Gene modified cell including modified human t cell receptor alpha steady region gene

Abstract

To provide a gene modified cell including a modified T cell receptor (TCR) alpha steady region gene in a genome of the gene modified cell.SOLUTION: There is provided a gene modified cell including in a genome thereof, a modified human T cell receptor (TCR) alpha steady region gene, the modified human TCR alpha steady region gene includes, from 5' to 3', a 5' region of the human TCR alpha steady region gene, an exogenous polynucleotide, and a 3' region of the human TCR alpha steady region gene. The modified human TCR alpha steady region gene is a gene modified human T cell or a gene modified cell derived from a human T cell, and in comparison with a non-modified control cell, expression of an endogenous TCR on a cell surface is suppressed.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application relates to the "Modified Human T-Cell Receptor Alpha Constant Region" filed on February 19, 2016. U.S. Provisional Patent Application No. 62 / 297,426, entitled "Genetically Modified Cells Containing a Multiregion Gene," and the "Modified Human T-Cell Receptor Alpha Constant Region Gene" filed on October 5, 2015. In U.S. Provisional Patent Application No. 62 / 237,394, entitled "Genetically Modified Cells Containing a Gene," The disclosures of which are incorporated herein by reference in their entireties. be absorbed.

[0002] The present invention relates to the fields of oncology, cancer immunotherapy, molecular biology, and recombinant nucleic acid technology. The present invention provides an endogenous T cell receptor alpha constant region gene comprising a modified human T cell receptor alpha constant region gene in its genome. The present invention further relates to genetically modified cells having reduced cell surface expression of a T cell receptor. Methods for producing such genetically modified cells and for treating cancer in a subject with such cells are also provided. and methods for using the same to treat diseases including:

[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 created ASCII copy is 2000706_00180WO1.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 or It may involve the expression of an exogenous T cell receptor. In contrast to an exogenous T cell receptor, a chimeric antigen receptor may be The specificity of the antibody is derived from the variable domains of the monoclonal antibody. T cells expressing antigen receptors (CAR T cells) target tumors at the major histocompatibility complex. To date, T cell adoptive immunotherapy has not been limited to the treatment of B cell malignancies. (e.g., acute lymphoblastic leukemia (ALL), B-cell non-Hodgkin's lymphoma (NHL), and chronic lymphocytic leukemia), multiple myeloma, neuroblastoma, glioblastoma, advanced neurological It is used as a clinical treatment for a number of cancers, including glioma, ovarian cancer, mesothelioma, melanoma, and pancreatic cancer. It is becoming more and more popular.

[0005] Despite their potential utility as cancer treatments, adoptive immunotherapy with CAR T cells has To some extent, this was 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 CART cells, but this approach the time and expense required to generate patient-specific CART cells after a patient's cancer is diagnosed Constrained by both.

[0006] Therefore, it reduces the expression of endogenous T cell receptors and prevents the development of GVHD upon administration. It may be beneficial to develop "off-the-shelf" CART cells prepared using T cells from three donors. Such products are likely to be useful in the future, as they are developed 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 is engineered to recognize DNA sequences at a desired locus. This can be done using engineered, site-specific, rare-cutting endonucleases. Methods for generating engineered site-specific nucleases are known in the art. For example, zinc finger nucleases (ZFNs) target specific sites in the genome. ZFNs can be engineered to recognize and cleave the nuclease of the FokI restriction enzyme. Chimeric proteins containing a zinc finger DNA-binding domain fused to a phosphodiesterase domain The zinc finger domain binds to a specific DNA sequence approximately 18 base pairs in length. Proteins that are specific for the target gene can be redesigned by rational or experimental means to generate proteins that are specific for the target gene. By fusing this engineered protein domain to the FokI nuclease, ZFNs are widely used in a wide range of eukaryotic cells, enabling targeted DNA cleavage with genome-level specificity. It has been widely used to target gene addition, removal, and replacement in organisms ( Similarly, TAL-effector nucleases (TALs) are EN) to cut specific sites in genomic DNA. TALENs are engineered site-specific TALENs fused to the FokI nuclease domain. It contains a DNA-binding domain (reviewed in Non-Patent Document 2). The A-binding domains are TAL-effector domains that each specifically recognize a single DNA base pair. The present invention is limited by the use of ZFNs and TALENs. The key feature is that they are heterodimers, resulting in a single functional nuclear transporter within the cell. The production of this enzyme requires the co-expression of two protein monomers.

[0008] Compact TALENs are alternative endonuclease structures that avoid the need for dimerization. Compact TALENs have a structure similar to that of the I-TevI ​​homing end (Non-Patent Document 3). Engineered site-specific TAL-fused to the nuclease domain from a nuclease It contains an effector DNA-binding domain. Unlike FokI, I-TevI ​​binds double-stranded DNA. Compact TALENs act as monomers because they do not need to dimerize to perform cleavage. It works like this.

[0009] Engineered endonucleases based on the CRISPR / Cas9 system are also known in the art. CRISPR endonucleases are characterized by two Components: (1) a caspase effector nuclease, typically a microbial Cas9; and ( 2) a targeting sequence of approximately 20 nucleotides that directs the nuclease to the desired location in the genome; A short "guide RNA" containing multiple guides, each with a different targeting sequence. By expressing RNA in the same cell, DNA can be targeted to multiple sites in the genome. Therefore, it is possible to simultaneously target cleavages. The major drawback of the CRISPR / Cas9 system is the reported high The frequency of off-target DNA breaks may explain the usefulness of the system for treating human patients. This may limit sexual activity (Non-Patent Document 6).

[0010] Homing endonucleases are commonly found in plant and fungal genomes. It is a group of naturally occurring nucleases that recognize cleavage sites of ~40 base pairs. have reported that parasite DNA elements, such as group 1 self-splicing introns and introns, They are frequently associated with chromosomal repair proteins that recruit the cell's DNA repair machinery. by homologous recombination or genetic engineering at a specific location in the host genome by generating a double-stranded break that Homing endonucleases are usually L AGLIDADG (SEQ ID NO: 7) family, GIY-YIG family, His-Cy They are classified into four families: the s-box family and the HNH family. The family is characterized by structural motifs that influence catalytic activity and recognition sequences. For example, members of the LAGLIDADG (SEQ ID NO: 7) family contain the conserved LAGLI characterized by having either one or two copies of the DADG (SEQ ID NO: 7) motif. (See Non-Patent Document 8). LAGLIDADG (SEQ ID NO: 7) homing endonuclease with one copy It forms homodimers but contains two copies of the LAGLIDADG (SEQ ID NO: 7) motif. Members having the nucleotide sequence are found as monomers.

[0011] I-CreI (SEQ ID NO: 6) is a gene encoding the alga Chlamydomonas reinhardtii. The 22 base pair recognition sequence in the chloroplast chromosome of Domonas reinhardtii Recognizes and cleaves the homing endonuclease LAGLIDADG (SEQ ID NO: 7) It is a member of the family. To alter the preference of the wild-type I-CreI cleavage site, Genetic selection techniques have been used (Non-Patent Document 3, Non-Patent Document 4, Non-Patent Document 5, Non-Patent Document 6) More recently, I-CreI and other homing endonucleases have been used to A wide variety of DNA sites, including sites in the genomes of animals, yeast plants, bacteria, and viruses Mono-LAGLIDADG (SEQ ID NO: 7) holoproteinases that can be comprehensively redesigned to target A method for rationally designing binding endonucleases has been described (Patent Document 1).

[0012] 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. A linker can be used to fuse the two proteins into a single polypeptide (Non-Patent Document 13; Therefore, functional "single-chain" meganucleases can be generated from a single transcript. It can be expressed.

[0013] 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.

[0014] 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.

[0015] 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. [Prior art documents] [Patent documents]

[0016] [Patent Document 1] International Publication No. 2007 / 047859 [License 2] International Publication No. 2009 / 059195 [License 3] International Publication No. 2014 / 191527 [License 4] U.S. Patent No. 895,828 [Patent Document 5] U.S. Patent and Trademark Publication No. 2014 / 034902 [License 6] U.S. Patent and Trademark Office Publication No. 2014 / 0301990 [Non-licensed literature]

[0017] [Non-licensed Document 1] Nucleic Acids Res 33(2005)、5978 [Non-licensed Document 2] Curr Opin Struct Biol.23(2013):93~9 [Non-licensed Document 3] Nat Commun.4(2013):1762 [Non-licensed Document 4] Nat Protoc.8(2013):2281~2308 [Non-licensed Document 5] Nat Methods 10(2013):957~63 [Non-licensed Document 6] Nat Biotechnol. 31(2013):822~6 [Non-licensed Document 7] Q Rev. Biophys. 38(2006): 49~95 [Non-licensed Document 8] Nucleic Acids Res.29(18)(2001):3757~3774 [Non-licensed Document 9] J.Mol.Biol.342(2004):31~41 [Non-licensed Document 10] Nucleic Acids Res.33(2005):e178 [Non-Patent Document 11] Nucleic Acids Res.30(2002):3870~9 [Non-Patent Document 12] J. Mol. Biol. 355(2006):443~58 [Non-Patent Document 13] Nucleic Acids Res.37(2009):1650~62 [Non-Patent Document 14] Nucleic Acids Res.37(2009):5405~19 Summary of the Invention [Problem to be solved by the invention]

[0018] The present invention relates to a method for treating a subject comprising administering to a subject a method for treating a subject therapies ... therapies comprising administering to a subject therapies Genetically modified cells are provided. Such cells may be genetically modified human T cells or human T cells. Furthermore, such cells exhibit a high level of phenotypic expression compared to unmodified control cells. In this case, the cell surface expression of endogenous TCR is reduced. The present invention also relates to the creation of genetically modified cells. The present invention further provides a method for treating cancer by administering genetically modified cells. The present invention provides immunotherapy methods for: [Means for solving the problem]

[0019] Thus, in one aspect, the present invention provides a recombinant human TCR comprising a modified human TCR alpha constant region in its genome. and a genetically modified cell comprising a gene, wherein the modified human TCR alpha constant region gene is selected from the group consisting of: From ' to 3': (a) the 5' region of the human TCR alpha constant region gene; (b) exogenous polynucleotides nucleotides; and (c) the 3' region of the human TCR alpha constant region gene. The modified cells are genetically modified human T cells or genetically modified cells derived from human T cells. Furthermore, the genetically modified cells have a significantly lower expression of endogenous TCRs compared to unmodified control cells. The surface expression is reduced.

[0020] In one embodiment, the exogenous polynucleotide is a nucleic acid encoding a chimeric antigen receptor. The chimeric antigen receptor comprises an extracellular ligand-binding domain and one or more cellular Contains an intracellular signaling domain.

[0021] In one such embodiment, the chimeric antigen receptor comprises at least 8 sequences selected from SEQ ID NO: 112 and SEQ ID NO: 113. 0%, at least 85%, at least 90%, at least 95%, or up to 100% an extracellular ligand-binding domain having sequence identity with the extracellular ligand-binding domain; binds to CD19.

[0022] In another such embodiment, the chimeric antigen receptor has at least one sequence selected from the group consisting of SEQ ID NO: 113 and SEQ ID NO: 114. 80%, at least 85%, at least 90%, at least 95%, or up to 100% They contain intracellular cytoplasmic signaling domains with sequence identity.

[0023] In another such embodiment, the chimeric antigen receptor has at least one sequence selected from the group consisting of SEQ ID NO: 114 and SEQ ID NO: 115. 80%, at least 85%, at least 90%, at least 95%, or up to 100% They contain intracellular costimulatory signaling domains with sequence identity.

[0024] In another such embodiment, the chimeric antigen receptor further comprises a signal peptide. In some embodiments, the signal peptide has at least 80% identical sequence identity to SEQ ID NO:115. , at least 85%, at least 90%, at least 95%, or up to 100% sequence identity It can have oneness.

[0025] In another such embodiment, the chimeric antigen receptor further comprises a hinge domain. In some embodiments, the hinge domain is at least 80% identical to SEQ ID NO:116, at least 80% identical to SEQ ID NO:116. at least 85%, at least 90%, at least 95%, or at most 100% sequence identity It has.

[0026] In another such embodiment, the chimeric antigen receptor further comprises a transmembrane domain. In some embodiments, the transmembrane domain is at least 80% identical to SEQ ID NO:117, at least 80% identical to SEQ ID NO:117. at least 85%, at least 90%, at least 95%, or at most 100% sequence identity It has.

[0027] In another such embodiment, the chimeric antigen receptor has at least one sequence selected from the group consisting of SEQ ID NO: 111 and SEQ ID NO: 112. 80%, at least 85%, at least 90%, at least 95%, or up to 100% They share sequence identity.

[0028] In another embodiment, the exogenous polynucleotide is a polypeptide that mediates expression of the exogenous polynucleotide. In one such embodiment, the promoter sequence includes a promoter sequence that drives Column number 118 and at least 80%, at least 85%, at least 90%, at least 9 5%, or up to 100% sequence identity.

[0029] In another embodiment, the nucleic acid sequence of the exogenous polynucleotide is at least as sequenced as SEQ ID NO:119. at least 80%, at least 85%, at least 90%, at least 95%, or at most 10 It has 0% sequence identity.

[0030] In another embodiment, the exogenous polynucleotide comprises a position within the recognition sequence comprising SEQ ID NO:3. In one such embodiment, the modified human TCR The alpha constant region gene may have at least 80%, at least 85%, or at least a small fraction identical to SEQ ID NO:120. Nucleic acid sequences with at least 90%, at least 95%, or up to 100% sequence identity. include.

[0031] In another embodiment, the exogenous polynucleotide comprises a position within the recognition sequence comprising SEQ ID NO:4. In one such embodiment, the TCR alpha constant region gene is inserted in the The modified human TCR alpha constant region gene has at least 80% identical sequence to SEQ ID NO: 121, and at least at least 85%, at least 90%, at least 95%, or up to 100% sequence identity The present invention includes a nucleic acid sequence having the following structure:

[0032] In another embodiment, the exogenous polynucleotide comprises a position within the recognition sequence comprising SEQ ID NO:5. In one such embodiment, the TCR alpha constant region gene is inserted in the The modified human TCR alpha constant region gene has at least 80% identical sequence to SEQ ID NO: 122, and at least at least 85%, at least 90%, at least 95%, or up to 100% sequence identity The present invention includes a nucleic acid sequence having the following structure:

[0033] In another aspect, the present invention provides the genetically modified cells and pharmaceutically acceptable carriers described herein. The present invention provides a pharmaceutical composition comprising a carrier comprising:

[0034] In another aspect, the present invention provides a method for producing a gene as described herein for use as a pharmaceutical. The present invention further provides modified cells for treating a disease in a subject in need thereof. The present invention also provides the use of a genetically modified cell as described herein in the manufacture of a medicament for the treatment of a cancer. In one aspect, the medicament is useful for treating cancer. The treatment for cancer is immunotherapy.

[0035] In another aspect, the present invention provides a genetically modified human TCR comprising a modified human TCR alpha constant region gene. A method for producing a mutant cell, comprising: (a) introducing into the cell: (i) a gene encoding an engineered nuclease; (ii) an engineered nuclease protein; The nuclease generates a cleavage site at the recognition sequence within the human TCR alpha constant region gene. and (b) introducing into the cell a second nucleic acid sequence comprising an exogenous polynucleotide. In such methods, the cells are human T cells or human T cells. Furthermore, the exogenous polynucleotide sequence is derived from a human T cell. The R alpha constant region gene is inserted into the gene. Furthermore, the genetically modified cells show a significantly higher β-amyloid activity compared to unmodified control cells. In comparison, cell surface expression of endogenous TCR is reduced.

[0036] In various embodiments of the method, the first nucleic acid sequence or the engineered nuclease protein The protein may be introduced into the cell before or after the second nucleic acid is introduced. can be done.

[0037] In one embodiment of this method, the second nucleic acid sequence comprises, 5' to 3': (a) at the cleavage site (b) a 5' homology arm homologous to the adjacent 5' upstream sequence; (c) an exogenous polynucleotide; and c) a 3' homology arm that is homologous to the 3' downstream sequence adjacent to the cleavage site. In this form, the sequence of the exogenous polynucleotide is inserted at the cleavage site by homologous recombination. It is inserted into the human TCR alpha constant region gene.

[0038] In another embodiment of the method, the second nucleic acid lacks substantial homology to the cleavage site. The sequence of the exogenous polynucleotide is linked to the human TCR alpha constant region by non-homologous end joining. It is inserted into the region gene.

[0039] In another embodiment of this method, the exogenous polynucleotide encodes a chimeric antigen receptor. It contains a nucleic acid sequence that encodes

[0040] In another embodiment of this method, the exogenous polynucleotide is The promoter sequence includes a first promoter sequence that drives expression of the

[0041] In another embodiment of the method, the first nucleic acid encoding the engineered nuclease comprises: In some embodiments, mRNA is introduced into the cell. a coding sequence for at least one engineered nuclease as described in the document and at least one Polycistronic nucleic acids containing coding sequences for additional proteins (e.g., second nucleases) In certain embodiments, polycistronic mRNAs may be composed of the same Targeting distinct recognition sequences within genes (e.g., T cell receptor alpha constant region genes) The nucleic acid sequence may encode two or more engineered nucleases as described herein. In one embodiment, the polycistronic mRNA comprises an engineered nucleotide sequence as described herein. leukemia, and different leukemias within the same gene (e.g., the T cell receptor alpha constant region gene). Recognize and cleave the recognition sequence, or a different recognition sequence within another gene of interest in the genome. A second nuclease can be encoded that recognizes and cleaves the sequence. In the form of a genetic modification made using such a polycistronic mRNA A cell can have multiple genes knocked out simultaneously. In the present invention, the polycistronic mRNA comprises at least one engineered mRNA as described herein. The target protein encodes a nuclease and one additional protein beneficial to the cell, which targets the cleavage site. This may improve the efficiency of insertion of exogenous sequences and / or be beneficial in treating diseases.

[0042] In another embodiment of the method, the at least second nucleic acid sequence comprises the second nucleic acid sequence The virus is introduced into cells by contacting the cells with the virus vector. In some embodiments, both the first nucleic acid sequence and the second nucleic acid sequence are The nucleic acid sequences are introduced by contacting the cells with a single viral vector containing both the Alternatively, the cell is infected with a first viral vector comprising a first nucleic acid sequence and a second nucleic acid sequence. The vector can be contacted with a second viral vector containing the sequence.

[0043] In such an embodiment of the method, the second nucleic acid sequence is introduced by a viral vector. wherein the second nucleic acid is located 5' upstream of the 5' homologous arm or 3' upstream of the 3' homologous arm. It may further comprise a second promoter sequence located downstream. In embodiments where the promoter is located 3' downstream of the homologous arm, the promoter may be inverted.

[0044] In another particular embodiment of the method, the at least second nucleic acid sequence comprises the second nucleic acid sequence by contacting the cells with a recombinant adeno-associated virus (AAV) vector comprising In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are introduced into a cell. a single recombinant AAV containing both the first and second nucleic acid sequences and a cell; Alternatively, the cells may be transfected by contacting the cells with a first vector containing the first nucleic acid sequence. The recombinant AAV can be contacted with a second recombinant AAV comprising a second nucleic acid sequence.

[0045] Such embodiments of the method wherein the second nucleic acid sequence is introduced by a recombinant AAV vector wherein the second nucleic acid is located 5' upstream of the 5' homologous arm or 3' upstream of the 3' homologous arm. It may further comprise a second promoter sequence located 3' downstream. In embodiments where the promoter is located 3' downstream of the 3' homology arm, the promoter may be inverted. .

[0046] In another such embodiment of the method, the recombinant AAV vector comprises a self-complementary AA V vector.

[0047] In another such embodiment of the method, the recombinant AAV vector is of any serotype. In certain embodiments of the method, the recombinant AAV vector can have the AA In another specific embodiment of the method, the recombinant AAV vector has a serotype of V2. , with the serotype AAV6.

[0048] In another embodiment of this method, at least the second nucleic acid sequence is synthesized using a single-stranded DNA template. It is introduced into cells using

[0049] In certain embodiments of the method, a nucleic acid encoding the engineered nuclease described herein is provided. The first nucleic acid sequence is introduced into the cell by mRNA, and the second nucleic acid sequence containing the exogenous polynucleotide is The nucleic acid sequence of 2 is introduced into cells 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 such an embodiment, the method comprises a chimeric antigen receptor, and To generate genetically modified T cells with reduced cell surface expression of endogenous T cell receptors.

[0050] In another embodiment of this method, the engineered nuclease is a recombinant meganuclease. , recombinant zinc finger nucleases (ZFNs), recombinant transcription activator-like effectors nuclease (TALEN), CRISPR / Cas nuclease, or megaTAL nuclease In certain embodiments of the method, the engineered nuclease is a recombinant nuclease. It is a meganuclease.

[0051] In such an embodiment of the method, the recombinant meganuclease is a human T cell receptor It recognizes and cleaves a recognition sequence within residues 93-208 of the alpha constant region (SEQ ID NO: 1). Such a recombinant meganuclease comprises a first subunit and a second subunit. The first subunit binds to the first recognition half-site of the recognition sequence and binds to the first hypervariable (HV) R1) region, and the second subunit binds to a second recognition half-site of the recognition sequence and It contains the hypervariable (HVR2) region of

[0052] In one such embodiment of the method, the recognition sequence is SEQ ID NO:3 (i.e., TRC1-2 recognition sequence).

[0053] In another such embodiment of the method, the first meganuclease subunit comprises: Residues 198 to 344 of any one of SEQ ID NOs: 8 to 18 or any one of SEQ ID NOs: 19 to 27 one of residues 7-153 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 selected from residues 7 to 153 of any one of SEQ ID NOs: 8 to 18 or SEQ ID NO: 19 Residues 198-344 of any one of 27 sequences are at least 80%, at least 85%, or at least It comprises an amino acid sequence having at least 90%, or at least 95% sequence identity.

[0054] In another such embodiment of the method, the HVR1 region is selected from the group consisting of: (a) SEQ ID NOs: 8-18 or (b) position 215 of any one of SEQ ID NOs: 19 to 27; or (b) position 24 of any one of SEQ ID NOs: 19 to 27 In another such embodiment, the HVR1 region comprises a Y at the corresponding position. (b) position 233 of any one of SEQ ID NOs: 8 to 18; or (b) any one of SEQ ID NOs: 19 to 27 In another such embodiment, the HVR1 region comprises a G at a position corresponding to position 42 of (a) positions 215 and 233, respectively, of any one of SEQ ID NOs: 8 to 18; or (b) 19 to 27, at positions corresponding to positions 24 and 42, respectively, and G.

[0055] In another such embodiment of the method, the HVR2 region is selected from the group consisting of: (a) 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 T at the corresponding position. (b) position 28 of any one of SEQ ID NOs: 8 to 18; or (b) position 28 of any one of SEQ ID NOs: 19 to 27 In another such embodiment, the HV The R2 region is (a) position 38 of any one of SEQ ID NOs: 8 to 18; or (b) SEQ ID NO: 19 In another such embodiment, the amino acid sequence of any one of 1 to 27 contains an F at a position corresponding to position 229. wherein the HVR2 region is (a) position 44 of any one of SEQ ID NOs: 8 to 18; or (b) It contains S at the position corresponding to position 235 of any one of SEQ ID NOs: 19 to 27. In one embodiment, the HVR2 region comprises: (a) position 46 of any one of SEQ ID NOs: 8 to 18; or (b) F or at the position corresponding to position 237 of any one of SEQ ID NOs: 19 to 27. Y. In another such embodiment, the HVR2 region comprises (a) SEQ ID NOs: 8-18 (b) any one of SEQ ID NO: 26, 28, 38, 44, and 46, respectively; or (b) SEQ ID NO: 19 ~27, corresponding to positions 217, 219, 229, 235, and 237, respectively At positions corresponding to the amino acid sequence, the amino acid sequence may include one or more of T, F or Y, F, S and F, or Y and R.

[0056] In another such embodiment of the method, the HVR1 region is any of SEQ ID NOs: 8-18. Residues 215 to 270 of any one of SEQ ID NOs: 19 to 27 or residues 24 to 7 of any one of SEQ ID NOs: 19 to 27 In another such embodiment, the HVR2 region comprises any of SEQ ID NOs: 8-18. Residues 24 to 79 of any one of SEQ ID NOs: 19 to 27 or residues 215 to 270 of any one of SEQ ID NOs: 19 to 27 Includes.

[0057] In another such embodiment of the method, the first meganuclease subunit comprises: 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 meganuclear The enzyme subunit is selected from residues 7 to 153 of any one of SEQ ID NOs: 8 to 18 or SEQ ID NO: 1 9-27 and containing residues 198-344.

[0058] In another such embodiment of the method, the recombinant meganuclease comprises a linker. A single-chain meganuclease comprising a linker and a first subunit and a second subunit. and covalently bonded.

[0059] In another such embodiment of the method, the recombinant meganuclease is selected from the group consisting of SEQ ID NOs: 8 to 10. It contains any one of 27 amino acid sequences.

[0060] In a further embodiment of the method, the recognition sequence is SEQ ID NO:4 (i.e., the TRC3-4 recognition sequence). column).

[0061] In one such embodiment of the method, the first meganuclease subunit comprises a sequence Residues 7 to 153 of sequence numbers 28 or 29 are at least 80%, at least 85%, or at least and a second megaprotein comprising an amino acid sequence having at least 90% or at least 95% sequence identity. The nuclease subunit comprises at least one nucleotide sequence identical to residues 198-344 of SEQ ID NO: 28 or 29. 80%, at least 85%, at least 90%, or at least 95% sequence identity It contains an amino acid sequence that

[0062] In another such embodiment of the method, the HVR1 region is selected from the group consisting of SEQ ID NO: 28 or 29 In another such embodiment, the HVR1 region comprises a Y at a position corresponding to position 24. In another such embodiment, the amino acid sequence of SEQ ID NO: 30 or 31 contains a T at a position corresponding to position 26 of SEQ ID NO: 30 or 31. In this case, the HVR1 region contains a Y at a position corresponding to position 46 of SEQ ID NO: 28 or 29. In such embodiments, the HVR1 region comprises a region at position 2 of SEQ ID NO: 28 or 29, respectively. and one or more of Y, T, and Y at positions corresponding to 4, 26, and 46.

[0063] In another such embodiment of the method, the HVR2 region is selected from the group consisting of SEQ ID NO: 28 or 29 In another such embodiment, the HVR2 region comprises an H at a position corresponding to position 215. , comprising a T at a position corresponding to position 266 of SEQ ID NO: 28 or 29. wherein the HVR2 region contains a C at a position corresponding to position 268 of SEQ ID NO: 28 or 29. In another such embodiment, the HVR2 region comprises 215 of SEQ ID NO: 28 or 29, It contains one or more of H, T, and C at positions corresponding to 266 and 268.

[0064] In another such embodiment of the method, the HVR1 region is selected from the group consisting of SEQ ID NO: 28 or 29 In another such embodiment, the HVR2 region comprises residues 24 to 79. 8 or 29 containing residues 215-270.

[0065] In another such embodiment of the method, the first meganuclease subunit comprises: In another such embodiment, the second The meganuclease subunit comprises residues 198 to 344 of SEQ ID NO: 28 or 29. .

[0066] In another such embodiment of the method, the recombinant meganuclease comprises a linker. A single-chain meganuclease comprising a linker and a first subunit and a second subunit. and covalently bonded.

[0067] In another such embodiment of the method, the recombinant meganuclease is selected from the group consisting of SEQ ID NO: 28 or a 29 amino acid sequence.

[0068] In a further embodiment of the method, the recognition sequence is SEQ ID NO:5 (i.e., the TRC7-8 recognition sequence). column).

[0069] In one such embodiment of the method, the first meganuclease subunit comprises a sequence Residues 7 to 153 of SEQ ID NO: 30 or residues 198 to 344 of SEQ ID NO: 31 or 32 at least 80%, at least 85%, at least 90%, or at least 95% sequence identity and the second meganuclease subunit comprises an amino acid sequence having the properties of SEQ ID NO: 30. or residues 7 to 153 of SEQ ID NO: 31 or 32 and at least 80 %, at least 85%, at least 90%, or at least 95% sequence identity Contains the amino acid sequence.

[0070] In another such embodiment of the method, the HVR1 region comprises (a) the 2'-nucleotide sequence of SEQ ID NO: 30 or (b) containing a Y at a position corresponding to position 215 of SEQ ID NO: 31 or 32.

[0071] In another such embodiment of the method, the HVR2 region comprises (a) the 2'-nucleotide sequence of SEQ ID NO: 30 or (b) Y or W at the position corresponding to position 24 of SEQ ID NO: 31 or 32. In another such embodiment, the HVR2 region comprises: (a) 231 of SEQ ID NO: 30 or (b) 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) amino acid sequence 23 of SEQ ID NO: 30; or (b) a Y at position corresponding to position 46 of SEQ ID NO: 31 or 32. In such embodiments, the HVR2 region comprises (a) positions 215 and 216 of SEQ ID NO: 30, respectively; (b) positions 24 and 40 of SEQ ID NO: 31 or 32, respectively; and at the position corresponding to position 46, one or more of Y or W, M, L or W and Y include.

[0072] In another such embodiment of the method, the HVR1 region is selected from residues 24 and 25 of SEQ ID NO: 30. to 79 or residues 215 to 270 of SEQ ID NO: 31 or 32. In one embodiment, the HVR2 region is a region consisting of residues 215 to 270 of SEQ ID NO: 30 or residues 215 to 270 of SEQ ID NO: 31 or or 32 residues 24 to 79.

[0073] In another such embodiment of the method, the first meganuclease subunit comprises: containing residues 7 to 153 of SEQ ID NO: 30 or residues 198 to 344 of SEQ ID NO: 31 or 32 In another such embodiment, the second meganuclease subunit has SEQ ID NO: 30 or residues 7 to 153 of SEQ ID NO: 31 or 32.

[0074] In another such embodiment of the method, the recombinant meganuclease comprises a linker. A single-chain meganuclease comprising a linker and a first subunit and a second subunit. and covalently bonded.

[0075] In another such embodiment of the method, the recombinant meganuclease is selected from the group consisting of SEQ ID NO: 30 It contains any one of the following 32 amino acid sequences:

[0076] In another aspect, the present invention provides an immunosuppressant for treating cancer in a subject in need thereof. In some embodiments, the method comprises administering to a subject a gene or gene product as described herein. and administering to a subject a pharmaceutical composition comprising the modified cells and a pharmaceutically acceptable carrier. In some embodiments, the method provides a method for producing a gene according to the methods described herein. The method includes administering to a subject a pharmaceutical composition comprising genetically modified cells and a pharmaceutically acceptable carrier. nothing.

[0077] In another embodiment of this method, the cancer to be treated is a cancer of B-cell origin, breast cancer, gastric cancer, neuronal cancer, or the like. Blastocystic sarcoma, osteosarcoma, lung cancer, melanoma, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, and Hodgkin's lymphoma.

[0078] In another embodiment of this method, the cancer of B-cell origin is B-lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, and B-cell non-Hodgkin's lymphoma .

[0079] 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 in accordance with the present invention. Included.

[0080] 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]

[0081] [Figure 1]Figure 1A shows the TRC recognition sequences in the human TRC alpha constant region gene. Each recognition sequence targeted by the 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]2A-2B show amino acid alignments of TRC1-binding subunits. 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. Pat. 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). 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 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. Pat. No. 8,021,867) and the R residue at position 330 (gray shaded and underlined) in meganucleases TRC1-2x.87 EE, 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). The 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]6A-6B show amino acid alignments of TRC7 binding subunits. 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 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 TRC7 binding subunits provided in Figure 6 share at least 90% sequence identity with the TRC7 binding subunit (residues 7-153) of the TRC 7-8x.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 TRC 7-8x.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. Figure 9D shows 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] 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] Figure 13A shows cleavage of the TRC1-2 recognition sequence in human T cells. 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, and the percentages of CD3-positive and CD3-negative T cells were determined. [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 product 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 is a map of the plasmid 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] Figure 1 shows 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 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 AAV408HDR 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]Diagram of digital PCR. 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 the insertion of the 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 the analysis of the insertion after transduction with AAV421. Figure 32B shows the 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. 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 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 two incubations with mitomycin C-inactivated IM-9 cells. [Figure 35] Figure 35 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 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 the addition of IL-7 (10 ng / mL) and IL-15 (10 ng / mL). Figure 36B shows the addition of IL-7 (10 ng / mL) and IL-15 (10 ng / mL) and incubation with mitomycin C-inactivated IM-9 cells. Figure 36C shows the addition of 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 expression of chimeric antigen receptors after transduction of linearized DNA donor templates. 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. Figure 39B shows 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] FIG. 10 is a diagram of a 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 42 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. Figure 42A shows mock-electroporated, mock-transduced cells (MOI-0), and mock-electroporated, transduced cells with increasing amounts of AAV423. Figure 42B shows TRC1-2x.87EE electroporated, mock-transduced cells (MOI-0), and TRC1-2x.87EE electroporated, transduced cells with increasing amounts of AAV423. [Figure 43]Figure 1 shows insertion of the chimeric antigen receptor coding sequence. 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-CD19CAR 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-CD19CAR 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-CD19CAR 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 reanalyzed for cell surface expression of CD3 and anti-CD19CAR. C) The purified population of CD3-CD19CAR 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-CD19CAR 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 injected intravenously 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.

[0082] 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 ...

[0083] SEQ ID NO: 2 is the amino acid sequence encoded by the human T cell receptor alpha constant region Shows.

[0084] SEQ ID NO: 3 shows the amino acid sequence of the TRC1-2 recognition sequence.

[0085] SEQ ID NO: 4 shows the nucleotide sequence of the TRC3-4 recognition sequence.

[0086] SEQ ID NO: 5 shows the nucleotide sequence of the TRC7-8 recognition sequence.

[0087] SEQ ID NO: 6 shows the amino acid sequence of I-CreI.

[0088] SEQ ID NO: 7 shows the amino acid sequence of the LAGLIDADG motif.

[0089] SEQ ID NO: 8 shows the amino acid sequence of the TRC1-2x.87EE meganuclease.

[0090] SEQ ID NO: 9 shows the amino acid sequence of the TRC1-2x.87QE meganuclease.

[0091] SEQ ID NO: 10 shows the amino acid sequence of the TRC1-2x.87EQ meganuclease.

[0092] SEQ ID NO: 11 shows the amino acid sequence of the TRC1-2x.87 meganuclease.

[0093] SEQ ID NO: 12 shows the amino acid sequence of the TRC1-2x.6 meganuclease.

[0094] SEQ ID NO: 13 shows the amino acid sequence of the TRC1-2x.20 meganuclease.

[0095] SEQ ID NO: 14 shows the amino acid sequence of the TRC1-2x.55 meganuclease.

[0096] SEQ ID NO: 15 shows the amino acid sequence of the TRC1-2x.60 meganuclease.

[0097] SEQ ID NO: 16 shows the amino acid sequence of the TRC1-2x.105 meganuclease.

[0098] SEQ ID NO: 17 shows the amino acid sequence of the TRC1-2x.163 meganuclease.

[0099] SEQ ID NO: 18 shows the amino acid sequence of TRC1-2x.113_3 meganuclease .

[0100] SEQ ID NO: 19 shows the amino acid sequence of the TRC1-2x.5 meganuclease.

[0101] SEQ ID NO: 20 shows the amino acid sequence of the TRC1-2x.8 meganuclease.

[0102] SEQ ID NO: 21 shows the amino acid sequence of the TRC1-2x.25 meganuclease.

[0103] SEQ ID NO: 22 shows the amino acid sequence of the TRC1-2x.72 meganuclease.

[0104] SEQ ID NO: 23 shows the amino acid sequence of the TRC1-2x.80 meganuclease.

[0105] SEQ ID NO: 24 shows the amino acid sequence of the TRC1-2x.84 meganuclease.

[0106] SEQ ID NO: 25 shows the amino acid sequence of the TRC1-2x.120 meganuclease.

[0107] SEQ ID NO: 26 shows the amino acid sequence of TRC1-2x.113_1 meganuclease .

[0108] SEQ ID NO: 27 shows the amino acid sequence of TRC1-2x.113_2 meganuclease .

[0109] SEQ ID NO: 28 shows the amino acid sequence of the TRC3-4x.3 meganuclease.

[0110] SEQ ID NO: 29 shows the amino acid sequence of the TRC3-4x.19 meganuclease.

[0111] SEQ ID NO: 30 shows the amino acid sequence of the TRC7-8x.7 meganuclease.

[0112] SEQ ID NO: 31 shows the amino acid sequence of the TRC7-8x.9 meganuclease.

[0113] SEQ ID NO: 32 shows the amino acid sequence of the TRC7-8x.14 meganuclease.

[0114] SEQ ID NO: 33 identifies residues 198 to 344 of the TRC1-2x.87EE meganuclease. show.

[0115] SEQ ID NO: 34 identifies residues 198 to 344 of the TRC1-2x.87QE meganuclease. show.

[0116] SEQ ID NO: 35 identifies residues 198 to 344 of the TRC1-2x.87EQ meganuclease. show.

[0117] SEQ ID NO: 36 shows residues 198 to 344 of the TRC1-2x.87 meganuclease .

[0118] SEQ ID NO: 37 shows residues 198 to 344 of the TRC1-2x.6 meganuclease.

[0119] SEQ ID NO: 38 shows residues 198 to 344 of the TRC1-2x.20 meganuclease .

[0120] SEQ ID NO: 39 shows residues 198 to 344 of the TRC1-2x.55 meganuclease .

[0121] SEQ ID NO: 40 shows residues 198 to 344 of the TRC1-2x.60 meganuclease .

[0122] SEQ ID NO: 41 represents residues 198 to 344 of the TRC1-2x.105 meganuclease. vinegar.

[0123] SEQ ID NO: 42 represents residues 198 to 344 of the TRC1-2x.163 meganuclease. vinegar.

[0124] SEQ ID NO: 43 identifies residues 198 to 344 of the TRC1-2x.113_3 meganuclease. Shows.

[0125] SEQ ID NO: 44 shows residues 7 to 153 of the TRC1-2x.5 meganuclease.

[0126] SEQ ID NO: 45 shows residues 7 to 153 of the TRC1-2x.8 meganuclease.

[0127] SEQ ID NO: 46 shows residues 7 to 153 of the TRC1-2x.25 meganuclease.

[0128] SEQ ID NO: 47 shows residues 7 to 153 of the TRC1-2x.72 meganuclease.

[0129] SEQ ID NO: 48 shows residues 7 to 153 of the TRC1-2x.80 meganuclease.

[0130] SEQ ID NO: 49 shows residues 7 to 153 of the TRC1-2x.84 meganuclease.

[0131] SEQ ID NO: 50 shows residues 7 to 153 of the TRC1-2x.120 meganuclease.

[0132] SEQ ID NO: 51 represents residues 7 to 153 of the TRC1-2x.113_1 meganuclease. vinegar.

[0133] SEQ ID NO: 52 represents residues 7 to 153 of the TRC1-2x.113_2 meganuclease. vinegar.

[0134] SEQ ID NO: 53 shows residues 7 to 153 of the TRC3-4x.3 meganuclease.

[0135] SEQ ID NO: 54 shows residues 7 to 153 of the TRC3-4x.19 meganuclease.

[0136] SEQ ID NO: 55 shows residues 7 to 153 of the TRC7-8x.7 meganuclease.

[0137] SEQ ID NO: 56 shows residues 198 to 344 of the TRC7-8x.9 meganuclease.

[0138] SEQ ID NO: 57 shows residues 198 to 344 of the TRC7-8x.14 meganuclease.

[0139] SEQ ID NO: 58 shows residues 7 to 153 of the TRC1-2x.87EE meganuclease .

[0140] SEQ ID NO: 59 shows residues 7 to 153 of the TRC1-2x.87QE meganuclease .

[0141] SEQ ID NO: 60 shows residues 7 to 153 of the TRC1-2x.87EQ meganuclease .

[0142] SEQ ID NO: 61 shows residues 7 to 153 of the TRC1-2x.87 meganuclease.

[0143] SEQ ID NO: 62 shows residues 7 to 153 of the TRC1-2x.6 meganuclease.

[0144] SEQ ID NO: 63 shows residues 7 to 153 of the TRC1-2x.20 meganuclease.

[0145] SEQ ID NO: 64 shows residues 7 to 153 of the TRC1-2x.55 meganuclease.

[0146] SEQ ID NO: 65 shows residues 7 to 153 of the TRC1-2x.60 meganuclease.

[0147] SEQ ID NO: 66 shows residues 7 to 153 of the TRC1-2x.105 meganuclease.

[0148] SEQ ID NO: 67 shows residues 7 to 153 of the TRC1-2x.163 meganuclease.

[0149] SEQ ID NO: 68 represents residues 7 to 153 of the TRC1-2x.113_3 meganuclease. vinegar.

[0150] SEQ ID NO: 69 shows residues 198 to 344 of the TRC1-2x.5 meganuclease.

[0151] SEQ ID NO: 70 shows residues 198 to 344 of the TRC1-2x.8 meganuclease.

[0152] SEQ ID NO: 71 shows residues 198 to 344 of the TRC1-2x.25 meganuclease.

[0153] SEQ ID NO: 72 shows residues 198 to 344 of the TRC1-2x.72 meganuclease .

[0154] SEQ ID NO: 73 shows residues 198 to 344 of the TRC1-2x.80 meganuclease .

[0155] SEQ ID NO: 74 represents residues 198 to 344 of the TRC1-2x.84 meganuclease .

[0156] SEQ ID NO: 75 represents residues 198 to 344 of the TRC1-2x.120 meganuclease. vinegar.

[0157] SEQ ID NO: 76 identifies residues 198 to 344 of the TRC1-2x.113_1 meganuclease Shows.

[0158] SEQ ID NO: 77 identifies residues 198 to 344 of the TRC1-2x.113_2 meganuclease Shows.

[0159] SEQ ID NO: 78 shows residues 198 to 344 of the TRC3-4x.3 meganuclease.

[0160] SEQ ID NO: 79 shows residues 198 to 344 of the TRC3-4x.19 meganuclease .

[0161] SEQ ID NO: 80 shows residues 198 to 344 of the TRC7-8x.7 meganuclease.

[0162] SEQ ID NO: 81 shows residues 7 to 153 of the TRC7-8x.9 meganuclease.

[0163] SEQ ID NO: 82 shows residues 7 to 153 of the TRC7-8x.14 meganuclease.

[0164] SEQ ID NO: 83 shows the nucleotide sequence of the antisense strand of the TRC1-2 recognition sequence.

[0165] SEQ ID NO: 84 shows the nucleotide sequence of the antisense strand of the TRC3-4 recognition sequence.

[0166] SEQ ID NO: 85 shows the nucleotide sequence of the antisense strand of the TRC7-8 recognition sequence.

[0167] SEQ ID NO: 86 represents nucleotides 162 to 233 of SEQ ID NO:1.

[0168] SEQ ID NO: 87 is nucleotide 1 of SEQ ID NO: 1, including the deletion due to truncation and NHEJ. Indicates 62 to 233.

[0169] SEQ ID NO: 88 is nucleotide 1 of SEQ ID NO: 1, including the deletion due to truncation and NHEJ. Indicates 62 to 233.

[0170] SEQ ID NO: 89 is nucleotide 1 of SEQ ID NO: 1, including the deletion due to truncation and NHEJ. Indicates 62 to 233.

[0171] SEQ ID NO: 90 is nucleotide 1 of SEQ ID NO: 1, including the deletion due to truncation and NHEJ. Indicates 62 to 233.

[0172] SEQ ID NO: 91 is nucleotide 1 of SEQ ID NO: 1, including the deletion due to truncation and NHEJ. Indicates 62 to 233.

[0173] SEQ ID NO: 92 is nucleotide 1 of SEQ ID NO: 1, including the deletion due to truncation and NHEJ. Indicates 62 to 233.

[0174] SEQ ID NO: 93 is nucleotide 1 of SEQ ID NO: 1, including the deletion due to truncation and NHEJ. Indicates 62 to 233.

[0175] 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.

[0176] 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.

[0177] SEQ ID NO: 96 is nucleotide 1 of SEQ ID NO: 1, including the deletion due to truncation and NHEJ. Indicates 62 to 233.

[0178] SEQ ID NO: 97 is nucleotide 1 of SEQ ID NO: 1, including the deletion due to truncation and NHEJ. Indicates 62 to 233.

[0179] SEQ ID NO: 98 is nucleotide 1 of SEQ ID NO: 1, including the deletion due to truncation and NHEJ. Indicates 62 to 233.

[0180] SEQ ID NO: 99 is nucleotide 1 of SEQ ID NO: 1, including the deletion due to truncation and NHEJ. Indicates 62 to 233.

[0181] 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.

[0182] 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.

[0183] 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.

[0184] 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.

[0185] 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.

[0186] SEQ ID NO: 105 represents nucleotides 181 to 214 of SEQ ID NO: 1.

[0187] 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.

[0188] SEQ ID NO: 107 is the nucleic acid sequence of the plasmid used to generate the AAV405 vector. The nucleotide sequence is shown.

[0189] SEQ ID NO: 108 is the nucleic acid sequence of the plasmid used to generate the AAV406 vector. The nucleotide sequence is shown.

[0190] SEQ ID NO: 109 is used to generate the AAV-CAR100 (AAV408) vector. The nucleotide sequences of the plasmids used are shown.

[0191] SEQ ID NO: 110 is used to generate the AAV-CAR763 (AAV412) vector. The nucleotide sequences of the plasmids used are shown.

[0192] SEQ ID NO: 111 shows the amino acid sequence of an anti-CD19 chimeric antigen receptor.

[0193] SEQ ID NO: 112 shows the amino acid sequence of the anti-CD19 extracellular ligand-binding domain.

[0194] SEQ ID NO: 113 is the amino acid sequence of the chimeric antigen receptor intracellular cytoplasmic signaling domain Indicates a column.

[0195] SEQ ID NO: 114 shows the amino acid sequence of the chimeric antigen receptor intracellular costimulatory domain.

[0196] SEQ ID NO: 115 shows the amino acid sequence of the chimeric antigen receptor signal peptide domain .

[0197] SEQ ID NO: 116 shows the amino acid sequence of the chimeric antigen receptor hinge region.

[0198] SEQ ID NO: 117 shows the amino acid sequence of the chimeric antigen receptor transmembrane domain.

[0199] SEQ ID NO: 118 shows the nucleotide sequence of the EF-1 alpha core promoter.

[0200] SEQ ID NO: 119 shows the nucleotide sequence of the exogenous polynucleotide insert.

[0201] 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.

[0202] 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.

[0203] 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.

[0204] SEQ ID NO: 123 is the nucleic acid sequence of the plasmid used to generate the AAV421 vector. The acid sequence is shown.

[0205] SEQ ID NO: 124 is the nucleic acid sequence of the plasmid used to generate the AAV422 vector. The acid sequence is shown.

[0206] 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

[0207] 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.

[0208] 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. These do not depart from the invention.

[0209] 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.

[0210] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. It is incorporated herein by reference.

[0211] 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.

[0212] 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.

[0213] 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 meganuclear activity were observed as measured using the methods described herein. The cells were transfected and incubated at 37°C without observing a significant decrease in the enzyme cleavage activity. can be maintained in

[0214] As used herein, the term "single-chain meganuclease" refers to a single-chain meganuclease linked by a linker. refers to a polypeptide containing a pair of nuclease subunits. The enzyme has the structure of N-terminal subunit-linker-C-terminal subunit. The meganuclease subunits are generally not identical in amino acid sequence. Therefore, single-chain meganucleases are typically pseudopalindole-binding enzymes. Single-chain meganucleases are actually dimeric. Although it is not a single-chain heterodimer, it is called a "single-chain heterodimer meganuclease." For clarity, unless otherwise specified, the term "meganuclease" is used. The term can refer to dimeric or single-chain meganucleases.

[0215] 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.

[0216] As used herein, the term "TALEN" refers to a FokI nuclease domain DNA-binding domains containing 16 to 22 TAL domain repeats fused to any moiety It refers to an endonuclease containing

[0217] As used herein, the term "compact TALEN" refers to an I-TevI ​​host Any catalytically active part of the nuclease domain of a taming endonuclease can be oriented in any direction. Contains a DNA-binding domain with 16 to 22 TAL domain repeats fused together Refers to an endonuclease.

[0218] As used herein, the term "CRISPR" refers to a combination of a caspase, such as Cas9, and a gene encoding a gene encoding a gene encoding a gene. By hybridizing to recognition sites in the genome, caspase-mediated DNA cleavage is inhibited. The term refers to a caspase-based endonuclease that contains a guide RNA that guides the endonuclease.

[0219] As used herein, the term "megaTAL" refers to an engineered sequence-specific homing Activator-like effector with endonuclease (TALE) DNA-binding domain refers to a single-chain nuclease comprising:

[0220] 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.

[0221] 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 that is a wild-type gene. The polypeptide encoded by the wild-type allele has its original function. " also refers to a polypeptide encoded by a wild-type allele. That is, polynucleotides and polypeptides contain one or more mutations relative to the wild-type sequence. Mutant or variant alleles and polypeptides containing mutations and / or substitutions are distinguished from A wild-type allele or polypeptide confers a normal phenotype in an organism. However, mutant or variant alleles or polypeptides may be Wild-type nucleases can be recombinant or non-naturally occurring nucleases. It is distinguished from aze.

[0222] 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.

[0223] As used herein, the term "recognition sequence" refers to a sequence that is bound and recognized by an endonuclease. In the case of meganucleases, the recognition sequence is a sequence of four base pairs. Thus, it contains a pair of inverted 9 base pair "half-sites" separated by a single chain of meganucleases. The N-terminal domain of the protein contacts the first half-site, and the C-terminal domain of the protein contacts the second half-site. The meganuclease contacts the 2 half-sites. Cleavage by the meganuclease occurs at the 4 base pair 3' overhang. "Overhangs" or "sticky ends" are the endonucleases of double-stranded DNA sequences. It is a short single-stranded DNA segment that can be generated by protease cleavage with I-CreI. In the case of the derived meganuclease and single-chain meganuclease, the overhang is It contains bases 10 to 13 of the base pair recognition sequence. In the case of compact TALENs, the recognition sequence is I- The first CNNNGN sequence recognized by the TevI domain, followed by a sequence of 4 to 16 bases in length. A non-specific spacer, which is a base pair, followed by a TAL-effector domain that is recognized by the TAL A second sequence of 16-22 bp in length (this sequence typically has a 5' T base) Cleavage by compact TALENs generates a 2-base pair 3' overhang. In the case of SPR, the recognition sequence is typically a 16-24 base pair sequence, to which a guide R NA binds and induces Cas9 cleavage. CRISPR cleavage generates blunt ends. .

[0224] As used herein, the term "target site" or "target sequence" refers to a recognition site for a nuclease. A region of a cell's chromosomal DNA that contains a specific sequence.

[0225] As used herein, the term "DNA binding affinity" or "binding affinity" refers to The tendency of the cleavage enzyme to bind non-covalently to the reference DNA molecule (e.g., the recognition sequence or any sequence). The binding affinity is expressed as the dissociation constant K d As used herein, If K of the nuclease against the reference recognition sequence, d However, statistically significant differences were observed compared to the reference nuclease. A nuclease is said to have "altered" its binding affinity if the binding affinity is increased or decreased by a significant percent change. It is "transforming" it.

[0226] 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.

[0227] 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, engineered A modified nuclease can be used.

[0228] 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 this case, the ligand-binding domain is specific for CD19.

[0229] 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.

[0230] 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 an extracellular ligand-binding domain and a spacer sequence. The polypeptide may further comprise additional structural elements, including a transmembrane domain linked to the polypeptide.

[0231] 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 can be The exogenous TCR useful in the present invention may comprise any antigen-specific TCR. It may have specificity for the original or desired epitope.

[0232] 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.

[0233] 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. 25:3389~340 2);Zhang et al. (2000), J.Comput.Biol.7(1-2):20 3-14. As used herein, the similarity between two amino acid sequences is The percentage is a score based on the following parameters for the BLASTp algorithm: There are: word size = 3; gap opening penalty = -11; gap extension penalty = -1; and score matrix = BLOSUM62. As used herein, two nucleic acid sequences The percent similarity of a column is based on the following parameters for the BLASTn algorithm: The score is: word size = 11; gap opening penalty = -5; gap extension penalty = Nulty = -2; Match Reward = 1; Mismatch Penalty = -3.

[0234] 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.

[0235] As used herein, the terms "recognition half-site," "recognition sequence half-site," or simply "half-site" are used interchangeably. The "site" may be formed by a monomer of a homodimeric or heterodimeric meganuclease or by a single A nucleus in a double-stranded DNA molecule recognized by one subunit of a single strand meganuclease It means the acid sequence.

[0236] 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 .

[0237] 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).

[0238] 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.

[0239] 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 for successfully transforming, selecting, and growing host cells containing any of the nucleic acid sequences , are fully aware of the genetic elements that must be present on the vector.

[0240] 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.

[0241] 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.

[0242] 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.

[0243] 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.

[0244] 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 0, 1, or 2 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 take the value 0.001, or any other real value >= 0 and <= 2.

[0245] 2.1 Principles of the present invention The present invention relates to the mechanism by which NHEJ at the cleavage site regulates the expression and ultimate expression of TCR alpha chain subunits. The engineered nuclease is then added to the target cell to ultimately disrupt the expression of the T cell receptor on the cell surface. Recognition and cleavage of the recognition sequence found in the human TCR alpha constant region gene (SEQ ID NO: 1) Further, in accordance with the present invention, exogenous polynucleotides can be used to The nucleotide sequence is then co-expressed in the cell with the sequence of interest, for example by homologous recombination. The fragment is inserted into the TCR alpha constant region gene at the nuclease cleavage site. Such exogenous sequences may be, for example, chimeric antigen receptors, exogenous TCR receptors, or any other exogenous sequences. It can encode a polypeptide of interest.

[0246] Thus, the present invention allows for the targeting of a single recognition site with a single engineered nuclease. This allows for the knockout of endogenous T cell receptors and the introduction of exogenous nucleic acid sequences (e.g., chimeric antigen receptors). The chimeric antigen receptor (CAR) coding sequence allows for both expression of endogenous and exogenous TCRs. In certain embodiments, where a TCR alpha constant region gene is inserted, the present invention provides Allogeneic TCR-expressing mice expressing specific CARs and reducing or completely knocking out endogenous TCR expression The present invention provides a simplified method for generating allogeneic T cells. Such cells can be administered to allogeneic subjects. When administered, the compound may exhibit reduced induction of or may induce graft-versus-host disease (GVHD). do not have.

[0247] 2.2 Nucleotide sequences for recognizing and cleaving recognition sequences within the T cell receptor alpha constant region gene Crease 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 be amplified or may trigger mechanisms such as nonsense-mediated mRNA decay. The use of nucleases to induce NHEJ-mediated mutagenesis has the potential to can be used to target sequences present in natural mutations or wild-type alleles The use of nucleases to induce double-strand breaks at target loci has been particularly useful in the detection of genomic Stimulates homologous recombination of transgenic DNA sequences flanked by sequences homologous to the target 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. It may encode a sequence or a polypeptide.

[0248] In different embodiments, a variety of different types of nucleases are useful in the practice of the present invention. In one embodiment, the present invention can be practiced using recombinant meganucleases. In another embodiment, the present invention provides a method for detecting CRISPR nucleases or CRISPR This can be achieved using Nickase. CRIS recognizes a specific DNA site. Methods for generating PR and CRISPR nicksases are known in the art. For example, Ran et al. (2013) Nat Protoc. 8:2281-308 In another embodiment, the present invention provides TALENs or compact TALEs. This can be done using N. Creating a TALE domain that binds to a specific DNA site Methods for preparing hydroxybenzoates are known in the art and are described, for example, in Reyon et al. (2012) N See Biotechnol. 30:460-5. In further embodiments, Thus, the present invention can be practiced using MegaTAL.

[0249] 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.

[0250] 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.

[0251] 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.

[0252] 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.

[0253] 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.

[0254] Table 1. Exemplary polypeptides engineered to recognize and cleave the TRC1-2 recognition sequence (SEQ ID NO: 3) Recombinant meganucleases [Table 1]

[0255] * "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.

[0256] Table 2. Exemplary polypeptides engineered to recognize and cleave the TRC3-4 recognition sequence (SEQ ID NO: 4) Recombinant meganucleases [Table 2]

[0257] *"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. Expresses identity.

[0258] Table 3. Exemplary polypeptides engineered to recognize and cleave the TRC7-8 recognition sequence (SEQ ID NO: 5) Recombinant meganucleases [Table 3]

[0259] *"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 domains of the ganuclease. Expresses identity.

[0260] 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. Genetically modified cells are engineered using engineered nucleases that recognize and cleave the recognition sequences expressed. 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 The break in the sequence allows for direct homologous recombination of the exogenous nucleic acid sequence into the TCR alpha constant region gene. This can further enable

[0261] The engineered nucleases of the present invention may be used in the form of proteins or, preferably, engineered Such nucleic acids can be delivered to cells as nucleic acids encoding the nuclease. (e.g., circular or linearized plasmid DNA or PCR product) or RNA. For embodiments in which the engineered nuclease coding sequence is delivered in DNA form, see Mega It must be operably linked to a promoter to promote transcription of the nuclease gene. Mammalian promoters suitable for the present invention include cytomegalovirus early (CMV) promoters. promoter (Thomsen et al., (1984), Proc Natl Acad Sci USA.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.

[0262] 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.

[0263] 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

[0264] Purified nuclease proteins can be delivered to cells to cleave genomic DNA. This allows the production of a target sequence by a variety of different mechanisms known in the art. This allows homologous recombination or non-homologous end joining at the cleavage site.

[0265] In some embodiments, an engineered nuclease protein or an engineered nuclease 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) Biochemistry43: 7698-7706), and HSV-1 VP-22 (Deshayes et al., (2005) ) Cell Mol Life Sci.62:1839-49) is another example. In embodiments, the engineered nuclease or a D encoding the engineered nuclease NA / mRNA is a nuclease protein / DNA / mRNA that binds to a target cell, Recognizes specific cell surface receptors expressed on target cells, thereby allowing it to be internalized Alternatively, the nuclease may be covalently or non-covalently linked to an antibody that binds the nuclease. The protein / DNA / mRNA is the natural ligand (or natural ligands) for such cell surface receptors. (McCall et al., (2014) T issue Barriers.2(4):e944449;Dinda et al., (2013 )Curr Pharm Biotechnol.14:1264~74;Kang et al. (2014)Curr Pharm Biotechnol.15(3):220~30 ;Qian et al., (2014) Expert Opin Drug Metab Toxi col.10(11):1491~508).

[0266] In some embodiments, an engineered nuclease protein or an engineered nuclease 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 the protein / mRNA / DNA delivered to the cell, thus increasing the number of copies of each individual Increasing intracellular expression of the engineered nuclease increases the likelihood of cleaving the target recognition sequence. The surface of such nanoparticles can be coated with polymers or lipids (e.g., chitosan, cationic The surface can be further modified with anionic polymers or cationic lipids to provide additional functionality. to form core-shell nanoparticles that enhance cellular delivery and payload uptake. (Jian et al., (2012) Biomaterials. 33(30):762 1-30) 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 targeting molecules include antibodies specific for cell surface receptors and natural targeting molecules for cell surface receptors. Ligands (or parts of natural ligands) are included.

[0267] In some embodiments, an engineered nuclease protein or an engineered nuclease The DNA / mRNA encoding the enzyme was encapsulated in liposomes using cationic lipids. embedded or complexed (e.g., Lipofectamine™, Life Technologies, chnologies Corp., Carlsbad, CA; Zuris et al. (201 5) Nat Biotechnol. 33:73~80; Mishra et al. (2011 ) See J Drug Deliv. 2011:863734. Liposomes and The lipoplex formulation protects the payload from degradation and facilitates fusion with the cell membrane and / or can enhance cellular uptake and delivery efficiency by disruption.

[0268] In some embodiments, an engineered nuclease protein or an engineered nuclease The DNA / mRNA encoding the enzyme is encapsulated within a polymer scaffold (e.g., PLGA). or complexed with cationic polymers (e.g., PEI, PLL) ( Tamboli et al. (2011) Ther Deliv.2(4):523~536).

[0269] In some embodiments, an engineered nuclease protein or an engineered nuclease The DNA / mRNA encoding the cleavage enzyme is combined with amphiphilic molecules that self-assemble into micelles. (Tong et al., (2007). J Gene Med. 9(11):956~ 66) Polymeric micelles prevent aggregation, mask charge interactions, and prevent nonspecific extracellular transport. Hydrophilic polymers (e.g., polyethylene glycol) that can reduce interactions The micelle shell may include a micelle shell formed by

[0270] In some embodiments, an engineered nuclease protein or an engineered nuclease 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. These 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). They also often contain one or more surfactants. Nanoemulsion formulations are well known. See, for example, U.S. Patent Application Nos. 2002 / 0045667 and 2004 / 00430 No. 41, as well as U.S. Patent Nos. 6,015,832, 6,506,803, and 6,63 Nos. 5,676 and 6,559,189, which are and is incorporated herein by reference in its entirety.

[0271] In some embodiments, an engineered nuclease protein or an engineered nuclease 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.

[0272] In some embodiments, the gene encoding the engineered nuclease is a viral vector. 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 The purpose of this method is to allow viral transduction of the cells and insertion of the nuclease gene into the cellular genome. In certain embodiments, the recombinant AAV vector can have any serotype. Recombinant AAV vectors have the serotype AAV2 or AAV6. It can be self-complementary so as not to require second-strand DNA synthesis in the cell (McCart (2001) Gene Ther. 8:1248~54).

[0273] The engineered nuclease gene may be in DNA form (e.g., a plasmid) and / or in a viral vector. 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 nuclease gene is expressed in a target cell (e.g., human T The gene is operably linked to a promoter that preferentially drives gene expression in the target cell.

[0274] The present invention further provides a method for preparing a nucleic acid sequence comprising the steps of: In some embodiments, the method provides for the introduction of exogenous nucleic acid into a cell so that the exogenous nucleic acid is inserted into the cell. wherein the exogenous nucleic acid comprises a 5' homologous arm and a 3' homologous arm and a nuclease cleavage site. This promotes the recombination of the nucleic acid sequence into the cellular genome.

[0275] 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.

[0276] 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.

[0277] In another particular embodiment, the endonuclease of the invention and / or the exogenous The gene encoding the nucleic acid sequence is transfected into cells by transfection of a linearized DNA template. In some embodiments, the endonuclease and / or exonuclease can be introduced into the The plasmid DNA encoding the inducible nucleic acid sequence is transferred to the cell via the circular plasmid DNA. digestion with one or more restriction enzymes to linearize the fragment prior to transfection. This can be done.

[0278] 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.

[0279] 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.

[0280] 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 recognized by the anti-CD20 mAb rituximab and suicide genes The recombinant antigenic polypeptide containing the epitope allows for the selection of cells expressing the gene. For example, two rituximab-binding epitopes and one QBEnd10-binding epitope can be used. RQR8 described in International Publication No. 2013153391, including a hybrid epitope For such genes, rituximab may be administered as needed. It can be administered to a subject to induce cell depletion.

[0281] 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. Pharmaceutical compositions containing the genetically modified cells of the present invention may contain additional drugs or biological agents. The compound can be administered in the same composition as the molecule, or can be co-administered in separate compositions. can.

[0282] 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.

[0283] 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 The viral particles containing the packaged genome (ITRs and one or more intervening genes of interest) are then followed by freeze-thaw cycles, sonication, detergents, or other means known in the art. The particles are then isolated from the cells by cesium chloride density gradient centrifugation or affinity chromatography. Purification using chromatographic chromatography followed by cloning of the gene(s) of interest. , delivered to a cell, tissue, or organism, such as a human patient.

[0284] 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 lead to reduced packaging efficiency and / or fragmented gels. Several approaches are used to package the Preventing endonuclease expression in stimulating cells:

[0285] 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 (Pa1AT, etc.), and hemopexin (Phpx, etc.) (Kramer , 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).

[0286] 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) .

[0287] 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 and including a gene encoding a transcription factor in the genotyped viral genome. This step results in the loss of target expression after recombinant AAV delivery if transcriptional activators are not supplied to the same cells. This is necessary because the endonuclease is not expressed in the cells or tissues. Transcription activators are expressed only in cells or tissues treated with their cognate small molecule activators. This approach allows us to determine when and how small molecule inducers induce cleavage gene expression. Spatio-temporal regulation of endonuclease gene expression by selecting tissues 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.

[0288] 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 R The SV promoter was modified to contain the Lac repressor operator, and the modified promoter We showed that gene expression from the ATPase inhibitor was significantly attenuated in cells expressing the repressor. Chang BD and Roninson IB (1996) Gene 183:13 7-42) The use of a non-human transcriptional repressor is necessary to prevent transcription of the endonuclease gene from being inhibited by the repressor. The resulting recombinant AAV vector is suppressed only in packaging cells that express the repressor. This ensures that the gene is not suppressed in the transduced target cells or tissues.

[0289] 2.6 Engineered nuclease mutants An embodiment of the present invention is directed to the use of engineered nucleases, particularly recombinant meganucleases, as described herein. Further embodiments of the present invention include the use of the compounds described herein. Isolated polynucleotides comprising nucleic acid sequences encoding recombinant meganucleases and Modified forms of the polynucleotide are included.

[0290] 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; i.e., for example, the TRC1-2 recognition sequence (SEQ ID NO: 3), TRC3- Human T cell receptor agonist (TCR) 4 recognition (SEQ ID NO: 4), and TRC7-8 recognition sequence (SEQ ID NO: 5) Possesses the ability to recognize and cleave the recognition sequence found in the human alpha constant region (SEQ ID NO: 1) Such variants may result, for example, from human manipulation. Biologically active variants of the polypeptides (e.g., SEQ ID NOS: 8-32), or any of the polypeptides described herein. Biologically active variants of the recognition half-site binding subunits (e.g., SEQ ID NOS: 33-82) described above. Variants are identified using sequence alignment programs and parameters described elsewhere herein. The data indicates that the polypeptide or subunit is about 40%, about 45%, or about 50%, approx. 55%, approx. 60%, approx. 65%, approx. 70%, approx. 75%, approx. 80%, approx. 85%, approx. 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, or about 99% sequence identity. A biologically active variant of the polypeptide or subunit of amino acid residues, only about 1-20, only about 1-10, only about 5, only 4 , 3, 2, or even 1 amino acid residue may differ.

[0291] 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 See Dayhoff et al. (1978) Atlas of Protein Sequences. ce 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.

[0292] 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.

[0293] [Table 4]

[0294] 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 In the case of polynucleotides, conservative variants include, due to the degeneracy of the genetic code, The variant polypeptides include sequences encoding the amino acid sequence of one of the polypeptides of the embodiments. Nucleotides may be generated, for example, by using site-directed mutagenesis, Synthetically derived fragments that still encode the recombinant meganucleases of the embodiments. Generally, variants of specific polynucleotides of the embodiments are Determined by sequence alignment programs and parameters described elsewhere in this specification. and the specific polynucleotide is at least about 40%, about 45%, about 50%, about 55%, or %, approx. 60%, approx. 65%, approx. 70%, approx. 75%, approx. 80%, approx. 85%, approx. 90%, approx. 91 %, approx. 92%, approx. 93%, approx. 94%, approx. 95%, approx. 96%, approx. 97%, approx. 98%, approx. 99 % or more sequence identity. A variant of (i.e., a reference polynucleotide) is a sequence encoded by a variant polynucleotide. and the polypeptide encoded by the reference polynucleotide. It can also be assessed by comparing the percent identity.

[0295] 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]

[0296] 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:

[0297] Example 1: Characterization of meganucleases that recognize and cleave TRC recognition sequences

[0298] 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.

[0299] 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), as well as the meganucleic acid residues Rease TRC1-2x.87EE, TRC1-2x.87QE, TRC1-2x.87 Position 330 (including R residue) of EQ, TRC1-2x.87, and TRC1-2x.163 The HVR1 region and the HVR2 region are identical except for the regions (shaded grey and underlined). Similarly, the HVR2 region is highly conserved.

[0300] 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.

[0301] 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).

[0302] 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.

[0303] 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.

[0304] 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).

[0305] 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.

[0306] 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.

[0307] 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.

[0308] 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."

[0309] 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.

[0310] 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 .

[0311] 5. Conclusion These studies have led to the development of the TRC1-2 meganuclease, TRC3-4 meganuclease, and The nuclease and TRC7-8 meganuclease have their respective recognition sequences. We demonstrated that it can be efficiently targeted and cleaved intracellularly.

[0312] Example 2: Cleavage of the TRC recognition sequence in T cells and suppression of cell surface T cell receptor expression

[0313] 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.

[0314] 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.

[0315] 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 further evidence of loss of T-cell receptor expression. did.

[0316] 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.

[0317] 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.

[0318] [Table 5]

[0319] [Table 6]

[0320] 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.

[0321] Example 3: Recombinant AAV vectors for introducing exogenous nucleic acids into human T cells

[0322] 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) prepared using the transfection protocol (Cots D, Bosch A, C hillon M (2013) Curr.Gene Ther.13(5):370~ (See page 81.) Figure 15 illustrates the insertion of an exogenous nucleic acid sequence into the cellular genome at a nuclease cleavage site. Figure 1 shows a general approach for using recombinant AAV vectors to transduce cells into mice.

[0323] 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. We further demonstrate the use of an AAV vector (GFP-AAV) that encodes GFP and is integrated into the vector. It was included in.

[0324] 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 Amaxa 4D-Nucleofector (Lonza) was used according to the manufacturer's instructions. mRNA (1 μg) encoding the TRC1-2x.87EE meganuclease was 2, 4, or 8 hours after transfection, cells were electroporated with GFP-AAV. (1e 5 The cells were transduced with 1000 viral genomes / cell. 72 hours after transduction, the cells were FP expression was analyzed 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 time increased, the number of GFP-positive cells decreased significantly, reaching 78% at 4 hours and GFAP at 8 hours. P-positive cells were 65%.

[0325] 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.

[0326] 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.

[0327] 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. 1B shows that some PCR products were cut with EagI (e.g., Fig. 20B, second row, left These fragments (lanes 1 to 6) show that the expected fragments of approximately 700 and 800 bp are produced. From the gel, EagI insertion was approximately 25% for AAV405 and AAV406, respectively. and 6% (adjusted for empty vector).

[0328] 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).

[0329] 3. Improving AAV transduction efficiency AAV transduction was performed 2 hours after transfection, whereas later transduction In light of the observation that transfection and transformation are more efficient than Experiments were conducted to optimize the timing of transfection. + T cells were treated with anti-CD3 antibodies The cells were stimulated with the antibody and anti-CD28 antibody for 3 days, and then transfected with Amaxa 4D-Nucleofect or (Lonza) according to the manufacturer's instructions, Immediately after transfection or immediately after transfection, cleavage (1 μg) was electroporated. Two hours after transfection, cells were transfected with GFP-AAV (1e 5 transduction of viral genomes / cells Furthermore, unstimulated cells were transfected with GFP-AAV(1e 5 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 transfection performed 2 hours after transfection. Transduction resulted in 90% GFP-positive cells, but transduction immediately after transfection Resting T cells were not amenable to AAV transduction. The non-transduced cells also showed approximately 0% GFP positive cells. Positive cells were indicated.

[0330] 4. Overview These studies used AAV vectors in conjunction with recombinant meganucleases to Through recombination, exogenous nucleic acid sequences can be incorporated into the cleavage site of the TCR alpha constant region. Demonstrate that this is possible.

[0331] Example 4: To introduce exogenous nucleic acids encoding chimeric antigen receptors in human T cells Recombinant AAV vectors

[0332] 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.

[0333] 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).

[0334] 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 activating the endogenous TCR Determine the efficiency of using recombinant AAV vectors to knock out cell surface expression of receptors Conduct research to achieve this.

[0335] To confirm transduction efficiency, human CD3 + T cells were obtained and subjected to anti-CD3 and anti-CD28 The cells were stimulated with antibody for 3 days, and then transfected with Amaxa 4D-Nucleofector (Lonz a) was used according to the manufacturer's instructions to conjugate the TRC1-2x.87EE meganuclease. The cells were electroporated with 1 μg of mRNA to be transfected as described above. Immediately after transfection, GFP-AAV (1e 5 The cells are transduced with the viral genome (1000 bp / cell). 72 hours after transduction, cells were analyzed by flow cytometry for GFP expression. Determine transfection efficiency.

[0336] 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.

[0337] 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.

[0338] Example 5: Insertion and expression of chimeric antigen receptors

[0339] 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 6cells) 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.

[0340] 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, AAV412 HDR, anneals within the CAR region of the HDR template Contains one primer that anneals to the human genome outside the 5' homology arm of the R template , 1872bp only when the CAR gene was successfully inserted within the TRC1-2 recognition sequence. The third primer set (referred to as the "outer 3' homology arm" in Table 7) amplifies a fragment of The CAR region of the AAV412 HDR template is annealed to the CAR region of the AAV412 HDR template. mer, and annealed to the human genome outside the 3' homology arm of the AAV412 HDR template The second primer set contains one primer that matches the first primer. The 1107b gene was expressed only when the CAR gene was successfully inserted into the TRC1-2 recognition sequence. In summary, the PCR products from all three primer sets were Whether the AR sequence is present in cells (primer set 1) and whether it is present in TRC1-2 Indicates whether or not the recognition sequence is inserted (primer sets 2 and 3).

[0341] 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.

[0342] 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.

[0343] 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.

[0344] 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 PCR products in mock-electroporated cells are shown. The HDR template generated by AAV408 was not found to be TRC1-2x. The CAR gene cannot be inserted into the TRC1-2 recognition sequence in the absence of 87EE mRNA. Lane 6 shows the results of electroporation of TRC1-2x.87EE mRNA and mock transfection. No PCR product was observed, and the TRC1-2 recognition sequence did not contain CAR. Lanes 7 to 10 show the TRC1-2x.87 vector. Each P represents a sample electroporated with EE mRNA and transduced with increasing amounts of AAV408. A band of the appropriate size for CR was evident, indicating that AAV408 recognizes TRC1-2. HDR donors for sequence repair can be generated, resulting in the insertion of a CAR gene. It shows.

[0345] [Table 7]

[0346] [Table 8]

[0347] 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.

[0348] 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.

[0349] 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-CD19CA R (anti-Fab-Alexa647) or CD3 (CD3-BB515) The cells were labeled and analyzed by flow cytometry. Figure 29A shows the anti-CAR labeling on the Y-axis. A, B shows flow cytometry plots with anti-CD3 labeling on the X-axis. Transduced cells (MOI-0) predominantly expressed CD3 + / CAR - (lower right quadrant Cells mock-electroporated and then transduced with increasing amounts of AAV408 showed a significantly higher IL-1 expression level compared with controls. It appears to be essentially identical to irradiated cells and CD3 + / CAR - The population is 98.8%, 99, 99%, Therefore, the present inventors concluded that the AAV408 virus alone was not detectable. It does not drive CAR expression at levels sufficient to disrupt T cell receptor expression. We conclude that

[0350] 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 in the population ranged from 2.09% to 5.9%. , 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 + CAR in the population + The cause of the cell increase 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. .

[0351] 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

[0352] Example 6: Further AAV Vector Characterization

[0353] 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.

[0354] 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, AAV422 M The OI was significantly higher than AAV421 in this experiment than in the previous experiment (approximately MO I is 1.25e 4 , 2.5e 4 , 5e 4 , and 1e 5 viral genome / cell). The AAV421 virus stock was large enough to allow for significantly higher titers than in previous experiments. As a control, cells were electroporated (mock or TRC1-2x.87). As an additional component of this experiment, the cells were transduced with "large-scale" Conditions are carried out and 6 TRC1-2x.87 cells (10x more than in a typical experiment) EE mRNA was electroporated and then transfected with AAV422 (2.5e 4 viral genome / cell) Finally, we also used the first viral stock for comparison. Therefore, a second viral stock of AAV421 was tested.

[0355] 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 products 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 and then Samples 1 to 5 were mock-transduced with AAV421, while samples 2 to 5 were mock-electroporated and then transduced with AAV421. The gel showed that none of these samples produced PCR products, and TRC1-2x.87 AAV421 insertion of the CAR gene into the TRC1-2 recognition sequence in the absence of EE mRNA Furthermore, electroporation of TRC1-2x.87EE mRNA showed that it was unable to drive The control sample (sample 6), which was then mock transduced, showed no PCR product. Samples 7 to 10 in Figure 32A were electroporated with TRC1-2x.87EE mRNA. Next, increasing amounts of AAV421 were transduced. The PCR bands of the extended products are shown (the two bands below each sample number), and the CAR This demonstrates the integration of the gene into the TRC1-2 recognition sequence. , lanes 11 and 12, 1e 6 or 10e 6 Start with either cells or samples TRC1-2x.87EE mRNA was electroporated and then transduced with AAV422. Both PCR bands (which represent different primers considering the long homology arms) represent the same sample. The presence of the CAR gene (larger in the first set because primers were used) This indicates that insertion of into the TRC1-2 recognition sequence was successful.

[0356] 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 PCR products, indicating the absence of TRC1-2x.87EE mRNA. In the presence of α-aminobutyric acid, AAV422 was unable to drive insertion of the CAR gene into the TRC1-2 recognition sequence. Samples 7 to 10 in Figure 32B contain TRC1-2x.87EE mRNA. were electroporated and then transduced with increasing amounts of AAV422. The PCR bands of the products extended beyond both the CAR gene and the TRC1-2 gene. Finally, sample 11 demonstrates integration into the TRC1-2x.87E EmRNA was electroporated and then incubated with a different viral stock solution than the sample shown in Figure 32A. The presence of a band indicates the expression of the CAR gene. The insertion 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 in T cells. We clearly demonstrated that we can generate HDR templates suitable for insertion into the RC1-2 recognition sequence. There are.

[0357] [Table 9]

[0358] [Table 10]

[0359] 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 perforated and then transduced with increasing amounts of AAV421 appeared essentially identical to control cells. I, 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 induce detectable levels of C We conclude that it neither drives AR expression nor disrupts T cell receptor expression. .

[0360] 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. Samples electroporated with mRNA and then transduced with increasing amounts of AAV412 were D3 - The population showed significant amounts of CAR labeling ranging from 4.99% to 13.4%. 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 mean fluorescence intensity was better defined, and the JeF promoter was more abundant than the eF1α core promoter. It was suggested that the expression of α-glucan also drives high expression.

[0361] 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 above and the experiment shown in Figure 33, the inventors have determined that TRC1-2x.8 7EE mRNA (2 μg) was electroporated followed by AAV421 (3.13 e 4 Virus The control samples were cells transduced with the genotype (genome / cells). 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 The cells were then incubated for 6 days in complete growth medium supplemented with 10 ng / mL of ATP. Cells were labeled with anti-CD19CAR and antibodies against CD3 and analyzed by flow cytometry. The mock-electroporated and mock-transduced cells were analyzed (Fig. 34A). - / CAR + There was a low level of background staining in the CD3 quadrant (0.13%). - / CA R +The populations were mock-electroporated and then AAV-transduced samples, or TRC1-2 x.87EE mRNA was electroporated and then mock-transduced in samples The results were similar (0.16% and 0.55%, respectively). A. Electroporated and mock-transduced cells expressed 53.2% CD3 - / CAR - Having a group, This is very close to the amount stained in the first part of this experiment shown in Figure 33B (56.7%). TRC1-2x.87EE electroporated and AAV transduced cells showed a CD of 12.6%. 3 - / CAR + cells, nearly identical to the original labeling of these cells shown in Figure 33. (13.4%), a mixture of IL-7 and IL-15, and specific CD3 - / CAR + cell population demonstrated that the concentration or proliferation of

[0362] 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.

[0363] In cells electroporated with TRC1-2x.87EE and then transduced with AAV421, , 24.2% of the CD3 population expressed CAR before expansion + (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.

[0364] 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 ( Pretreated with mitomycin C and incubated for another 6 days. The cells were then stained for CD3 and anti-CD19CAR and analyzed by flow cytometry. The CD3 in either control sample was analyzed by ELISA (Figure 34C). - / CAR + The percentage of cells is essentially the same as that of the first round of enrichment for IM-9 cells. It didn't change much.

[0365] 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.

[0366] 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 + / C AR - (lower right quadrant, 98.8%). 22-transduced cells appeared essentially identical to control cells and showed CD3 + / CAR - group The results were 98.6%, 98.6%, 98.9%, and 98.4%. showed that the AAV422 vector alone did not drive detectable levels of CAR expression, and Neither can it disrupt the expression of cellular receptors.

[0367] 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.

[0368] 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; In contrast, the efficacies of titrated AAV421 were 74.9%, 77.8%, and 74.4%. 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.

[0369] 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 cells) Only enrichment of the transduced cells was attempted in new experiments. Flow cytometry plots are shown approximately 4 days after transduction. Mock-electroporated / transduced cells is 0.13% CD3 - / CAR + The background staining of the cells is shown. TRC1-2x.87EE cells were electroporated and transduced with AAV422. 0.44% CD3 - / CAR +The cells were treated with IL-7 and IL- Incubation with IM-9 cells (pretreated with mitomycin) in the presence of 15 for 6 days followed by The results were analyzed by flow cytometry after incubation with IM-9 cells. tion, CD3 in AAV422-transduced cells. - / CAR + Dramatically increased the population to 35.8% This indicates that the CAR + The cells were found to be 6.69% total CD3 - collection As mentioned above, we also investigated the IM-9 cells. This was further enriched by a second addition of IM-9 cells (Fig. 36C). As a result of the inoculation, 65.1% of CD3 - / CAR + CAR cells were obtained. + The cells are all CD3 - Constituting 78.25% of the population, CD3 - / CAR + Significant antigen-dependent enrichment of cells This shows:

[0370] 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

[0371] 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 injected 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%).

[0372] 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%).

[0373] 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 noteworthy that the percent increase in knockout efficiency was nearly identical among the three experiments. Taken together, these data support the efficacy of single-stranded AAV vectors for cell transduction. However, our nuclease knockdown assays were independent of the nuclease or AAV cargo. This strongly indicates that it increases the efficiency of the output.

[0374] 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.

[0375] 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.

[0376] 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.

[0377] Example 7: Linearized Plasmid DNA

[0378] 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 5' homology arm) or a "long" (5' homology 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 named "pDS" and the one with the long homologous arms was named "pDI". Additionally, some plasmids contained an intron upstream of the CAR gene.

[0379] 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 antibodies against CD19CAR and anti-CD19 and analyzed by flow cytometry (Figure 3). 9). Figure 39A shows the results of a 0.15% background CD3 - / CAR + Showing staining. Blackground 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 60.8% 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-1 or IL-2 enhancer (with or without enhancer). In contrast, linearized CAR donors with only the EF1α core promoter showed 2.38% CD3 - / CAR + A population was generated containing the EF1α core promoter and the HTLV enhancer. The vector inhibited a significant percentage of CD3 - / CAR + No cells were produced. Cells electroporated with these two vectors in the absence of C1-2x.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.

[0380] Figures 39G and 39H show two vectors containing the MND promoter, both of which drive the expression of CAR. Figure 39G shows long homology arm constructs of the CAR gene. Surprisingly, the MND promoter and intron The long homology arm plasmid showed significant CAR expression (Figure 39G, 4.14%), whereas the CD3 - / CAR + ), whereas the intronless construct (Figure 39H) is TRC1-2x Co-electroporation with .87EE mRNA did not result in detectable CAR expression. A short homology arm plasmid containing the D promoter but no intron was also produced by 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. Which CAR + No cells were produced (Figures 39J, 39K, and 39L).

[0381] 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 This did not result in any significant changes in the TRC1-2x.87EE mRNA expression in cells (Figures 39O and 39P). When co-electroporated, the JeT-containing constructs inhibited 2.69% of CD3 - / CAR + Shows cells, The CMV-containing construct inhibited 2.7% of CD3 - / CAR + The cells were grown.

[0382] 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.

[0383] Insertion of CAR using linearized DNA constructs is specific to the TRC1-2 recognition sequence locus To confirm this, we investigated primers located within CAR and outside the homologous arms. Cells were analyzed as described above using the marker (Figure 40, Table 11). Samples 1 and 2 were Either mock electroporation or electroporation with mRNA encoding TRC1-2x.87EE alone Consistent with the above results, the PCR bands were not present. The absence of the CAR gene in the TRC1-2 recognition site was observed in samples 3, 4, and 5. 5. Cells Co-electroporated with TRC1-2x.87EE and Linearized CAR Homology Plasmid (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 into the TRC1-2 recognition site. Samples 6, 7, and 8 contain the same linearized CAR homology plasmid as samples 3, 4, and 5. derives from electroporated cells that do not contain TRC1-2x.87EE mRNA. Samples 9 and 10 were either pseudo-electroporated or TRC1- PCs derived from cells electroporated with either mRNA encoding 2x.87EE alone or The PCR bands are not shown. Samples 11, 12, 13, and 14 Derived from cells co-electroporated with TRC1-2x.87EE and linearized CAR homology plasmid (The sample names are those in Figure 40.) Each sample contains the TRC1-2 Two PCR bands of the expected size are shown, indicating insertion into the recognition site. Samples 15, 16, and 17 , and 18 are the same linearized CAR homology plasmids as samples 11, 12, 13, and 14. derived from cells electroporated with TRC1-2x.87EE but not containing TRC1-2x.87EE mRNA. As expected, there are no PCR bands.

[0384] Figures 39 and 40 show mRNA encoding TRC1-2x.87EE and linearized CAR. Homology plasmids were cloned into human CD3 + Co-electroporation of T cells delivers the CAR gene to TRC1 This clearly demonstrates that this is an effective method for inserting into the -2 recognition sequence.

[0385] [Table 11]

[0386] Example 8: Further AAV Vector Characterization

[0387] 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 The overall gene modification efficiency at the recognition site increased, and the total CD3- population increased by 4% compared to 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.

[0388] 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).

[0389] [Table 12]

[0390] 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:

[0391] Given the ability of AAV423 to insert the CAR sequence into the TRC1-2 recognition site after cleavage , which can be engineered with ATRC1-2 recognition sites to encode an anti-CD19 CAR The AV423 promoter was used to transfect T cells with linearized DNA templates. The smid (Figure 41) is linearized by digestion with one restriction enzyme and amplified by one or more restriction enzymes. It is further envisioned that the ATP may be delivered to cells via digestion.

[0392] Example 9: In vivo efficacy of anti-CD19 TCR-negative CAR T cells

[0393] 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. The CD3- cells were analyzed by flow cytometry as previously described (Fig. 44A). 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). 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).

[0394] Studies utilizing the Raji disseminated lymphoma model are being conducted at the Charles River Laboratory borators International Inc.(Morrisville) The experiment was carried out by the University of California, San Diego, NC, USA. Firefly luciferase (ffLuc)44 was used as a marker. 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.

[0395] 2.Results As shown in Figure 45, CD19 + Raji cell growth was observed in all mice by day 8. Low levels were evident in the untreated and TCR-control groups, which increased significantly by day 11 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.

[0396] 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 gene containing a modified human T cell receptor (TCR) alpha constant region gene in its genome 1. A modified cell comprising: The modified human TCR alpha constant region gene is, 5' to 3': (a) the 5' region of the human TCR alpha constant region gene; (b) an exogenous polynucleotide; and (c) the 3' region of the human TCR alpha constant region gene; Including, are genetically modified human T cells or genetically modified cells derived from human T cells, and are non- The gene has reduced cell surface expression of endogenous TCR when compared to modified control cells. Modified cells.

2. the exogenous polynucleotide comprises a nucleic acid sequence encoding a chimeric antigen receptor, A chimeric antigen receptor comprises an extracellular ligand-binding domain and one or more intracellular signal transduction domains. The genetically modified cell of claim 1 , comprising a domain.

3. The chimeric antigen receptor has at least 80% sequence identity with SEQ ID NO:

112. an extracellular ligand-binding domain, wherein the extracellular ligand-binding domain binds to CD19; The genetically modified cell of claim 2.

4. The chimeric antigen receptor has at least 80% sequence identity with SEQ ID NO:

113. The gene according to any one of claims 2 to 3, comprising an intracellular cytoplasmic signaling domain. Modified cells.

5. The chimeric antigen receptor has at least 80% sequence identity with SEQ ID NO:

114. The gene according to any one of claims 2 to 4, comprising an intracellular costimulatory signaling domain. Modified cells.

6. 6. Any one of claims 2 to 5, wherein the chimeric antigen receptor further comprises a signal peptide. Item 1. The genetically modified cell according to item 1.

7. The signal peptide has at least 80% sequence identity with SEQ ID NO:

115. The genetically modified cell of claim 6.

8. 8. Any one of claims 2 to 7, wherein the chimeric antigen receptor further comprises a hinge domain. The genetically modified cell according to claim 1.

9. The hinge domain has at least 80% sequence identity with SEQ ID NO:

116. The genetically modified cell of claim 8.

10. 10. The method of claim 2, wherein the chimeric antigen receptor further comprises a transmembrane domain. The genetically modified cell described herein.

11. wherein the transmembrane domain has at least 80% sequence identity with SEQ ID NO:

117.

11. The genetically modified cell of claim 10.

12. The chimeric antigen receptor has at least 80% sequence identity with SEQ ID NO:

111. A genetically modified cell according to any one of claims 2 to 11.

13. The exogenous polynucleotide may comprise a promoter that drives expression of the exogenous polynucleotide. The genetically modified cell of any one of claims 1 to 12, comprising a data sequence.

14. The promoter sequence has at least 80% sequence identity with SEQ ID NO:

118.

14. The genetically modified cell of claim 13.

15. The exogenous polynucleotide has at least 80% sequence identity with SEQ ID NO:

119. The genetically modified cell according to any one of claims 1 to 14.

16. The exogenous polynucleotide comprises the TC at a position within the recognition sequence comprising SEQ ID NO:

3. The gene according to any one of claims 1 to 15, which is inserted into the R alpha constant region gene. Modified cells.

17. the modified human TCR alpha constant region gene has at least 80% identity with SEQ ID NO: 120 17. The genetically modified cell of claim 16, comprising a nucleic acid sequence having sequence identity.

18. The exogenous polynucleotide comprises the TC at a position within the recognition sequence comprising SEQ ID NO:

4. The gene according to any one of claims 1 to 15, which is inserted into the R alpha constant region gene. Modified cells.

19. the modified human TCR alpha constant region gene has at least 80% identity with SEQ ID NO: 121 20. The genetically modified cell of claim 18, comprising a nucleic acid sequence having sequence identity.

20. The exogenous polynucleotide comprises the TC at a position within the recognition sequence comprising SEQ ID NO:

5. The gene according to any one of claims 1 to 15, which is inserted into the R alpha constant region gene. Modified cells.

21. the modified human TCR alpha constant region gene has at least 80% identity with SEQ ID NO: 122 22. The genetically modified cell of claim 21, comprising a nucleic acid sequence having sequence identity.

22. 1. A method for producing a genetically modified cell comprising a modified human TCR alpha constant region gene, comprising: (a) In cells (i) a first nucleic acid sequence encoding an engineered nuclease; or (ii) engineered nuclease proteins; introducing: wherein the engineered nuclease is a nucleotide sequence encoding a nucleotide sequence within the human TCR alpha constant region gene. generating a cleavage site in the recognition sequence; and (b) introducing into said cell a second nucleic acid sequence comprising an exogenous polynucleotide; Including, the cells are human T cells or cells derived from human T cells; a sequence of oligonucleotides in the human TCR alpha constant region gene at the cleavage site; and wherein the genetically modified cells exhibit a reduced expression of endogenous TCRs when compared to unmodified control cells. The method has reduced cell surface expression.

23. The second nucleic acid sequence comprises, from 5' to 3': (a) a 5' homology arm that is homologous to a 5' upstream sequence adjacent to the cleavage site; (b) the exogenous polynucleotide; and (c) a 3' homology arm that is homologous to a 3' downstream sequence adjacent to the cleavage site; Including, The sequence of the exogenous polynucleotide is inserted into the human genome at the cleavage site by homologous recombination.

23. The method of claim 22, wherein the gene is inserted into the TCR alpha constant region gene.

24. the exogenous polynucleotide comprises a nucleic acid sequence encoding a chimeric antigen receptor, A chimeric antigen receptor comprises an extracellular ligand-binding domain and one or more intracellular signal transduction domains.

24. The method of claim 22 or 23, comprising a domain.

25. The exogenous polynucleotide comprises a first gene that drives expression of the exogenous polynucleotide. The method of any one of claims 22 to 24, comprising a promoter sequence.

26. At least the second nucleic acid sequence is a recombinant adeno-associated virus comprising the second nucleic acid sequence. The cell is introduced by contacting the cell with an AAV vector. The method according to any one of claims 22 to 25.

27. The second nucleic acid is located 5' upstream of the 5' homologous arm or 5' upstream of the 3' homologous arm.

27. The method of claim 26, further comprising a second promoter sequence located 3' downstream of the promoter sequence.

28. 28. The method of claim 26 or 27, wherein the recombinant AAV vector is a self-complementary AAV vector. The method described.

29. 26-2, wherein the recombinant AAV vector has the serotype AAV2 or AAV6.

9. The method according to any one of claims 8 to 8.

30. The engineered nuclease may be a recombinant meganuclease, a recombinant zinc finger nuclease, or nucleases (ZFNs), recombinant transcription activator-like effector nucleases (TALENs) ), CRISPR / Cas nuclease, or MegaTAL nuclease.

30. The method of any one of 2 to 29.

31. 31. The method of claim 22, wherein the engineered nuclease is a recombinant meganuclease. The method according to any one of claims 1 to 4.

32. The recombinant meganuclease comprises a residue of the wild-type human TCR alpha constant region (SEQ ID NO: 1). The recombinant meganuclease recognizes and cleaves the recognition sequence within residues 93-208. a first subunit and a second subunit, wherein the first subunit is a first subunit of the recognition sequence The second subunit binds to the first recognition half-site and comprises the first hypervariable (HVR1) region. a second recognition half-site of said recognition sequence, said second recognition half-site comprising a second hypervariable (HVR2) region; 32. The method of claim 31 .

33. 10. The method of claim 1, wherein the recognition sequence within the human TCR alpha constant region gene comprises SEQ ID NO:

3.

32. The method according to claim 32.

34. 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 an amino acid sequence having the sequence of any one of SEQ ID NOs: 8 to 18. residues 7-153 of one of SEQ ID NOs: 19-27 or residues 198-344 of any one of SEQ ID NOs: 19-27 and at least 34. The method of claim 33, comprising an amino acid sequence having at least 80% sequence identity.

35. 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 35. The method of claim 33 or 34, comprising Y at the position corresponding to:

36. the HVR1 region is selected from residues 215 to 270 of any one of SEQ ID NOs: 8 to 18, or the sequence Any one of claims 33 to 35, comprising residues 24 to 79 of any one of numbers 19 to 27. The method described in paragraph .

37. the HVR2 region is selected from residues 24 to 79 of any one of SEQ ID NOs: 8 to 18 or SEQ ID NO: Any one of claims 33 to 36, comprising residues 215 to 270 of any one of 19 to 27 The method described in paragraph .

38. 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 33 to 37, comprising residues 7 to 153 of any one of SEQ ID NOs: 19 to 27; The method according to any one of claims 1 to 10.

39. The second subunit comprises residues 7 to 153 of any one of SEQ ID NOs: 8 to 18, or Any of claims 33 to 38, comprising residues 198 to 344 of any one of sequence numbers 19 to 27 The method according to any one of claims 1 to 10.

40. The recombinant meganuclease is a single-chain meganuclease containing a linker, 33. A Car covalently links the first subunit and the second subunit.

40. The method according to any one of claims 1 to 39.

41. The recombinant meganuclease comprises any one of the amino acid sequences of SEQ ID NOs: 8 to 27. The method according to any one of claims 33 to 40.

42. 10. The method of claim 1, wherein the recognition sequence within the human TCR alpha constant region gene comprises SEQ ID NO:

4.

32. The method according to claim 32.

43. 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.

43. The method of claim 42, comprising:

44. the HVR1 region comprises a Y at a position corresponding to position 24 of SEQ ID NO: 28 or 29. Item 44. The method according to item 42 or 43.

45. the HVR1 region comprises a T at a position corresponding to position 26 of SEQ ID NO: 28 or 29. Item 44. The method according to item 42 or 43.

46. the HVR1 region comprises a Y at a position corresponding to position 46 of SEQ ID NO: 28 or 29. Item 44. The method according to item 42 or 43.

47. the HVR2 region comprises an H at a position corresponding to position 215 of SEQ ID NO: 2 or 29. Item 44. The method according to item 42 or 43.

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

44. The method according to claim 42 or 43.

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

44. The method according to claim 42 or 43.

50. 4. Claims 42-4, wherein the HVR1 region comprises residues 24 to 79 of SEQ ID NO: 28 or 29.

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

51. 42. The HVR2 region comprising residues 215 to 270 of SEQ ID NO: 28 or 29.

51. The method according to any one of claims 1 to 50.

52. Claim 4, wherein the first subunit comprises residues 7 to 153 of SEQ ID NO: 28 or 29.

52. The method of any one of claims 2 to 51.

53. the second subunit comprises residues 198 to 344 of SEQ ID NO: 28 or 29. Item 53. The method according to any one of Items 42 to 52.

54. The recombinant meganuclease is a single-chain meganuclease containing a linker, 42. A Car covalently links the first subunit and the second subunit.

54. The method according to any one of claims 1 to 53.

55. 20. The method of claim 19, wherein the recombinant meganuclease comprises the amino acid sequence of SEQ ID NO: 28 or 29.

55. The method of any one of claims 42 to 54.

56. 10. The method of claim 9, wherein the recognition sequence within the human TCR alpha constant region gene comprises SEQ ID NO:

5.

32. The method according to claim 32.

57. 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.

57. The method of claim 56.

58. the HVR1 region is (a) position 24 of SEQ ID NO: 30, or (b) position 215 of SEQ ID NO: 31 or 32; 58. The method of claim 56 or 57, comprising Y at the position corresponding to:

59. the HVR1 region is a sequence selected from residues 24 to 79 of SEQ ID NO: 30 or residues 31 or 32 59. The method of any one of claims 56 to 58, comprising residues 215 to 270.

60. the HVR2 region is selected from residues 215 to 270 of SEQ ID NO: 30 or SEQ ID NO: 31 or 3 60. The method of any one of claims 56 to 59, comprising residues 24 to 79 of SEQ ID NO:

2.

61. the first subunit is selected from residues 7 to 153 of SEQ ID NO: 30 or SEQ ID NO: 31 or 61. The method of any one of claims 56 to 60, comprising 32 residues 198 to 344.

62. the second subunit is a sequence of residues 198 to 344 of SEQ ID NO: 30 or SEQ ID NO: 31 or 62. The method of any one of claims 56 to 61, comprising residues 7 to 153 of at least 32.

63. The recombinant meganuclease is a single-chain meganuclease containing a linker, 57. A Car covalently links the first subunit and the second subunit.

63. The method according to any one of claims 1 to 62.

64. The recombinant meganuclease has the amino acid sequence of any one of SEQ ID NOs: 30 to 32.

64. The method of any one of claims 56 to 63, comprising:

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

22. A method for producing a pharmaceutical composition comprising the genetically modified cell of claim 21 and a pharmaceutically acceptable carrier. administering a pharmaceutical composition to said subject.

66. 1. A method of immunotherapy for treating cancer in a subject in need thereof, comprising: Genetically modified cells and pharmaceuticals prepared according to the method of any one of claims 2 to 64. administering to said subject a pharmaceutical composition comprising an acceptable carrier.

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

67. The method of any one of claims 65 and 66.

68. 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 67. The method of any one of claims 65 and 66, selected from the group:

69. 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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