrated into the cell's genome. The reporter cassette consisted of, in 5' to 3' order: the SV40 early promoter; the 5' two-thirds of the GFP gene; the recognition sequence for an engineered meganuclease of the present invention (e.g., the TRC1-2 recognition sequence, the TRC3-4 recognition sequence, or the TRC7-8 recognition sequence); the recognition sequence for the CHO-23 / 24 meganuclease (WO / 2012 / 167192); and the 3' two-thirds of the GFP gene. Cells stably transfected with this cassette did not express GFP in the absence of a DNA break-inducing agent. Meganucleases were introduced by transduction of plasmid DNA or mRNA encoding the respective meganuclease. When a DNA break is induced at either of the meganuclease recognition sequences, the overlapping regions of the GFP gene recombine with each other to generate a functional GFP gene. The percentage of GFP-expressing cells can then be determined by flow cytometry as an indirect measure of the frequency of genome cleavage by the meganuclease. [Figure 9]Figure 9 shows the efficiency of recombinant meganucleases that recognize and cleave recognition sequences in the human T cell receptor alpha constant region (SEQ ID NO: 1) in a CHO cell reporter assay. Each of the recombinant meganucleases shown in SEQ ID NOs: 8-32 was engineered to target the TRC1-2 recognition sequence (SEQ ID NO: 3), the TRC3-4 recognition sequence (SEQ ID NO: 4), or the TRC7-8 recognition sequence (SEQ ID NO: 5) and screened for efficacy in a CHO cell reporter assay. The results shown provide the percentage of GFP-expressing cells observed in each assay, demonstrating the efficacy of each meganuclease for cleaving the TRC target recognition sequence or the CHO-23 / 24 recognition sequence. A negative control (RHO1-2bs) was also included in each assay. Figures 9A-9C show meganucleases targeting the TRC1-2 recognition sequence. D) Meganucleases targeting the TRC3-4 recognition sequence. Figures 9E-9F show meganucleases targeting the TRC7-8 recognition sequence. Figure 9G shows mutants of TRC1-2x.87 meganuclease in which Q at position 271 is substituted with E (TRC1-2x.87QE), Q at position 80 is substituted with E (TRC1-2x.87EQ), or both Q at position 80 and Q at position 271 are substituted with E (TRC1-2x.87EE). [Figure 10] Figure 1 shows the time course of recombinant meganuclease efficacy in a CHO cell reporter assay. TRC1-2x.87QE, TRC1-2x.87EQ, and TRC1-2x.87EE meganucleases were evaluated in a CHO reporter assay using the percentage of GFP-expressing cells determined 1, 4, 6, 8, and 12 days after transfection of CHO reporter cells with mRNA encoding the meganucleases. [Figure 11] Figure 1 shows the analysis of Jurkat cell genomic DNA after transfection with TRC1-2 meganuclease. 72 hours after transfection with mRNA encoding TRC1-2 meganuclease, genomic DNA was harvested and a T7 endonuclease assay was performed to estimate genetic modifications in the endogenous TRC1-2 recognition sequence. [Figure 12]Figure 1 shows the dose response of TRC1-2 meganuclease expression in Jurkat cells for genetic modifications in the endogenous TRC1-2 recognition sequence. Jurkat cells were transfected with either 3 μg or 1 μg of a given TRC1-2 meganuclease mRNA. At 96 hours, genomic DNA was analyzed using a T7 endonuclease assay. [Figure 13] Cleavage of the TRC1-2 recognition sequence in human T cells is shown in Figure 13A. CD3+ T cells were stimulated with anti-CD3 and anti-CD28 antibodies for 3 days and then electroporated with mRNA encoding the TRC1-2x.87EE meganuclease. Genomic DNA was harvested 3 and 7 days after transfection and analyzed using a T7 endonuclease assay. Figure 13B shows that to determine whether mutation of the endogenous TRC1-2 recognition sequence was sufficient to eliminate surface expression of the T cell receptor, cells were analyzed by flow cytometry using an anti-CD3 antibody. Control cells (water-transfected) and TRC1-2x.87EE-transfected cells were analyzed 3 and 7 days after transfection to determine the percentages of CD3-positive and CD3-negative T cells. [Figure 14] Representative nucleic acid sequence deletions observed in the TRC1-2 recognition sequence in human T cells after expression of the TRC1-2 meganuclease are shown. [Figure 15] FIG. 1 shows sequence elements of a recombinant AAV vector and its use in combination with an engineered nuclease to insert an exogenous nucleic acid sequence into an endogenous TCR alpha constant region gene. [Figure 16] 1 shows a map of the plasmid used to generate the AAV405 vector. [Figure 17] 1 shows a map of the plasmid used to generate the AAV406 vector. [Figure 18]Figure 1 shows the determination of the timing of meganuclease mRNA transfection and recombinant AAV transduction to improve AAV transduction efficiency. Human CD3+ T cells were electroporated with mRNA encoding the TRC1-2x.87EE meganuclease, and 2, 4, or 8 hours post-transfection, the cells were transduced with a recombinant AAV vector encoding GFP (GFP-AAV). T cells were analyzed by flow cytometry for GFP expression 72 hours post-transduction to determine transduction efficiency. [Figure 19] Analysis of human T cells for insertion of exogenous nucleic acid sequences using recombinant AAV vectors is shown. CD3+ T cells were transfected with TRC1-2x.87EE mRNA and subsequently transduced (2 hours post-transfection) with AAV405 or AAV406. Transduction-only controls were mock transfected (with water) and transduced with either AAV405 or AAV406. Meganuclease-only controls were transfected with TRC1-2x.87EE and then mock transduced (with water) 2 hours post-transfection. Genomic DNA was harvested from T cells, and the TRC1-2 locus was amplified by PCR using primers that recognize sequences beyond the homology region in the AAV vector. PCR primers outside the homology region only allowed amplification of the T cell genome, not the AAV vector. The PCR products were purified and digested with EagI. The PCR products were then analyzed for cleavage. [Figure 20]Figure 20A shows the characterization of the EagI insertion into the TRC1-2 recognition sequence in human T cells using AAV405. In Figure 20A, the undigested PCR product generated in the previous experiment was cloned into pCR-blunt vector. Colony PCR was performed using M13 forward and reverse primers, and portions of the PCR products from cells transfected with TRC1-2x.87EE and AAV405 were analyzed by gel electrophoresis. The analysis shows a mixture of full-length PCR product (approximately 1600 bp), smaller inserts, and empty plasmid (approximately 300 bp). In Figure 20B, in parallel, another portion of the PCR product was digested with EagI to determine the percentage of clones containing the EagI recognition site inserted into the TRC1-2 recognition sequence. The EagI-cleaved PCR product generated the expected fragments of approximately 700 and 800 bp. [Figure 21] Figure 21A shows the characterization of the EagI insertion into the TRC1-2 recognition sequence in human T cells using AAV406. In Figure 21A, the undigested PCR product generated in the previous experiment was cloned into pCR-blunt vector. Colony PCR was performed using M13 forward and reverse primers, and portions of the PCR products from cells transfected with TRC1-2x.87EE and AAV406 were analyzed by gel electrophoresis. The analysis shows a mixture of full-length PCR product (approximately 1600 bp), smaller inserts, and empty plasmid (approximately 300 bp). In Figure 21B, in parallel, another portion of the PCR product was digested with EagI to determine the percentage of clones containing the EagI recognition site inserted into the TRC1-2 recognition sequence. The EagI-cleaved PCR product generated the expected fragments of approximately 700 and 800 bp. [Figure 22] Figure 22A shows representative nucleic acid sequence deletions and insertions (i.e., indels) observed in the TRC1-2 recognition sequence in human T cells after expression of the TRC1-2 meganuclease. Figure 22B shows the nucleic acid sequence of the TRC1-2 recognition sequence confirming insertion of an exogenous nucleic acid sequence containing an EagI restriction site. [Figure 23]Figure 1 shows the improvement of recombinant AAV transduction efficiency. Transduction efficiency was further analyzed by optimizing the timing of meganuclease mRNA transfection and subsequent AAV transduction. Human CD3+ T cells were electroporated with mRNA encoding TRC1-2x.87EE meganuclease immediately after transfection or 2 hours after transfection, and subsequently transduced with GFP-AAV. Additionally, unstimulated resting T cells were transduced with GFP-AAV. Mock-transduced cells were also analyzed. 72 hours after transduction, cells were analyzed by flow cytometry for GFP expression to determine AAV transduction efficiency. [Figure 24] 1 shows maps of the plasmids used to generate the AAV-CAR100 (AAV408) vector. [Figure 25] 1 is a map of the plasmid used to generate the AAV-CAR763 (AAV412) vector. [Figure 26]

[0033] Figure 1 shows a diagram depicting insertion of a chimeric antigen receptor coding sequence at the TRC1-2 recognition site in human T cells. A PCR-based assay was developed to determine whether the AAV412 HDR template was utilized to repair the double-strand break in the TRC1-2 recognition sequence. [Figure 27]

[0023] Figure 27 shows a diagram illustrating insertion of a chimeric antigen receptor coding sequence at the TRC1-2 recognition site in human T cells. A PCR-based assay was developed to determine whether the AAV408 HDR template was utilized to repair the double-strand break in the TRC1-2 recognition sequence. Figure 27A shows PCR products generated using a primer pair that amplifies a product only at the 5' end of the TRC1-2 recognition sequence locus when a CAR gene is inserted at that locus. Figure 27B shows PCR products generated using a primer pair that amplifies a product only at the 3' end of the TRC1-2 recognition sequence locus when a CAR gene is inserted at that locus. [Figure 28]Figure 28A shows a schematic diagram of a digital PCR assay developed to quantitatively determine the insertion efficiency of a chimeric antigen receptor coding sequence into the TRC1-2 recognition site in human T cells. Figure 28B shows the results of digital PCR on genomic DNA from human T cells electroporated with TRC1-2x.87EE meganuclease mRNA and / or increasing amounts of AAV408. [Figure 29] Figure 29 shows cell surface expression of CD19 chimeric antigen receptor on human T cells. Expression levels of anti-CD19 chimeric antigen receptor were determined in cells with a CAR gene inserted into the TRC1-2 recognition sequence using AAV408 as an HDR template. Cell surface expression was analyzed by flow cytometry. Figure 29A shows mock-electroporated, mock-transduced cells (MOI-0), and mock-electroporated, transduced cells with increasing amounts of AAV408. Figure 29B shows TRC1-2x.87EE electroporated, mock-transduced cells (MOI-0), and TRC1-2x.87EE electroporated, transduced cells with increasing amounts of AAV408. [Figure 30] 1 shows a map of the plasmid used to generate the AAV421 vector. [Figure 31] 1 shows a map of the plasmid used to generate the AAV422 vector. [Figure 32] Figure 32 shows a diagram illustrating the insertion of a chimeric antigen receptor coding sequence. PCR was used to determine whether the chimeric antigen receptor coding sequence introduced by AAV421 or AAV422 was inserted into the TRC1-2 recognition site cleaved by the TRC1-2x.87EE meganuclease. Figure 32A shows an analysis of the insertion after transduction with AAV421. Figure 32B shows an analysis of the insertion after transduction with AAV422. [Figure 33]Figure 33 shows cell surface expression of CD19 chimeric antigen receptor on human T cells. The expression level of anti-CD19 chimeric antigen receptor was determined in cells in which a CAR gene was inserted into the TRC1-2 recognition sequence using AAV421 as an HDR template. Cell surface expression was analyzed by flow cytometry. Figure 33A shows mock-electroporated, mock-transduced cells (MOI-0), and mock-electroporated, transduced cells with increasing amounts of AAV421. Figure 33B shows TRC1-2x.87EE electroporated, mock-transduced cells (MOI-0), and TRC1-2x.87EE electroporated, transduced cells with increasing amounts of AAV421. [Figure 34] Figure 34 shows the proliferation of human T cells expressing cell surface chimeric antigen receptors. Several methods were used to determine the preferential expansion and enrichment of CD3- / CAR+ T cell populations after electroporation of mRNA for TRC1-2x.87EE meganuclease and transduction with AAV421. Figure 34A shows supplementation with IL-7 (10 ng / mL) and IL-15 (10 ng / mL). Figure 34B shows supplementation with IL-7 (10 ng / mL) and IL-15 (10 ng / mL) and incubation with mitomycin C-inactivated IM-9 cells. Figure 34C shows supplementation with IL-7 (10 ng / mL) and IL-15 (10 ng / mL) and incubation twice with mitomycin C-inactivated IM-9 cells. [Figure 35] Figure 35 shows cell surface expression of CD19 chimeric antigen receptor on human T cells. The expression level of anti-CD19 chimeric antigen receptor was determined in cells in which a CAR gene was inserted into the TRC1-2 recognition sequence using AAV422 as an HDR template. Cell surface expression was analyzed by flow cytometry. Figure 35A shows mock-electroporated, mock-transduced cells (MOI-0), and mock-electroporated, transduced cells with increasing amounts of AAV422. Figure 35B shows TRC1-2x.87EE electroporated, mock-transduced cells (MOI-0), and TRC1-2x.87EE electroporated, transduced cells with increasing amounts of AAV422. [Figure 36]Figure 36 shows the proliferation of human T cells expressing cell surface chimeric antigen receptors. Several methods were used to determine the preferential expansion and enrichment of CD3- / CAR+ T cell populations after electroporation of mRNA for TRC1-2x.87EE meganuclease and transduction with AAV422. Figure 36A shows supplementation with IL-7 (10 ng / mL) and IL-15 (10 ng / mL). Figure 36B shows supplementation with IL-7 (10 ng / mL) and IL-15 (10 ng / mL) and incubation with mitomycin C-inactivated IM-9 cells. Figure 36C shows supplementation with IL-7 (10 ng / mL) and IL-15 (10 ng / mL) and incubation twice with mitomycin C-inactivated IM-9 cells. [Figure 37] Figure 37 shows meganuclease knockout efficiency using single-stranded AAV. Experiments were performed to investigate the knockout efficiency of two meganucleases in human T cells when co-transduced with single-stranded AAV vectors. Figure 37A shows cells electroporated with mRNA for TRC1-2x.87EE and transduced with increasing amounts of single-stranded AAV412. Figure 37B shows cells electroporated with mRNA for a meganuclease targeting the beta-2 microglobulin gene and transduced with increasing amounts of single-stranded AAV412. Figure 37C shows cells electroporated with mRNA for TRC1-2x.87EE and transduced with increasing amounts of single-stranded AAV422. [Figure 38] Figure 38 shows the functional activity of anti-CD19 CAR T cells. Figure 38A shows an IFN-γ ELISPOT assay with either CD19+ Raji cells or CD19- U937 cells as the target population. Figure 38B shows a cell killing assay targeting luciferase-labeled CD19+ Raji cells. [Figure 39]Figure 39 shows the expression of a chimeric antigen receptor after transduction of a linearized DNA donor template. These experiments generated plasmids containing an anti-CD19 CAR gene flanked by homology arms homologous to the TRC1-2 recognition sequence locus. Different promoters were used in some plasmids, and the homology arms were either "short" (200 bp on the 5' homology arm and 180 bp on the 3' homology arm) or "long" (985 bp on the 5' homology arm and 763 bp on the 3' homology arm). CAR donor plasmids were linearized at restriction sites in the vector backbone and gel-purified. Figure 39A shows background CD3- / CAR+ staining. B) Cells electroporated with TRC1-2x.87EE mRNA alone. Figure 39C shows cells co-electroporated with TRC1-2x.87EE mRNA and a long homology arm vector with the EF1α core promoter and HTLV enhancer. Figure 39D shows cells electroporated with TRC1-2x.87EE mRNA and a short homology arm vector with the EF1α core promoter (enhancerless). Figure 39E shows cells electroporated with a long homology arm vector with the EF1α core promoter and HTLV enhancer in the absence of TRC1-2x.87EE mRNA. Figure 39F shows cells electroporated with a short homology arm vector with the EF1α core promoter (enhancerless) in the absence of TRC1-2x.87EE mRNA. Figure 39G shows cells electroporated with TRC1-2x.87EE mRNA and a long homology arm construct containing the MND promoter and an intron driving CAR expression at the 5' end of the CAR gene. Figure 39H shows cells electroporated with TRC1-2x.87EE mRNA and a long homology arm construct containing the MND promoter and no intron driving CAR expression. Figure 39I shows cells electroporated with the short homology arm plasmid containing the MND promoter and no intron and TRC1-2x.87EE mRNA. Figure 39J shows cells electroporated with the long homology arm construct containing the MND promoter and intron driving expression of CAR at the 5' end of the CAR gene, but not TRC1-2x.87EE mRNA.Figure 39K shows cells electroporated with an intronless long homology arm construct containing the MND promoter driving CAR expression, but not TRC1-2x.87EE mRNA. Figure 39L shows cells electroporated with an intronless short homology arm plasmid containing the MND promoter, but not TRC1-2x.87EE mRNA. Figure 39M shows cells electroporated with a short homology arm construct containing the JeT promoter and TRC1-2x.87EE mRNA. Figure 39N shows cells electroporated with a long homology arm construct containing the CMV promoter and TRC1-2x.87EE mRNA. Figure 39O shows cells electroporated with a short homology arm construct containing the JeT promoter, but not TRC1-2x.87EE mRNA. Figure 39P shows cells electroporated with the long homology arm construct containing the CMV promoter, but not the TRC1-2x.87EE mRNA. [Figure 40] 1 shows PCR analysis to determine whether the chimeric antigen receptor coding region delivered by the linearized DNA construct was inserted into the TRC1-2 recognition sequence in human T cells. [Figure 41] 1 shows a map of the plasmid used to generate the AAV423 vector. [Figure 42] Figure 1 shows cell surface expression of CD19 chimeric antigen receptor on human T cells. Expression levels of anti-CD19 chimeric antigen receptor were determined in cells in which a CAR gene was inserted into the TRC1-2 recognition sequence using AAV423 as an HDR template. Cell surface expression was analyzed by flow cytometry. A) Mock-electroporated, mock-transduced cells (MOI-0) and mock-electroporated, transduced with increasing amounts of AAV423. B) TRC1-2x.87EE electroporated, mock-transduced cells (MOI-0) and TRC1-2x.87EE electroporated, transduced with increasing amounts of AAV423. [Figure 43]Insertion of the chimeric antigen receptor coding sequence is shown. PCR was used to determine whether the chimeric antigen receptor coding sequence introduced by AAV423 was inserted into the TRC1-2 recognition site cleaved by the TRC1-2x.87EE meganuclease. [Figure 44] Phenotypic analysis of anti-CD19 CAR T cells is shown. In Figure 44A, activated T cells were electroporated with TRC1-2x.87EE mRNA and then transduced with an AAV6 vector containing an anti-CD19 CAR expression cassette driven by the JeT promoter and flanked by homology arms. After 5 days of culture with IL-2 (10 ng / mL), cells were analyzed for cell surface CD3 and anti-CD19 CAR expression by flow cytometry. In Figure 44B, CD3- cells were enriched by depleting CD3+ cells using anti-CD3 magnetic beads. The depleted cells were then cultured in IL-15 (10 ng / mL) and IL-21 (10 ng / mL) for 3 days and re-analyzed for cell surface expression of CD3 and anti-CD19 CAR. In Figure 44C, the purified population of CD3-CD19-CAR T cells was analyzed by flow cytometry to determine the percentage of cells that were CD4+ and CD8+. In Figure 44D, the purified population of CD3-CD19-CAR T cells was further analyzed by flow cytometry to determine whether they were central memory T cells, transitional memory T cells, or effector memory T cells by staining for CD62L and CD45RO. [Figure 45]The Raji disseminated lymphoma model is shown. Raji cells stably expressing firefly luciferase (ffLuc)44 were intravenously injected into 5-6 week-old female NSG mice at a dose of 2.0 x 10 cells / mouse on day 1. On day 4, mice were intravenously injected with PBS, PBS containing gene-edited control TCR KO T cells prepared from the same healthy donor's PBMCs, or PBS containing the indicated dose of CAR T cells prepared from the same donor. On the indicated days, surviving mice were intraperitoneally injected with luciferin substrate (150 mg / kg saline), anesthetized, and luciferase activity was measured 7 minutes later using an IVIS SpectrumCT® (Perkin Elmer, Waltham, MA). Data were analyzed and exported using Living Image software 4.5.1 (Perkin Elmer, Waltham, MA). Luminescence signal intensity is expressed as luminance in p / sec / cm2 / sr.

[0089] A brief description of arrays SEQ ID NO: 1 is the human T cell receptor alpha constant region gene (NCBI Gene ID The nucleotide sequence of the gene encoding the nucleotide sequence of ...

[0090] SEQ ID NO: 2 is the amino acid sequence encoded by the human T cell receptor alpha constant region Shows.

[0091] SEQ ID NO: 3 shows the amino acid sequence of the TRC1-2 recognition sequence.

[0092] SEQ ID NO: 4 shows the nucleotide sequence of the TRC3-4 recognition sequence.

[0093] SEQ ID NO: 5 shows the nucleotide sequence of the TRC7-8 recognition sequence.

[0094] SEQ ID NO: 6 shows the amino acid sequence of I-CreI.

[0095] SEQ ID NO: 7 shows the amino acid sequence of the LAGLIDADG motif.

[0096] SEQ ID NO: 8 shows the amino acid sequence of the TRC1-2x.87EE meganuclease.

[0097] SEQ ID NO: 9 shows the amino acid sequence of the TRC1-2x.87QE meganuclease.

[0098] SEQ ID NO: 10 shows the amino acid sequence of the TRC1-2x.87EQ meganuclease.

[0099] SEQ ID NO: 11 shows the amino acid sequence of the TRC1-2x.87 meganuclease.

[0100] SEQ ID NO: 12 shows the amino acid sequence of the TRC1-2x.6 meganuclease.

[0101] SEQ ID NO: 13 shows the amino acid sequence of the TRC1-2x.20 meganuclease.

[0102] SEQ ID NO: 14 shows the amino acid sequence of the TRC1-2x.55 meganuclease.

[0103] SEQ ID NO: 15 shows the amino acid sequence of the TRC1-2x.60 meganuclease.

[0104] SEQ ID NO: 16 shows the amino acid sequence of the TRC1-2x.105 meganuclease.

[0105] SEQ ID NO: 17 shows the amino acid sequence of the TRC1-2x.163 meganuclease.

[0106] SEQ ID NO: 18 shows the amino acid sequence of TRC1-2x.113_3 meganuclease .

[0107] SEQ ID NO: 19 shows the amino acid sequence of the TRC1-2x.5 meganuclease.

[0108] SEQ ID NO: 20 shows the amino acid sequence of the TRC1-2x.8 meganuclease.

[0109] SEQ ID NO: 21 shows the amino acid sequence of the TRC1-2x.25 meganuclease.

[0110] SEQ ID NO: 22 shows the amino acid sequence of the TRC1-2x.72 meganuclease.

[0111] SEQ ID NO: 23 shows the amino acid sequence of the TRC1-2x.80 meganuclease.

[0112] SEQ ID NO: 24 shows the amino acid sequence of the TRC1-2x.84 meganuclease.

[0113] SEQ ID NO: 25 shows the amino acid sequence of the TRC1-2x.120 meganuclease.

[0114] SEQ ID NO: 26 shows the amino acid sequence of TRC1-2x.113_1 meganuclease .

[0115] SEQ ID NO: 27 shows the amino acid sequence of TRC1-2x.113_2 meganuclease .

[0116] SEQ ID NO: 28 shows the amino acid sequence of the TRC3-4x.3 meganuclease.

[0117] SEQ ID NO: 29 shows the amino acid sequence of the TRC3-4x.19 meganuclease.

[0118] SEQ ID NO: 30 shows the amino acid sequence of the TRC7-8x.7 meganuclease.

[0119] SEQ ID NO: 31 shows the amino acid sequence of the TRC7-8x.9 meganuclease.

[0120] SEQ ID NO: 32 shows the amino acid sequence of the TRC7-8x.14 meganuclease.

[0121] SEQ ID NO: 33 identifies residues 198 to 344 of the TRC1-2x.87EE meganuclease. show.

[0122] SEQ ID NO: 34 identifies residues 198 to 344 of the TRC1-2x.87QE meganuclease. show.

[0123] SEQ ID NO: 35 identifies residues 198 to 344 of the TRC1-2x.87EQ meganuclease. show.

[0124] SEQ ID NO: 36 shows residues 198 to 344 of the TRC1-2x.87 meganuclease .

[0125] SEQ ID NO: 37 shows residues 198 to 344 of the TRC1-2x.6 meganuclease.

[0126] SEQ ID NO: 38 shows residues 198 to 344 of the TRC1-2x.20 meganuclease .

[0127] SEQ ID NO: 39 shows residues 198 to 344 of the TRC1-2x.55 meganuclease .

[0128] SEQ ID NO: 40 shows residues 198 to 344 of the TRC1-2x.60 meganuclease .

[0129] SEQ ID NO: 41 represents residues 198 to 344 of the TRC1-2x.105 meganuclease. vinegar.

[0130] SEQ ID NO: 42 represents residues 198 to 344 of the TRC1-2x.163 meganuclease. vinegar.

[0131] SEQ ID NO: 43 identifies residues 198 to 344 of the TRC1-2x.113_3 meganuclease. Shows.

[0132] SEQ ID NO: 44 shows residues 7 to 153 of the TRC1-2x.5 meganuclease.

[0133] SEQ ID NO: 45 shows residues 7 to 153 of the TRC1-2x.8 meganuclease.

[0134] SEQ ID NO: 46 shows residues 7 to 153 of the TRC1-2x.25 meganuclease.

[0135] SEQ ID NO: 47 shows residues 7 to 153 of the TRC1-2x.72 meganuclease.

[0136] SEQ ID NO: 48 shows residues 7 to 153 of the TRC1-2x.80 meganuclease.

[0137] SEQ ID NO: 49 shows residues 7 to 153 of the TRC1-2x.84 meganuclease.

[0138] SEQ ID NO: 50 shows residues 7 to 153 of the TRC1-2x.120 meganuclease.

[0139] SEQ ID NO: 51 represents residues 7 to 153 of the TRC1-2x.113_1 meganuclease. vinegar.

[0140] SEQ ID NO: 52 represents residues 7 to 153 of the TRC1-2x.113_2 meganuclease. vinegar.

[0141] SEQ ID NO: 53 shows residues 7 to 153 of the TRC3-4x.3 meganuclease.

[0142] SEQ ID NO: 54 shows residues 7 to 153 of the TRC3-4x.19 meganuclease.

[0143] SEQ ID NO: 55 shows residues 7 to 153 of the TRC7-8x.7 meganuclease.

[0144] SEQ ID NO: 56 shows residues 198 to 344 of the TRC7-8x.9 meganuclease.

[0145] SEQ ID NO: 57 shows residues 198 to 344 of the TRC7-8x.14 meganuclease.

[0146] SEQ ID NO: 58 shows residues 7 to 153 of the TRC1-2x.87EE meganuclease .

[0147] SEQ ID NO: 59 shows residues 7 to 153 of the TRC1-2x.87QE meganuclease .

[0148] SEQ ID NO: 60 shows residues 7 to 153 of the TRC1-2x.87EQ meganuclease .

[0149] SEQ ID NO: 61 shows residues 7 to 153 of the TRC1-2x.87 meganuclease.

[0150] SEQ ID NO: 62 shows residues 7 to 153 of the TRC1-2x.6 meganuclease.

[0151] SEQ ID NO: 63 shows residues 7 to 153 of the TRC1-2x.20 meganuclease.

[0152] SEQ ID NO: 64 shows residues 7 to 153 of the TRC1-2x.55 meganuclease.

[0153] SEQ ID NO: 65 shows residues 7 to 153 of the TRC1-2x.60 meganuclease.

[0154] SEQ ID NO: 66 shows residues 7 to 153 of the TRC1-2x.105 meganuclease.

[0155] SEQ ID NO: 67 shows residues 7 to 153 of the TRC1-2x.163 meganuclease.

[0156] SEQ ID NO: 68 represents residues 7 to 153 of the TRC1-2x.113_3 meganuclease. vinegar.

[0157] SEQ ID NO: 69 shows residues 198 to 344 of the TRC1-2x.5 meganuclease.

[0158] SEQ ID NO: 70 shows residues 198 to 344 of the TRC1-2x.8 meganuclease.

[0159] SEQ ID NO: 71 shows residues 198 to 344 of the TRC1-2x.25 meganuclease.

[0160] SEQ ID NO: 72 shows residues 198 to 344 of the TRC1-2x.72 meganuclease .

[0161] SEQ ID NO: 73 shows residues 198 to 344 of the TRC1-2x.80 meganuclease .

[0162] SEQ ID NO: 74 represents residues 198 to 344 of the TRC1-2x.84 meganuclease .

[0163] SEQ ID NO: 75 represents residues 198 to 344 of the TRC1-2x.120 meganuclease. vinegar.

[0164] SEQ ID NO: 76 identifies residues 198 to 344 of the TRC1-2x.113_1 meganuclease Shows.

[0165] SEQ ID NO: 77 identifies residues 198 to 344 of the TRC1-2x.113_2 meganuclease Shows.

[0166] SEQ ID NO: 78 shows residues 198 to 344 of the TRC3-4x.3 meganuclease.

[0167] SEQ ID NO: 79 shows residues 198 to 344 of the TRC3-4x.19 meganuclease .

[0168] SEQ ID NO: 80 shows residues 198 to 344 of the TRC7-8x.7 meganuclease.

[0169] SEQ ID NO: 81 shows residues 7 to 153 of the TRC7-8x.9 meganuclease.

[0170] SEQ ID NO: 82 shows residues 7 to 153 of the TRC7-8x.14 meganuclease.

[0171] SEQ ID NO: 83 shows the nucleotide sequence of the antisense strand of the TRC1-2 recognition sequence.

[0172] SEQ ID NO: 84 shows the nucleotide sequence of the antisense strand of the TRC3-4 recognition sequence.

[0173] SEQ ID NO: 85 shows the nucleotide sequence of the antisense strand of the TRC7-8 recognition sequence.

[0174] SEQ ID NO: 86 represents nucleotides 162 to 233 of SEQ ID NO:1.

[0175] SEQ ID NO: 87 is nucleotide 1 of SEQ ID NO: 1, including the deletion due to truncation and NHEJ. Indicates 62 to 233.

[0176] SEQ ID NO: 88 is nucleotide 1 of SEQ ID NO: 1, including the deletion due to truncation and NHEJ. Indicates 62 to 233.

[0177] SEQ ID NO: 89 is nucleotide 1 of SEQ ID NO: 1, including the deletion due to truncation and NHEJ. Indicates 62 to 233.

[0178] SEQ ID NO: 90 is nucleotide 1 of SEQ ID NO: 1, including the deletion due to truncation and NHEJ. Indicates 62 to 233.

[0179] SEQ ID NO: 91 is nucleotide 1 of SEQ ID NO: 1, including the deletion due to truncation and NHEJ. Indicates 62 to 233.

[0180] SEQ ID NO: 92 is nucleotide 1 of SEQ ID NO: 1, including the deletion due to truncation and NHEJ. Indicates 62 to 233.

[0181] SEQ ID NO: 93 is nucleotide 1 of SEQ ID NO: 1, including the deletion due to truncation and NHEJ. Indicates 62 to 233.

[0182] SEQ ID NO: 94 is nucleotide 1 of SEQ ID NO: 1, including the insertion resulting from the cleavage and NHEJ. Indicates 62 to 233.

[0183] SEQ ID NO: 95 is nucleotide 1 of SEQ ID NO: 1, including the insertion resulting from the cleavage and NHEJ. Indicates 62 to 233.

[0184] SEQ ID NO: 96 is nucleotide 1 of SEQ ID NO: 1, including the deletion due to truncation and NHEJ. Indicates 62 to 233.

[0185] SEQ ID NO: 97 is nucleotide 1 of SEQ ID NO: 1, including the deletion due to truncation and NHEJ. Indicates 62 to 233.

[0186] SEQ ID NO: 98 is nucleotide 1 of SEQ ID NO: 1, including the deletion due to truncation and NHEJ. Indicates 62 to 233.

[0187] SEQ ID NO: 99 is nucleotide 1 of SEQ ID NO: 1, including the deletion due to truncation and NHEJ. Indicates 62 to 233.

[0188] SEQ ID NO: 100 is the nucleotide sequence of SEQ ID NO: 1 containing the deletion due to truncation and NHEJ. Indicates 162 to 233.

[0189] SEQ ID NO: 101 is the nucleotide sequence of SEQ ID NO: 1 containing the deletion due to truncation and NHEJ. Indicates 162 to 233.

[0190] SEQ ID NO: 102 is the nucleotide sequence of SEQ ID NO: 1 containing the deletion due to truncation and NHEJ. Indicates 162 to 233.

[0191] SEQ ID NO: 103 is the nucleotide sequence of SEQ ID NO: 1 containing the deletion due to cleavage and NHEJ. Indicates 162 to 233.

[0192] SEQ ID NO: 104 is the nucleotide sequence of SEQ ID NO: 1 containing the deletion due to truncation and NHEJ. Indicates 162 to 233.

[0193] SEQ ID NO: 105 represents nucleotides 181 to 214 of SEQ ID NO: 1.

[0194] SEQ ID NO: 106 is a sequence of SEQ ID NO: 1 containing an exogenous nucleic acid sequence inserted by homologous recombination. Nucleotides 181 to 214 are shown.

[0195] SEQ ID NO: 107 is the nucleic acid sequence of the plasmid used to generate the AAV405 vector. The nucleotide sequence is shown.

[0196] SEQ ID NO: 108 is the nucleic acid sequence of the plasmid used to generate the AAV406 vector. The nucleotide sequence is shown.

[0197] SEQ ID NO: 109 is used to generate the AAV-CAR100 (AAV408) vector. The nucleotide sequences of the plasmids used are shown.

[0198] SEQ ID NO: 110 is used to generate the AAV-CAR763 (AAV412) vector. The nucleotide sequences of the plasmids used are shown.

[0199] SEQ ID NO: 111 shows the amino acid sequence of an anti-CD19 chimeric antigen receptor.

[0200] SEQ ID NO: 112 shows the amino acid sequence of the anti-CD19 extracellular ligand-binding domain.

[0201] SEQ ID NO: 113 is the amino acid sequence of the chimeric antigen receptor intracellular cytoplasmic signaling domain Indicates a column.

[0202] SEQ ID NO: 114 shows the amino acid sequence of the chimeric antigen receptor intracellular costimulatory domain.

[0203] SEQ ID NO: 115 shows the amino acid sequence of the chimeric antigen receptor signal peptide domain .

[0204] SEQ ID NO: 116 shows the amino acid sequence of the chimeric antigen receptor hinge region.

[0205] SEQ ID NO: 117 shows the amino acid sequence of the chimeric antigen receptor transmembrane domain.

[0206] SEQ ID NO: 118 shows the nucleotide sequence of the EF-1 alpha core promoter.

[0207] SEQ ID NO: 119 shows the nucleotide sequence of the exogenous polynucleotide insert.

[0208] SEQ ID NO: 120 is a human TRC1-2 recognition sequence containing an exogenous nucleic acid sequence inserted within the TRC1-2 recognition sequence. The nucleotide sequence of the CR alpha constant region gene is shown.

[0209] SEQ ID NO: 121 is a human TRC3-4 recognition sequence containing an exogenous nucleic acid sequence inserted within the TRC3-4 recognition sequence. The nucleotide sequence of the CR alpha constant region gene is shown.

[0210] SEQ ID NO: 122 is a human TRC7-8 gene encoding an exogenous nucleic acid sequence inserted within the TRC7-8 recognition sequence. The nucleotide sequence of the CR alpha constant region gene is shown.

[0211] SEQ ID NO: 123 is the nucleic acid sequence of the plasmid used to generate the AAV421 vector. The acid sequence is shown.

[0212] SEQ ID NO: 124 is the nucleic acid sequence of the plasmid used to generate the AAV422 vector. The acid sequence is shown.

[0213] SEQ ID NO: 125 is the nucleic acid sequence of the plasmid used to generate the AAV423 vector. The acid sequence is shown. DETAILED DESCRIPTION OF THE INVENTION

[0214] 1.1 References and Definitions The patent and scientific literature referred to herein establishes knowledge that is available to those skilled in the art. Issued U.S. patents, granted patents, and related patent applications containing GenBank database sequences cited in the document. Each patent application, published foreign application, and bibliographic reference is specifically and individually incorporated by reference. are incorporated herein by reference to the same extent as if incorporated.

[0215] This invention may be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are to be construed as an allegation that this disclosure will be thorough and complete. These and other related drawings are provided so that the scope of the present invention will be fully conveyed to those skilled in the art. Features illustrated in one embodiment may be incorporated into other embodiments and may be used interchangeably with those illustrated in one embodiment. Any feature that is not specifically mentioned may be omitted from the embodiment. Many variations and additions to the embodiments will be apparent to those skilled in the art in light of this disclosure. They do not depart from the invention.

[0216] Unless otherwise defined, all technical and scientific terms used herein are within the meaning of the present invention. has the same meaning as commonly understood by a person skilled in the art to which it pertains. The terminology used to describe the invention herein is intended to describe particular embodiments only. and is not intended to limit the invention.

[0217] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. It is incorporated herein by reference.

[0218] As used herein, "a," "an," or "this" refers to "A" cell can mean one or more. For example, "a" cell means a single It can refer to one cell or many cells.

[0219] As used herein, unless specifically indicated otherwise, the word "or" means "And / or" is used in the inclusive sense and "either / or" is used in the exclusive sense. It is not something that can be done.

[0220] As used herein, the term "meganuclease" refers to a nuclease with a recognition sequence of more than 12 base pairs. It refers to an endonuclease that binds to double-stranded DNA. Preferably, the meganuclease of the present invention The recognition sequence of the enzyme is 22 base pairs. It may be a nuclease, e.g., having DNA binding specificity, DNA cleavage activity, DNA binding affinity, or engineered I-CreI that is modified compared to the native I-CreI with respect to its dimerization properties. To create such an engineered mutant of I-CreI, Methods for this purpose are known in the art (see, for example, WO 2007 / 0478 As used herein, meganucleases are heterodimers. or a pair of DNA-binding domains linked into a single polypeptide using a peptide linker The term "homing enzyme" is used to describe a single-stranded meganuclease that binds to double-stranded DNA. The term "nuclease" is synonymous with the term "meganuclease." The enzyme is substantially non-toxic when expressed in cells, particularly human T cells, and therefore, No adverse effects on cell viability or meganuclei were observed when measured using the methods described herein. Cells were transfected and incubated at 37°C without observing a significant decrease in cleavage activity of the ATPase. It can be maintained in

[0221] As used herein, the term "single-chain meganuclease" refers to a single-chain meganuclease linked by a linker. It refers to a polypeptide comprising a pair of coupled meganuclease subunits. The ase has the structure N-terminal subunit-linker-C-terminal subunit. The two meganuclease subunits generally have identical, but not identical, amino acid sequences. Therefore, single-chain meganucleases typically recognize DNA sequences that are not pseudovaline. Single-chain meganucleases cleave palindromic or non-palindromic recognition sequences. Although it is not a dimer, it is referred to as a "single-chain heterodimer" or "single-chain heterodimer meganuclease." For clarity, unless otherwise specified, we refer to them as "meganucleases." The term can refer to dimeric or single-chain meganucleases.

[0222] As used herein, the term "linker" refers to a molecule that connects two meganuclease subunits. A linker refers to an exogenous peptide sequence used to link a single polypeptide. The polypeptide may have a sequence found in the naturally occurring protein, or may have a sequence not found in the naturally occurring protein. The linker may be flexible and lacking secondary structure, or may be a naturally occurring artificial sequence. The linker may have a tendency to form a particular three-dimensional structure under physical conditions. , including but not limited to those contained in U.S. Pat. No. 8,445,251. In some embodiments, the linker can be any of SEQ ID NOs: 8-32. It can have an amino acid sequence comprising one residue 154-195.

[0223] As used herein with respect to proteins, the term "recombinant" refers to a protein that is produced by a method that encodes the protein. The result of applying genetic engineering techniques to the nucleic acid that encodes the protein and the cells or organisms that express the protein. With respect to nucleic acids, the term "recombinant" means having an altered amino acid sequence. means having a nucleic acid sequence that has been modified as a result of the application of genetic engineering techniques. Genetic engineering techniques include, but are not limited to, PCR and DNA cloning techniques; Transfection, transformation, and other gene transfer techniques; homologous recombination; site-specific recombination and gene fusion. It has the same amino acid sequence as the protein, but is produced by cloning and expression in a heterologous host. Proteins produced using recombinant methods are not considered recombinant.

[0224] As used herein, the term "wild type" refers to a gene that is a member of a population of alleles of the same species. refers to the most common naturally occurring alleles (i.e., polynucleotide sequences) of a gene in the wild The polypeptide encoded by the wild-type allele retains its original function. "Type" also refers to a polypeptide encoded by a wild-type allele. (i.e., polynucleotides) and polypeptides may have one or more substitutions relative to the wild-type sequence. Mutant or variant alleles and polypeptides containing natural mutations and / or substitutions are distinct from A wild-type allele or polypeptide is one that confers a normal phenotype in an organism. Although mutant or variant alleles or polypeptides may be The wild-type homing endonuclease can be expressed in recombinant or non-naturally occurring meganucleases.

[0225] As used herein with respect to recombinant proteins, the term "modification" refers to a modification of a reference sequence (e.g., any insertion of amino acid residues in the recombinant sequence compared to the wild-type or native sequence , deletion, or substitution.

[0226] As used herein, the term "recognition sequence" refers to a sequence that is bound and recognized by a meganuclease. The recognition sequence refers to the DNA sequence to be cleaved. In the case of the recombinant meganuclease of the present invention, the recognition sequence is inversions, separated by 1 base pair, containing a pair of 9 base pair "half sites" or "recognition half sites" In the case of single-chain meganucleases, the N-terminal subunit of the protein is attached to the first half-site. The C-terminal subunit of the protein contacts the second half-site. Cleavage by the cleavage gene results in a 4-base pair 3' overhang. "Ends" are short single-stranded DNA fragments that can be produced by meganuclease cleavage of double-stranded DNA sequences. In the case of the meganuclease of the present invention, the overhang is a 22 base pair A segment. It contains bases 10 to 13 of the recognition sequence.

[0227] As used herein, the term "target site" or "target sequence" refers to a target site or sequence that is targeted by a meganuclease. It refers to the region of a cell's chromosomal DNA that contains the recognition sequence for the gene.

[0228] 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 measured by the dissociation constant Kd. In this case, the Kd of the recombinant meganuclease for the reference recognition sequence is If the percent change in the IL-100 expression level is increased or decreased by a statistically significant amount compared to the percent change in the IL-100 expression level, the meganuclease is This "changes" the binding affinity.

[0229] As used herein, the term "homologous recombination" or "HR" refers to the use of a homologous DNA fragment as a repair template. It refers to the natural cellular process of repairing double-stranded DNA breaks using DNA sequences (e.g., C ahill et al. (2006), Front.Biosci.11:1958~1976) The homologous DNA sequence can be an endogenous chromosomal sequence or an exogenous nucleic acid delivered to the cell.

[0230] As used herein, the term "non-homologous end joining" or "NHEJ" refers to the cleavage of a double-stranded DNA fragment. A natural cellular process in which DNA breaks are repaired by direct joining of two non-homologous DNA segments. refers to processes (e.g., Cahill et al. (2006), Front. Biosci. 1 1:1958-1976). DNA repair by non-homologous end joining prevents errors from occurring. The repair is prone to breakage, frequently resulting in non-templated addition or deletion of DNA sequences at the repair site. In examples, cleavage at the target recognition sequence results in NHEJ at the target recognition site. Nuclease-induced cleavage of a target site in the coding sequence of a gene, followed by DNA repair by NHEJ It is possible to introduce mutations into the coding sequence that disrupt gene function, such as frameshift mutations. Therefore, to effectively knock out a gene in a cell population, An engineered nuclease such as a nuclease can be used.

[0231] As used herein, a "chimeric antigen receptor" or "CAR" refers to an immune effector Engineered receptors that confer or graft specificity for antigens onto cells (e.g., human T cells) A chimeric antigen receptor typically consists of an extracellular ligand-binding domain or portion, and and an intracellular domain containing one or more stimulatory domains that transmit signals necessary for T cell activation. In some embodiments, the extracellular ligand-binding domain or portion comprises a molecule. These may be in the form of single chain variable fragments (scFv) derived from monoclonal antibodies, epitopes or antigens (e.g., on the surface of cancer cells or other disease-causing cells or particles) Provides specificity for a preferentially occurring epitope or antigen. Extracellular ligand binding The domain may be specific for any antigen or epitope of interest. In another embodiment, the ligand binding domain is specific for CD19. v can be humanized.

[0232] The extracellular domain of the chimeric antigen receptor also binds to autoantigen-specific B cell receptors on B lymphocytes. can be recognized by the body and thus autoreactive B lymphocytes in antibody-mediated autoimmune diseases These include self-antigens that direct T cells to specifically target and kill leukocytes (Payne et al., (2016), Science 353(6295):179-184). Such CARs are called chimeric autoantibody receptors (CAARs), and their use is in accordance with the present invention. Included.

[0233] The scFvs can be linked via a linker sequence. One or more cytoplasmic signaling molecules that transmit activation signals to immune effector cells after binding to the target cells. Such cytoplasmic signaling domains may include, but are not limited to, The intracellular stimulatory domain also induces proliferative responses after ligand binding. and / or may contain one or more intracellular costimulatory domains that transmit cell survival signals. Such intracellular costimulatory domains include, but are not limited to, the CD28 domain. , 4-1BB domain, OX40 domain, or a combination thereof. Chimeric antigen receptors are composed of extracellular ligand-binding domains connected via hinge or spacer sequences. The protein may further comprise additional structural elements, including a transmembrane domain linked to the ribosomal domain.

[0234] As used herein, an "exogenous T cell receptor" or "exogenous TCR" refers to a TCR Gating of immune effector cells (e.g., human T cells) that may or may not endogenously express This refers to a TCR whose sequence is introduced into the genome. Expression of exogenous TCRs on immune effector cells Currently, specific epitopes or antigens (e.g., cancer cells or other disease-causing cells or particles) are targeted to It can confer specificity for epitopes or antigens that are preferentially present on the surface of the molecule. Such exogenous T cell receptors can include alpha and beta chains, or The exogenous TCR useful in the present invention may comprise any It may have specificity for an antigen or epitope of interest.

[0235] As used herein, the term "down-regulation" refers to the reduction in expression of a gene modified in a cell compared to a control cell. This refers to any reduction in the expression of endogenous T cell receptors on the cell surface of transformed cells. The term also refers to the expression of endogenous polypeptides (i.e., endogenous polypeptides) at the cell surface when compared to a population of control cells. It can refer to a decrease in the percentage of cells in a cell population that express the T cell receptor (T cell receptor). Such reductions can occur at 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 2 It can be 0%, 80%, 90%, 95%, or even up to 100%. " encompasses both partial and complete knockdown of endogenous T cell receptors.

[0236] As used herein with respect to both amino acid and nucleic acid sequences, the term "identity pattern" refers to a sequence of a nucleic acid molecule. "Percent," "Sequence Identity," "Percent Similarity," "Sequence Similarity," etc. Identical or similar residues are used to maximize the similarity between the amino acid residues or nucleotides involved. The number of residues or nucleotides, the total number of residues or nucleotides and the gaps in the sequence alignment The degree of similarity between two sequences based on sequence alignment, which is a function of the presence and length of fragments It refers to a measure of sequence similarity. Various algorithms for determining sequence similarity using standard parameters are Rhythms and computer programs are available. Sequence similarity was calculated using the BLASTp program for amino acid sequences and B These were measured using the LASTn program, both of which were National Cen ter for Biotechnology Information(www.nc bi.nlm.nih.gov / ), e.g., Altschul et al. (1990), J. Mol. Biol. 215:403-410; Gish and St ates(1993), Nature Genet.3:266~272;Madden (1996), Meth.Enzymol.266:131~141;Altsch ul et al. (1997), Nucleic Acids Res.25:3389~3402 );Zhang et al., (2000), J.Comput.Biol.7(1-2):203 As used herein, the similarity pattern of two amino acid sequences is The score is based on the following parameters for the BLASTp algorithm: Word size = 3; Gap opening penalty = -11; Gap extension penalty = - 1; and scoring matrix = BLOSUM62. As used herein, two nucleic acid sequences The percent similarity is based on the following parameters for the BLASTn algorithm: Scores are: word size = 11; gap opening penalty = -5; gap extension penalty = Luti = -2; Match Reward = 1; Mismatch Penalty = -3.

[0237] As used herein in reference to two protein or amino acid sequence modifications, the first A particular modification in a protein has the same amino acid residue position as a modification in a second protein. The two proteins are then aligned by standard sequence alignment (e.g., using the BLASTp program). When the first protein is subjected to a genomic DNA sequencing (using a DNA sequencing program), the amino acid position of the modification in the first protein is "Pair" is used to indicate that the amino acid position of the modification in the protein corresponds to or aligns with the amino acid position of the modification. Therefore, if residues X and Y correspond to each other in the sequence alignment, If the corresponding amino acid is 'X', then the modification of residue 'X' in the first protein to amino acid 'A' is X and Y correspond to the modification of residue "Y" in the second protein to amino acid "A." In practice, the number may be different.

[0238] As used herein, the terms "recognition half-site," "recognition sequence half-site," or simply "half-site" are used interchangeably. The "position" can be determined by a monomer of a homodimeric or heterodimeric meganuclease or by a single chain A nucleic acid in a double-stranded DNA molecule that is recognized by one subunit of a meganuclease It means an array.

[0239] As used herein, the term "hypervariable region" refers to an amino acid sequence having a relatively high degree of variability. Hypervariable region refers to a localized sequence within a meganuclease monomer or subunit that contains a nucleotide. is about 50-60 consecutive residues, about 53-57 consecutive residues, or preferably about 56 consecutive residues. In some embodiments, the hypervariable region residues can include residues set forth in SEQ ID NO: The hypervariable region may correspond to any one of positions 24 to 79 or positions 215 to 270 of any one of 8 to 32. may contain one or more residues that contact a DNA base in the recognition sequence, and may be a monomer or The hypervariable regions can also be modified to alter the base selection of the subunits. When a ganucleases binds to a double-stranded DNA recognition sequence, it binds to one or more of the DNA backbones. Such residues may include a number of residues relative to the DNA backbone and the target recognition sequence. The meganucleases can be modified to change their binding affinity. In some embodiments, the hypervariable region can contain between 1 and 20 residues that exhibit variability. These can be modified to affect base selection and / or DNA binding affinity. In certain embodiments, the hypervariable region comprises between about 15 and 18 residues that exhibit variability. These can be modified to affect group selection and / or DNA binding affinity. In some embodiments, the variable residues within the hypervariable region are selected from any one of SEQ ID NOs: 8-32. 24, 26, 28, 29, 30, 32, 33, 38, 40, 42, 44, 46, 66, 6 It corresponds to one or more of positions 8, 70, 72, 73, 75, and 77. The variable residues in the hypervariable region are 215, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 19, 221, 223, 224, 229, 231, 233, 235, 237, 248, 2 corresponding to one or more of positions 57, 259, 261, 263, 264, 266, and 268 .

[0240] As used herein, the terms "T cell receptor alpha constant region gene" and "TC The "R alpha constant region gene" is used interchangeably and is designated NCBI GenID NO. 287 It refers to the human gene identified by SEQ ID NO: 55 (SEQ ID NO: 1).

[0241] The terms "recombinant DNA construct," "recombinant construct," "expression cassette," and "expression construct" "Chimeric construct," "construct," and "recombinant DNA fragment" are used interchangeably herein. Recombinant constructs include, but are not limited to, single-stranded or double-stranded polynucleotides. single- or double-stranded sequences containing regulatory and coding sequences that are not found together in nature, but For example, a recombinant DNA construct may be a combination of different strands of polynucleotides. Regulatory and coding sequences derived from the same source, or derived from the same source and found in nature. Such constructs may contain regulatory and coding sequences arranged in a manner different from that shown. The entities may be used alone or in combination with a vector.

[0242] As used herein, a "vector" or "recombinant DNA vector" refers to a Replication systems and sequences capable of transcribing and translating the polypeptide-encoding sequence in a host cell When a vector is used, the selection of the vector is well known to those skilled in the art. The vectors may be any of a variety of vectors, depending on the method used to transform the host cells. Although not intended to be limiting, plasmid vectors and recombinant AAV vectors, or megagenes of the present invention, Any suitable method known in the art for delivering the gene encoding the nuclease to the target cells can be used. Those skilled in the art can easily understand the isolated nucleotide or to successfully transform, select and grow host cells containing any of the nucleic acid sequences, Be fully aware of the genetic elements that must be present on the vector.

[0243] As used herein, "vector" can also refer to a viral vector. Viral vectors include, but are not limited to, retroviral vectors, Adenovirus vectors, adenovirus vectors, and adeno-associated virus vectors (AA V) can be mentioned.

[0244] As used herein, a "polycistronic" mRNA is one that contains two or more coding sequences. A single messenger that contains (i.e., a cistron) and encodes two or more proteins Polycistronic mRNA refers to RNA containing, but not limited to, an IRES element. , T2A element, P2A element, E2A element, and F2A element, from the same mRNA molecule The present invention also provides a method for the translation of two or more genes of a gene encoding a nucleotide sequence, comprising the steps of: This can be done.

[0245] As used herein, "human T cells" or "T cells" refer to cells isolated from a human donor. Human T cells and cells derived from them include single cells that have not been subcultured. Isolated T cells, T cells passaged and maintained under cell culture conditions without immortalization, and immortalized T cells These include T cells that can be isolated and maintained indefinitely in cell culture.

[0246] As used herein, a "control" or "control cell" refers to a control cell that is a genetically modified cell of a similar genotype or refers to cells that provide a reference point for measuring changes in phenotype. For example: (a) wild-type cells, i.e., the same as the starting material for the genetic modification that gave rise to the genetically modified cells; (b) cells of the same genotype as the genetically modified cells but containing a null construct Transformed with a construct (i.e., carrying a construct that has no known effect on the trait of interest) (c) cells that are genetically identical to the genetically modified cells but have an altered genotype or expression This includes cells that have not been exposed to conditions or stimuli that induce expression of the present gene or to further genetic modification. It is possible.

[0247] As used herein, the recitation of a numerical range for a variable indicates that the invention contemplates the use of any or all of the values ​​within that range. The intent is to convey that the invention may be practiced with variables equal to either one. For a discrete variable, this variable can be equal to any integer value within the numeric range, including the end points of the range. Similarly, for a variable that is inherently continuous, the variable may be expressed as a range of values, including the endpoints of the range. By way of example and not limitation, the value may be equal to any real value between 0 and 2. A variable described as having a value between 0, 1, or 2 is not necessarily a sine wave if the variable is discrete in nature. If the variable is continuous in nature, it can take on values ​​of 0.0, 0.1, 0.01, It can be 0.001 or any other real value >= 0 and <= 2.

[0248] 2.1 Principles of the present invention The present invention relates to a method for producing a recombinant meganuclease encoding a human TCR alpha constant region gene (SEQ ID NO: 1). 1) engineered to recognize and cleave a recognition sequence found specifically within residues 93-208 Cleavage at such a recognition sequence is based in part on the discovery that Allows NHEJ at the cleavage site and expression of the human T cell receptor alpha chain subunit disruption of T cell receptors, resulting in decreased expression and / or function of T cell receptors on the cell surface.

[0249] Furthermore, cleavage at such recognition sequences may be achieved by targeting the TCR alpha constant region of the exogenous nucleic acid sequence. Such exogenous nucleic acid sequences can further enable direct homologous recombination into genes. , a chimeric antigen receptor, an exogenous TCR receptor, or any other polypeptide of interest. Thus, the present invention provides a method for producing a single engineered nucleic acid sequence, which can include a sequence of interest, such as a sequence that Knockout of endogenous T cell receptors by targeting a single recognition site with ribosomal clease and expression of an exogenous nucleic acid sequence (e.g., a chimeric antigen receptor or an exogenous TCR). Such cells, when administered to allogeneic subjects, can prevent graft-versus-host disease (GVHD). ) may show reduced or no induction.

[0250] 2.2 Mechanism for recognizing and cleaving the recognition sequence in the T cell receptor alpha constant region gene Ganuclease

[0251] Using site-specific nucleases to produce DNA breaks in the genome of living cells Such DNA breaks can be repaired through mutagenic NHEJ repair or by transfection. can result in permanent alterations of the genome through homologous recombination with transgenic DNA sequences. It is known in the art that NHEJ results in mutagenesis at the cleavage site, resulting in allelic NHEJ-associated mutagenesis leads to the generation of premature stop codons, abnormal inactivating alleles via frameshift mutations, which generate non-functional proteins may induce mutations that may lead to mutations in the nucleoside analogues, or may trigger mechanisms such as nonsense-mediated mRNA decay. The use of meganucleases to induce NHEJ-mediated mutagenesis has been shown to can be used to target sequences present in natural mutations or wild-type alleles The use of meganucleases to induce double-strand breaks at target loci is particularly useful in genome editing. Sequences homologous to the NOM target stimulate homologous recombination of adjacent transgenic DNA sequences In this way, an exogenous nucleic acid sequence can be inserted into a target locus. Such exogenous nucleic acids may be, for example, chimeric antigen receptors, exogenous TCRs, or any other nucleic acid of interest. The gene may encode any sequence or polypeptide.

[0252] In a preferred embodiment, the nuclease used to practice the present invention is a single-stranded Single-chain meganucleases consist of an N-terminal subunit and a linker peptide. Each of the two domains contains a C-terminal subunit linked by a recognition peptide. The recognition sequence is half (i.e., the recognition half-site), and the site of DNA cleavage is determined by the interaction of two subunits. The DNA strand break occurs in the middle of the recognition sequence near the junction of the DNA fragments. The cleavage is reversed by four base pairs so that a four base pair 3' single-stranded overhang is generated. Be killed.

[0253] In some embodiments, the recombinant meganuclease of the present invention comprises a TRC1-2 recognition sequence. Such a recombinant meganucleotide has been engineered to recognize and cleave the sequence (SEQ ID NO: 3). The nucleases are collectively referred to herein as "TRC1-2 meganucleases." Exemplary TRC1-2 meganucleases are provided in SEQ ID NOs: 8-27.

[0254] In a further embodiment, the recombinant meganuclease of the present invention comprises the TRC3-4 recognition sequence ( Such a recombinant meganuclear fragment is engineered to recognize and cleave the sequence of SEQ ID NO: 4. The enzymes are collectively referred to herein as "TRC3-4 meganucleases." The TRC3-4 meganuclease is provided in SEQ ID NOs: 28 and 29.

[0255] In a further embodiment, the recombinant meganuclease of the present invention comprises the TRC7-8 recognition sequence ( Such a recombinant meganuclear fragment is engineered to recognize and cleave the sequence of SEQ ID NO: 5. The enzymes are collectively referred to herein as "TRC7-8 meganucleases." The TRC7-8 meganuclease is provided in SEQ ID NOs: 30-32.

[0256] The recombinant meganuclease of the present invention comprises a first hypervariable region (HVR1) region. and a second subunit comprising a second hypervariable region (HVR2). The first subunit binds to the first recognition half-site of the recognition sequence (e.g., TRC1, TRC3, or TRC7 half-site), and the second subunit binds to the second recognition half-site of the recognition sequence ( For example, it binds to a TRC2, TRC4, or TRC8 half-site. In embodiments where the enzyme is a single-chain meganuclease, the enzyme comprises an HVR1 region and a first half-site The first subunit that binds to the HVR2 region is located as the N-terminal subunit. The second subunit that binds to the second half-site is positioned as the C-terminal subunit. In another embodiment, the first and second subunits can be oriented such that The first subunit comprises the HVR1 region and binds to the first half-site. a second subunit arranged as a subunit containing the HVR2 region and binding to the second half-site; orienting the first and second subunits so that is positioned as the N-terminal subunit Exemplary TRC1-2 meganucleases of the present invention are shown in Table 1. Exemplary TRC3-4 meganucleases are shown in Table 2. Exemplary TRC7-8 meganucleases of the present invention are shown in Table 2. The ganucleases are listed in Table 3.

[0257] Table 1. Exemplary polypeptides engineered to recognize and cleave the TRC1-2 recognition sequence (SEQ ID NO: 3) Recombinant meganucleases [Table 1]

[0258] * "TRC1 subunit %" and "TRC2 subunit %" are the percentages of each meganuclear cell line. TRC1-binding and TRC2-binding subunit regions of the clease and TRC1-2x.87 Amino acid residues between the TRC1-binding and TRC2-binding subunit regions of EE meganuclease The amino acid sequence identity is shown.

[0259] Table 2. Exemplary polypeptides engineered to recognize and cleave the TRC3-4 recognition sequence (SEQ ID NO: 4) Recombinant meganucleases [Table 2]

[0260] * "TRC3 subunit %" and "TRC4 subunit %" are the percentages of each meganuclear cell line. TRC3-binding and TRC4-binding subunit regions of the clease and the TRC3-4x.3 membrane Amino acid sequence between the TRC3-binding and TRC4-binding subunit domains of the ganuclease. Represents identity.

[0261] Table 3. Exemplary polypeptides engineered to recognize and cleave the TRC7-8 recognition sequence (SEQ ID NO: 5) Recombinant meganucleases [Table 3]

[0262] * "TRC7 subunit %" and "TRC8 subunit %" are the percentages of each meganuclear cell line. TRC7-binding and TRC8-binding subunit regions of the cleavage enzyme and the TRC7-8x.7 membrane Amino acid sequence between the TRC7-binding and TRC8-binding subunit regions of the ganuclease. Represents identity.

[0263] 2.3 Methods for producing genetically modified cells The present invention relates to a method for identifying a T cell receptor (TCR) alpha constant region gene (SEQ ID NO: 1) containing a T cell receptor (TCR) alpha constant region gene. The recombinant meganuclease that recognizes and cleaves the recognition sequence expressed by the gene is used to generate the gene-modified cells. Cleavage at such a recognition sequence results in a N at the cleavage site. This allows HEJ and disrupts the expression of the human T cell receptor alpha chain subunit on the cell surface. This results in a decrease in the expression and / or function of T cell receptors in the target cells. The excision in the TCR alpha constant region gene allows for direct homologous recombination of the exogenous nucleic acid sequence into the TCR alpha constant region gene. This can be made possible.

[0264] The recombinant meganuclease of the present invention can be prepared in the form of a protein or, preferably, a recombinant meganuclease. The nucleic acid may be delivered into the cell as a nucleic acid encoding the ganucleases. A (e.g., circular or linear plasmid DNA or PCR product) or RNA. do.

[0265] For embodiments in which the recombinant meganuclease coding sequence is delivered in DNA form, It must be operably linked to a promoter to promote transcription of the ganucleases gene. Mammalian promoters suitable for the present invention include cytomegalovirus early (CMV) promoters. ) promoter (Thomsen et al., (1984), Proc Natl Acad Sc iUSA.81(3):659-63) or SV40 early promoter (Benoist and and Chambon (1981), Nature. 290(5804):304-10) etc. Constitutive promoter of , as well as tetracycline-inducible promoter (Dingermann (1992), Mol Cell Biol. 12(9):4038-45) Examples include inducible promoters.

[0266] In some embodiments, the mRNA encoding the engineered nuclease is Reduce the likelihood that the gene encoding the modified nuclease will be integrated into the cell's genome Such mRNA encoding the engineered nuclease is then delivered to the cell. They can be made using methods known in the art, such as in vitro transcription. In some embodiments, the mRNA is capped with 7-methyl-guanosine. In some embodiments, the mRNA may be polyadenylated.

[0267] In certain embodiments, the mRNA encoding the engineered nuclease of the invention comprises: Polycistronic mRNAs encoding two or more nucleases that are simultaneously expressed in cells Polycistronic mRNAs can have different recognition sequences in the same target gene. The target gene may encode two or more nucleases of the invention. The lysistronic mRNA can be expressed in a similar manner to at least one nuclease described herein. Target distinct recognition sequences located in the genes, or cleavage sites are located in both genes. at least one targeting a second recognition sequence located in a second gene to be generated; Polycistronic mRNAs can encode additional nucleases. However, the IRES element, the T2A element, the P2A element, the E2A element, and the F2A element elements, allowing translation of two or more genes (i.e., cistrons) from the same mRNA molecule The invention may include any element known in the art that enables the

[0268] Purified recombinant meganuclease proteins are delivered to cells to cleave genomic DNA. This allows homologous recombination to occur by a variety of different mechanisms known in the art. This allows for non-homologous end joining at the cleavage site containing the desired sequence.

[0269] In some embodiments, the recombinant meganuclease protein or recombinant meganuclease DNA / mRNA encoding the enzyme is conjugated to a cell-penetrating peptide or targeting ligand. Examples of cell-penetrating peptides known in the art include: Poly-arginine (Jearawiriyapaisarn et al. (2008) Mol T her.16:1624~9), TAT peptide derived from HIV virus (Hudecz et al. , (2005), Med.Res.Rev.25:679~736), MPG(Sime Oni et al. (2003) Nucleic Acids Res.31:2717-272 4), Pep-1 (Deshayes et al. (2004) Biochemistry 43 :7698-7706), and HSV-1VP-22 (Deshayes et al., (2005 ) Cell Mol Life Sci.62:1839-49) is another example. In embodiments, a recombinant meganuclease or a D encoding a recombinant meganuclease NA / mRNA is the binding of the meganuclease protein / DNA / mRNA to the target cell. binds to specific cell surface receptors expressed on target cells so that it is internalized Alternatively, the recombinant meganuclease may be covalently or non-covalently bound to an antibody that recognizes the Proteins / DNA / mRNA are natural ligands (or natural ligands) for such cell surface receptors. The ligands can be covalently or non-covalently bound to the ATP-binding site (part of the natural ligand) (McCall et al., (2014) )Tissue Barriers.2(4):e944449;Dinda et al. (20 13)Curr Pharm Biotechnol.14:1264~74;Kang et al. (2014) Curr Pharm Biotechnol.15(3):220~ 30;Qian et al., (2014) Expert Opin Drug Metab T oxicol.10(11):1491~508).

[0270] In some embodiments, the recombinant meganuclease protein or recombinant meganuclease The DNA / mRNA encoding the enzyme can be synthesized into nanoparticles using methods known in the art. Covalently or preferably non-covalently bound to the molecule or encapsulated within such nanoparticles. (Sharma et al. (2014) Biomed Res Int. 2014). The nanoparticles are nanoscale particles whose length scale is <1 μm, preferably <100 nm. Such nanoparticles can be made of metals, lipids, polymers, or biopolymers. The core can be designed using recombinant meganuclease proteins, mRNA Alternatively, multiple copies of DNA can be attached or encapsulated in the nanoparticle core. Increase the number of copies of protein / mRNA / DNA delivered to each cell, thus increasing the number of copies of each Increasing intracellular expression of the recombinant meganuclease maximizes the likelihood of cleaving the target recognition sequence. The surface of such nanoparticles is coated with polymers or lipids (e.g., chitosan, cations, etc.). The surface can be further modified with additional functional groups (e.g., cationic polymers, or cationic lipids) to provide additional functionality. This allows the formation of core-shell nanoparticles that enhance cellular delivery and payload uptake. (Jian et al., (2012) Biomaterials. 33(30):7621 ~30) Nanoparticles can be targeted to the appropriate cell type and / or facilitate cellular uptake. To enhance the activity, it may be advantageous to further bind to a targeting molecule. Examples of immunomodulating molecules include antibodies specific for cell surface receptors and natural ligands for cell surface receptors. Examples include ligands (or portions of natural ligands).

[0271] In some embodiments, the meganuclease protein or meganuclease is The DNA / mRNA to be loaded is encapsulated or complexed within liposomes using cationic lipids. (e.g., Lipofectamine™, Life Technologies, gies Corp. Carlsbad, CA; Zuris et al. (2015) Nat B iotechnol.33:73~80;Mishra et al. (2011) J Drug Deliv. 2011:863734). Liposomes and Lipoplexes The formulation protects the payload from degradation and prevents cell death by fusing with and / or disrupting the cell membrane. This can enhance cellular uptake and delivery efficiency.

[0272] In some embodiments, the recombinant meganuclease protein or recombinant meganuclease The enzyme is encapsulated in a polymer (e.g., PLGA) or in a cationic polymer (e.g., P EI, PLL) (Tamboli et al., (2011) Ther Deliv.2(4):523~536).

[0273] In some embodiments, the recombinant meganuclease protein or the recombinant meganuclease The DNA / mRNA encoding the cleavage enzyme is combined with amphiphilic molecules that self-assemble into micelles. Combined (Tong et al. (2007) J Gene Med. 9(11):956-6 6) Polymer micelles prevent aggregation, mask charge interactions, and disperse nonspecific extracellular phases. Hydrophilic polymers (e.g., polyethylene glycol) that can reduce interactions It may include a formed micellar shell.

[0274] In some embodiments, the recombinant meganuclease protein or the recombinant meganuclease The DNA / mRNA encoding the nuclease is formulated in an emulsion or It is formulated into a nanoemulsion (i.e., with an average particle size <1 nm). "John" includes, but is not limited to, any oil-in-water, water-in-oil, water-in-oil-in-water, or It refers to an oil-in-water dispersion or droplets in which the water-immiscible phase is mixed with the aqueous phase, resulting in the formation of non-polar residues ( as a result of hydrophobic forces that direct the polar head groups (e.g., long hydrocarbon chains) away from water and the polar head groups towards water These other lipid structures include, but are not limited to, lipid structures that can be formed. Emulsions include unilamellar, paucilamellar, and multilamellar lipid vesicles, micelles, and lamellar phases. An emulsion consists of an aqueous phase and a lipophilic phase (typically containing oil and an organic solvent). Nanoemulsion formulations are well known. and, for example, U.S. Patent Application Nos. 2002 / 0045667 and 2004 / 0043 041, as well as U.S. Patent Nos. 6,015,832, 6,506,803, and 6,6 35,676 and 6,559,189, which are incorporated herein by reference. each of which is incorporated herein by reference in its entirety.

[0275] In some embodiments, the recombinant meganuclease protein or the recombinant meganuclease The DNA / mRNA encoding the cleavage enzyme is conjugated to a multifunctional polymer conjugate, DNA dendrimers, and polymers covalently or non-covalently bound to dendrimers (Masto rakos et al. (2015) Nanoscale.7(9):3845~56; Chen g et al. (2008) J Pharm Sci. 97(1):123-43). Dendrimers The generation of the charge can control the capacity and size of the payload, achieving high payload capacity. Furthermore, the display of multiple surface groups can be utilized to improve stability and reduce non-specific binding. This can reduce the potential interactions.

[0276] In some embodiments, the gene encoding the recombinant meganuclease is Such vectors are known in the art and are incorporated into cells using vectors. , lentiviral vectors, adenoviral vectors, and adeno-associated viruses (AAV) ) vectors (Vannucci et al., (2013 New Microbio Recombinant AAV vectors useful in the present invention are This allows for viral transduction of the cells and insertion of the meganuclease gene into the cellular genome. In certain embodiments, the recombinant AAV vector can have any serotype. The recombinant AAV vectors have the serotypes AAV2 or AAV6. It can be self-complementary so that second-strand DNA synthesis is not required in the host cell (McCa rty et al. (2001) Gene Ther. 8:1248-54).

[0277] The recombinant meganuclease gene may be in DNA form (e.g., a plasmid) and / or in a virus. When delivered via a vector (e.g., AAV), they are promoter-operable. In some embodiments, this is a viral vector. Endogenous promoter or known site (e.g., LTR of lentiviral vector) Viral promoters such as the early promoter of megalovirus or SV40 virus. In a preferred embodiment, the meganuclease gene is expressed in a target cell (e.g., a human The gene is operably linked to a promoter that preferentially drives gene expression in human T cells.

[0278] The present invention further provides a method for the preparation of a nucleic acid sequence comprising the step of: In some embodiments, the invention provides for the introduction of exogenous nucleic acid into a cell so that it is inserted into a gene. wherein the exogenous nucleic acid comprises a 5' homologous arm and a 3' homologous arm, and a nuclease cleavage site The nucleic acid sequence at the target site is then promoted to be recombined into the cell genome.

[0279] The exogenous nucleic acid of the invention can be introduced into a cell by any of the means discussed above. In certain embodiments, the exogenous nucleic acid is a lentivirus, retrovirus, adenovirus, or The vector is introduced using a viral vector such as a rhesus monkey virus, or preferably a recombinant AAV vector. Recombinant AAV vectors useful for introducing exogenous nucleic acids are those that express the virus into cells. Any serotype that allows for transduction and insertion of exogenous nucleic acid sequences into the cell genome. In certain embodiments, the recombinant AAV vector is AAV2 or AAV6. The recombinant AAV vectors also have the serotype of It may be self-complementary so as not to require A synthesis.

[0280] In another specific embodiment, the exogenous nucleic acid is introduced into the cell using a single-stranded DNA template. The single-stranded DNA can include exogenous nucleic acids, and in a preferred embodiment to promote insertion of a nucleic acid sequence into a nuclease cleavage site by homologous recombination in The single-stranded DNA can contain 5' and 3' homologous arms. The 5' AAV inverted terminal repeat (ITR) sequence is located upstream, and the 3' AA sequence is located 3' downstream of the 3' homologous arm. It may further comprise a V ITR sequence.

[0281] In another particular embodiment, the recombinant meganuclease of the invention and / or other compounds of the invention The gene encoding the causative nucleic acid sequence is transfected by transfection of a linearized DNA template. In some embodiments, the recombinant meganuclease and and / or the plasmid DNA encoding the exogenous nucleic acid sequence is transferred to the cell via a circular plasmid DNA. The DNA is linearized prior to transfection and digested with one or more restriction enzymes. It can be made into

[0282] When delivered to a cell, the exogenous nucleic acid of the invention induces the expression of the encoded polypeptide in the cell. Any promoter suitable for expression of the gene, including the mammalian promoters and inducible promoters mentioned above, may be used. The exogenous nucleic acids of the invention can be operably linked to a synthetic promoter. Synthetic promoters include, but are not limited to, JeT promoter (International Publication No. 2002 / 012514) can.

[0283] In an embodiment in which the genetically modified cell of the invention is a human T cell or a cell derived therefrom, Such cells require activation prior to introduction of meganucleases and / or exogenous nucleic acid sequences. For example, T cells may be soluble or conjugated to a support (i.e., beads). The cells can be contacted with the anti-CD3 and anti-CD28 antibodies for a time sufficient to activate the cells. can.

[0284] The genetically modified cells of the present invention may be further modified to express one or more inducible suicide genes. The induction of these proteins can cause cell death and can be used to treat cells 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 or can encode a polypeptide that activates a cytotoxic gene pathway in a cell. That is, suicide genes encode products that cause cell death, either alone or in the presence of other compounds. A typical example of such a suicide gene is the thymidine locus of herpes simplex virus. A further example is the varicella-zoster virus thymidine kinase. The gene encoding the enzyme and the reaction of 5-fluorocytosine with the highly toxic compound 5-fluorouracil The bacterial gene for cytosine deaminase can be converted into cytosine deaminase. Further examples include, but are not limited to, caspase-9, caspase-8, or cytosine deaminase. In some instances, caspase-9 is a dimer Suicide genes can also be activated using specific chemical inducers of cytotoxicity (CID). at the surface of the cell, rendering the cell sensitive to therapeutic and / or cytotoxic monoclonal antibodies In a further example, the suicide gene may encode a polypeptide that is expressed. Antigenic motifs and suicide genes recognized by the anti-CD20 mAb, rituximab. A recombinant antigenic polypeptide encoding an epitope that allows for the selection of cells expressing the For example, two rituximab-binding epitopes and one QBEnd10-binding epitope can be used. The RQR8 fragment described in International Publication No. 2013153391 contains an epitope. For such genes, rituximab may be used as needed. It can be administered to elephants to induce cell depletion.

[0285] 2.4 Pharmaceutical Compositions In some embodiments, the present invention provides a genetically modified cell of the present invention, or a genetically modified cell of the present invention. A pharmaceutical composition is provided comprising a population of modified cells and a pharmaceutical carrier. Such a pharmaceutical composition comprises: It can be prepared according to known techniques, e.g., Remington, The Sc ience and Practice of Pharmacy (21st ed., 2005 In the manufacture of pharmaceutical formulations according to the present invention, the cells are typically The composition is then mixed with an acceptable carrier and the resulting composition is administered to a subject. Therefore, the carrier must be acceptable in the sense of being compatible with the other ingredients in the formulation, In some embodiments, the pharmaceutical compositions of the present invention include: It may further comprise one or more additional agents useful in treating the disease of interest. In further embodiments, the cells are genetically modified human T cells (or cells derived therefrom). The pharmaceutical compositions of the present invention contain cytokines (e.g., cytokines) that promote cell proliferation and engraftment in vivo. The present invention further includes biomolecules such as IL-2, IL-7, IL-15, and / or IL-21. The pharmaceutical compositions comprising the genetically modified cells of the present invention may contain additional drugs or biological agents. The molecule may be administered in the same composition or may be co-administered in separate compositions. do.

[0286] The pharmaceutical compositions of the present invention can be used to treat any disease state that can be targeted by T cell adoptive immunotherapy. In certain embodiments, the pharmaceutical compositions of the present invention may be useful for treating cancer. Such cancers include, but are not limited to, carcinoma, lymphoma, leukemia, and leukemia. tumor, blastoma, leukemia, cancer 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 In certain embodiments, cancers of B-cell origin include, but are not limited to, However, B-lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, and B-cell non-Hodgkin lymphoma Examples include lymphoma.

[0287] 2.5 Methods for producing recombinant AAV vectors In some embodiments, the present invention provides recombinant proteins for use in the methods of the present invention. AAV vectors are provided. Recombinant AAV vectors are typically derived from vectors such as HEK-293. The viral cap and rep genes are expressed in mammalian cell lines such as Preventing its self-replication and delivering a therapeutic gene (e.g., an endonuclease gene) These are then transferred to the packaging cell line. Furthermore, the "helpers" ( For example, adenoviruses) need to be provided (Cots D, Bosch A, Chillon M (2013) Curr. Gene Ther. 13(5):3 70-81) Often, recombinant AAV vectors are engineered to encode “helper” components in cell lines. The first plasmid contains the cap and rep genes, the second plasmid contains the virus A third plasmid containing the viral ITRs containing the intervening DNA sequence is packaged into the plasmid. The capsid is produced using triple transfection, where the capsid is transfected with the Viral particles containing the packaged genome (ITRs of interest and intervening gene(s)) are , followed by freeze-thaw cycles, sonication, detergents, or other procedures known in the art. The particles are then isolated from the cells by cesium chloride density gradient centrifugation or af Purification using affinity chromatography followed by isolation of the gene(s) of interest. The drug is delivered to a cell, tissue, or organism, such as a human patient.

[0288] Recombinant AAV particles are typically produced (manufactured) in cells, so they are site-specific. To ensure that the endonuclease is not expressed in the packaging cells, Precautions must be taken in the practice of the invention. The viral genome of the invention is The nucleotide sequence contains a recognition sequence for a nucleotide sequence encoding ... This endonuclease is required to cleave the viral genome before it is packaged into a viral particle. This can result in reduced packaging efficiency and / or fragmentation. This results in the packaging of the genome. Several approaches can be used, including: can be used to prevent endonuclease expression in packaging cells:

[0289] 1. The endonuclease is a tissue-specific protein that is not active in the packaging cells. It can be under the control of a motor, for example, to insert (one or more) endons into muscle tissue. If a viral vector is developed for delivery of the lyase gene, a muscle-specific promoter will be generated. Examples of muscle-specific promoters include C5-12 (Liu et al., (2004) Hum Gene Ther. 15:783-92), muscle-specific creatine Kinase (MCK) promoter (Yuasa et al. (2002) Gene Ther. 9: 1576-88), or the smooth muscle 22 (SM22) promoter (Haase et al., (2013 )BMC Biotechnol.13:49-54) Examples of gene-specific promoters include the NSE, synapsin, and MeCP2 promoters ( Lentz et al. (2012) Neurobiol Dis. 48:179-88) Examples of liver-specific promoters include the albumin promoter (Palb, etc.), human α1-antitrypsin (e.g., Pa1AT) and hemopexin (e.g., Phpx) (Krame r, MG et al. (2003) Mol. Therapy 7:375-85). Examples of specific promoters include opsin and the corneal epithelium-specific K12 promoter (Ma rtin KRG, Klein RL and Quigley HA (2002)Metho. ds(28):267~75) (Tong Y et al. (2007) J Gene Med, 9::956-66). These promoters or other combinations known in the art can be used. The tissue-specific promoter is not highly active in HEK-293 cells, and therefore, When incorporated into a clear viral vector, significant levels of The virus of the present invention is not expected to produce endonuclease gene expression. Vectors may be used in other cell lines and incompatible tissue-specific promoters (i.e., well-known The HeLa cell line (human epithelial cells) and the liver-specific hemopexin promoter were used. Other examples of tissue-specific promoters include PDZD4 (cerebellum) in synovial sarcoma, C6 (liver), ASB5 (muscle), PPP1R12B (heart), SLC5A12 (kidney), Cholesterol-regulated APOM (liver), ADPRHL1 (heart), and monogenic malformation syndromes Group TP73L (muscle) is one example. (Jacox E et al. (2010) PLoS On e v.5(8):e12274).

[0290] 2. Alternatively, the vector can be transfected into cells from a different species in which the endonuclease is poorly expressed. For example, viral particles can be packaged in non-mammalian packaging media. The well-known early promoter of cytomegalovirus or SV40 virus, which is not active in cells, It can be produced in microbial, insect, or plant cells using mammalian promoters, such as the In a preferred embodiment, the viral particle is a baculovirus as described by Gao et al. It is produced in insect cells using the system (Gao, H. et al. (2007) J. Biotech hnol.131(2):138-43). Endonucleases under the control of mammalian promoters The cleavage enzyme is unlikely to be expressed in these cells (Airenne, KJ et al., (20 13) Mol. Ther. 21(4):739~49). Additionally, insect cells can be used in mammalian cells. Therefore, human growth hormone ( a mammalian intron, such as a human HGH intron or an SV40 large T antigen intron, These introns can be incorporated into the coding sequence of the endonuclease. Insect cells cannot function because they are not efficiently spliced ​​from pre-mRNA transcripts in cells. In contrast, the resulting Mammalian cells into which recombinant AAV particles are delivered properly splice pre-mRNA. Haifeng Chen is the author of Toxic proteins barnase and diphtheria toxin fragment A in insect packaging cells This attenuates the expression of these toxin genes, allowing the production of recombinant AAV vectors carrying these toxin genes. reported the use of HGH and the SV40 large T antigen intron to induce HIV transmission (Chen, H(2012)Mol Ther Nucleic Acids.1(11):e57) .

[0291] 3. The endonuclease gene requires a small molecule inducer for endonuclease expression. If desired, it can be operably linked to an inducible promoter. An example of such data is the Tet-On system (Clontech; Chen H. et al., (2012) 015)BMC Biotechnol.15(1):4)) and RheoSwitch System (Intrexon; Sowa G. et al., (2011) Spine, 36(10 ):E623-8). Both systems, as well as similar systems known in the art, tem is transcribed in response to small molecule activators (doxycycline or ecdysone, respectively) Ligand-inducible transcription factors that activate the Tet repressor and ecdysone receptor, respectively The implementation of the present invention using such a ligand-inducible transcription activator depends on the type of the target gene (variant of the target gene). The method comprises: 1) introducing an endonuclease gene having a binding site(s) for a transcription factor(s); 1) placing the gene under the control of a promoter responsive to the corresponding transcription factor; and 2) packaging the gene. The latter includes the inclusion of a gene encoding a transcription factor in the viral genome. The process is similar to that described above, but the target cells are not transfected after recombinant AAV delivery if the transcriptional activator is not provided to the same cells. This is necessary because the endonuclease is not expressed in the cells or tissues. The activator induces endonuclease activity only in cells or tissues treated with the cognate small molecule activator. This approach allows us to determine when and in what combination small molecule inducers are present. Spatio-temporal regulation of endonuclease gene expression by selecting the tissue to be delivered However, the loading capacity is significantly limited. The need to include the inducer in the viral genome presents a drawback to this approach.

[0292] 4. In another preferred embodiment, the recombinant AAV particles are It is produced in mammalian cell lines that express a transcriptional repressor that prevents Repressors are known in the art and include Tet-repressor, Lac-repressor, Cro repressor and Lambda repressor. Ecdysone receptor, etc. Many nuclear hormone receptors in the human genome undergo transcriptional repression in the absence of their cognate hormone ligands. In order to practice the present invention, packaging cells are The virus is transfected / transduced with a vector encoding the promoter, which then inserts the promoter into the viral genome. The nuclease gene (packaging vector) is inserted into the promoters modified to contain binding sites for repressors to enhance transcription. The gene encoding the transcriptional repressor can be placed in various locations. The gene can be encoded on a separate vector and can contain, in addition to the ITR sequences, It can be incorporated into a packaging vector on the side, and can be used in cap / rep vectors or adenoviruses. It can be incorporated into a viral helper vector, or most preferably, it can be constitutively It can be stably integrated into the genome of the packaging cell so that it is expressed. Methods for modifying common mammalian promoters to incorporate lesser sites are known in the art. For example, Chang and Roninson have demonstrated that potent constitutive CMV and RS The V promoter was modified to contain the Lac repressor operator, showed that gene expression from the TA was greatly attenuated in cells expressing the repressor. (Chang BD and Roninson IB (1996) Gene 183:137~ 42) The use of a non-human transcriptional repressor prevents transcription of the endonuclease gene from being blocked by the repressor. The resulting recombinant AAV vector is suppressed only in packaging cells that express the enzyme. Ensure that the transduced target cells or tissues are not repressed.

[0293] 2.6 Recombinant meganuclease variants Embodiments of the present invention include the recombinant meganucleases and variants thereof described herein. A further embodiment of the present invention is a recombinant meganuclease encoding the enzyme described herein. The present invention also includes isolated polynucleotides comprising nucleic acid sequences as well as variants of such polynucleotides. do.

[0294] As used herein, "variant" is intended to mean a substantially similar sequence. A "variant" polypeptide is one that has a mutation at one or more internal sites of the native protein. or deletion or addition of several amino acids, and / or one or more of the naturally occurring polypeptides Derived from the "native" polypeptide by substitution of one or more amino acids at positions As used herein, "native" polypeptides are intended to mean polypeptides that are naturally occurring. The oligonucleotide or polypeptide includes the parent sequence from which the variant is derived. The variant polypeptides encompassed are biologically active, i.e., they are similar to the native protein. the desired biological activity of the TRC1-2 recognition sequence (SEQ ID NO: 3), the TRC3-4 recognition sequence, (SEQ ID NO: 4), and human T cell receptor α containing the TRC7-8 recognition sequence (SEQ ID NO: 5). The nucleotide sequence of the ... Such variants may result, for example, from human manipulation. Biologically active variants of the peptides (e.g., SEQ ID NOS: 8-32), or the recognized peptides described herein. Biologically active variants of the recognition site-binding subunits (e.g., SEQ ID NOS: 33-82) are , using the sequence alignment program and parameters described elsewhere herein. about 40%, about 45%, about 50%, or about 60% of the naturally occurring polypeptide or naturally occurring subunit, as determined by Approximately 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%, or about 99% sequence identity. An environmentally active variant may have only about 1 to 40 amino acids in addition to the polypeptide or subunit. acid residues, only about 1-20, only about 1-10, only about 5, only 4, 3, 2 One, or even one, amino acid residue may differ.

[0295] 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. For example, amino acid sequence variants can be prepared by mutations in DNA. Methods for mutagenesis and polynucleotide modification are well known in the art. For example, Kunkel (1985) Proc. Natl. Acad. Sc i.USA 82:488-492; Kunkel et al. (1987) Methods i n Enzymol. 154:367-382; U.S. Pat. No. 4,873,192 ;Walker and Gaastra (eds.), (1983) Techniques in Mo lecular Biology(MacMillan Publishing Com See the following publications: Guidance on appropriate amino acid substitutions that do not affect the biological activity of the protein The reference is Dayhoff et al. (1978) Atlas of Protein Sequences. nce and Structure(Natl.Biomed.Res.Found. Washington, DC), which is incorporated by reference. Replacing one amino acid with another amino acid with similar properties Conservative substitutions such as may be optimal.

[0296] Amino acid modifications to the DNA recognition domain of wild-type I-CreI meganuclease Qualitative numbers have been previously identified (e.g., U.S. Pat. No. 8,021,867), These can be used alone or in combination to produce rationally designed meganuclei. within the DNA recognition sequence half-site so that the enzyme has a different half-site specificity than the wild-type enzyme. Recombinant meganucleases with altered specificity at individual bases are produced. , which enhances specificity based on the base present at each half-site position (-1 to -9) of the recognition half-site. We present promising substitutions that can be made in recombinant meganuclease monomers or subunits to provide.

[0297] [Table 4]

[0298] In the case of polynucleotides, a "variant" refers to one or more portions of a naturally occurring polynucleotide. The nucleic acid of the embodiment may include one or more deletions and / or additions of nucleotides at positions 1 and 2. It is recognized that mutants of the nucleotide sequence are constructed such that the open reading frame is maintained. In the case of polynucleotides, conservative variants include those that are different due to the degeneracy of the genetic code. includes a sequence encoding the amino acid sequence of one of the polypeptides of the embodiments. The polynucleotides may be generated, for example, by using site-directed mutagenesis. but still encode the recombinant meganuclease of the embodiments. Generally, variants of particular polynucleotides of the embodiments include: Using the sequence alignment program and parameters described elsewhere herein. and the determination of the identity of the particular polynucleotide is 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 sequence identity. A variant of a sequence (i.e., a reference polynucleotide) is encoded by a variant polynucleotide. between the polypeptide encoded by the reference polynucleotide and the polypeptide encoded by the reference polynucleotide. It can also be assessed by comparing the percent sequence identity.

[0299] Deletions, insertions, and substitutions in the protein sequences encompassed herein are intended to be indicative of the characteristics of the polypeptide. However, prior to its implementation, replacement When it is difficult to predict the exact effect of a deletion or insertion, one skilled in the art can Selectively recognizes and recognizes the recognition sequence found within the receptor alpha constant region gene (SEQ ID NO: 1). The effectiveness of the polypeptides can be determined by screening them for their ability to cleave the polypeptides. will understand that will be evaluated. [Example]

[0300] The present invention is further illustrated by the following examples, which are not to be construed as limiting the invention. Those skilled in the art will be able to implement the present invention using no more than routine experimentation. Many equivalents to the specific substances and procedures described in this document are recognized or can be identified. Such equivalents may be deemed to be encompassed by the claims following the examples below. It is intended that:

[0301] Example 1: Characterization of meganucleases that recognize and cleave TRC recognition sequences

[0302] 1. Meganuclease that recognizes and cleaves the TRC1-2 recognition sequence Recombinant meganucleases (collectively referred to herein as "TRC1-2 meganucleases") SEQ ID NOs: 8 to 27 are sequences encoding the TRC1-2 recognition domains present in the human T cell receptor alpha constant region. Each TRC1-2 recombinant was engineered to recognize and cleave the recognition sequence (SEQ ID NO: 3). The meganuclease contains an SV40-derived N-terminal nuclease localization signal, a meganuclease subunit, a linker sequence, and a second meganuclease subunit The first subunit of each TRC1-2 meganuclease contains the TRC1 recognition sequence of SEQ ID NO: 3. The first subunit binds to the TRC2 recognition half-site, and the second subunit binds to the TRC2 recognition half-site (see Figure 1A). I want to be.

[0303] As shown in Figures 2 and 3, the TRC1-binding subunit and the TRC2-binding subunit Each contains a 56 base pair hypervariable region called HVR1 and HVR2, respectively. The C1-binding subunit contains the HVR1 residue except for positions 80 and 271 (including Q or E residues). They are identical outside the HVR1 region and highly conserved within the HVR1 region. The binding subunits also contain residues at positions 80 or 271 (including Q or E residues) and the meganucleoside Aze TRC1-2x.87EE, TRC1-2x.87QE, TRC1-2x.87E Q, TRC1-2x.87, and position 330 (including R residue) of TRC1-2x.163 ( The sequences are identical outside the HVR2 region except for the gray (shaded and underlined) and the HVR1 region. Similarly, the HVR2 region is also highly conserved.

[0304] The TRC1 binding regions of SEQ ID NOs: 8 to 27 are shown in FIG. 2 and are designated as SEQ ID NOs: 33 to 52, respectively. SEQ ID NOs: 33 to 52 are sequences encoding meganuclease TRC1-2x.87E, respectively. At least 90% sequence identity with SEQ ID NO: 33, the TRC1 binding region of E (SEQ ID NO: 8) The TRC2-binding regions of SEQ ID NOs: 8 to 27 are shown in FIG. SEQ ID NOs: 58 to 77 are provided as meganuclease TRC1- SEQ ID NO: 58, which is the TRC2 binding region of 2x.87EE (SEQ ID NO: 8), and at least 90 % sequence identity.

[0305] 2. Meganuclease that recognizes and cleaves the TRC3-4 recognition sequence Recombinant meganucleases (collectively referred to herein as "TRC3-4 meganucleases") SEQ ID NOs: 28 and 29) are sequences encoding the TRC3- Each TRC3-4 pair was engineered to recognize and cleave the 4 recognition sequence (SEQ ID NO: 4). The recombinant meganuclease contains an SV40-derived N-terminal nuclease localization signal, a nuclease subunit, a linker sequence, and a second meganuclease subunit The first subunit of each TRC3-4 meganuclease comprises the TRC3 subunit of SEQ ID NO: 4. The first subunit binds to the recognition half-site, and the second subunit binds to the TRC4 recognition half-site (see Figure 1A). I want to be illuminated).

[0306] As shown in Figures 4 and 5, the TRC3-binding subunit and the TRC4-binding subunit Each contains a 56 base pair hypervariable region called HVR1 and HVR2, respectively. The C3-binding subunit contains the HVR1 residues except for positions 80 and 271 (including Q or E residues). They are identical outside the HVR1 region and highly conserved within the HVR1 region. The binding subunit also contains HVR2 except for positions 80 or 271 (containing Q or E residues). They are identical outside the region and highly conserved within the HVR2 region.

[0307] The TRC3 binding regions of SEQ ID NOs: 28 and 29 are shown in Figure 4, and SEQ ID NOs: 53 and 55, respectively. SEQ ID NOs: 53 and 54 share 96.6% sequence identity. The TRC4 binding regions of numbers 28 and 29 are shown in FIG. 5 and are designated as SEQ ID NOs: 78 and 79, respectively. SEQ ID NOs: 78 and 79 also share 96.6% sequence identity.

[0308] 3. Meganuclease that recognizes and cleaves the TRC7-8 recognition sequence Recombinant meganucleases (collectively referred to herein as "TRC7-8 meganucleases") SEQ ID NOs: 30 to 32) are sequences encoding TRC7-8, which are present in the human T cell receptor alpha constant region. Each TRC7-8 recombinant was engineered to recognize and cleave the recognition sequence (SEQ ID NO: 5). The meganuclease contains an SV40-derived N-terminal nuclease localization signal, a meganuclease subunit, a linker sequence, and a second meganuclease subunit. The first subunit of each TRC7-8 meganuclease contains the TRC7 recognition sequence of SEQ ID NO: 5. The first subunit binds to the TRC8 recognition half-site, and the second subunit binds to the TRC8 recognition half-site (see Figure 1A). (I want to be).

[0309] As shown in Figures 6 and 7, the TRC7-binding subunit and the TRC8-binding subunit Each contains a 56 base pair hypervariable region called HVR1 and HVR2, respectively. The C7-binding subunit contains the HVR1 residues except for positions 80 and 271 (including Q or E residues). They are identical outside the HVR1 region and highly conserved within the HVR1 region. The binding subunit also contains HVR2 except for positions 80 or 271 (containing Q or E residues). They are identical outside the region and highly conserved within the HVR2 region.

[0310] The TRC7-binding regions of SEQ ID NOs: 30 to 32 are shown in FIG. 6, and the TRC7-binding regions of SEQ ID NOs: 55 to 57 and 58 are shown in FIG. SEQ ID NOs: 55 to 57 are provided as the sequences for meganuclease TRC7-8x.7( At least 90% sequence identity with SEQ ID NO: 55, the TRC7 binding region of SEQ ID NO: 30 The TRC8-binding regions of SEQ ID NOs: 30 to 32 are shown in FIG. SEQ ID NOs: 80-82 are provided as sequences encoding meganuclease TRC7- 8x.7 (SEQ ID NO: 30) TRC8-binding region SEQ ID NO: 80 and at least 90% They share sequence identity.

[0311] 4. Human T-cell receptor alpha constant region recognition sequence in CHO cell reporter assay Disconnection of TRC1-2, TRC3-4, and TRC7-8 meganucleases each recognize a specific sequence. Determine whether the sequences (SEQ ID NOS: 3, 4, and 5, respectively) can be recognized and cleaved. To detect the activity of each recombinant meganuclease, we used the previously described CHO cell reporter assay ( See WO / 2012 / 167192 and Figure 8) To perform the assay, a non-functional green fluorescent We constructed a CHO cell reporter strain carrying a protein (GFP) gene expression cassette. The GFP gene of each cell line was expressed by intracellular cleavage of one of the recognition sequences by meganuclease. A pair of recognition sequences stimulates a homologous recombination event that results in a functional GFP gene. They were divided into two groups.

[0312] In the CHO reporter cell line developed for this study, The recognition sequences selected are the TRC1-2 recognition sequence (SEQ ID NO: 3), the TRC3-4 recognition sequence ( The sequence was the TRC7-8 recognition sequence (SEQ ID NO: 4) or the TRC7-8 recognition sequence (SEQ ID NO: 5). The second recognition sequence identified was identified by a control meganuclease called "CHO-23 / 24." The CHO-23 / 24 recognition sequence was recognized and cleaved by the TRC1-2 recognition sequence. The CHO reporter cells containing the CHO-23 / 24 recognition sequence are referred to herein as "TRC1 These cells are called "TRC3-4 cells" and "CHO-23 / 24 cells." The HO reporter cells are referred to herein as "TRC3-4 cells." The CHO reporter cells containing the recognition sequence and the CHO-23 / 24 recognition sequence are referred to herein as " These cells are called "TRC7-8 cells."

[0313] CHO reporter cells were transfected with plasmids encoding their corresponding recombinant meganucleases. Transfect the smid DNA (e.g., TRC1-2 cells with TRC1-2 meganucleotides). transfected with a plasmid DNA encoding the nuclease) or CHO-23 Plasmid DNA encoding the / 34 meganuclease was transfected into each of the In the experiment, Lipofectamine 2000 (Therm) was used according to the manufacturer's instructions. 4e in a 96-well plate using a Fisher 5 CHO Reporter Cells were transfected with 50 ng of plasmid DNA. After 8 hours, cells were assessed by flow cytometry to identify untransfected negative The percentage of GFP-positive cells compared to the control (TRC1-2bs) was determined. As shown in Figure 9, the TRC1-2, TRC3-4, and TRC7-8 meganucleases In cell lines containing the corresponding recognition sequence, GFP-positive cells were detected at frequencies significantly higher than those of the negative control. It was found to produce cells.

[0314] TRC1-2x.87QE, TRC1-2x.87EQ, and TRC1-2x.87E The efficacy of E meganuclease was also determined in a time-dependent manner. Cell(1e 6 ) at 1e per cell 6 The meganuclease mRNA copies were Electroporation was performed using a BioRad Gene Pulser Xcell according to the instructions. At 1, 4, 6, 8, and 12 days after transfection, cells were analyzed by flow cytometry. The percentage of GFP-positive cells was determined by the evaluation. TRC1-2 meganuclease showed high efficiency, reaching 50% two days after transfection. More than 100 GFP-positive cells were observed. This effect persisted for 12 days, and no cytotoxicity was observed. No evidence of .

[0315] 5. Conclusion These studies have led to the development of the TRC1-2 meganuclease, TRC3-4 meganuclease, and The nuclease and TRC7-8 meganuclease bind to their respective recognition sequences. We demonstrated that it can be efficiently targeted and cleaved within the cell.

[0316] Example 2: Cleavage of the TRC recognition sequence in T cells and suppression of cell surface T cell receptor expression

[0317] 1. Cleavage of the TRC1-2 recognition sequence in Jurkat cells This study demonstrated that the TRC1-2 meganuclease encompassed by the present invention inhibits the expression of β-glucan in Jurkat cells. Demonstration of ability to cleave the TRC1-2 recognition sequence in (immortalized human T lymphocyte cell line) I did. 1e 6 Jurkat cells were treated with 8e per cell. 6 Given the TRC1-2 meganuclei The mRNA copy number was measured using the BioRad Gene Pulser Xcell. 72 hours after transfection, the genome was electroporated using the following instructions. The DNA (gDNA) was harvested from the cells and subjected to a T7 endonuclease I (T7E) assay. The gene alterations in the endogenous TRC1-2 recognition sequence (Figure 11) were estimated. In the assay, primers flanking the TRC1-2 recognition site were used to identify the TRC1-2 gene. The locus is amplified by PCR. Indels (random insertions or deletions) are detected within the TRC1-2 locus. If there is a mutation, the resulting PCR product will be a mixture of wild-type and mutant alleles. The PCR product is likely to consist of a mixture of denatured and slowly reannealed. Rapid reannealing results in the formation of heteroduplexes consisting of wild-type and mutant alleles. The T7E1 enzyme allows synthesis of mismatched bases and / or bulges. The TR gene cleaves at the cleavage site, producing cleavage products that can be visualized by gel electrophoresis. Thirteen different versions of C1-2 meganuclease gave positive results in the T7E1 assay. This resulted in the efficient generation of indels in the endogenous TRC1-2 recognition sequence. It was clearly demonstrated.

[0318] To further investigate the cleavage properties of the TRC1-2 meganuclease, a dose-response experiment was performed. This was performed using kat cells. 6 Jurkat cells were given 3 μg or 1 μg per cell. TRC1-2 meganuclease mRNA copies were obtained from BioRad Gene Pulse Transfection was performed using er Xcell according to the manufacturer's instructions. After 96 hours, gDNA was harvested and T7E1 assays were performed as described above. Contains three different versions of the TRC1-2x.87 meganuclease, allowing Fifteen different TRC1-2 meganucleases cleave at the endogenous TRC1-2 recognition site. TRC1-2x.87EE performed particularly well, demonstrating a T7E1 assay. It produced a strong signal and had little or no toxicity in Jurkat cells.

[0319] 2. Cleavage of the TRC1-2 recognition sequence in human T cells This study demonstrates that the TRC1-2 meganuclease encompassed by the present invention was obtained from a donor. We demonstrated that the TRC1-2 recognition sequence can be cleaved in human T cells. + T thin The cells were stimulated with anti-CD3 and anti-CD28 antibodies for 3 days, and then treated with Amaxa4D-Nucl TRC1-2x.8 was transfected using a eofector (Lonza) according to the manufacturer's instructions. mRNA encoding the 7EE meganuclease was electroporated. After 3 and 7 days, gDNA was harvested and T7E1 assays were performed as described above. A shows that TRC1-2x.87EE targets the endogenous TRC1-2 recognition sequence in human T cells. The mutations were effectively introduced, allowing the meganuclease to recognize and cleave the TRC1-2 recognition sequence. The intensity of the cleavage product was significantly higher after 3 days of transfection and after 10 days of transfection. There appears to be no change between day 7 and day 8, indicating that TRC1-2x.87EE meganuclease This suggests that there is little or no toxicity associated with the endogenous TRC1-2 recognition sequence. To determine whether the mutations present are sufficient to eliminate surface expression of the T cell receptor Next, the cells were analyzed by flow cytometry using an anti-CD3 antibody. Approximately 50% of the transfected T cells stained negatively for CD3. The CD3-negative population is shown after transfection. There was no significant change between days 3 and 7, and the TRC1-2x.87EE meganuclease There is little or no associated toxicity or even loss of T cell receptor expression. showed.

[0320] To verify that the loss of CD3 expression is due to a mutation in the TRC1-2 recognition site To do this, gDNA was collected from transfected T cells and cloned into the TRC1-2 recognition site. The gene locus was amplified by PCR. The PCR products were cloned using Zero Blunt PCR. pCR- Individual colonies were picked and miniprep plasmids were prepared. Figure 14 shows some representative deletions observed in the TRC1-2 recognition sequence. The observed sequence is the result of the DNA duplex generated by the endonuclease. This is typical of deletions resulting from non-homologous end joining repair of strand breaks.

[0321] In addition to the TRC1-2x.87EE, there are also the TRC1-2x.55 and TRC1-2x.7 Other TRC1-2 meganucleases, including 2, have been shown to knock down T cell receptors in human T cells. Although we were able to knock out the knockouts previously observed for TRC1-2x.87EE, TRC1-2x.72Q47E was a megakaryon. It has a mutation in the active site of the nuclease (amino acid 47) and serves as a negative control.

[0322] [Table 5]

[0323] [Table 6]

[0324] 3. Conclusion These studies demonstrate that the TRC1-2 meganuclease encompassed by the present invention is Both T cells (immortalized T lymphocyte cell lines) and T cells obtained from human donors We demonstrated that the RC1-2 recognition sequence can be recognized and cleaved. The study showed that NHEJ occurs at the meganuclease cleavage site, as evidenced by the appearance of indels. Furthermore, the TRC1-2 meganuclease was obtained from a donor. It has been shown to reduce cell surface expression of the T cell receptor on human T cells.

[0325] Example 3: Recombinant AAV vectors for introducing exogenous nucleic acids into human T cells

[0326] 1. Recombinant AAV Vectors In this study, two recombinant AAV vectors (referred to as AAV405 and AAV406) were used. The exogenous nucleic acid sequence containing the EagI restriction site is inserted into the TRC1-2 recognition region by homologous recombination. Each recombinant AAV vector was designed to be introduced into the genome of human T cells in the following sequence: The cell line is then infected with the "helper" components (e.g., adenovirus) necessary to support replication. The first plasmid contains the cap and rep genes, the second plasmid contains the cap and rep genes, and the A virus that contains an intervening DNA sequence (e.g., an exogenous nucleic acid sequence) that is packaged into the virus. Triple transfection of a third plasmid containing a heterologous inverted terminal repeat (ITR) The transfection protocol is prepared using the method described in (Cots D, Bosch A, Chillon M(2013)Curr.Gene Ther.13(5):370~ 81). Figure 15 shows the insertion of an exogenous nucleic acid sequence into the cellular genome at a nuclease cleavage site. We present a general approach for using recombinant AAV vectors to transduce cells into the host.

[0327] The plasmid shown in Figure 16 was used to prepare AAV405 (SEQ ID NO: 107). As shown, AAV405 plasmids generally contain the 5'ITR, CMV enhancer and and a nucleic acid sequence containing a promoter sequence, a 5' homology arm, an EagI restriction site, SV40 poly( A) The plasmid shown in Figure 17 contains a signal sequence, a 3' homology arm, and a 3' ITR. AAV406 (SEQ ID NO: 108) was prepared using the following: The plasmid contains sequences similar to AAV405, but lacks the CMV endonucleases upstream of the 5' homology arm. This AAV study was conducted to determine the transduction efficiency of AAV. Further, the use of an AAV vector encoding GFP (GFP-AAV) integrated into the vector has been described. It included.

[0328] 2. Introduction of an exogenous nucleic acid sequence into the TRC1-2 recognition sequence AAV template undergoes homologous recombination repair after double-strand break generation by TRC1-2 meganuclease. Is it suitable for homology directed repair (HDR)? To test whether T cells stimulate the immune response, a series of experiments were performed using human T cells. Determine the timing of RC1-2 RNA electroporation and transduction of recombinant AAV vectors Human CD3 + T cells were stimulated with anti-CD3 and anti-CD28 antibodies for 3 days, and then Amaxa4D-Nucleofector (Lonza) was used according to the manufacturer's instructions. Then, mRNA (1 μg) encoding the TRC1-2x.87EE meganuclease was electrophoresed. Two, four, or eight hours after transfection, the cells were transfected with GFP-AAV ( 1e 5 The cells were transduced with 1000 viral genomes / cell. 72 hours after transduction, the cells were resuspended in GFAP. The transfected cells were analyzed for P expression by flow cytometry. The highest transduction efficiency was observed when cells were transduced 2 hours after transfection ( GFP-positive cells (88%). Transduction efficiency was measured by the time between transfection and transduction. As the time increased, the number of GFP-positive cells decreased significantly, reaching 78% at 4 hours and 10% at 8 hours. The positive cells were 65%.

[0329] Efficient viral transduction when cells were transduced 2 hours after transfection Therefore, we investigated the effects of AAV405 and AAV406 vectors on human T cells. CD3 was used as the HDR template. + Stimulate T cells with 1 µg of TRCs as described above. 1-2x.87EE mRNA was transfected. 2 hours after transfection Then, cells were transfected with AAV405 or AAV406 (1e 5 of viral genomes / cells) As a transduction-only control, cells were mock transfected (with water). AAV405 or AAV406(1e 5 (viral genome / cell) For the meganuclease-only control, cells were transfected with TRC1-2x.87EE. Transfection, followed by mock transduction (with water) 2 hours after transfection. did.

[0330] To determine whether the AAV vectors could function as HDR templates, gDNA was transfected into cells. The cells were collected and analyzed using primers that recognize sequences beyond the homologous region in the AAV vector. The TRC1-2 locus was amplified by PCR using PCR primers outside the homologous region. This allowed amplification of only the T cell genome, not the AAV vector. The resulting vector was purified and digested with EagI. PCR products amplified from cells transduced with either AAV vector were transduced. The fragment is shown (see arrow) and indicates the insertion of an EagI site into the TRC1-2 recognition sequence. PCR products from all control cell populations were not cleaved by EagI. Site insertion requires generation of a DNA double-strand break by the TRC1-2 meganuclease This proves that.

[0331] To further clarify the insertion of the EagI site into human T cells, The undigested PCR products generated from the above experiments were analyzed using Zero Blun PCR cloning kit (Thermo Fisher) according to the manufacturer's instructions. The clone was cloned into the pCR-blunt vector using colony PCR. The primers were forward and reverse (pCR blunt ends were located adjacent to the insert). 13 containing forward and reverse priming sites), TRC1-2x.87EE and A portion of the PCR products from cells transfected with AAV405 or AAV406 was The full-length nucleotides were analyzed by gel electrophoresis (Figs. 20A and 21A, respectively). PCR product (approximately 1600 bp), smaller inserts, and several empty plasmids In this assay, the empty plus fragments (approximately 300 bp) are mixed together. Bands larger than mid-mid often represent sequences containing large deletions within the TRC1-2 recognition sequence. In parallel, another part of the PCR product was digested with EagI to amplify the TRC1-2 recognition sequence. The percentage of clones containing the EagI recognition site inserted into the ribosome was determined. 21B shows that some PCR products were cut with EagI (e.g., Fig. 20B, second row, left (lanes 1 to 6) show that the expected fragments of approximately 700 and 800 bp are produced. From the gels of these authors, it was found that the EagI insertion occurred at approximately 25°C for AAV405 and AAV406, respectively. % and 6% (adjusted for the empty vector).

[0332] Confirms observations from gel electrophoresis of uncut and EagI-digested PCR products The remaining portion of each PCR product was sequenced to identify the TRC1-2 recognition sequence. The sequences of some representative deletions and insertions observed in the endonuclease are shown. to sequences resulting from non-homologous end joining repair of DNA double-strand breaks generated by cleavage. Typical results are shown in Table 1. All PCR products digested with EagI contained the TRC1-2 recognition sequence. It contained an inserted EagI site (Figure 22B).

[0333] 3. Improving AAV transduction efficiency AAV transduction was performed 2 hours after transfection, whereas later transduction In view of the observation that transfection and transduction are more efficient than Experiments were conducted to optimize the timing of injection. + T cells were isolated using an anti-CD3 antibody and anti-CD28 antibody for 3 days, and then transfected with Amaxa4D-Nucleofector (Lonza) according to the manufacturer's instructions. The transfection was carried out immediately after transfection or immediately after transfection. Two hours after transfection, cells were transfected with GFP-AAV (1e 5 transduced with 100 viral genomes / cell Furthermore, unstimulated cells were transfected with GFP-AAV(1e 5 Transduction of viral genomes / cell 72 hours after transduction, cells were analyzed for GFP expression by flow cytometry. Figure 23 shows the results of GFP-AAV transduction performed 2 hours after transfection. Transduction immediately after transfection resulted in 98% GFP-positive cells, whereas transduction immediately after transfection resulted in 90% GFP-positive cells. Resting T cells were not amenable to AAV transduction. Non-transduced cells also showed approximately 0% GFP-positive cells. showed the cells.

[0334] 4. Overview These studies used AAV vectors in conjunction with recombinant meganucleases to The exogenous nucleic acid sequence can be integrated into the cleavage site of the TCR alpha constant region via recombination. Demonstrate that.

[0335] Example 4: To introduce exogenous nucleic acids encoding chimeric antigen receptors in human T cells Recombinant AAV vectors

[0336] 1. Recombinant AAV Vectors In this study, two recombinant AAV vectors (AAV-CAR100 and AAV-C AR763) by homologous recombination with an exogenous nucleic acid sequence encoding a chimeric antigen receptor. These were designed to be introduced into the genome of human T cells via the TRC1-2 recognition sequence. Recombinant AAV vectors were transfected using the triple transfection protocol described previously. Prepared.

[0337] AAV-CAR100 (also referred to herein as AAV408) is a vector containing the plus shown in FIG. As indicated, AAV-CAR100 ( AAV408) is designed to generate self-complementary AAV vectors and is generally ITR, 5' homology arm, nucleic acid sequence encoding anti-CD19 chimeric antigen receptor, SV4 0 poly(A) signal sequence, 3' homology arms, and 3' ITR. 110) was used to express AAV-CAR763 (referred to herein as AAV412 As indicated, AAV-CAR763 (AAV412) plus The mide generally contains the same sequence as AAV-CAR100 (AAV408), but with a single A strand. Single-stranded AAV vectors are designed to generate larger payloads. The 5' homologous arm and the 3' homologous arm can accommodate the AAV-CAR 763 (AAV412) is longer than AAV-CAR100 (AAV408). AAV studies have incorporated a GFP-encoding vector as a positive control for AAV transduction efficiency. Further included is the use of an AAV vector (GFP-AAV).

[0338] 2. Introduction of a chimeric antigen receptor sequence into the TRC1-2 recognition sequence Insertion of a chimeric antigen receptor sequence into the TCR alpha constant region gene, simultaneously replicating the endogenous TCR Determine the efficiency of using recombinant AAV vectors to knock out cell surface expression of receptors Conduct research to achieve this.

[0339] To confirm transduction efficiency, human CD3 + T cells were obtained and subjected to anti-CD3 and anti-CD28 The cells were stimulated with antibodies for 3 days, and then transfected with Amaxa4D-Nucleofector (Lonza). ) according to the manufacturer's instructions to coat the TRC1-2x.87EE meganuclease. The cells were electroporated with mRNA (1 μg) containing the desired nucleotides. Immediately after transfection with GFP-AAV (1e 5 The cells are transduced with the viral genome (1000 cells / cell). 72 hours after transduction, GFP expression was analyzed by flow cytometry. Determine the input efficiency.

[0340] AAV-CAR100 (AAV408) and AAV-CAR763 (AAV412) vectors The vector was designed to generate HDR templates in human T cells for insertion of anti-CD19 chimeric antigen receptor sequences. Used as a template. Human CD3 + Stimulate T cells with 1 µg of TRC1-2 as described above. x.87EE mRNA is transfected. Then, immediately after transfection or The cells were transfected with AAV-CAR100 ( AAV408) or AAV-CAR763 (AAV412) (1e 5 viral genome / As a transduction-only control, cells were mock transfected (with water). AAV-CAR100 (AAV408) or AAV-CAR763 (AAV4 12)(1e 5 The cells were transduced with either the viral genome or the meganuclease. For the control, cells were transfected with mRNA encoding TRC1-2x.87EE alone. The cells are transfected with PBS and then mock transduced (with water) immediately after transfection.

[0341] Insertion of the chimeric antigen receptor sequence requires sequencing of the cleavage site in the TCR alpha constant region gene. The cell surface expression of the chimeric antigen receptor is confirmed by the measurement of anti-Fab or anti-CD19 antibody. Confirm by flow cytometry using the endogenous T cell receptor (TCR) assay. Knockout is determined by flow cytometry as described above.

[0342] Example 5: Insertion and expression of chimeric antigen receptors

[0343] 1. Insertion of chimeric antigen receptor sequences into the TRC1-2 recognition sequence In this study, AAV inserted a chimeric antigen receptor sequence into the TCR alpha constant region gene. and can be used to simultaneously knock out cell surface expression of endogenous TCR receptors. In the first experiment, we tested whether human CD3 R-templates could be provided. + T Cell(1e 6 cells) and encodes the TRC1-2x.87EE meganuclease mRNA (2 μg) was electroporated and then transfected with AAV412 (1e 5 viral genome / cell) As a control, cells were mock electroporated and then transduced with AAV412. or electroporation with mRNA encoding TRC1-2x.87EE, and then An additional control of mock-electroporated and mock-transduced cells was also included.

[0344] Was the AAV HDR template utilized to repair the double-strand break in the TRC1-2 recognition sequence? A PCR-based assay was developed to determine whether the genus P. cerevisiae was a genotype or not. Three primer pairs were used: The first set was used for PCR analysis. This first primer set (referred to as "internal homolog arm / The CAR region (referred to as the "CAR region") is located within the homologous region and therefore corresponds to the unmodified TRC1-2 recognition sequence of the genome. AAV412 vector input (2603 bp), or CAR gene The second gene is inserted into the TRC1-2 recognition sequence (2603 bp) and amplified. The primer set (referred to as "outer 5' homology arms" in Table 7) was One primer anneals within the CAR region of the HDR template, AAV412HDR template It contains one primer that anneals to the human genome outside the 5' homology arm of type C Only when the AR gene was successfully inserted within the TRC1-2 recognition sequence did an 1872 bp fragment emerge. The third primer set (referred to as the "outer 3' homology arm" in Table 7) amplifies the 3' fragment. ) is one primer that anneals within the CAR region of the AAV412HDR template, and 1, which anneals to the human genome outside the 3' homology arm of the AAV412 HDR template. Similar to the second primer set, the third primer set comprises: A 1107-bp fragment was generated only when the CAR gene was successfully inserted into the TRC1-2 recognition sequence. In summary, the PCR products from all three primer sets amplified the CAR sequence. Whether or not the TRC1-2 recognition sequence is present in the cells (primer set 1), and whether or not it is present in the cells The inserts (primer sets 2 and 3) are shown.

[0345] Four days after transduction, cells were analyzed using the PCR primer pair described above. Approximately 3000 cells were harvested, pelleted, lysed, and PCR was performed to detect the CAR gene. The PCR products were then analyzed to determine whether the TRC1-2 recognition sequence was inserted into the TRC1-2 recognition sequence. The results were separated on an agarose gel (lane descriptions are shown in Table 7). Lanes 1 to 3 are TR mRNA encoding C1-2x.87EE was extracted from electroporated and mock-transduced samples. This is the original PCR product.

[0346] As expected, the first primer pair ("internal homolog arm / CAR region") was The RC1-2 recognition sequence locus was amplified to generate the 349 bp band shown in lane 1. Sequences 2 and 3 are produced only when the CAR gene is inserted into the TRC1-2 recognition sequence. Lanes 7-9 correspond to primer pairs that generate α- and β-glucan-1-phosphate dehydrogenase (GD), and products are not shown. Pore, and mock-transduced samples represent TRC1-2x.87EE mRNA as described above. Lanes 4–6 show the same bands as the control. Lane 4 shows the PCR product from the electroporated AAV412-transduced sample. The first primer pair ("internal homolog / CAR region") was used to generate the two bands. The unmodified TRC1-2 recognition sequence locus (349 bp) and the AAV412 vector are shown. TRC1-2 recognition sequence with target input (2603 bp) or CAR gene inserted (2603 bp). Lanes 5 and 6 show the amplification of the CAR nucleic acid sequence TRC1-2x. The primer pair amplifies the product only if it is inserted into the 87EE recognition site. Both bands are of the expected size (1872 and 1107, respectively). bp). Lanes 10–12 show mock-electroporated, AAV412-transduced samples. Lane 10 represents the amplification by the first primer pair ("internal homolog / CAR region"). The two bands generated by the TRC1-2 gene are shown, and the unmodified TRC1-2 recognition sequence locus (349 bp) of the genome is shown. ) and AAV412 vector input (2603 bp). 12 produces a product only when the CAR gene is inserted into the TRC1-2 recognition sequence. Lanes 11 and 12 (outside the homologous arms) correspond to the primer pairs shown, and products are not shown. The absence of a band in lane 10 (including the primer on the other side) indicates the 2603 bp band in lane 10. were generated from amplification of AAV412 input.

[0347] In summary, PCR analysis demonstrated that both TRC1-2x.87EE mRNA and AAV412 If either of these is present in the cell, the CAR gene is introduced into the TRC1-2x.87EE recognition site. Therefore, the present inventors have demonstrated that AAV412 inhibits TRC1-2 Generate a suitable HDR template that can be used to insert the CAR gene into the x.87EE recognition sequence We conclude that this is useful.

[0348] In the second experiment, human CD3 + Stimulates T cells and TRC1-2x.87E Immediately after electroporation with mRNA encoding E meganuclease, increasing amounts of AAV40 8 (0 μL, 3.125 μL, 6.25 μL, 12.5 μL, or about 25 μL, which is about 0, 3.125e 3 , 6.250e 3 , 1.25e 4 , and 2.5e 4 Viral genome / As a control, cells were mock electroporated and then transduced with increasing amounts of A Further controls included mock-electroporated and mock-transduced cells, and TRC1-2x.87EE mRNA electroporated and then mock-transduced. Four days after transduction, cells were harvested and analyzed as described above, except that the CAR gene was expressed in the Only use primer pairs that amplify products only when inserted into the TRC1-2 recognition sequence. The PCR products were separated on an agarose gel as shown in Figure 27A. only when the CAR gene is inserted into the TRC1-2 recognition sequence locus. Using the above primer pair ("outer 5' homology arm"), amplify a product at the 5' end of Figure 27B shows the PCR products generated. The above primer pair amplifies a product at the 3' end of the locus only if the gene is inserted. ("external 3' homology arm"). Lane legends are given in Table 1. Lanes 1 to 5 in both Figures 27A and 27B show the results of either sham electroporation or sham electroporation. The figure represents a sample that was electroporated and then mock-transduced. It is not possible to see the difference between the HDR template generated by AAV408 and the TRC1-2x Inability to insert the CAR gene into the TRC1-2 recognition sequence in the absence of .87EE mRNA Lane 6 shows TRC1-2x.87EE mRNA electroporated and pseudotransformed. The sample shown is a transfected sample. No PCR product was observed, and CA was not found in the TRC1-2 recognition sequence. Lanes 7 to 10 show the TRC1-2x.8 vector without the R gene inserted. Samples electroporated with 7EE mRNA and transduced with increasing amounts of AAV408 are represented. A band of the appropriate size was evident in each PCR, indicating that AAV408 was encoding TRC1-2 It is possible to generate HDR donors for repair of the recognition sequence, resulting in the insertion of the CAR gene. This shows that:

[0349] [Table 7]

[0350] [Table 8]

[0351] The PCR-based assay described above demonstrated that the CAR gene was inserted into the TRC1-2 recognition sequence. This is useful for determining whether the CAR insertion is efficient, but does not provide information on the efficiency. To determine the α- and β-amyloid nucleotide sequence, we developed a digital PCR-based assay (Figure 28). (Shown in Figure A). This assay uses two primer sets. The first set The first set amplifies an unrelated gene sequence and provides a reference sequence to control the number of templates. In this study, a product was amplified only when the CAR gene was inserted into the TRC1-2 recognition sequence. One primer anneals within the CAR gene and the other anneals outside the 3' homology arm. The VIC-labeled probe consists of one primer that anneals to the first primer. The FAM-labeled probe anneals to the amplicon generated from the second set. Anneals within the amplicon generated by the primer set. FAM label The number of amplicons detected by the probe was calculated as the number of amplicons detected by the VIC-labeled probe. Divide the number of amplicons modified by the insertion of the CAR gene by the number of reference sequences. This allows for accurate quantification of the percentage of TRC1-2 recognition sequence loci.

[0352] Figure 28B shows a mock-electroporated and then transduced sample, TRC1-2x.87E Samples electroporated with EmRNA and then mock transduced, or TRC1-2x.87 EE mRNA was electroporated and then transduced with increasing amounts of AAV408. The digital PCR results are shown below. Digital PCR was performed approximately one week after transduction. The PCR observations described in Figure 27 were performed using genomic DNA isolated from cells. Consistent with this, both control samples (transduction only or electroporation only) showed a significant increase in TRC1-2 It was found that 0% of the CAR gene was inserted into the x.87EE recognition sequence. mRNA encoding 1-2x.87EE was electroporated, followed by increasing amounts of AAV408. The transduced samples were found to have approximately 1.5% to 7%. The assay consisted of two This digital PCR-based assay was performed on different instruments (trademarks QX200 and QS3D). Significant agreement was observed demonstrating the sensitivity and precision of the assay.

[0353] 2. Expression of anti-CD19 chimeric antigen receptors on T cells In addition to determining whether CAR insertion occurred at the molecular level, we also investigated the role of AAV408 In cells in which the CAR gene was inserted into the TRC1-2 recognition sequence using CAR as an HDR template, We sought to determine the expression level of the anti-CD19 chimeric antigen receptor (CAR). The efficiency of insertion into the C1-2x.87EE recognition sequence to knock out T cell receptors The samples analyzed above and in Figures 27 and 28 were also analyzed by flow cytometry. Approximately 4 days after transduction, cells were transfected with anti-CD19C AR (anti-Fab-Alexa647) or CD3 (CD3 - BB515) and analyzed by flow cytometry. Figure 29A shows anti-CAR labeling on the Y-axis. Flow cytometry plots are shown with anti-CD3 labeling on the X-axis. Isotransduced cells (MOI-0) predominantly expressed CD3+ / CAR - (bottom right elephant) Cells mock-electroporated and then transduced with increasing amounts of AAV408 showed appear essentially identical to the control cells and CD3 + / CAR - The population is 98.8%, 99, 99% , and 99.1%. Therefore, the present inventors concluded that the AAV408 virus alone was not detectable. It does not drive CAR expression at feasible levels and is also unable to disrupt T cell receptor expression. I conclude that there is no.

[0354] Figure 29B shows the results of electroporation of mRNA encoding TRC1-2x.87EE followed by mock After electroporation of the transduced samples or TRC1-2x.87EE, increasing amounts of AA Flow cytometry plots of V408-transduced cells are shown. Mock-transduced cells had 47.1% CD3 - cells and the efficiency of the T cell receptor complex The background of anti-CD19CAR labeling was very low, demonstrating effective knockout. CD3 - 0.6% of the population, CD3 + The incidence was 0.78% in the population. E-encoding mRNA was electroporated into samples, which were then transduced with increasing amounts of AAV408. Pull CD3 - The CAR markers ranged from 2.09% to 5.9% in the population. , CD3 + There was also a slight increase in CAR labeling in the population, from 1.08% to 1.91% The present inventors have investigated the CD3 + The cause of the increase in CAR+ cells in the population was not identified. However, it is possible that the CAR was inserted into a non-expressing T cell receptor allele (T cell receptor allele Only one allele of the rFA chain is expressed and incorporated into the T cell receptor complex. .

[0355] These data correlated well with the quantitative digital PCR-based assay described above. For example, the highest MOI of AAV408 (2.5e 4 virus genome / cell) Digital PCR assay showed approximately 6% CAR insertion, and flow cytometry assay showed 5. 9% of CARs + / CD3 - Cells were shown. CAR + / CD3 + Taking the population into account, The data are based on a flow cytometry assay showing approximately 7.8% CAR + is digital PCR This is still exactly the same as the 6% achieved by

[0356] Example 6: Further AAV Vector Characterization

[0357] 1. Insertion of chimeric antigen receptor sequences into the TRC1-2 recognition sequence The AAV vector is suitable for inserting the CAR gene into the TRC1-2x.87EE recognition sequence. Therefore, we have further refined the construction of AAV vectors. The present inventors sought to optimize the TRC1-2 recognition sequence locus and the AAV ITR. The CAR gene expression cassette is driven by the JeT promoter and is flanked by short homologous regions. We have constructed a vector that can be used to generate a self-complementary AAV genome containing the This vector is called AAV421 (Figure 30; SEQ ID NO: 123). Due to the limited packaging capacity, short homology arms were required. The CAR gene, driven by the CMV promoter, is flanked by the same arms and AAV ITRs. Create a vector that can be used to generate a single-stranded AAV genome containing a gene expression cassette. This vector is called AAV422 (Figure 31; SEQ ID NO: 124). Because the V genome has a larger cargo capacity, it can carry longer homologous archaea than self-complementary vectors. We were able to use the system.

[0358] AAV421 and AAV422 target insertion of the CAR gene into the TRC1-2 recognition sequence To test whether this would be useful for identifying human C D3 + In the first experiment, human CD3 + T cells (1e 6 Cells) perforated and then transduced with increasing amounts of AAV421 or 422, or TRC1- 2x.87EE mRNA (2 μg) was electroporated, followed by increasing amounts of AAV421 or AA The aforementioned experiments with AAV408 were performed using MO Since higher I suggested more efficient CAR insertion, AAV422MO The MOI was significantly higher than that of AAV421 in this experiment than in the previous experiment (approximate MOI is 1.25e 4 , 2.5e 4 , 5e 4 , and 1e 5 (The virus genome / cell). A The AV421 virus stock was sufficiently large to allow for significantly higher titers than in previous experiments. As a control, cells were electroporated (mock or TRC1-2x.87E As an additional component of this experiment, the cells were transduced with 'large-scale' conditions. The items were carried out and 10e 6 TRC1-2x.87E cells (10x more than in a typical experiment) EmRNA was electroporated and then transfected with AAV422 (2.5e 4 virus genome / cell) Finally, we also transduced the first virus stock with A second viral stock of AAV421 was tested.

[0359] The CAR gene was inserted into the TRC1-2x.87EE recognition sequence. by PCR as above using a primer pair that amplifies a product only if The PCR results were separated by agarose gel as shown in Figures 32A and 32B. (Lane descriptions are provided in Tables 9 and 10.) Sample 1 in Figure 32A was mock-electroporated; Samples 2 to 5 were then mock-transduced and mock-electroporated and then transduced with AAV421. The gel showed that none of these samples produced PCR products, and TRC1-2x.8 In the absence of 7EE mRNA, AAV421 inserted the CAR gene into the TRC1-2 recognition sequence. Furthermore, electroporation of TRC1-2x.87EE mRNA showed that it was unable to drive the The control sample (sample 6), which was thawed and then mock transduced, showed no PCR product. Samples 7 to 10 in Figure 32A were electroporated with TRC1-2x.87EE mRNA. The cells were then transduced with increasing amounts of AAV421. The PCR bands of the extended products are shown (the two bands below each sample number), and CA This demonstrates the integration of the R gene into the TRC1-2 recognition sequence. Lanes 11 and 12 are 1e 6 or 10e 6 Cells / samples The TRC1-2x.87EE mRNA was electroporated and then transduced with AAV422. Both PCR bands (different bands due to the long homology arms) represent the input samples. The presence of 2 primers (larger than the first set) indicates a link between the CAR gene and the This indicates successful insertion into the TRC1-2 recognition sequence.

[0360] Sample 1 in Figure 32B was mock-electroporated and then mock-transduced, while samples 2-5 were mock-electroporated and then mock-transduced. were mock-electroporated and then transduced with increasing amounts of AAV422 (Table 10). None of these samples produced any PCR products, and no TRC1-2x.87EE mRNA was detected. In its absence, AAV422 fails to drive insertion of the CAR gene into the TRC1-2 recognition sequence Samples 7 to 10 in Figure 32B contain TRC1-2x.87EEmRN A was electroporated and then transduced with increasing amounts of AAV422. PCR bands of products extending beyond both arms were observed, indicating that the CAR gene TRC1-2 Finally, sample 11 demonstrates incorporation of the TRC1-2x.87 recognition sequence. EE mRNA was electroporated and then subjected to a different viral strain than the sample shown in Figure 32A. The presence of a band indicates the presence of the CAR gene. The results show that the insertion of α-glucan into the TRC1-2 recognition sequence confirms the reproducibility between different virus stocks. In summary, Figure 32 shows that both AAV421 and AAV422 express the CAR gene. We clearly demonstrated that we can generate HDR templates suitable for insertion into the TRC1-2 recognition sequence. are.

[0361] [Table 9]

[0362] [Table 10]

[0363] 2. Expression of anti-CD19 chimeric antigen receptors on T cells using AAV421 Here, cells in which the CAR gene was inserted into the TRC1-2 recognition sequence using AAV421 We sought to determine the expression level of anti-CD19 chimeric antigen receptors in mice. Samples analyzed in 32A were also analyzed for CAR and CD3 expression by flow cytometry. Approximately 4 days after transduction, the cells were analyzed by incubating with anti-CD19CAR or an antibody that recognizes CD3. The cells were labeled with α- and β-AAV and analyzed by flow cytometry. 421-transduced cells and mock-electroporated and mock-transduced control cells. Flow cytometry plots are shown. Mock-electroporated and mock-transduced cells (MOI- 0) is overwhelmingly CD3 + / CAR - (lower right quadrant, 98.8%). Cells porated and then transduced with increasing amounts of AAV421 appeared essentially identical to control cells. CD3 + / CAR - The populations were 98.8%, 98.6%, 98.8%, and 97.9%. Therefore, the present inventors found that the AAV421 virus alone did not produce detectable levels of CA. We conclude that the IL-16 receptor agonist does not drive R expression and is unable to disrupt T cell receptor expression.

[0364] Figure 33B shows electroporation of TRC1-2x.87EE mRNA followed by mock transduction. After electroporation of the sample or TRC1-2x.87EE, increasing amounts of AAV421 were injected. Flow cytometry plots of transduced cells are shown. Electroporation followed by mock transduction. The cells were 56.7% CD3 - cells and efficient knockdown of the T cell receptor complex The background of the anti-CD19CAR labeling was very low, and the CD3 - group 0.48%, CD3 + The incidence was 0.36% in the population. The samples were electroporated with mRNA and then transduced with increasing amounts of AAV412. CD3 - The population showed significant amounts of CAR labeling ranging from 4.99% to 13.4%. Also, CD3 + There was also a slight increase in CAR labeling in the population, from 1.27% to 3.95%. As mentioned above, the CAR gene is inserted into a non-expressing T cell receptor allele. In contrast to the AAV408 experiment, CAR + The group is higher The JeT promoter is better defined with a higher mean fluorescence intensity than the eF1α core promoter. These results suggest that it drives higher expression than

[0365] To evaluate the insertion of the CAR gene using AAV421 in conjunction with TRC1-x.87EE On the other hand, the inventors have - / CAR + Determine how to preferentially grow and enrich the population. From the experiments described above and shown in FIG. 33, the present inventors have found that TRC1-2x.87 EE mRNA (2 μg) was electroporated and then transfected with AAV421 (3.13 e 4 Virus Genome The control samples were cells transduced with the IgG1 gene (gamma / cells). Harvested, mock electroporation and mock transduction, mock electroporation and AAV421 transduction, or electroporated and mock transduced with TRC1-2x.87EE. As a result of the enrichment and proliferation process, these cells were stimulated with IL-7 and IL-15 (both of which were 1 The cells were then incubated for 6 days in complete growth medium supplemented with 0.05 mg / mL of ATP. The cells were labeled with antibodies against anti-CD19CAR and CD3 and analyzed by flow cytometry. The mock-electroporated and mock-transduced cells expressed CD3 - / CAR + elephant There was a low level of background staining at the CD3 limit (0.13%). - / CAR + The populations were mock-electroporated and then AAV-transduced samples, or TRC1-2x .87EE mRNA was electroporated and then mock-transduced, resulting in essentially the same The TRC1-2x.87EE mRNA levels were similar to those of the control (0.16% and 0.55%, respectively). Electroporated and mock-transduced cells were 53.2% CD3 - / CAR - With a population,Fig. This is very close to the amount stained in the first part of this experiment shown in Figure 33B (56.7%). RC1-2x.87EE electroporated and AAV-transduced cells showed 12.6% CD3 - / CAR + cells, which are nearly identical to the original labeling of these cells shown in Figure 33 ( 13.4%), and a mixture of IL-7 and IL-15 inhibited specific CD3 - / CAR + Cell populations demonstrated that it was insufficient to enrich or grow the cells.

[0366] Next, the four samples were mixed with IM-9 cells, which express CD19 on their cell surface. Incubation with CD3 in an antigen-specific manner - / CAR + Concentrate the population IM-9 cells were inactivated by pretreatment with mitomycin C, and then incubated at 1: 1 ratio with the samples in the presence of IL-7 and IL-15 (10 ng / mL) for 6 days. The cells were then labeled with antibodies against CD3 and anti-CD19CAR. and analyzed by flow cytometry (Figure 34B). The cells were CD3 - / CAR + showed low levels of background staining in the quadrants (0.2%). CD3 - / CAR + The population was mock-electroporated and then transduced with AAV. The same was true in the samples (0.2%), and TRC1-2x.87EE was electroporated. Slightly higher in mock-transduced cells (1.24%). CD3 in EE alone control - / CAR + The increase in cells is due to the fact that CAR nucleic acid has been introduced into the system. TRC1-2x.87EE mRNA was electroporated and therefore considered background. The porated and mock-transduced cells were 42.5% CD3 - / CAR - population, and before proliferation ( 56.7%, significantly lower than that of CD + Cells have a growth advantage in this system However, TRC1-2x.87EE was electroporated and AAV Transduced cells were 49.9% CD3 - / CAR + This is shown in Figure 33. The expression of IL-7 and IL-15 was dramatically increased compared to the original labeling of these cells (13.4%). Incubation of this sample with IM-9 cells in the presence of CD3 - / CA R + have proven highly effective in enriching and expanding CD3 populations. + / C AR + The population was also grown under the same conditions and the mock-electroporated / AAV-transduced samples and T The RC1-2x.87EE electroporated / AV transduced samples showed 2.53% and 1.0% HIV-1 mediated cytotoxicity, respectively. 5.3% CD3 + / CAR + Shows.

[0367] In cells electroporated with TRC1-2x.87EE and then transduced with AAV421, , CD3 - 24.2% of the population had CAR before proliferation + (Figure 33B). IL-7 and I After incubation in medium supplemented with L-15, CD3 - 25.3% of cells are CA R + (Figure 34A), and the ratio of gene knock-in to gene knock-out did not change. However, in addition to IL-7 and IL-15, the ink was also After the incubate, CD3 - More than 80% of the cells (80.35%, Figure 34B) were CAR + Yes demonstrated that incubation with IM-9 cells resulted in antigen-specific enrichment. do.

[0368] Mitomycin C very potently activates cells, and IM-9 cells A second injection of IM-9 cells further increased the CD3 - / CAR +We reasoned that this may increase the concentration of cells. Some of the cells were cultured on fresh IM-9 cells ( The cells were then mixed with mitomycin C (pretreated) and incubated for an additional 6 days. were stained for CD3 and anti-CD19CAR and analyzed by flow cytometry ( (Figure 34C). CD3 in either control sample - / CAR + Percentage of cells was essentially unchanged compared to the first round of enrichment on IM-9 cells.

[0369] However, cells electroporated with TRC1-2x.87EE and transduced with AAV421 , CD3 - / CAR + showed significant enrichment of cells, 49.9% (incubation with IM-9 cells) After the first round of incubation, the IR increased from 6.5% (Figure 34B) to 65.7% (Figure 34C). Importantly, CD3 - 93.75% of the population is CAR + and further antigen-specific amplification This indicates reproduction.

[0370] 3. Expression of anti-CD19 chimeric antigen receptors on T cells using AAV422 We used AAV422 to provide HDR templates in cells (see PCR results above). We also examined the expression of anti-CD19 CAR from the 16C- and 16D-cells (shown in Figure 32B). Approximately 4 days after transduction, the cells The cells were labeled with anti-CD19CAR or CD3-recognizing antibodies and analyzed by flow cytometry. Figure 35A shows the results of the analysis of cells that were mock electroporated and transduced with increasing amounts of AAV422, and mock electroporated and transduced with increasing amounts of AAV422. Flow cytometry plots for mock-electroporated and mock-transduced control cells are shown. Mock-electroporated and mock-transduced cells (MOI-0) predominantly expressed CD3 + / CA R - The results were as follows: (lower right quadrant, 98.8%). 2-transduced cells appeared essentially identical to control cells and showed CD3 + / CAR - The group The results were 98.6%, 98.6%, 98.9%, and 98.4%. However, the AAV422 vector alone did not drive detectable levels of CAR expression, and T cells Nor can it disrupt the expression of cell receptors.

[0371] Figure 35B shows electroporation of TRC1-2x.87EE mRNA followed by mock transduction. After electroporation of the samples or TRC1-2x.87EE, increasing amounts of AAV422 were injected. Flow cytometry plots of transduced cells are shown. Electroporation followed by mock transduction. The cells were 59.3% CD3 - cells and efficient knockdown of the T cell receptor complex The background of the anti-CD19CAR labeling was very low, and the CD3 - group 1.47%, CD3 + The incidence was 0.52% in the population. Samples electroporated with mRNA and then transduced with increasing amounts of AAV422 were D3 - The population showed significant amounts of CAR labeling ranging from 14.7% to 20.3%. CD3 + There was also a slight increase in CAR labeling in the population, ranging from 2.3% to 2.7%. Boo.

[0372] Surprisingly, we observed a T cell receptor knockout efficiency in the presence of AAV422. We observed a significant increase in CD3 knockout efficiency with titrated AAV422. 71.6%, compared with 59.3% for RC1-2x.87EE electroporation alone; The efficacies of the titrated AAV421 were 74.9%, 77.8%, and 74.4%, respectively. The overall CD3 knockout efficiency was 57.5% with TRC1-2x.87EE electroporation alone. 18%, while the rates were 56.99%, 56.62%, 57.4%, and 55.4%. Therefore, TRC1-2x.87E in the presence of a single-stranded AAV genome Electroporation of E. coli significantly increased the overall knockout efficiency of TRC1-2x.87EE nuclease. This appears to result in an increase in the expression of the AAV genome, but not in the presence of the self-complementary AAV genome. For the increase, CAR + CD3 - The percentage of cells is CD3 - / CAR + Cell Despite the higher numbers, there was no significant difference between AAV421- and AAV422-transduced cells. Not significantly different. CAR using AAV421 + CD3 - The highest percentage of cells The MOI was 24.18% (MOI=3.13e 4 viral genome / cell), compared with AAV422 was 26.48% (MOI = 1e 5 viral genome / cell) This observation takes into account the large difference in MOI between AAV421 and AAV422. and particularly interesting.

[0373] Cells from this experiment were used to generate CD3 - / CAR + Cell specificity Again, rather than testing the entire panel, the concept of selectively enriching The authors performed mock electroporation and then transduced cells with AAV422, or TRC1-2 x.87EE was electroporated and then transfected with AAV422 (2.5e 4 viral genome / cell) Only enrichment of transduced cells was attempted in new experiments. Flow cytometry plots are shown approximately 4 days after transduction. The cells were 0.13% CD3 - / CAR + The background staining of the cells was shown. In comparison, TRC1-2x.87EE electroporated and AAV422 transduced cells 4.44% CD3 - / CAR + The cells were treated with IL-7 and IL-1 as described above. Incubated with IM-9 cells (pretreated with mitomycin) in the presence of -15 for 6 days. The results were then analyzed by flow cytometry. tion, CD3 in AAV422-transduced cells. - / CAR + Dramatically reduce the population to 35.8% This indicates a significant increase in + The cells were found to be 6.69% total CD3 - They comprised 45.2% of the population (Figure 36A). As mentioned above, we also A second addition of cells further enriched the cells (Figure 36C). As a result of incubation, 65.1% of CD3 - / CAR + CAR cells were obtained. + Cells All CD3 - Constituting 78.25% of the population, CD3 - / CAR + Significant antigen-dependent concentration of cells This shows shrinkage.

[0374] These data, together with those above, support the anti-CD3000 antibody inserted into the TRC1-2 recognition sequence. Cells carrying the 19CAR gene were cultured in IM-9 cells in the presence of IL-7 and IL-15. The enrichment was achieved by incubation with β-glucan, and the enrichment was achieved after just 12 days of culture. Over 90% are CARs + CD3 - This clearly demonstrates that it can bring about a collective

[0375] 4. Increased knockout efficiency observed when using single-stranded AAV vectors In this study, a single-stranded AAV vector knocked out the TRC1-2x.87EE nuclease. In the first experiment, we followed up on the observation that TRC1-2x cells increased the efficiency of transfection. 0.87EE (2 μg) was electroporated and either mock transduced or with increasing amounts of AAV412 (6.25 e 4 , 1.25e 4 , 2.5e 4 , or 5e 4 Transduction of viral genome / cell Four days after transduction, cells were labeled with an antibody against CD3 and analyzed by flow cytometry. In mock-transduced cells, 20.7% were CD3 - in compared with 21.6% and 23.7% in cells transduced with titrated AAV412. , 25.5%, and 25%, respectively, and the knockout efficiency of TRC1-2x.87EE was AA. up to 23% higher in the presence of V412 (25.5% compared to 20.7%).

[0376] To determine whether this increase in knockout efficiency was nuclease specific, In further experiments, cells were treated with a nuclease targeting the β2-microglobulin gene. mRNA (2 μg) encoding the vector was electroporated, mock transduced, or injected with increasing amounts of AAV412 Four days after transduction, the cells were transduced with either β2-microglobulin or β2-microglobulin. The cells were stained for IgG and analyzed by flow cytometry (Figure 37B). The β2-microglobulin knockout efficiency was 64.5%, and increasing doses of AAV41 In cells transduced with 2, the percentage increased to 68.6%, 70.7%, 77.2%, and 82.5%. However, knockout efficiency increased by up to 27.9% (82.5% compared to 64.5%).

[0377] In parallel experiments, cells were electroporated with TRC1-2x.87EE mRNA and pseudotransfected. transduced with either AAV412 or AAV422 (using the same MOI as AAV412). The cells were labeled with an antibody against CD3 and analyzed by flow cytometry ( (Figure 37C). Mock-transduced cells showed 62.2% T cell receptor knockout, with increasing amounts In the AAV-induced T cell receptor knockout cases, the T cell receptor knockout frequencies were 72.6%, 75.5%, and 78%. .3% and 75.1%, respectively. Here, the presence of AAV422 increased TRC1-2x. The knockout efficiency of 87EE was increased by 25.8% (compared to 62.2% from 78.3%). These were compared using two different nucleases and two different AAV vectors. It is notable that the percent increase in knockout efficiency was nearly identical among the three experiments. Taken together, these data suggest that transduction of cells by single-stranded AAV vectors is However, our nuclease knockdown assays, regardless of the nuclease or AAV cargo, This strongly suggests that the ion exchange rate increases the efficiency of the ion exchange.

[0378] 5. Activity of T cells expressing anti-CD19 chimeric antigen receptors The above experiments were carried out by electroporating cells with TRC1-2x.87EE mRNA and then immediately transfecting the cells with TRC1-2x.87EE mRNA. Generation of CAR T cells by transduction with AAV421 and CD19-expressing IM- These cells were co-cultured with CD3 - / CAR + Enrichment for populations Next, we investigated the activity of these CAR T cells against target cells. In the first experiment, the cells described above and shown in Figure 34C were transfected with CD19 + Raji cells In an IFN-γ ELISPOT assay, either CD19- or CD19-U937 cells were used as the target population. As shown in Figure 38A, anti-CD19 CAR T cells were used together with U937 cells. When incubated, they secreted IFN-γ regardless of the target:effector ratio. However, when CAR T cells were incubated with Raji cells, high levels of IFN-γ secretion by the bell occurs in a dose-dependent manner and is antigen-specific. was shown.

[0379] These CAR T cells were then used to target luciferase-labeled Raji cells and induce cell-killing Briefly, CAR T cells were co-administered with luciferase-labeled Raji cells. At several time points, cells were washed, lysed, and Luciferase activity was measured as a measure of remaining cells. Control cells were incubated for 55 min. The luciferase activity exceeded 0.000 arbitrary units (Figure 38B). After 1 h and 5 h of co-incubation, the luciferase activity increased to 4598 , 3292, 2750, and 1932 arbitrary units. Within 5 hours of treatment, luciferase activity decreased by approximately 65%, demonstrating the potent cellular response of CAR T cells. showed cytolytic activity.

[0380] Taken together, these data support the conclusion that anti-CD19C antibodies generated according to the methods described herein AR T cells express CD19 + It has been demonstrated to be effective in killing cells.

[0381] Example 7: Linearized Plasmid DNA

[0382] 1. Expression of Chimeric Antigen Receptor from Linearized Plasmid DNA The HDR templates generated by AAV are linear DNA molecules and therefore can be derived from any source. The linear DNA is a suitable HDR template for inserting the CAR gene into the TRC1-2 recognition sequence. To test this, we located the TRC1-2 recognition sequence locus. Several plasmids containing the anti-CD19 CAR gene flanked by homology arms that are homologous to In some plasmids, different promoters were used and the homologous arms were "Short" (200 bp in the 5' homology arm and 3' homology arm) mimics the self-complementary AAV vector. 180 bp in the same arm), or a "long" (5' homologous arm) that mimics a single-stranded AAV vector. The short homologous arm was either 985 bp in the 3' homologous arm or 763 bp in the 4' homologous arm. The plasmid with the long homologous arms was designated "pDS" and the one with the long homologous arms was designated "pDI". Furthermore, some plasmids contain an intron upstream of the CAR gene. Ta.

[0383] The CAR donor plasmid was linearized at the restriction site in the vector backbone and gel purified. Human CD3 + T cells were injected with linearized CAR donor plasmid alone (purified linearized plasmid) Electroporate the cells (500ng to 1000ng depending on the concentration of plasmid) or As a control, cells were co-electroporated with TRC 1-2.87EE mRNA (2 μg). were subjected to mock electroporation or electroporation with TRC1-2x.87EE alone. Approximately 4 days after electroporation, the cells were transfected with CD3 and The cells were labeled with an antibody against anti-CD19CAR and analyzed by flow cytometry (Figure 39 Figure 39A shows the results of a 0.15% background CD3 - / CAR + Showing staining. Back Ground CD3 + / CAR + It should be noted that staining was unusually high at 4.31%. Figure 39B shows cells electroporated with TRC1-2x.87EE mRNA alone, Figures 39C and 39D show a 0.8% CD3 knockout in TRC1-2. x.87EE mRNA and a long gene containing the EF1α core promoter and HTLV enhancer A long homology arm vector or a short homology arm vector with the EF1α core promoter (EF1α) The figures show samples co-electroporated with either the IL-11 promoter (with or without enhancer). , linearized CAR donors with only the EF1α core promoter increased CD3 - / CAR + The population was generated using the EF1α core promoter and HTLV enhancer. The vector inhibited a significant percentage of CD3 - / CAR + No cells were generated. Cells electroporated with these two vectors in the absence of 1-2x.87EE mRNA CD3 - / CAR + There was no significant increase in the TRC population (Figures 39E and 39F). CD3 expression by EF1α core promoter vector in the presence of 1-2x.87EE - / CA R + Population increase: Linearized plasmid repairs double-strand break in TRC1-2 recognition sequence These results suggest that the nucleotide sequence can function as an HDR template for the nucleotide sequence.

[0384] Figure 39G and Figure 39H show two CAR-containing vectors, both containing the MND promoter driving expression of CAR. Two long homology arm constructs are shown. One of these constructs, shown in Figure 39G, contains the CAR gene. The gene also contains an intron at the 5' end. Surprisingly, the MND promoter and intron The long homology arm plasmid carrying CAR showed significant CAR expression (Figure 39G, 4.14 % CD3 - / CAR + ), whereas the intronless construct (Figure 39H) Co-electroporation with x.87EE mRNA did not result in detectable CAR expression. A short homology arm plasmid with the ND promoter but no intron also encodes TRC1. Testing with -2x.87EE mRNA did not demonstrate CAR expression (Figure 39I ) Both MND promoter-containing constructs expressed TRC1-2x.87EE mRNA in the absence of TRC1-2x.87EE mRNA. So which CAR + No cells were produced (Figures 39J, 39K, and 39L).

[0385] Finally, in this experiment, a short homologous archaeon containing the JeT promoter driving the expression of CAR was inserted. The CAR construct and the "long" homology arm construct with the CMV promoter driving the expression of CAR. Alone, neither of these linearized plasmids produced significant CAR. + Cells The expression of TRC1-2x.87EE mRNA in cells was not significant (Fig. 39O and Fig. 39P). When co-electroporated with JeT, the JeT-containing construct inhibited 2.69% of CD3 - / CAR + Shows cells , and the CMV-containing construct resulted in 2.7% CD3 - / CAR + The cells were grown.

[0386] The flow plot shown in Figure 39 shows the linearized promoter encoding the CAR flanked by homologous arms. Plasmid DNA repairs DNA breaks caused by TRC1-2x.87EE We clearly demonstrated that the CAR nucleic acid acts as an HDR template for the insertion of the CAR nucleic acid. The promoter strength plays an important role in the expression of CAR. It is clear that some promoters drive more efficient expression when the nucleotide sequence is present. be.

[0387] Insertion of CAR using linearized DNA constructs is specific to the TRC1-2 recognition sequence locus To confirm this, we used primers located within the CAR and outside the homologous arms. Cells were analyzed as described above using the ELISA (Figure 40, Table 11). Samples 1 and 2 were Either electroporation with the mRNA encoding TRC1-2x.87EE alone or electroporation with the mRNA encoding TRC1-2x.87EE alone Consistent with the above results, no PCR bands were present. Samples 3, 4, and 5 show the absence of the CAR gene at the TRC1-2 recognition site. TRC1-2x.87EE and linearized CAR homology plasmids were co-electroporated into cells. (The sample names are those in Figure 40.) Each sample contained a CAR gene expression cassette. Two PCR bands of the expected size are shown, indicating insertion of the nucleotide sequence into the TRC1-2 recognition site. Pulls 6, 7, and 8 were electroporated with the same linearized CAR homology plasmid as samples 3, 4, and 5. As expected, the cells were perforated but did not contain TRC1-2x.87EE mRNA. Samples 9 and 10 were obtained by either mock electroporation or TRC1-2. PCR from cells that were either electroporated with mRNA encoding x.87EE alone The PCR bands are not shown. Samples 11, 12, 13, and 14 were T RC1-2x.87EE and linearized CAR homology plasmid co-electroporated cells (Sample names are those in Figure 40). Each sample contained the TRC1-2 recognition Two PCR bands of the expected size are shown, indicating insertion into the recognition site. Samples 15, 16, and 17 and 18 contain the same linearized CAR homology plasmid as samples 11, 12, 13, and 14. derives from electroporated cells that do not contain TRC1-2x.87EE mRNA. and no PCR bands are present.

[0388] Figures 39 and 40 show mRNA encoding TRC1-2x.87EE and linearized CA R homology plasmid to human CD3 + Co-electroporation of T cells delivers the CAR gene to TRCs This clearly demonstrates that this is an effective method for inserting into the 1-2 recognition sequence.

[0389] [Table 11]

[0390] Example 8: Further AAV vector characterization

[0391] 1. Use of AAV with the JeT promoter and long homology arms Taken together, the above data suggest that vectors utilizing the JeT promoter are able to express high levels of CAR. This demonstrates that longer homology arms can drive consistent expression and increase gene insertion efficiency. We have demonstrated that the JeT promoter drives the expression of long homologous arms and anti-CD19 CAR. The AAV423 gene was used to generate a single-stranded AAV (herein AAV423) carrying the nucleotide sequence shown in Figure 1. The vector shown in Figure 41 (SEQ ID NO: 125) was designed and constructed. + TR to T cells mRNA encoding C1-2x.87EE was electroporated and transduced with increasing amounts of AAV423. The above data suggested that a higher MOI could increase the insertion efficiency. The inventors used 1.875e 4 From 1.5e 5 As a control, titers ranging from Cells were electroporated with mRNA encoding TRC1-2x.87EE and then pseudotransfected. Cells were either transduced or mock electroporated and then transduced with increasing amounts of AAV423. Approximately 6 days after transfection, the cells were labeled with antibodies that recognize CD3 or anti-CD19CAR and analyzed by flow cytometry. As shown in Figure 42, sham electroporation was followed by increasing amounts of AAV423-transduced cells were predominantly CD3 + / CAR - (96.6%~98. Electroporation of mRNA encoding TRC1-2x.87EE resulted in a pseudo- The transduced cells expressed CD3 - 39%, which indicates efficient knockout of T cell receptors In these cells, background CAR staining was very low (approximately 2 %). mRNA encoding TRC1-2x.87EE was electroporated, followed by increasing amounts of A AV423-transduced cells showed dramatic CAR staining in conjunction with CD3 knockout. CD3 - / CAR + The population ranged from 21.6% to 22.7%, while CD3 + / CAR + As mentioned above, the presence of single-stranded AAV significantly increased the TRC1-2 Increases overall gene modification efficiency at the recognition site and increases total CD3 - The population is 4 in control cells 5 in cells electroporated from 1.44% and then transduced with increasing amounts of AV423 7.6%, 59.2%, 58.7%, and 56.1%. + CD3 - The percentage of cells ranged from 37.5% to 39.9%, which is in line with the data above. showed a dramatic increase in insertion efficiency.

[0392] The insertion of CAR using AAV423 is specific to the TRC1-2 recognition sequence locus. To confirm this, we used primers located within the CAR and outside the homologous arms. Cells were analyzed as described above using the ELISA kit (Figure 43, Table 12).

[0393] [Table 12]

[0394] Samples 1 and 2 are PCR products from mock-electroporated cells. Consistently, no PCR band was present, indicating the absence of the CAR gene at the TRC1-2 recognition site. Samples 3-6 were mock electroporated and then transduced with increasing amounts of AAV423. Consistent with the results above, no PCR bands were present. Sample 7 , mRNA encoding TRC1-2x.87EE was electroporated and then mock transduced. Samples 8-11 are derived from cells that have been transfected with TRC1-2x.87 and do not show PCR bands. Cells were electroporated with mRNA encoding EE and then transduced with increasing amounts of AAV423. PCR band expected if CAR is inserted into the TRC1-2 recognition sequence. This shows:

[0395] Given the ability of AAV423 to insert the CAR sequence into the TRC1-2 recognition site after cleavage , a line that can encode an anti-CD19 CAR, incorporating a TRC1-2 recognition site AV423 Plus was used to transfect T cells with the engineered DNA template. The mide (Figure 41) is linearized by digestion with one restriction enzyme and then digested with one or more restriction enzymes. It is further envisioned that it may be delivered to cells upon digestion.

[0396] Example 9: In vivo efficacy of anti-CD19 TCR-negative CAR T cells

[0397] 1. Mouse Model of Disseminated B-cell Lymphoma The efficacy of gene-edited anti-CD19 CAR T cells was assessed in a mouse model of disseminated B-cell lymphoma. Activated T cells were transfected with TRC1-2x.87EE mRNA as described above. Electroporation followed by the JeT promoter-driven anti-CD19C vector flanked by homology arms The cells were transduced with an AAV6 vector containing an AR expression cassette. IL-2 (10 ng / mL) After 5 days of culture with α-glucan, cells were analyzed for cell surface CD3 and anti-CD19 CAR expression. , and analyzed by flow cytometry as previously described (Figure 44A). CD3 - Thin The cells were then purified using anti-CD3 magnetic beads. + The cells were enriched by depletion. Depleted cells were incubated with IL-15 (10 ng / mL) and IL-21 (10 ng / mL). The cells were cultured at RT for 3 days and reanalyzed for cell surface expression of CD3 and anti-CD19 CAR (Figure 4 4B). CD3 - Isolation of the CD3 population is highly efficient. + After cell depletion, flow cytometry A purity of 99.9% was obtained as determined by chromatometry (Figure 44B). - collection The group had a CD4 count of 56%. + and 44% CD8 + cells (Fig. 44C), CD62L and Central memory, transitional memory, as determined by staining for CD45RO phenotype (Fig. 44D).

[0398] Studies utilizing the Raji disseminated lymphoma model are being conducted at the Charles River Laboratory borators International Inc.(Morrisville) Firefly luciferase (ffLuc) was used. 44 Cheap Constantly expressed CD19 + Raji cells, 2.0 × 10 5 Dose of cells / mouse, Day 1 The mice were intravenously injected with PBS or the same healthy donors at 4 days after the injection. -PBS containing gene-edited control TCR KO T cells prepared from PBMCs or PBS containing the indicated doses of CAR T cells prepared from the same donor. On the indicated days, surviving mice were treated with luciferin substrate (150 mg / kg saline). Saline) was injected intraperitoneally to anesthetize the animals, and 7 minutes later, the animals were anesthetized using the IVIS SpectrumCT (Perk Luciferase activity was measured using a ELISA kit (Elmer, Waltham, MA). Living Image software 4.5.1 (Perkin Elmer, Wal Data were analyzed and exported using a fluorometric analyzer (Tham, MA). Luminescence signal intensity was calculated as p / seconds / cm 2 It is expressed in luminance in / sr.

[0399] 2.Results As shown in Figure 45, CD19 + Raji cell growth was observed in all mice by day 8. Low levels were evident in untreated and TCR by day 11. - Significant increase in the control group In the control group, significant tumor growth was observed by day 15, and continued until day 18 or 19. All control groups were euthanized at 0°C. In contrast, mice treated with anti-CD19 CAR T cells All groups showed no signs of tumor growth by day 11, except for a single mouse in the low dose group. The animals remained tumor-free up to day 29 of the study. Tumor regrowth was observed by day 36. This was observed in three mice in the low-dose cohort, one of which died at day 42. Although the animal died shortly after the procedure, imaging revealed only low levels of tumor in this animal, and the death was not considered. The death is unlikely to be tumor-related.

[0400] 3. Conclusion These results demonstrate that gene-edited CD3-CAR T cells express CD19 + tumor cells This study provides clear evidence of in vivo clearance of CAR T cells and demonstrates its potential for allogeneic CAR T cell therapy. Support further preclinical development of the platform.

Claims

1. A recombinant meganuclease that recognizes and cleaves a recognition sequence comprising SEQ ID NO:3, comprising 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, The second subunit binds to a second recognition half-site of the recognition sequence and forms a second hypervariable ( HVR2) region, The first subunit is selected from residues 198 to 344 of any one of SEQ ID NOs: 8 to 18, or has at least 80% sequence identity with residues 7-153 of any one of SEQ ID NOs: 19-27 and The second subunit comprises residues 7 to 153 of any one of SEQ ID NOs: 8 to 18, or At least 80% sequence identity with residues 198-344 of any one of sequences 19-27 1. A recombinant meganuclease comprising an amino acid sequence having the formula:

2. the HVR1 region is (a) position 215 of any one of SEQ ID NOs: 8 to 18; or (b) position 24 of any one of SEQ ID NOs: 19 to 27 2. The recombinant meganuclease of claim 1, comprising Y at the position corresponding to:

3. the HVR1 region is selected from residues 215 to 270 of any one of SEQ ID NOs: 8 to 18, or the sequence The recombinant DNA of claim 1 or 2, comprising residues 24 to 79 of any one of numbers 19 to 27. Meganuclease.

4. the HVR2 region is selected from residues 24 to 79 of any one of SEQ ID NOs: 8 to 18 or SEQ ID NO: 19-27, comprising residues 215-270 of any one of claims 1 to 3.

1. A recombinant meganuclease as described.

5. The first subunit is selected from residues 198 to 344 of any one of SEQ ID NOs: 8 to 18, or any one of claims 1 to 4, comprising residues 7 to 153 of any one of SEQ ID NOs: 19 to 27 2. A recombinant meganuclease according to claim 1.

6. The second subunit comprises residues 7 to 153 of any one of SEQ ID NOs: 8 to 18, or Any of claims 1 to 5, comprising residues 198 to 344 of any one of sequence numbers 19 to 27 2. A recombinant meganuclease according to claim 1.

7. a single-chain meganuclease comprising a linker, said linker being linked to said first subunit; and the second subunit are covalently linked to the recombinant DNA molecule of any one of claims 1 to 6. Meganuclease.

8. Any one of claims 1 to 7, comprising an amino acid sequence of any one of SEQ ID NOs: 8 to 27 A recombinant meganuclease according to claim 1.

9. A recombinant meganuclease that recognizes and cleaves a recognition sequence comprising SEQ ID NO: 4, 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, wherein the second subunit binds to a second recognition half-site of the recognition sequence. and a second hypervariable (HVR2) region, the first subunit is a nucleotide sequence identical to residues 7 to 153 of SEQ ID NO: 28 or 29 and at least 80 % sequence identity, and the second subunit comprises an amino acid sequence having SEQ ID NO:28 or an amino acid sequence having at least 80% sequence identity with residues 198-344 of 29.

1. A recombinant meganuclease comprising:

10. the HVR1 region comprises a Y at a position corresponding to position 24 of SEQ ID NO: 28 or 29. Item 10. The recombinant meganuclease according to Item 9.

11. the HVR1 region comprises a T at a position corresponding to position 26 of SEQ ID NO: 28 or 29. Item 11. The recombinant meganuclease according to Item 9 or 10.

12. the HVR1 region comprises a Y at a position corresponding to position 46 of SEQ ID NO: 28 or 29. Item 12. The recombinant meganuclease according to any one of Items 9 to 11.

13. the HVR2 region comprises H at a position corresponding to position 215 of SEQ ID NO: 28 or 29.

13. The recombinant meganuclease according to any one of claims 9 to 12.

14. the HVR2 region comprises a T at a position corresponding to position 266 of SEQ ID NO: 28 or 29.

14. The recombinant meganuclease according to any one of claims 9 to 13.

15. the HVR2 region comprises a C at a position corresponding to position 268 of SEQ ID NO: 28 or 29.

15. The recombinant meganuclease according to any one of claims 9 to 14.

16. 9 to 15, wherein the HVR1 region comprises residues 24 to 79 of SEQ ID NO: 28 or 29.

10. The recombinant meganuclease according to any one of claims 1 to 9.

17. The HVR2 region comprises residues 215 to 270 of SEQ ID NO: 28 or 29.

17. A recombinant meganuclease according to any one of claims 16.

18. 9. The method of claim 8, wherein the first subunit comprises residues 7 to 153 of SEQ ID NO: 28 or 29.

18. A recombinant meganuclease according to any one of claims 1 to 17.

19. the second subunit comprises residues 198 to 344 of SEQ ID NO: 28 or 29. Item 19. The recombinant meganuclease according to any one of Items 9 to 18.

20. a single-chain meganuclease comprising a linker, said linker being linked to said first subunit; and the second subunit are covalently bonded to the combination of any one of claims 9 to 19. Recombinant meganucleases.

21. The method according to any one of claims 9 to 20, comprising the amino acid sequence of SEQ ID NO: 28 or 29. Recombinant meganucleases.

22. A recombinant meganuclease that recognizes and cleaves a recognition sequence comprising SEQ ID NO: 5, 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, wherein the second subunit binds to a second recognition half-site of the recognition sequence. and a second hypervariable (HVR2) region, the first subunit is selected from residues 7 to 153 of SEQ ID NO: 30 or SEQ ID NO: 31 or 32 residues 198-344 of which contain an amino acid sequence having at least 80% sequence identity. the second subunit is a sequence of residues 198 to 344 of SEQ ID NO: 30 or SEQ ID NO: 31 or and an amino acid sequence having at least 80% sequence identity with residues 7-153 of 32. , recombinant meganucleases.

23. the HVR1 region is (a) position 24 of SEQ ID NO: 30, or (b) position 215 of SEQ ID NO: 31 or 32; 23. The recombinant meganuclease of claim 22, comprising a Y at the position corresponding to:

24. the HVR1 region is a sequence selected from residues 24 to 79 of SEQ ID NO: 30 or residues 31 or 32 24. The recombinant meganuclease of claim 22 or 23, comprising residues 215-270.

25. the HVR2 region is selected from residues 215 to 270 of SEQ ID NO: 30 or SEQ ID NO: 31 or 3 25. The recombinant meganucleic acid of claim 22, comprising residues 24 to 79 of SEQ ID NO:

2. Aze.

26. the first subunit is selected from residues 7 to 153 of SEQ ID NO: 30 or SEQ ID NO: 31 or 26. The recombinant megakaryon according to any one of claims 22 to 25, comprising 32 residues 198 to 344. Nuclease.

27. the second subunit is a sequence of residues 198 to 344 of SEQ ID NO: 30 or SEQ ID NO: 31 or 27. The recombinant megakaryon according to any one of claims 22 to 26, comprising residues 7 to 153 of at least 32 of the Nuclease.

28. a single-chain meganuclease comprising a linker, said linker being linked to said first subunit; and the second subunit are covalently linked to each other according to any one of claims 22 to 27. Recombinant meganucleases.

29. Any one of claims 22 to 28, comprising the amino acid sequence of any one of SEQ ID NOs: 30 to 32. A recombinant meganuclease according to any one of claims 1 to 14.

30. A nucleic acid encoding the recombinant meganuclease according to any one of claims 1 to 29. An isolated polynucleotide comprising the sequence:

31. 31. A recombinant DNA construct comprising the isolated polynucleotide of claim 30.

32. 32. The recombinant adeno-associated virus (AAV) vector of claim 31 . DNA constructs.

33. 31. A recombinant AAV vector comprising the isolated polynucleotide of claim 30.

34. Generating genetically modified eukaryotic cells containing an exogenous sequence of interest inserted into the chromosome of the eukaryotic cell 1. A method for transfecting a eukaryotic cell with a (a) a gene encoding a recombinant meganuclease according to any one of claims 1 to 29; 1, and (b) a second nucleic acid sequence comprising the sequence of interest; transfecting one or more nucleic acids comprising The recombinant meganuclease comprises a recognition sequence comprising SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO:

5. a cleavage site is generated in the chromosome at a sequence, and the sequence of interest is located at the cleavage site. The method is inserted into a chromosome.

35. the second nucleic acid further comprises a sequence homologous to a sequence adjacent to the cleavage site, 35. The method of claim 34, wherein the sequence is inserted into the cleavage site by homologous recombination.

36. 36. The method according to claim 34 or 35, wherein the eukaryotic cell is a human T cell or a cell derived therefrom. How to post.

37. 37. The method according to any one of claims 34 to 36, wherein the sequence of interest encodes a chimeric antigen receptor. The method described.

38. At least the second nucleic acid sequence is introduced into the eukaryotic cell by a recombinant AAV vector. The method according to any one of claims 34 to 37,

39. Generating genetically modified eukaryotic cells containing an exogenous sequence of interest inserted into the chromosome of the eukaryotic cell 1. A method for: (a) infecting a eukaryotic cell with the recombinant meganuclease of any one of claims 1 to 29; introducing into (b) transfecting said eukaryotic cell with a nucleic acid comprising said sequence of interest; Including, The recombinant meganuclease comprises a recognition sequence comprising SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO:

5. a cleavage site is generated in the chromosome at a sequence, and the sequence of interest is located at the cleavage site. The method by which the gene is inserted into the chromosome.

40. the nucleic acid further comprises a sequence homologous to a sequence adjacent to the cleavage site, and the sequence of interest is 40. The method of claim 39, wherein the insertion occurs at the cleavage site by homologous recombination.

41. 41. The method according to claim 39 or 40, wherein the eukaryotic cell is a human T cell or a cell derived therefrom. How to post.

42. 42. The method according to any one of claims 39 to 41, wherein the sequence of interest encodes a chimeric antigen receptor. The method described.

43. 3. The method of claim 2, wherein the nucleic acid sequence is introduced into the eukaryotic cell by a recombinant AAV vector.

43. The method according to any one of claims 9 to 42.

44. Generating genetically modified eukaryotic cells by disrupting target sequences in the chromosomes of eukaryotic cells A method comprising administering to a eukaryotic cell the recombinant meganucleotide sequence of any one of claims 1 to 29. transfecting a nucleic acid encoding a lyase, The recombinant meganuclease comprises a recognition sequence comprising SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO:

5. a cut site is generated in the chromosome at a sequence, and the target sequence is heterologous at the cut site. The method is destroyed by homo-terminal joining.

45. 45. The method of claim 44, wherein the eukaryotic cell is a human T cell or a cell derived therefrom. 。

46. and transfecting said eukaryotic cell with a second nucleic acid comprising an exogenous sequence of interest.

46. ​​The method of claim 44 or 45, further comprising:

47. the second nucleic acid further comprises a sequence homologous to a sequence adjacent to the cleavage site, 47. The method of claim 46, wherein the sequence is inserted into the cleavage site by homologous recombination.

48. 48. The method of claim 46 or 47, wherein the sequence of interest encodes a chimeric antigen receptor.

49. At least the second nucleic acid is introduced into the eukaryotic cell by a recombinant AAV vector. The method according to any one of claims 46 to 48,

50. Method for producing genetically modified eukaryotic cells by disrupting a target sequence in the chromosome of a eukaryotic cell 30. A method for producing a recombinant meganuclease according to any one of claims 1 to 29 in a eukaryotic cell. introducing the vector into a cell, The meganuclease has a recognition sequence comprising SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:

5. a cleavage site is generated in the chromosome by non-homologous end-binding of the target sequence at the cleavage site. How it is destroyed by merging.

51. 51. The method of claim 50, wherein the eukaryotic cell is a human T cell or a cell derived therefrom. 。

52. further comprising the step of transfecting said eukaryotic cell with a nucleic acid comprising an exogenous sequence of interest.

52. The method of claim 50 or 51.

53. the nucleic acid further comprises a sequence homologous to a sequence adjacent to the cleavage site, is inserted into the cleavage site by homologous recombination.

54. 54. The method of claim 52 or 53, wherein the sequence of interest encodes a chimeric antigen receptor.

55. 52 to 54, wherein the nucleic acid is introduced into the eukaryotic cell by a recombinant AAV vector.

54. The method of any one of claims 54 to 54.

56. 1. A method of immunotherapy for treating cancer in a subject in need thereof, comprising:

56. The genetically modified cell and the pharmaceutical produced by the method according to any one of 4 to 55. administering to said subject a pharmaceutical composition comprising an aseptically acceptable carrier.

57. The cancer is selected from the group consisting of carcinoma, lymphoma, sarcoma, blastoma, and leukemia.

57. The method of claim 56.

58. The cancer is selected from the group consisting of cancer of B-cell origin, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, melanoma, prostate cancer, and the like. Consists of adenocarcinoma, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, and Hodgkin's lymphoma 57. The method of claim 56, wherein the compound is selected from the group:

59. The cancer of B-cell origin is B-lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, and B-cell non-Hodgkin's lymphoma.

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