Optimized and engineered nuclease having specificity for human t cell receptor alpha constant region gene

Abstract

To provide an engineered meganuclease that recognizes and cleaves the recognition sequence in the human T cell receptor alpha constant region gene.SOLUTION: Provided herein is an engineered meganuclease comprising a first subunit and a second subunit, where the first subunit binds to a first recognition half site of a specific sequence in human T cell receptor alpha constant region gene and comprises a first hypervariable region, and the second subunit binds to a second recognition half site of the specific sequence comprising the specific sequence and comprises a second hypervariable region, where the second hypervariable region has (a) at least 81% sequence homology to an amino acid sequence corresponding to residues 24 to 79 of a specific sequence or (b) at least 86% sequence homology to an amino acid sequence corresponding to residues 24 to 79 of a specific sequence.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the fields of oncology, cancer immunotherapy, molecular biology and recombinant nucleic acid technology. The present invention provides a method for detecting a specific recognition sequence in the human T cell receptor alpha constant region gene. The present invention further relates to an optimized engineered nuclease having the properties of a gene modified Methods for producing T cells and methods for treating diseases, including cancer, in a subject The present invention also relates to the use of such recombinant meganucleases in methods that employ cells containing such recombinant meganucleases.

[0002] Reference to sequence listing submitted as a text file via EFS-WEB This application contains a Sequence Listing submitted in ASCII format via EFS-WEB and is incorporated by reference. This AS, created on April 11, 2019, is hereby incorporated by reference in its entirety. The CII copy is named P109070028WO00-SEQ and is 2 in size. It is 700 bytes. [Background technology]

[0003] T cell adoptive immunotherapy is a promising approach for cancer treatment. This strategy targets specific Isolated human T cells that have been genetically modified to enhance their specificity for tumor-associated antigens Genetic modification involves the use of chimeric antigen receptors or chimeric antigen receptors to transfer antigen specificity to T cells. 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 receptor derives its specificity from the variable domains of the monoclonal antibody. T cells expressing antigen receptors (CAR T cells) are major histocompatibility complex-restricted T cell adoptive immunotherapy induces tumor immune activity in a non-targeted manner. Acute lymphoblastic leukemia, B-cell non-Hodgkin's lymphoma, acute myeloid leukemia, and chronic lymphocytic leukemia leukemia), multiple myeloma, neuroblastoma, glioblastoma, progressive glioma, ovarian It is available as a clinical therapy for many cancers, including cancer, mesothelioma, melanoma, prostate cancer, and pancreatic cancer. It has been used.

[0004] Despite its potential utility as a cancer treatment, adoptive immunotherapy with CAR T cells The method is limited, in part, by the expression of endogenous T cell receptors on the cell surface. CAR T cells expressing endogenous T cell receptors have shown significant efficacy after administration to allogeneic patients. It may recognize numerous and minor histocompatibility antigens, which may lead to graft-versus-host disease (GVHD). As a result, clinical trials are underway to isolate and characterize patients' T cells. The vaccine is genetically modified to incorporate a marker antigen receptor and then reinjected into the same patient. The autologous approach focuses primarily on the use of autologous CAR T cells. However, this approach is not feasible until the patient's cancer is diagnosed. This is due to both the time and expense required to produce patient-specific CAR T cells after the initial Restricted.

[0005] Therefore, it reduces the expression of endogenous T cell receptors and does not cause GVHD when administered. Developing "off-the-shelf" CAR T cells prepared using T cells from healthy third-party donors It would be advantageous to develop such a product. Such a product would be capable of being generated and verified prior to diagnosis. Therefore, it is readily available to patients when needed. To achieve this, it is necessary to develop allogeneic CAR T cells that lack endogenous T cell receptors.

[0006] Genetic modifications of genomic DNA are engineered to recognize the DNA sequence of a desired locus. It can also be done using site-specific and rare-cutting endonucleases. Homing endonucleases are commonly found in plant and fungal genomes. A group of naturally occurring nucleases that recognize 0 base pair cleavage sites. The study involved parasitic DNA elements such as group 1 self-splicing introns and inteins. They induce double-strand breaks in the chromosome, which are essential for the host genome. naturally promotes homologous recombination or gene insertion at specific locations in the genome, and activates the cell's DNA repair machinery Homing endonucleases generally recruit the LAGLI DADG (SEQ ID NO: 2) family, GIY-YIG family, His-Cys box These are classified into four families: the HB family, the HB 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: 2) family contain conserved It is characterized by having one or two copies of the LAGLIDADG (SEQ ID NO: 2) motif. (See Non-Patent Document 2.) LAGLIDADG (SEQ ID NO: 2) homing endonuclease with a single copy of The enzyme forms a homodimer, but the two copies of the LAGLIDADG (SEQ ID NO: 2) motif Members with peaks are identified as monomers.

[0007] I-Crel (SEQ ID NO: 1) is a nucleotide sequence of the alga Chlamydomonas reinhardtii (Chl A 22-base pair sequence in the chloroplast chromosome of Amydomonas reinhardtii The homing endonuclease LAGLIDADG ( It is a member of the I-Crel family (SEQ ID NO: 2). It alters the wild-type I-Crel cleavage site preference. To achieve this, gene selection techniques are used (Non-Patent Documents 3 to 6). More recently, a comprehensive re-establishment of I-Crel and other homing endonucleases has been performed. In total, a wide variety of sequences were identified, including sites in mammalian, yeast, plant, bacterial, and viral genomes. Mono-LAGLIDADG (SEQ ID NO: 1) can target divergent DNA sites. 2) A method for rationally designing homing endonucleases has been described (Patent Document 1). 1).

[0008] As first described in U.S. Patent No. 5,929,999, I-Crel and its engineered derivatives is usually a dimer, with the C-terminus of the first subunit linked to the N-terminus of the second subunit. A short peptide linker can be used to fuse the two sequences into a single polypeptide ( Therefore, functional "single-chain" meganucleases are capable of cleaving a single transcript. It can be manifested from things.

[0009] Small hairpin RNA, zinc finger nuclease (ZFN), transcription activator-like factor vector nucleases (TALENs), megaTALs, and CRISPR systems (e.g. , Non-Patent Documents 9-10, Patent Documents 3-4) The use of nucleases for this purpose has been disclosed.

[0010] An engineered megagene for cleaving a DNA target in the human TCR alpha constant region gene. Specific uses of cleavage have also been disclosed. For example, Patent Document 5 discloses a method for the treatment of TCR antigens. It targets the recognition sequence in exon 1 of the lupus constant region gene (SEQ ID NO: 3 in Patent Document 5). The patent disclosed a variant of I-Onul meganuclease that was engineered to act as a nuclease inhibitor. Reference 5 discusses that chimeric antigen receptors can be expressed in TCR knockout cells. However, the authors do not disclose the insertion of the CAR coding sequence into the meganuclease cleavage site. .

[0011] Furthermore, in U.S. Patent No. 5,629,997 and U.S. Patent No. 5,629,997, Applicant discloses a TCR alpha constant region gene. We have disclosed an engineered meganuclease with specificity for the recognition sequence in exon 1 of the gene. These include the "T RC1-2 meganuclease." Publications in Patent Documents 6 and 7 are , a CAR coding sequence or an exogenous TCR coding sequence is inserted into the TCR1-2 meganuclease. A method for targeted insertion at the cleavage site is also disclosed. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] International Publication No. 2007 / 047859 [Patent Document 2] International Publication No. 2009 / 059195 [Patent Document 3] U.S. Patent No. 8,956,828 [Patent Document 4] U.S. Patent Publication No. 2012 / 0321667 [Patent Document 5] International Publication No. 2014 / 191527 [Patent Document 6] International Publication Gazette No. 2017 / 062439

Patent Document 7

Non-Patent Documents

[0013]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Summary of the Invention

Problems to be Solved by the Invention

[0014] In the present invention, applicants have improved upon the nucleases and methods taught in the prior art. Through extensive experimentation, applicants have identified a compound containing unique and unpredictable combinations of residues that are patentable. The first generation TRC1-2 meganucleases taught in Reference 6 and Patent Document 7 are more promising than the TRC1-2 meganucleases taught in Reference 6 and Patent Document 7. We have generated novel, second-generation TRC1-2 meganucleases that are surprisingly superior. The second generation TRC1-2 meganuclease of the present invention has improved (i.e., increased) It has specificity and reduced off-target cleavage, and after expression from mRNA, showed a decrease in maintenance time, and CAR T cells (e.g., enhanced / increased TCR knockout, Enhanced / increased CAR knock-in, enhanced / increased CAR T proliferation, improved CAR T cell phenotypes) and functionally superior in vitro. and when used in a full-scale CAR T cell manufacturing process, improved CAR T cell Produces a population of cells. [Means for solving the problem]

[0015] Summary of the Invention The present invention relates to the first sequence of the human T cell receptor (TCR) alpha constant region gene (SEQ ID NO: 3). Provides engineered meganucleases that recognize and cleave recognition sequences present within exons Such meganucleases disrupt the TCR alpha constant region gene, resulting in cellular These are useful for disrupting cell surface TCR expression and / or function. The breaks are caused by the mutagenic activity of non-homologous end joining or by exogenous insertion into the gene by homologous recombination. disrupt gene function by either facilitating the introduction of a causative polynucleotide In some embodiments, the meganuclease can be an allogeneic C that lacks an endogenous TCR. To be useful in generating AR T cells, the introduced exogenous polynucleotide must be chimeric. In some embodiments, the method of the present disclosure comprises a nucleic acid sequence encoding a CAR. The meganuclease developed is the first generation meganuclease, TRC1-2x.87. It exhibits at least one optimized feature compared to EE. reduces off-target cleavage and maintains the protein in the cell (e.g., after mRNA-derived expression). and enhance (i.e., increase) the efficiency of TCR alpha constant region gene modification. Furthermore, the engineered polypeptides of the present disclosure have improved (i.e., increased) specificity. Cells genetically modified with meganucleases exhibit improved characteristics. - Reduces target cleavage and its effectiveness, and reduces the maintenance time of meganucleases in cells Furthermore, the cells are capable of expressing CAR T cells in a manner that enhances (i.e., increases) their proliferation. The differentiation process was significantly improved when compared to cells genetically modified with C1-2x.87EE meganuclease. In addition, the meganucleases of the present disclosure (or nucleic acids encoding same) are introduced into The transfected cell population expresses the TRC1-2x.87EE meganuclease (or its encoding enzyme). A greater proportion of the modified cells are expressed in the modified gene compared to a population of cells into which a nucleic acid (which encodes the nucleic acid) has been introduced. They have large proportions of cells and a greater proportion of less differentiated cells.

[0016] The present invention further provides engineered meganuclear vectors for producing genetically modified eukaryotic cells. The gene encoding the enzyme protein, i.e., the engineered meganuclease, is then transfected into a eukaryotic cell. Thus, the present invention provides a method for producing genetically modified eukaryotic cells and their The present invention further provides a genetically modified eukaryotic cell and a pharmaceutical composition comprising the genetically modified eukaryotic cell and the population thereof. Immunotherapy to treat cancer by administering genetically modified T cells or populations thereof wherein the T cells are responsive to a tumor-specific antigen (e.g., a CAR or an exogenous TCR). Methods for expressing the signal are also provided.

[0017] Thus, in one aspect, the present invention provides a human TCR alpha constant region gene (SEQ ID NO: 3) An engineered membrane that recognizes and cleaves the TRC1-2 recognition sequence (SEQ ID NO: 5) in exon 1 of The engineered meganuclease comprises a first subunit and a second subunit. and a second subunit, the first of which is a first recognition half of the recognition sequence. The second subunit binds to the fusiform nuclei and contains the first hypervariable (HVR1) region, and the second subunit contains The second recognition half site of the recognition sequence binds to the TRC1-2L.1592 (sequence The amino acid sequence corresponding to residues 24 to 79 of the amino acid sequence designated as amino acid number 7 In contrast, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, At least 94%, at least 95%, at least 96%, at least 97%, at least or have 98%, at least 99%, or more sequence homology with the TRC of the present disclosure. 1-2L.1775 meganuclease (amino acid sequence set forth as SEQ ID NO:8) At least 86%, at least 87% of the amino acid sequence corresponding to residues 24 to 79 of , at least 88%, at least 89%, at least 90%, at least 91%, at least At least 92%, at least 93%, at least 94%, at least 95%, at least 96% %, at least 97%, at least 98%, at least 99%, or more sequence identity The second hypervariable (HVR2) region has the same sequence.

[0018] In certain embodiments, the HVR2 region comprises at most 1, 2, 3, 4, 5, 6, 7, 8, Residue 24 of SEQ ID NO:7 or SEQ ID NO:8 with 9, 10 or 11 amino acid substitutions Contains an amino acid sequence corresponding to ~79.

[0019] In some embodiments, the HVR2 region comprises residues 24, 26, 42, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 Contains residues corresponding to 6, 48, 50, 70, 71, 72 and 73.

[0020] In some embodiments, the HVR2 region comprises residues 24, 26, 38, 42, 44, 46, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 Contains residues corresponding to 6, 48, 50, and 70.

[0021] In some embodiments, the HVR2 region comprises residues 24, 26 of SEQ ID NO:7 or SEQ ID NO:8. , 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75 and 7 Contains residues corresponding to 7.

[0022] In some embodiments, the HVR2 region comprises residues 48, 50, 71, 72 and 73 of SEQ ID NO:7. and 73.

[0023] In some embodiments, the HVR2 region comprises residues corresponding to residues 48 and 50 of SEQ ID NO:8. Contains a group.

[0024] In some embodiments, the HVR2 region comprises residues 24, 26 of SEQ ID NO:7 or SEQ ID NO:8. , 28, 30, 32, 33, 38, 40, 42, 44, 46, 48, 50, 68, 70, Contains residues corresponding to 71, 72, 73, 75 and 77.

[0025] In some embodiments, the HVR2 region corresponds to residue 66 of SEQ ID NO:7 or SEQ ID NO:8. Residues that are present include Y, R, K, or D.

[0026] In certain embodiments, the HVR2 region is residues 24-7 of SEQ ID NO:7 or SEQ ID NO:8. Includes 9.

[0027] In certain embodiments, the second subunit comprises residues 7-8 of SEQ ID NO:7 or SEQ ID NO:8. At least 80%, at least 85%, at least At least 90%, at least 91%, at least 92%, at least 93%, at least 94% , at least 95%, at least 96%, at least 97%, at least 98%, at least In some embodiments, the amino acid sequence has 99% or more sequence identity with the The second subunit has an amino acid sequence corresponding to residues 7 to 153 of SEQ ID NO: 7. In some embodiments, the amino acid sequence has at least 93% sequence identity. The two subunits have at least one amino acid sequence corresponding to residues 7 to 153 of SEQ ID NO:8. It contains an amino acid sequence with at least 94% sequence identity.

[0028] In some embodiments, the second subunit is at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 , 23, 24, 25, 26, 27, 28, 29 or 30 amino acid substitutions It contains the amino acid sequence corresponding to residues 7 to 153 of SEQ ID NO:7 or SEQ ID NO:8.

[0029] In certain embodiments, the second subunit comprises residues of SEQ ID NO:7 or SEQ ID NO:8. At the residue corresponding to 19, it contains G, S or A.

[0030] In certain embodiments, the second subunit comprises residues of SEQ ID NO:7 or SEQ ID NO:8. The residue corresponding to 80 contains E, Q, or K.

[0031] In some embodiments, the second subunit comprises residue 80 of SEQ ID NO:7 or SEQ ID NO:8. It contains residues corresponding to:

[0032] In certain embodiments, the second subunit comprises residues of SEQ ID NO:7 or SEQ ID NO:8. Contains residues corresponding to 139.

[0033] In certain embodiments, the second subunit comprises residues 7-8 of SEQ ID NO:7 or SEQ ID NO:8. Includes 153.

[0034] In some such embodiments, the HVR1 region comprises residue 21 of SEQ ID NO:7 or SEQ ID NO:8. For the amino acid sequence corresponding to 5-270, at least 80%, at least 81%, or at least at least 82%, at least 83%, at least 84%, at least 85%, at least 8 6%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% In certain embodiments, the HV comprises an amino acid sequence having at least one amino acid sequence homology thereto. The R1 region may contain up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids. The amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 7 or SEQ ID NO: 8, with an amino acid substitution Contains columns.

[0035] In some embodiments, the HVR1 region corresponds to residues 219 and 231 of SEQ ID NO:7. It contains residues that

[0036] In certain embodiments, the HVR1 region comprises residue 215 of SEQ ID NO:7 or SEQ ID NO:8, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, Contains residues corresponding to 259, 261, 266 and 268.

[0037] In some embodiments, the HVR1 region is sequenced to sequence 257 of SEQ ID NO:7 or SEQ ID NO:8. The corresponding residues include Y, R, K, or D.

[0038] In certain embodiments, the HVR1 region comprises residues 215-27 of SEQ ID NO:7 or SEQ ID NO:8. Includes 0.

[0039] In some embodiments, the first subunit is selected from residues 19 of SEQ ID NO:7 or SEQ ID NO:8. For the amino acid sequence corresponding to 8 to 344, at least 80%, at least 85%, or at least at least 90%, at least 91%, at least 92%, at least 93%, at least 9 4%, at least 95%, at least 96%, at least 97%, at least 98%, It contains amino acid sequences with at least 99% or more sequence identity. In embodiments, the first subunit is a nucleotide sequence selected from residues 198-344 of SEQ ID NO:7 or SEQ ID NO:8. An amino acid sequence having at least 99% sequence identity to the amino acid sequence corresponding to In certain embodiments, the first subunit comprises at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, with 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions , comprising an amino acid sequence corresponding to residues 198 to 344 of SEQ ID NO:7 or SEQ ID NO:8.

[0040] In certain embodiments, the first subunit comprises residues of SEQ ID NO:7 or SEQ ID NO:8. At the residue corresponding to 210, it contains G, S, or A.

[0041] In certain embodiments, the first subunit comprises residues of SEQ ID NO:7 or SEQ ID NO:8. At the residue corresponding to 271, contains E, Q or K.

[0042] In certain embodiments, the first subunit comprises residues of SEQ ID NO:7 or SEQ ID NO:8. Contains residues corresponding to 271.

[0043] In certain embodiments, the first subunit is selected from residues 19 of SEQ ID NO:7 or SEQ ID NO:8. Includes 8 to 344.

[0044] In some embodiments, the first subunit of the engineered meganuclease is 7 or at least 8 for the amino acid sequence corresponding to residues 198 to 344 of SEQ ID NO: 8 0%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence homology, The second subunit contains amino acids corresponding to residues 7 to 153 of SEQ ID NO: 7 or SEQ ID NO: 8. At least 80%, at least 85%, at least 90%, at least 91% %, at least 92%, at least 93%, at least 94%, at least 95%, at least at least 96%, at least 97%, at least 98%, at least 99%, or more In certain embodiments, the engineered meganucleic acid comprises an amino acid sequence having sequence homology to the above. The first subunit of the enzyme is located at residues 198 to 344 of SEQ ID NO: 7 or SEQ ID NO: 8. A second subunit having at least 99% sequence identity to the corresponding amino acid sequence. The amino acid sequence corresponding to residues 7 to 153 of SEQ ID NO: 7 or SEQ ID NO: 8 is In certain embodiments, the first subunit comprises an amino acid sequence having at least 93% of the amino acid sequence. The first and / or second subunits are selected from the group consisting of residues SEQ ID NO: 7 and SEQ ID NO: 8, respectively. Up to 1, 2, 3, 4, 5, 6, 7, 8, 9 for residues 198–344 and 7–153 ,10,11,12,13,14,15,16,17,18,19,20,21,22, May contain 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions can.

[0045] In certain embodiments, the engineered meganuclease comprises a linker. - covalently links the first subunit and the second subunit.

[0046] In some embodiments, the engineered meganuclease has the sequence of SEQ ID NO: 7 or SEQ ID NO: 8. At least about 90%, 91%, 92%, 93%, 94%, 95%, It includes amino acid sequences with 96%, 97%, 98% or 99% sequence identity. In certain embodiments, the engineered meganuclease has the amino acid sequence of SEQ ID NO: 7: In certain embodiments, the amino acid sequence has at least 97% sequence identity. The engineered meganuclease has at least 98% identity to the amino acid sequence of SEQ ID NO:8. It includes amino acid sequences that have sequence homology.

[0047] In certain embodiments, the engineered meganuclease has the sequence of SEQ ID NO: 7 or SEQ ID NO: 8 Contains the amino acid sequence.

[0048] In certain embodiments, the engineered meganuclease is set forth as SEQ ID NO:9. Compared to the meganuclease TRC1-2x.87EE, which is Increased specificity, reduced retention time in cells, and human TCR alpha constant region gene At least one of the optimization features, such as enhancing (i.e., increasing) the efficiency of the modification of the child, Indicates one.

[0049] In a specific embodiment, a recognition sequence comprising SEQ ID NO:5 in a human TCR alpha constant region gene. The engineered meganuclease that recognizes and cleaves the sequence comprises a first subunit and a second subunit. the first subunit comprises (a) a residue of SEQ ID NO: 7 or SEQ ID NO: 8 For 198-344, at least 80%, at least 85%, at least 90%, (b) an amino acid sequence having at least 95% or more sequence identity with the sequence of SEQ ID NO: SEQ ID NO: 7 or the amino acid sequence corresponding to residues 215 to 270 of SEQ ID NO: 8, 80%, at least 85%, at least 90%, at least 95% or more the second subunit comprises an HVR1 region having sequence homology with (a) SEQ ID NO: 7 or is at least 80%, at least 85%, or at least amino acid sequences with at least 90%, at least 95%, or more sequence identity; and (b) an amino acid sequence corresponding to residues 24 to 79 of SEQ ID NO: 7 or SEQ ID NO: 8, At least 80%, at least 85%, at least 90%, at least 95%, or It contains the HVR2 region, which has at least one sequence homology with the HVR2 region.

[0050] In a specific embodiment, a recognition sequence comprising SEQ ID NO:5 in a human TCR alpha constant region gene. The engineered meganuclease that recognizes and cleaves the sequence comprises a first subunit and a second subunit. the first subunit comprises (a) a residue of SEQ ID NO: 7 or SEQ ID NO: 8 For 198-344, at least 80%, at least 85%, at least 90%, (b) an amino acid sequence having at least 95% or more sequence identity with the sequence of SEQ ID NO: SEQ ID NO: 7 or the amino acid sequence corresponding to residues 215 to 270 of SEQ ID NO: 8, 80%, at least 85%, at least 90%, at least 95% or more and has sequence homology with residues 215, 217, 219, 22 of SEQ ID NO:7 or SEQ ID NO:8 1, 223, 224, 229, 231, 233, 235, 237, 259, 261, 26 The second subunit contains the HVR1 region, which includes residues corresponding to 6 and 268, and a) at least 80%, at least have 85%, at least 90%, at least 95%, or more sequence homology and (b) an amino acid sequence corresponding to residues 24 to 79 of SEQ ID NO: 7 or SEQ ID NO: 8. For amino acid sequences, at least 80%, at least 85%, at least 90%, at least and has 95% or more sequence identity with residues of SEQ ID NO: 7 or SEQ ID NO: 8. 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 7 In such an embodiment, the HVR2 region comprises residues corresponding to 5 and 77. The two regions are comprised of residues corresponding to residues 48, 50, 71, 72 and 73 of SEQ ID NO: 7, and and / or may further comprise residues corresponding to residues 48 and 50 of SEQ ID NO:8.

[0051] In a specific embodiment, a recognition sequence comprising SEQ ID NO:5 in a human TCR alpha constant region gene. The engineered meganuclease that recognizes and cleaves the sequence comprises a first subunit and a second subunit. the first subunit comprises (a) a residue of SEQ ID NO: 7 or SEQ ID NO: 8 For 198-344, at least 80%, at least 85%, at least 90%, (b) an amino acid sequence having at least 95% or more sequence identity with the sequence of SEQ ID NO: SEQ ID NO: 7 or the amino acid sequence corresponding to residues 215 to 270 of SEQ ID NO: 8, 80%, at least 85%, at least 90%, at least 95% or more and has sequence homology with residues 215, 217, 219, 22 of SEQ ID NO:7 or SEQ ID NO:8 1, 223, 224, 229, 231, 233, 235, 237, 259, 261, 26 The second subunit contains the HVR1 region, which includes residues corresponding to 6 and 268, and a) at least 80%, at least have 85%, at least 90%, at least 95%, or more sequence homology and (b) an amino acid sequence corresponding to residues 24 to 79 of SEQ ID NO: 7 or SEQ ID NO: 8. For amino acid sequences, at least 80%, at least 85%, at least 90%, at least and has 95% or more sequence identity with residues of SEQ ID NO: 7 or SEQ ID NO: 8. 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 48, 50, 6 The HVR2 region contains residues corresponding to 8, 70, 71, 72, 73, 75, and 77. nothing.

[0052] In yet another embodiment, a recognition sequence comprising SEQ ID NO: 5 in the human TCR alpha constant region gene The engineered meganuclease that recognizes and cleaves the sequence comprises a first subunit and a second subunit. the first subunit comprises (a) a residue of SEQ ID NO: 7 or SEQ ID NO: 8 For groups 198 to 344, at least 80%, at least 85%, at least 90%, (b) an amino acid sequence having at least 95% or more sequence identity with the sequence HVR1 having an amino acid sequence corresponding to residues 215 to 270 of SEQ ID NO: 7 or SEQ ID NO: 8 the second subunit comprises a region comprising (a) residues 7-15 of SEQ ID NO: 7 or SEQ ID NO: 8; 3, at least 80%, at least 85%, at least 90%, at least 95% or more sequence homology, and (b) an amino acid sequence having SEQ ID NO: 7 or SEQ ID NO: It contains an HVR2 region having an amino acid sequence corresponding to residues 24 to 79 of sequence number 8.

[0053] In another aspect, the present invention provides a method for producing a nucleic acid encoding the engineered meganuclease described herein. A polynucleotide comprising a nucleic acid sequence that

[0054] In certain embodiments, the polynucleotide is mRNA.

[0055] In a further embodiment, the mRNA is synthesized using the engineered meganucleases and and a polycistronic molecule encoding at least one additional polypeptide or nucleic acid. It is RNA.

[0056] In another aspect, the present invention provides a recombinant DNA molecule comprising a polynucleotide described herein. A construct is provided.

[0057] In certain embodiments, the recombinant DNA construct encodes a viral vector. In this embodiment, the viral vector is an adenoviral vector, a lentiviral vector, or , retroviral vectors or adeno-associated viral (AAV) vectors. In certain embodiments, the viral vector is a recombinant AAV vector.

[0058] In another aspect, the invention provides a viral vector comprising a polynucleotide described herein. -Provide.

[0059] In certain embodiments, the viral vector is an adenoviral vector, a lentiviral vector, or a In certain embodiments, the vector is a viral vector, a retroviral vector, or an AAV vector. In the present invention, the viral vector is a recombinant AAV vector.

[0060] In another aspect, the present invention provides a method for the production of a gene encoding an exogenous sequence of interest, comprising: A method for producing a genetically modified eukaryotic cell is provided, the method comprising: (a) generating a gene encoding a gene encoding a gene for ... and encoding the engineered meganuclease of claim 1, wherein the engineered meganuclease is expressed in eukaryotic cells. (b) an engineered meganuclear cell containing a first nucleic acid expressed in the cell; and The enzyme creates a cleavage site in the chromosome at a recognition sequence containing SEQ ID NO: 5, and the target sequence is and a second nucleic acid to be inserted into the chromosome at the cleavage site. This includes introducing the compound into cells.

[0061] In certain embodiments of the method, the second nucleic acid contains a sequence homologous to a sequence adjacent to the cleavage site. The sequence further comprises a sequence, and a sequence of interest is inserted at the cleavage site by homologous recombination.

[0062] In certain embodiments of the method, the second nucleic acid contains a sequence homologous to a sequence adjacent to the cleavage site. The sequence does not contain the cleavage site, and the sequence of interest is inserted at the cleavage site by non-homologous insertion.

[0063] In certain embodiments of the method, endogenous T cell receptors (e.g., alpha / beta T Cell surface expression of the IL-1 receptor (IL-1 receptor) was significantly lower than that of unmodified control cells. It is down.

[0064] In some embodiments of the method, the eukaryotic cell is a human T cell or a cell derived from a human T cell. cells, or human NK cells, or cells derived from human NK cells.

[0065] In some embodiments of the method, the sequence of interest is a chimeric antigen receptor or an exogenous T cell receptor. In certain embodiments of the method, the chimeric antigen receptor or the exogenous antigen receptor is a coding sequence for the exogenous antigen receptor. T-cell receptors contain extracellular ligand-binding domains with specificity for tumor-specific antigens. Includes.

[0066] In some embodiments of the method, the at least first nucleic acid is expressed in a eukaryotic cell by mRNA. It will be introduced.

[0067] In certain embodiments of the method, the at least second nucleic acid is delivered by a viral vector. In certain embodiments of the method, the viral vector is an adenovirus. vectors, lentiviral vectors, retroviral vectors, or AAV vectors In certain embodiments of the method, the viral vector is a recombinant AAV vector. .

[0068] In another aspect, the present invention provides a method for the production of a gene encoding an exogenous sequence of interest, comprising: A method for producing a genetically modified eukaryotic cell is provided, the method comprising: (a) generating a gene encoding a gene encoding a gene for ... (b) introducing the engineered meganuclease described in (a) into a eukaryotic cell; and introducing into a eukaryotic cell a nucleic acid comprising the sequence of However, a cleavage site is created in the chromosome with a recognition sequence containing SEQ ID NO: 5, and the target sequence is and insertion into the chromosome at the break site.

[0069] In certain embodiments of the method, the nucleic acid further comprises a sequence homologous to the sequence adjacent to the cleavage site. and the sequence of interest is inserted at the cleavage site by homologous recombination.

[0070] In certain embodiments of the method, the nucleic acid comprises a sequence homologous to a sequence adjacent to the cleavage site. First, the sequence of interest is inserted at the cleavage site by non-homologous transfection.

[0071] In certain embodiments of the method, endogenous T cell receptors (e.g., alpha / beta T Cell surface expression of the IL-1 receptor (IL-1 receptor) was significantly lower than that of unmodified control cells. It is down.

[0072] In some embodiments of the method, the eukaryotic cell is a human T cell or a cell derived from a human T cell. cells, or human NK cells, or cells derived from human NK cells.

[0073] In some embodiments of the method, the sequence of interest is a chimeric antigen receptor or an exogenous T cell receptor. In certain embodiments of the method, the chimeric antigen receptor or the exogenous antigen receptor is a coding sequence for the exogenous antigen receptor. T-cell receptors contain extracellular ligand-binding domains with specificity for tumor-specific antigens. Includes.

[0074] In certain embodiments of the method, the nucleic acid is introduced into the eukaryotic cell by a viral vector. In certain embodiments of the method, the viral vector is an adenoviral vector, a lenthral vector, or a lenthral vector. The vector is an immunoviral vector, a retroviral vector, or an AAV vector. In certain embodiments, the viral vector is a recombinant AAV vector.

[0075] In another aspect, the present invention provides a method for disrupting a target sequence in a chromosome of a eukaryotic cell to induce genetic A method for producing a genetically modified eukaryotic cell is provided, the method comprising the steps of: and introducing into a eukaryotic cell a nucleic acid encoding the engineered meganuclease. The engineered meganucleases are expressed in eukaryotic cells, and the engineered meganucleases are A cleavage site is created in the chromosome using a recognition sequence containing SEQ ID NO: 5, and cleavage occurs by non-homologous end joining. Destroys the target sequence at the site.

[0076] In certain embodiments of the method, endogenous T cell receptors (e.g., alpha / beta T Cell surface expression of the IL-1 receptor (IL-1 receptor) was significantly lower than that of unmodified control cells. It is down.

[0077] In some embodiments of the method, the eukaryotic cell is a human T cell or a cell derived from a human T cell. cells, or human NK cells, or cells derived from human NK cells.

[0078] In some embodiments of the method, the nucleic acid is introduced into the eukaryotic cell by mRNA.

[0079] In another aspect, the present invention provides a method for disrupting a target sequence in a chromosome of a eukaryotic cell to induce genetic A method for producing a genetically modified eukaryotic cell is provided, the method comprising the steps of: and introducing the engineered meganuclease into a eukaryotic cell. The enzyme creates a cleavage site in the chromosome at a recognition sequence containing SEQ ID NO:5, and then cleaves the chromosome by non-homologous end joining. The target sequence is destroyed at the cleavage site.

[0080] In certain embodiments of the method, endogenous T cell receptors (e.g., alpha / beta T Cell surface expression of the IL-1 receptor (IL-1 receptor) was significantly lower than that of unmodified control cells. It is down.

[0081] In some embodiments of the method, the eukaryotic cell is a human T cell or a cell derived from a human T cell. cells, or human NK cells, or cells derived from human NK cells.

[0082] In another aspect, the present invention provides a method for detecting a human T cell receptor alpha constant region gene in a genome. and a genetically modified eukaryotic cell comprising a modified human T-cell receptor alpha constant region gene. The gene is inserted into exon 1 of SEQ ID NO:5 in the T cell receptor alpha constant region. Genetically modified eukaryotic cells containing an exogenous sequence of interest are referred to as engineered eukaryotic cells described herein. It is prepared by the methods described herein using meganucleases.

[0083] In certain embodiments, the genetically modified eukaryotic cells are genetically modified human T cells. or cells derived from human T cells, or human NK cells, or human NK cells These are induced cells.

[0084] In certain embodiments, the sequence of interest is a chimeric antigen receptor or an exogenous T cell receptor. In certain embodiments, the coding sequence for a chimeric antigen receptor or an exogenous T cell The receptor contains an extracellular ligand-binding domain with specificity for a tumor-specific antigen.

[0085] In certain embodiments, endogenous T cell receptors (e.g., alpha / beta T cell receptors) The cell surface expression of the gene-modified β-lactamase (GlcNAc) was significantly higher than that of the unmodified control cells. It is reduced in eukaryotic cells.

[0086] In certain embodiments, the genetically modified eukaryotic cell is set forth as SEQ ID NO: 9. Compared to the meganuclease TRC1-2x.87EE, the engineered meganuclease Reduction of off-target effects by enzymes and / or engineered meganucleases in cells This includes reducing the retention time of the enzyme.

[0087] In another aspect, the present invention provides a target sequence disrupted with a recognition sequence comprising SEQ ID NO:5. and a target sequence comprising a cleavage site. The genetically modified eukaryotic cells are disrupted by non-homologous end joining at the nucleotide level and subjected to the manipulations described herein. The antibody is prepared by the methods described herein using engineered meganucleases.

[0088] In certain embodiments, the genetically modified eukaryotic cells are genetically modified human T cells. or cells derived from human T cells, or human NK cells, or human NK cells These are induced cells.

[0089] In certain embodiments, endogenous T cell receptors (e.g., alpha / beta T cell receptors) The cell surface expression of the gene-modified β-lactamase (GlcNAc) was significantly higher than that of the unmodified control cells. It is reduced in eukaryotic cells.

[0090] In certain embodiments, the genetically modified eukaryotic cell is set forth as SEQ ID NO: 9. Compared to the meganuclease TRC1-2x.87EE, the engineered meganuclease These include reducing off-target effects of the enzyme and / or reducing its retention time in cells. nothing.

[0091] In another aspect, the invention includes a plurality of genetically modified eukaryotic cells as described herein, A population of genetically modified eukaryotic cells is provided.

[0092] In some embodiments, at least 10%, at least 15%, at least 2% of the population 0%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, At least 90%, at least 95%, at least 96%, at least 97%, at least At least 98%, at least 99%, or up to 100% of the cells are These are genetically modified eukaryotic cells such as:

[0093] In certain embodiments, the genetically modified eukaryotic cells of the population are genetically modified human T cells. human T cell-derived cells, or genetically modified NK cells, or are cells derived from these NK cells.

[0094] In certain embodiments, the genetically modified eukaryotic cells of the population are cell surface chimeric antigen receptors. In some of these embodiments, the antigen comprises a chimeric antigen receptor or an exogenous T cell receptor. Receptors or exogenous T cell receptors are extracellular ligands with specificity for tumor-specific antigens. It contains a nucleotide-binding domain.

[0095] In certain embodiments, the genetically modified eukaryotic cells of the population are isolated from an unmodified control eukaryotic cell. endogenous T cell receptors (e.g., alpha / beta T cell receptors) compared with human T cell ) cell surface expression.

[0096] In another aspect, the present invention provides a drug useful for treating a disease in a subject in need thereof. The pharmaceutical composition comprises a pharmaceutically acceptable carrier and a therapeutically effective amount of The present invention includes a genetically modified eukaryotic cell or population thereof as described herein.

[0097] In certain embodiments, the genetically modified eukaryotic cell or population thereof is a genetically modified eukaryotic cell. human T cells, or cells derived from such human T cells, or genetically modified N It consists of K cells or cells derived from NK cells.

[0098] In some embodiments, the target gene present in the genetically modified T cell or population thereof is The exogenous sequence includes a coding sequence for a chimeric antigen receptor or an exogenous T cell receptor. In certain embodiments, the chimeric antigen receptor or exogenous T cell receptor is a tumor-specific antigen receptor. It comprises an extracellular ligand-binding domain with specificity for

[0099] In some embodiments, endogenous T cell receptors (e.g., alpha / beta T cell receptors) The cell surface expression of the gene-modified β-lactamase (GlcNAc) was significantly higher than that of the unmodified control cells. It is reduced in eukaryotic cells.

[0100] In another aspect, the present invention provides at least one engineered meganuclear vector as described herein. The present invention provides a lipid nanoparticle or a lipid nanoparticle formulation comprising mRNA encoding a lipase. In some embodiments, the lipid nanoparticles are soluble in a composition that has increased transport and uptake by T cells. It has a composition.

[0101] In another aspect, the present invention provides a method for treating a disease in a subject in need thereof, comprising: The method includes administering a therapeutically effective amount of a genetically modified eukaryotic cell or population thereof described herein to a mammalian animal, comprising: administering to a subject.

[0102] In some embodiments, the methods include administering to a subject a pharmaceutical composition described herein. Includes.

[0103] In certain embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of a compound selected from the group consisting of acetaminophen, benzodiazepine ... In some such embodiments, the genetically modified eukaryotic cells are modified human T cells, or cells derived from such human T cells, or genetically modified Human NK cells or cells derived from such human NK cells, which have been genetically modified The exogenous sequence of interest present in the eukaryotic cell is used to express cells with specificity for tumor-specific antigens. For chimeric antigen receptors or exogenous T cell receptors containing extracellular ligand-binding domains a coding sequence for an endogenous T cell receptor (e.g., alpha / beta T cell receptor) Cell surface expression was significantly higher in the genetically modified thymocytes compared to unmodified control cells. It is reduced in nuclear cells.

[0104] In some embodiments of the method, the cancer is selected from the group consisting of carcinoma, lymphoma, sarcoma, blastoma, and leukemia. The cancer is selected from the group consisting of:

[0105] In certain embodiments of the method, the cancer is 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.

[0106] In certain embodiments of the method, the cancer of B-cell origin is B-cell acute lymphoblastic leukemia, B A group consisting of B-cell chronic lymphocytic leukemia, B-cell non-Hodgkin's lymphoma, and multiple myeloma are selected.

[0107] In certain embodiments of the methods, the subject may be a mammal, such as a human.

[0108] In another aspect, the present invention provides a compound as described herein for use as a medicament. The present invention provides genetically modified cells, or populations thereof, for use in a subject in need of treatment. In the manufacture of a drug for treating a disease in a mammal, the genetically modified In one such embodiment, the medicament is a It is useful.

[0109] In another aspect, the present invention provides a compound comprising: A genetically modified cell, or population thereof, as described herein is provided. [Brief explanation of the drawings]

[0110] [Figure 1] This is the TRC1-2 recognition sequence in the human T cell receptor alpha constant region gene. The TRC1-2 recognition sequence targeted by the engineered meganuclease of the present invention contains two recognition half-sites. Each recognition half-site contains 9 base pairs, which are separated by a 4 base pair central sequence. The TRC1-2 recognition sequence (SEQ ID NO: 5) contains two recognition half-sites designated TRC1 and TRC2. [Figure 2] The engineered meganucleases of the present invention comprise two subunits, with a first subunit comprising an HVR1 region binding to a first recognition half-site (e.g., TRC1), and a second subunit comprising an HVR2 region binding to a second recognition half-site (e.g., TRC2). In embodiments where the engineered meganuclease is a single-chain meganuclease, the first subunit comprising an HVR1 region can be positioned as either the N-terminal or C-terminal subunit. Similarly, the second subunit comprising an HVR2 region can be positioned as either the N-terminal or C-terminal subunit. [Figure 3]1 shows a schematic of a reporter assay in CHO cells to evaluate the engineered meganucleases of the present invention. CHO cells were generated in which reporter activity was stably integrated into the cellular genome. The reporter cassette contained, from 5' to 3', an SV40 early promoter, the 5' two-thirds of the GFP gene, a recognition sequence for the engineered meganuclease of the present invention (e.g., the TRC1-2 recognition sequence), a recognition sequence for the CHO23 / 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 cleavage-inducing agent. The meganucleases were introduced by transfecting plasmid DNA or mRNA encoding each meganuclease. When DNA cleavage was induced at either of the meganuclease recognition sequences, the overlapping regions of the GFP gene recombined with each other to produce a functional GFP gene. The proportion of GFP-expressing cells could then be determined by flow cytometry, which is an indirect measure of the frequency of genome cleavage by the meganuclease. [Figure 4] Figure 1 shows the efficiency of engineered meganucleases to recognize and cleave the TRC1-2 recognition sequence in a CHO cell reporter assay. The TRC1-2L.1592, TRC1-2L.1775, and TRC1-2L.1843 meganucleases were engineered to target the TRC1-2 recognition sequence (SEQ ID NO: 5) and screened for efficacy in a CHO cell reporter assay. The results shown provide the observed percentage of GFP-expressing cells, indicating the efficacy of each meganuclease to cleave the target recognition sequence or the CHO23 / 24 recognition sequence. A negative control (bs) and the first-generation TRC1-2x.87EE were also included in the comparative assays. A) CHO reporter assay for recognizing TRC1-2L.1592. B) CHO reporter assay for recognizing TRC1-2L.1775. C) CHO reporter assay for recognizing TRC1-2L.1843. [Figure 5]Figure 1 shows the efficiency of engineered meganucleases to recognize and cleave the TRC Off 1 recognition sequence (SEQ ID NO: 16) and the TRC Off 2 recognition sequence (SEQ ID NO: 17) in a CHO reporter assay. mRNA encoding the TRC1-2 meganuclease of the invention was transfected into CHO reporter cells containing the counterselected Off 1 or Off 2 recognition sequence between GFP direct repeats and the CHO23-24 recognition sequence. The second-generation meganucleases were compared in each assay to the first-generation TRC1-2x.87EE. A) Cleavage of the off-target recognition sequence by TRC1-2L.1592 and TRC1-2x.87EE. B) Cleavage of the off-target recognition sequence by TRC1-2L.1775 and TRC1-2x.87EE. C) Cleavage of the off-target recognition sequence by TRC1-2L.1843 and TRC1-2x.87EE. [Figure 6] Figure 1 shows the efficiency of engineered meganucleases to recognize and cleave the TRC1-2 recognition sequence in a CHO cell reporter assay. The TRC1-2x.87EE (first generation), TRC1-2L.1108 (intermediate), and TRC1-2L.1469 (intermediate) meganucleases were engineered to target the TRC1-2 recognition sequence (SEQ ID NO: 5) and screened for efficacy in a CHO cell reporter assay 2, 5, and 7 days after nucleofection to measure toxicity. The results shown provide the percentage of GFP-expressing cells observed over the 7-day analysis period. This shows the efficacy of each meganuclease to cleave the target recognition sequence or the CHO23 / 24 recognition sequence as a function of time. [Figure 7]Figure 1 shows the efficiency of engineered meganucleases to recognize and cleave the TRC1-2 recognition sequence in a CHO cell reporter assay. Second-generation meganucleases TRC1-2L.1592, TRC1-2L.1775, and TRC1-2L.1843 were optimized to target the TRC1-2 recognition sequence (SEQ ID NO: 5) and screened for efficacy in a CHO cell reporter assay 2, 5, and 7 days after nucleofection to measure toxicity. The first-generation TRC1-2x.87EE meganuclease and the intermediate TRC1-2L.1469 meganuclease were also included in the assay for comparison. The results shown provide the percentage of GFP-expressing cells observed over the 7-day analysis period. This shows the efficacy of each meganuclease to cleave the target recognition sequence or the CHO23 / 24 recognition sequence as a function of time. [Figure 8] Figure 1 shows the efficiency of engineered meganucleases to recognize and cleave the TRCOff1-Off2 recognition sequences in a CHO cell reporter assay. The first-generation TRC1-2x.87EE meganuclease, the intermediate TRC1-2L.1469 meganuclease, and the second-generation TRC1-2L.1592, TRC1-2L.1775, and TRC1-2L.1843 meganucleases were screened for efficacy in a CHO cell reporter assay 2, 5, and 7 days after nucleofection in CHO GFFP reporter cells containing the TRCOff1 (SEQ ID NO: 16) or Off2 (SEQ ID NO: 17) recognition sequences to measure toxicity. The results shown provide the percentage of GFP-expressing cells observed over the 7-day analysis period. A) Cleavage of the Off1 recognition sequence. B) Cleavage of the Off2 recognition sequence. [Figure 9] Graphical visualization of oligo capture data as a measure of the number of potentially plausible off-target sites. Each off-target cleavage generated by a specific nuclease is plotted based on the number of unique sequence reads for the probe oligo captured at that site. The target site (i.e., the TRC1-2 recognition sequence) has the highest aggregate read count for each meganuclease tested (circled). [Figure 10] Graphical visualization of oligo capture data in which off-target sites are plotted according to read number listed on the x-axis and the number of mismatches compared to the target site indicated by color, with darker colors indicating nearly complete matches between the off-target site and the target binding site. Boxes indicate regions of highest confidence. [Figure 11] This table summarizes the in vitro analysis of CAR T cells generated using the first-generation TRC1-2x.87EE meganuclease, the intermediate TRC1-2L.1469 meganuclease, or the second-generation TRC1-2L.1592, TRC1-2L.1775, and TRC1-2L.1843 meganucleases. Meganucleases were screened for gene editing efficiency, post-editing proliferation, and differentiation potential. CAR T cells were prepared from cells obtained from three different healthy human donors, and experiments were performed by three different operators. [Figure 12] Graphical visualization of oligo capture data generated on T cell populations obtained from three different healthy human donors. [Figure 13] In vitro analysis of CAR T cells generated with first-generation TRC1-2x.87EE meganuclease or second-generation TRC1-2L.1592, TRC1-2L.1775, and TRC1-2L.1843 meganucleases. A) Total number of cells after editing on days 0, 4, and 8. B) Total number of edited cells (i.e., TCR-negative) after editing on days 0, 4, and 8. C) Total number of TCR-negative / CAR-positive cells after editing on days 0, 4, and 8. [Figure 14] CAR T cell proliferation following co-culture with antigen-associated target cells. Proliferation was assessed after co-culture of CAR T cells with the CD19+ tumor lines Raji or Nalm6 at E:T ratios of 1:1 and 1:2 for 5 days. Cell input numbers are identified by dashed lines. [Figure 15]CAR T cell proliferation following co-culture with antigen-associated target cells. Proliferation was assessed after co-culture of CAR T cells with the CD19+ tumor line Raji at an E:T ratio of 1:2 for 5 days. A) Total number of CAR-positive cells in culture after co-culture with Raji cells. B) Total number of CD19-positive cells remaining in culture after co-culture of CAR T cells with Raji cells. [Figure 16] CAR T cell cytokine secretion into culture supernatant after co-culture with antigen-associated target cells for 2 days. Cytokine secretion was assessed after co-culture of CAR T cells with the CD19+ tumor lines Raji or Nalm6 at E:T ratios of 1:1 and 1:2. CD19-negative K562 myeloid leukemia cells were used as controls. A) IL-2 secretion B) TNF-alpha secretion C) INF-gamma secretion D) Granzyme B secretion E) Perforin secretion [Figure 17] Western blot analysis of meganuclease expression in CAR T cells. Cells were electroporated with mRNA encoding TRC1-2x.87EE or TRC1-2L.1592 meganuclease, and then transduced with a recombinant AAV6 vector carrying a donor template encoding an anti-CD19 CAR designed to be inserted at the TRC1-2 site. After electroporation at 6, 24, 48, 96, and 168 hours, meganuclease protein expression was measured by Western blot analysis. Mock cells from the same donor were activated as the nuclease treatment group and cultured in the same medium. These mock cells were also collected at 24 hours after the nuclease treatment group had been electroporated. [Figure 18] Total viable cell counts at days 0, 3, 8 (before and after CD3-positive cell depletion) and 13 of the large-scale CAR T manufacturing process using TRC1-2x.87EE or TRC1-2L.1592. [Figure 19]Total viable CD3-negative cell counts at day 8 of the large-scale CAR T manufacturing process using TRC1-2x.87EE or TRC1-2L.1592. [Figure 20] Percentage of CD3-negative cells that are CAR-positive at day 8 (before and after depletion of CD3-positive cells) and day 13 of the large-scale CAR T manufacturing process using TRC1-2x.87EE or TRC1-2L.1592.

[0111] A brief description of arrays SEQ ID NO: 1 is the sequence of Chlamydomonas reinhardtii The amino acid sequence of wild-type I-Crel meganuclease derived from Bacillus inhardtii was determined. is doing.

[0112] SEQ ID NO: 2 defines the amino acid sequence of the LAGLIDADG motif.

[0113] SEQ ID NO: 3 is the human T cell receptor alpha constant region gene (NCBI gene no. 287 55) nucleic acid sequence.

[0114] SEQ ID NO: 4 is the polypeptide encoded by the human T cell receptor alpha constant region gene. It defines the amino acid sequence of the peptide.

[0115] SEQ ID NO:5 defines the nucleic acid sequence of the sense strand of the TRC1-2 recognition sequence.

[0116] SEQ ID NO: 6 defines the nucleic acid sequence of the antisense strand of the TRC1-2 recognition sequence.

[0117] SEQ ID NO: 7 defines the amino acid sequence of the TRC1-2L.1592 meganuclease. There are.

[0118] SEQ ID NO: 8 defines the amino acid sequence of the TRC1-2L.1775 meganuclease. There are.

[0119] SEQ ID NO: 9 defines the amino acid sequence of the TRC1-2x.87EE meganuclease. There are.

[0120] SEQ ID NO: 10 is the TRC1-2L.1592 meganuclease TRC1 binding subunit It specifies the amino acid sequence of the

[0121] SEQ ID NO: 11 is the TRC1-2L.1775 meganuclease TRC1 binding subunit It specifies the amino acid sequence of the

[0122] SEQ ID NO: 12 is the TRC1-2x.87EE meganuclease TRC1 binding subunit It specifies the amino acid sequence of the

[0123] SEQ ID NO: 13 is the TRC1-2L.1592 meganuclease TRC2 binding subunit It specifies the amino acid sequence of the

[0124] SEQ ID NO: 14 is the TRC1-2L.1775 meganuclease TRC2 binding subunit It specifies the amino acid sequence of the

[0125] SEQ ID NO: 15 is the TRC1-2x.87EE meganuclease TRC2 binding subunit It specifies the amino acid sequence of the

[0126] SEQ ID NO: 16 defines the nucleic acid sequence of the Off1 recognition sequence.

[0127] SEQ ID NO: 17 defines the nucleic acid sequence of the Off2 recognition sequence.

[0128] SEQ ID NO: 18 defines the amino acid sequence of the polypeptide linker.

[0129] 1.1 References and Definitions The patent and scientific literature referenced herein establishes knowledge that is available to those skilled in the art. Genbank database sequences, public gene databases and protein databases Accession number or code (and its associated nucleic acid and / or amino acid sequence) issued U.S. and non-U.S. patents cited herein, which contain the amino acid sequence of Allowed applications, published U.S. applications, published non-U.S. applications, and PCT applications, shared and co-pending unpublished U.S. patent applications, published foreign applications, and scientific, technical, and medical references, each of which is incorporated by reference, are specific and individual and the like, all of which are incorporated herein by reference to the same extent as if set forth herein.

[0130] This invention may be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments should not be construed as construed as constituting an entire or complete disclosure. , are provided to fully convey the scope of the present invention to those skilled in the art. For example, with respect to one embodiment, The illustrated features may be incorporated into other embodiments and may not be illustrated with respect to a particular embodiment. Any feature may be omitted from the embodiment. Numerous variations and additions to the embodiments will be apparent to those skilled in the art in light of this disclosure. does not depart from the invention.

[0131] Unless otherwise defined, all technical and scientific terms used herein are defined by the principles of the present invention. The meaning is the same as that commonly understood by a person skilled in the art as of the priority date. The terminology used in the description of the invention herein has the same meaning as that of the particular implementation. They are for illustrative purposes only and are not intended to be limiting of the invention.

[0132] All publications, patent applications, patents, and other references mentioned herein are hereby incorporated by reference. No. 6,239,999, which is incorporated herein by reference in its entirety.

[0133] As used herein, "a," "an," or "this" refers to "the" can mean one or more. For example, "a" cell can refer to a single cell or multiple cells.

[0134] As used herein, unless otherwise indicated, the term "or" means "either" or It is used in the inclusive sense of "and / or" rather than the exclusive sense of "either / or."

[0135] As used herein, the term "endonuclease" refers to an enzyme that binds to a polynucleotide. Refers to an enzyme that breaks phosphodiester bonds within a chain.

[0136] As used herein, with respect to double-stranded DNA, the terms "cut" or "cleavage" refer to Such a term results in a double-stranded break within the target sequence, referred to herein as the "cleavage site." The target sequence is then subjected to endonuclease-mediated phospho-ligation within the backbone of the recognition sequence. This refers to the hydrolysis of phosphodiester bonds. Some endonucleases cleave the fragments to blunt ends. Double-stranded fragments with termini or fragments with 5' or 3' overhangs Fragmentation may occur.

[0137] As used herein, the term "meganuclease" refers to a nucleic acid having more than 12 base pairs. It refers to an endonuclease that binds to double-stranded DNA at a recognition sequence. The recognition sequence of the disclosed meganuclease is 22 base pairs. rel, e.g., an endonuclease having DNA binding specificity, D Comparison with native I-Crel in terms of NA cleavage activity, DNA binding affinity, or dimerization properties The term "I-Crel" may refer to an engineered variant of I-Crel that has been modified in such a way that it is Methods for producing such variants are known in the art (see, for example, International Publication No. (Patent Publication No. 2007 / 047859, which is incorporated by reference in its entirety). As used herein, a meganuclease binds to double-stranded DNA as a heterodimer. Meganucleases have also been developed in which a pair of DNA-binding domains is linked together using a peptide linker. It may be a "single-chain meganuclease" that is bound to a single polypeptide. The term "meganuclease" is synonymous with the term "meganuclease." The meganucleases of the present disclosure are expressed in the target cells described herein, particularly human T cells. are substantially non-toxic when administered and have an overall No substantial adverse effects on cell viability or significant changes in meganuclease cleavage activity Cells can be transfected and maintained at 37°C without any noticeable degradation.

[0138] As used herein, the term "single-chain meganuclease" refers to a single-chain meganuclease comprising a subunit The linker is inserted so that the two interact functionally as heterodimers to cleave the double-stranded recognition site. A single-chain polypeptide refers to a polypeptide that contains a pair of nuclease subunits bound by a single chain. Meganucleases are composed of an N-terminal subunit, a linker, and a C-terminal subunit. The two meganuclease subunits generally have the same amino acid sequence. It recognizes non-identical DNA half sites within the recognition sequence, rather than a single A multi-strand meganuclease typically recognizes pseudo-palindromic or non-palindromic sequences. Single-chain meganucleases are not actually dimeric but are "single-stranded heterodimers." These are sometimes called "dimeric" or "single-chain heterodimeric meganucleases." Therefore, unless otherwise specified, the term "meganuclease" refers to either a dimer or a single-chain It can refer to meganucleases.

[0139] As used herein, the term "linker" refers to a linker that connects two meganuclease subunits. Refers to an exogenous peptide sequence used to join units into a single polypeptide. The linker may have a sequence found in the naturally occurring protein, or may be a sequence that is unique to any naturally occurring protein. The linker may be an artificial sequence not found in any known molecule. The linker is flexible and lacks secondary structure. or may have a tendency to form a particular three-dimensional structure under physiological conditions. See, but not limited to, U.S. Patent Nos. 8,445,251, 9,340,777, 9,434,931, and any of those encompassed by 10,041,053 The present invention may include any of the following, each of which is incorporated by reference in its entirety: In embodiments, the linker is at least 80%, at least 85%, at least 90% , at least 91%, at least 92%, at least 93%, at least 94%, at least At least 95%, at least 96%, at least 97%, at least 98%, at least 99% %, or more, of sequence homology with SEQ ID NO: 18, which may be SEQ ID NO: 7 or defines residues 154-195 of SEQ ID NO: 8. In some embodiments, the linker has the sequence Amino acids containing SEQ ID NO: 18, defining residues 154-195 of SEQ ID NO: 7 or SEQ ID NO: 8 It may have an array.

[0140] As used herein, the terms "recombinant" or "engineered" with respect to proteins The term refers to a nucleic acid that encodes a protein and a cell or organism that expresses the protein. In contrast, it means that the amino acid sequence has been altered as a result of the application of genetic engineering techniques. With respect to nucleic acids, the terms "recombinant" or "engineered" refer to the use of genetic engineering techniques. It means that the nucleic acid sequence has been altered as a result of the application of genetic engineering techniques. PCR and DNA cloning techniques, gene transfer, transformation and other gene transfer Techniques include, but are not limited to, homologous recombination, site-directed mutagenesis, and gene fusion By this definition, a protein has the same amino acid sequence as a naturally occurring protein. However, proteins produced by cloning and expression in a heterologous host are considered recombinant. is not considered.

[0141] As used herein, the term "wild type" refers to a population of alleles of a homogenous gene. The most common naturally occurring alleles (i.e., polynucleotide sequences) in The polypeptide encoded by the wild-type allele has its native function. The term "wild-type" also refers to a polypeptide encoded by a wild-type allele. Wild-type alleles (i.e., polynucleotides) and polypeptides refer to wild-type Mutants or variants containing one or more mutations and / or substitutions to the sequence It is possible to distinguish between variant alleles and polypeptides. A polypeptide can confer a normal phenotype to an organism, but a mutant or mutated polypeptide can A variant allele or polypeptide can, in some cases, confer an altered phenotype. Wild-type nucleases are distinguished from recombinant or non-naturally occurring nucleases. The term "wild type" also refers to the wild-type allele of a particular gene. Cells, organisms, and / or subjects retained or used for comparative purposes; and / or can refer to an object.

[0142] As used herein, the term "genetic modification" refers to a process in which a genomic DNA sequence is recombined. Refers to a cell or organism that has been intentionally altered by technology, or in its ancestors. As used herein, the term "genetically modified" is also used interchangeably with "genetically recombinant." This includes terms such as:

[0143] As used herein with respect to recombinant proteins, the term "modified" refers to a modification of a reference sequence. Any insertion of an amino acid residue in a recombinant sequence relative to a sequence (e.g., a wild-type or native sequence) , deletion, or substitution.

[0144] As used herein, the term "recognition sequence" or "recognition site" refers to an endonuclease The term refers to the DNA sequence that is bound and cleaved by a meganuclease. The recognition sequence consists of a pair of inverted 9-base pair "half sites" separated by 4 base pairs. In the case of single-chain meganucleases, the N-terminal domain of the protein contains the first half-side The C-terminal domain of the protein contacts the second half-site. Cleavage by the ATPase produces a four base pair 3' "overhang." " or "sticky ends" are produced by endonuclease cleavage of double-stranded DNA sequences It is a short single-stranded DNA segment that is obtained by I-Crel-derived meganuclease. For single-stranded meganucleases, the overhang is 10–15 bp of the 22-base pair recognition sequence. Contains 13 bases.

[0145] As used herein, the term "target site" or "target sequence" refers to a region of a cell's chromosomal DNA that contains a recognition sequence for a nuclease.

[0146] As used herein, the term "DNA binding affinity" or "binding affinity" refers to The meganuclease binds non-covalently to a reference DNA molecule (e.g., a recognition sequence or any sequence). The binding affinity is measured by the dissociation constant Kd. As used herein, the Kd of a nuclease for a reference recognition sequence is the Kd of the reference nuclease A statistically significant percent change or a biologically significant amount (e.g., at least If the activity of the nuclease increases or decreases by only 2-fold, or 2-fold to 10-fold, then the nuclease is "transformed." It has a binding affinity similar to that of the ATP.

[0147] As used herein, the term "specificity" refers to the specificity of the base pairs called recognition sequences. recognize and cleave double-stranded DNA molecules only at specific sequences or only at specific sets of recognition sequences The set of recognition sequences is defined as a set of sequences that are conserved at certain positions or sequences. They share a common sequence motif but may be degenerate at one or more positions. Highly specific nucleases can cleave only one or a few recognition sequences. Sex can be determined by any method known in the art.

[0148] As used herein, a nuclease refers to a nuclease that, under physiological conditions, acts like a reference nuclease (e.g., When a protein binds to and cleaves a recognition sequence that has not been bound and cleaved by a target protein (e.g., a wild-type protein), or the rate of cleavage of the recognition sequence is biologically significant (e.g., , at least 2-fold, or 2-fold to 10-fold) increase or decrease, is "changing" the specificity.

[0149] In some embodiments, the engineered meganuclease of the present disclosure is TRC1-2x.87 When compared to EE meganuclease (amino acid sequence set forth as SEQ ID NO: 9) For target recognition sequences containing SEQ ID NO: 5 (i.e., TRC1-2), Thus, in certain embodiments, the disclosed procedures The constructed meganuclease was compared with the TRC1~2x.87EE meganuclease. This indicates that off-target cleavage is reduced. The cleavage can be performed using, for example, oligo capture analysis as described herein, T7 endonuclease I (T7 E) Assays, digital PCR, and targeted sequencing of specific off-target sites Exome sequencing, whole genome sequencing, and labeling on streptavidin Direct disruption of belling enrichment in situ and next-generation sequencing (BLESS) ), genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE -Seq), and high-throughput genome-wide translocation sequencing via linear amplification (L AM-HTGTS) (e.g. Zischewski et al. (2017) Bio See Technology Advances 35(1):95-104. using any method known in the art, including the use of a fluorophore (which is incorporated by reference in its entirety). It can be measured using

[0150] As used herein, the term "homologous recombination" or "HR" refers to the synthesis of double-stranded DNA. A refers to the natural cellular process by which breaks are repaired using homologous DNA sequences as repair templates (e.g., Cahill et al. (2006), Front. Biosci. 1 1:1958-1976). Homologous DNA sequences are endogenous sequences that are delivered to cells. It may be a chromosomal sequence or an exogenous nucleic acid.

[0151] As used herein, the term "non-homologous end joining" or "NHEJ" refers to a A natural method for repairing double-stranded DNA breaks by direct joining of two non-homologous DNA segments. Refers to cellular processes (e.g., Cahill et al. (2006), Front. Biosci. 11:1958-1976). DNA fragmentation by non-homologous end joining. A-repair is error-prone and involves the non-templated addition or deletion of DNA sequences at the repair site. In some cases, cleavage at the target recognition sequence results in target recognition failure. NHEJ occurs at the target site. Nucleases target the coding sequence of a gene. Induced breaks and subsequent DNA repair by NHEJ are essential for the repair of fragments that disrupt gene function. Mutations such as rEM shift mutations may be introduced into the coding sequence. Using engineered nucleases, we can effectively knock out genes in cell populations. As used herein, "disruption of a target sequence" refers to disruption of a gene function. impairing the expression and / or function of the polypeptide product / expression product encoded thereby This refers to the introduction of a mutation (e.g., a frameshift mutation) that prevents the

[0152] As used herein, "homology arms" or "arms adjacent to a meganuclease cleavage site" refers to The "sequence homologous to a sequence that cleaves a nucleic acid molecule at the cleavage site generated by the meganuclease" refers to a sequence that cleaves a nucleic acid molecule at the cleavage site generated by the meganuclease. It refers to the sequences adjacent to the 5' and 3' ends of a nucleic acid that facilitate the insertion of a homologous sequence. The arms are at least 50 base pairs, preferably at least 100 base pairs, and at most 20 The nucleic acid sequence may have a length of at least 900 base pairs, and at least 90%, preferably at least 9 It may have a sequence homology to the corresponding sequence in the genome of 5% or more.

[0153] As used herein, a "chimeric antigen receptor" or "CAR" refers to a chimeric antigen receptor (CAR) that is capable of inhibiting immune effectors. engineered cells (e.g., human T cells) that confer or graft specificity for an antigen Chimeric antigen receptors typically contain at least an extracellular ligand-binding domain. a domain or portion thereof, and one or more signaling and / or costimulatory domains domains, including intracellular domains.

[0154] In some embodiments, the extracellular ligand-binding domain or portion is a monoclonal antibody. The single chain variable fragment (scFv) form is derived from a specific epitope. A target molecule or antigen (e.g., a cancer cell or other disease-causing cell or particle) provides specificity for epitopes or antigens that are preferentially present on the cell surface. In some embodiments, the scFvs are attached via a linker sequence. The extracellular ligand-binding domain is specific for any antigen or epitope of interest. In some embodiments, the scFv is a murine, humanized, or fully human scFv. .

[0155] The extracellular domain of a chimeric antigen receptor can also contain a self-antigen (Payne et al. See t al.(2016),Science 353(6295):179-184 This is recognized by autoantigen-specific B cell receptors on B lymphocytes. Therefore, it is possible to specifically target autoreactive B lymphocytes in antibody-mediated autoimmune diseases. Such CARs induce T cells to target and kill specific antigens. AAR), the use of which is encompassed by the present invention.

[0156] The extracellular domain of a chimeric antigen receptor may also bind to a naturally occurring ligand for an antigen of interest. or fragments of naturally occurring ligands that retain the ability to bind to the antigen of interest. It may include

[0157] The intracellular stimulatory domain delivers activation signals to immune effector cells following antigen binding. Such cytoplasmic signaling domains may include one or more cytoplasmic signaling domains. Transduction domains include, but are not limited to, CD31. Intracellular stimulatory domains include and transducing proliferative and / or cell survival signals after ligand binding. The antibody may comprise one or more intracellular costimulatory domains. Such intracellular costimulatory domains are well known in the art. The antigen can be any of those known in the art, such as CD27, CD28, CD8, 4-1 BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H 3, and ligases that specifically bind to CD83, N1, N6, or any combination thereof. This may include, but is not limited to,

[0158] Chimeric antigen receptors (CARs) are composed of an extracellular ligand-binding domain attached via a hinge or spacer sequence. The protein may further comprise additional structural elements, including a transmembrane domain attached to the transmembrane domain. The domain may be derived from any membrane-bound or transmembrane protein For example, transmembrane polypeptides are the subunits of the T cell receptor (i.e., α, β, γ or ζ, a polypeptide that constitutes the CD3 complex), IL2 receptor p55 (chain), p75 (β chain) or γ chain, a subunit chain of an Fc receptor (e.g., Fcy receptor III) or The transmembrane domain may be a CD protein, such as the CD8 alpha chain. Alternatively, the transmembrane domain may be a synthetic The amino acid sequence may be structural and may contain primarily hydrophobic residues such as leucine and valine.

[0159] The hinge region functions to link the transmembrane domain to the extracellular ligand-binding domain. For example, the hinge region may be up to 300 amino acids long. Preferably, the amino acid sequence contains 10 to 100 amino acids, and most preferably 25 to 50 amino acids. The hinge region can be made of all or part of the extracellular domain of CD8, CD4, or CD28. all or part of a naturally occurring molecule, such as a portion of a polypeptide chain, or all or part of an antibody constant region Alternatively, the hinge region may be derived from a synthetic sequence corresponding to a naturally occurring hinge sequence. The hinge domain may be a synthetic sequence or may be a completely synthetic hinge sequence. The antibody may contain a portion of the human CD8 alpha chain, the FcγR111 receptor, or IgG1. This can be done.

[0160] As used herein, "exogenous T cell receptor" or "exogenous TCR" refers to a T cell receptor that is expressed by a T cell receptor. Immune effector cells (e.g., human) that may or may not endogenously express R The term refers to a TCR whose sequence is introduced into the genome of a target T cell. Expression of a specific TCR binds to a specific epitope or antigen (e.g., a cancer cell or other disease-causing specific for epitopes or antigens that are preferentially present on the surface of cells or particles that cause Such exogenous T cell receptors can provide an alpha chain and a beta chain. It may comprise a gamma chain and a delta chain, or alternatively, it may comprise a gamma chain and a delta chain. The exogenous TCR used can have specificity for any antigen or epitope of interest.

[0161] As used herein, the term "down-expression" refers to a decrease in expression of a gene in a cell compared to a control cell. In this case, the endogenous T cell receptor (e.g., alpha / beta) on the cell surface of the genetically modified T cell is expressed. The term "decreased" also refers to a decrease in the expression of T cell receptors (TCRs). endogenous polypeptides (i.e., endogenous T cell receptors) on the cell surface compared to This can refer to a decrease in the proportion of cells in a population of cells that express the Large 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, It can be 95%, 96%, 97%, 98%, 99%, or even up to 100%. The term "reduction" refers to both partial and complete knockdown of endogenous T cell receptors. This includes both knockout of cell surface expression of endogenous T cell receptors (i.e., (i.e., complete knockdown) was achieved by T cell proliferation using the engineered meganucleases described herein. It can result from genetic inactivation of the T-cell receptor alpha constant region gene. The alpha constant domain, encoded by the alpha constant region gene, binds endogenously to the cell surface. The engineered meganucleic acid described herein is therefore required for the assembly of the TCR complex. Knockout of the T cell receptor alpha constant region gene using ribosomal enzymes results in the formation of T cell surface This results in a knockout of T cell receptor expression.

[0162] As used herein with respect to both amino acid and nucleic acid sequences, "percent" means a number. Terms such as "homology," "sequence identity," "percent similarity," and "sequence similarity" refer to the degree to which two sequences The term also refers to a measure of the degree of similarity between aligned amino acid residues or nucleotides. The similarity between the two sequences is maximized, and the number of identical or similar residues or nucleotides, total residues or is a function of the number of nucleotides and the presence and length of gaps in the sequence alignment It refers to a measure of the degree of similarity between two sequences based on the alignment of the sequences. Various algorithms and computer programs are available to measure sequence similarity using data. As used herein, sequence similarity is measured using the BL This is measured using the ASTp program and the BLASTn program for nucleic acid sequences. Both are National Center for Biotechnology y Information (www.ncbi.nlm.nih.gov / ) It is possible, for example, to ol.215:403-410, Gish and States (1993), Nat. ure Genet.3:266-272, Madden et al. (1996), Meth.Enzymol.266:131-141;Altschul et al. (1997), Nucleic Acids Res. 25:33 89-3402), Zhang et al.(2000),J.Comput.Biol.7(1-2): As used herein, a part of two amino acid sequences is The centroid similarity is a score based on the following parameters of the BLASTp algorithm: Word size = 3, Gap opening penalty = -11, Gap extension penalty = -1, score matrix = BLOSUM62. The percent similarity is a score based on the following parameters of the BLASTn algorithm: Character size = 11, Gap opening penalty = -5, Gap extension penalty = -2 , match reward = 1, mismatch penalty = -3.

[0163] As used herein in reference to modifications of two proteins or amino acid sequences, "pair" refers to a combination of two or more proteins or amino acid sequences. The term "corresponds to" means that a particular modification of a first protein is the same as a modification of a second protein. substitution of amino acid residues, and the two proteins are subjected to standard sequence alignment If so, (e.g., using the BLASTp program) first identify the amino acid sequence of the modification of the protein. The amino acid positions correspond to or align with the amino acid positions of the modification in the second protein. Therefore, the address of residue "X" in the first protein is The modification to amino acid "A" is such that residues X and Y correspond to each other in a sequence alignment. In the case of the second protein, despite the fact that X and Y can be different numbers, This corresponds to the modification of residue "Y" in the nucleotide sequence with amino acid "A".

[0164] As used herein, "recognition half-site," "recognition sequence half-site," or The term "half site" simply refers to the homodimeric or heterodimeric meganuclear Recognition by a monomer of a single-chain meganuclease or by one subunit of a single-chain meganuclease "DNA fragment" refers to a nucleic acid sequence in a double-stranded DNA molecule.

[0165] As used herein, the term "hypervariable region" refers to a region having a relatively high degree of variability. Refers to a localized sequence within a meganuclease monomer or subunit that contains amino acids. The variable region may be about 50-60 consecutive residues, about 53-57 consecutive residues, or preferably about In some embodiments, the hypervariable region residues can comprise 56 residues. 7 or SEQ ID NO: 8. The region can contain one or more residues that contact the DNA base of the recognition sequence, and the monomer The base preference of a given subunit or subunit can be altered to change its base preference. also binds to the DNA backbone when the meganuclease binds to the double-stranded DNA recognition sequence. It may contain one or more residues. Such residues may be present in the DNA backbone and target recognition sequences. The present invention can be modified to alter the binding affinity of the meganuclease to In different embodiments, the hypervariable region may comprise 1 to 20 residues that exhibit variability, and may be base-preferred. The polypeptides can be modified to affect their affinity and / or DNA binding affinity. In certain embodiments, the hypervariable region comprises about 15-20 residues that exhibit variability and base preference. and / or can be modified to affect DNA binding affinity.

[0166] In some embodiments, the variable residues within the hypervariable region are at positions SEQ ID NO: 7 or SEQ ID NO: 8. 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 7 5 and 77. In some embodiments, the variable residues within the hypervariable region correspond to one or more of The group is located at one or more of positions 48, 50, 71, 72, and 73 of SEQ ID NO:7. In some embodiments, the variable residue in the hypervariable region corresponds to position 48 of SEQ ID NO:8. and 50. In some embodiments, the hypervariable region The variable residues are at positions 24, 26, 28, 30, 32, 33 of SEQ ID NO: 7 or SEQ ID NO: 8. , 38, 40, 42, 44, 46, 48, 50, 68, 70, 71, 72, 73, 75 and and 77.

[0167] In other embodiments, the variable residue within the hypervariable region is at position 2 of SEQ ID NO:7 or SEQ ID NO:8. 15, 217, 219, 221, 223, 224, 229, 231, 233, 235, 2 37, 259, 261, 266, and 268.

[0168] As used herein, "T cell receptor alpha gene" or "TCR alpha" The terms "gene" and "gene" are interchangeable and refer to genes encoding the T cell receptor alpha subunit. The T cell receptor alpha is a gene locus in T cells that controls the T cell receptor alpha before and after rearrangement. See gene number 6955. Following rearrangement, the T cell receptor alpha gene , endogenous promoter, rearranged V and J segments, endogenous splice donor site , introns, endogenous splice acceptor sites, and exon-coding sequences. The T cell receptor alpha constant region locus contains the subunit.

[0169] As used herein, "T cell receptor alpha constant region" or "TCR alpha" The term "T cell receptor alpha constant region" refers to the coding sequence of the T cell receptor alpha gene. The lupha constant region contains the wild-type sequence identified by NCBI Gen number 28755 and and functional variants thereof.

[0170] "recombinant DNA construct", "recombinant construct", "expression cassette", "expression construct", The terms "chimeric construct," "construct," and "recombinant DNA fragment" are used herein. These are used interchangeably herein and refer to single-stranded or double-stranded polynucleotides. The construct contains regulatory and coding sequences that are not found together in nature. This includes, but is not limited to, any artificial combination of nucleic acid fragments, e.g., recombinant DNA constructs. The present invention relates to a method for producing a recombinant vector comprising regulatory and coding sequences derived from different sources, or derived from the same source. may contain regulatory and coding sequences arranged differently than found in nature. Such constructs may be used alone or in combination with vectors. That's fine.

[0171] As used herein, a "vector" or "recombinant DNA vector" refers to a given A replication system capable of transcribing and translating a polypeptide-encoding sequence in a host cell. If a vector is used, the choice of vector is determined by The amount of time that the vector can be transformed will depend on the method used to transform the host cell, as is well known to those skilled in the art. Vectors include plasmid vectors and recombinant viral vectors (e.g., AAV vectors). ), or to deliver the gene encoding the meganuclease of the present invention to the target cell. The vector may include, but is not limited to, any other vector known in the art suitable for carrying out the Those skilled in the art will be able to identify and identify loci containing any of the isolated nucleotide or nucleic acid sequences of the present invention. must be present on the vector for successful transformation, selection and growth of host cells. We are fully aware of the genetic factors that may prevent this.

[0172] As used herein, "vector" can also refer to a viral vector. Viral vectors include retroviral vectors, lentiviral vectors, and These may include adenovirus vectors and adeno-associated virus vectors (AAV), Not limited to these.

[0173] As used herein, a "polycistronic" mRNA refers to a gene that contains two or more coding sequences. A single message contains a sequence (i.e., a cistron) that codes for two or more proteins. Polycistronic mRNA refers to a polycistronic mRNA that contains an IRES element, a T2A element, and This includes, but is not limited to, the P2A element, the E2A element, and the F2A element. The present invention provides a method for the translation of two or more genes from the same mRNA molecule, including, but not limited to, It may include any element known in the art.

[0174] As used herein, "human T cells" or "T cells" refers to cells from a donor, particularly a human donor. T cells and their derivatives are cultured in a variety of culture media. Unfed isolated T cells, passaged and maintained under cell culture conditions without immortalization These include T cells, and T cells that have been immortalized and can be maintained indefinitely under cell culture conditions.

[0175] As used herein, a "control" or "control cell" refers to a gene cells that provide a reference point for measuring genotypic or phenotypic changes in the modified cells. Control cells include, for example: (a) wild-type cells, i.e., genetically modified cells. (b) of a genotype similar to the starting material for the genetic modification that resulted in the genetic modification; Cells of the same genotype but transformed with a null construct (i.e., a gene known to encode a trait of interest) (c) a construct that is genetically identical to the genetically modified cell but does not have the effect of , has not been exposed to conditions that induce the expression of an altered genotype or phenotype, or The present invention can include cells that have not been exposed to a stimulus or further genetic modification.

[0176] As used herein, the terms "treatment" or "treating a subject" refer to the treatment of a disease. The term "immunoglobulin" refers to the administration of a genetically modified T cell or population of genetically modified T cells of the present invention to a subject having the disease. For example, the subject may have a disease such as cancer, and the treatment may include immunotherapy for the treatment of the disease. The desired effects of treatment include prevention of disease onset or recurrence, alleviation of symptoms, Relief, reduction of the direct or indirect pathological consequences of the disease, reduction of the rate of progression of the disease, These include, but are not limited to, improvement or palliation, and remission or improved prognosis. In some embodiments, the genetically modified eukaryotic cells or genetically modified eukaryotic cells described herein are The population of cells is administered during treatment in the form of a pharmaceutical composition of the present invention.

[0177] The terms "effective amount" or "therapeutically effective amount" refer to a beneficial or desirable biological and A therapeutically effective amount refers to an amount sufficient to produce a therapeutically effective and / or clinical result. The composition, the disease and its severity, and the age, weight, physical condition and response of the subject to be treated In certain embodiments, an effective amount of the genetically modified T cells of the present invention or The population of genetically modified T cells or the pharmaceutical compositions disclosed herein may be used to treat a variety of conditions, including: In these embodiments, where the disease is cancer, the present invention provides a method for treating cancer. An effective amount of the engineered meganuclease or pharmaceutical composition disclosed herein can be used to treat cancer. Reduce the level of proliferation or metastasis, resulting in a partial or complete response or remission of the cancer or reduces at least one of the following symptoms of cancer in a subject:

[0178] As used herein, the term "cancer" refers to a cancer that causes a malignant growth or tumor. Any neoplastic disease (invasive or metastatic) characterized by abnormal and uncontrolled cell division It should be understood to include:

[0179] As used herein, the term "cytoma" refers to a malignant growth composed of epithelial cells. Refers to reproduction.

[0180] As used herein, the term "leukemia" refers to a malignant tumor of the blood-forming organ / system. , generally refers to the abnormal proliferation and development of white blood cells and their precursors in the blood and bone marrow. It is characterized by:

[0181] As used herein, the term "sarcoma" refers to a tumor composed of a substance similar to embryonic connective tissue. They are generally made up of closely packed cells embedded in a fibrillar, heterogeneous, or homogeneous substance. It refers to a tumor composed of

[0182] As used herein, the term "melanoma" refers to melanoma of the skin and other organs. It refers to a tumor arising from the uterine system.

[0183] As used herein, the term "lymphoma" refers to a hematologic tumor arising from lymphocytes. Refers to a group of tumors.

[0184] As used herein, the term "blastoma" refers to the growth of progenitor cells or blasts (immature cells). It refers to a type of cancer caused by a malignant tumor of the esophagus (or embryonic tissue).

[0185] As used herein, the recitation of a numerical range for a variable does not limit the invention to any of the values ​​within that range. The intent is to convey that the method can be implemented with variables equivalent to either For a variable that is strictly discrete, the variable can be equal to any integer value within a numeric range, including the endpoints of the range. Similarly, for a variable that is inherently continuous, the variable can be any number, including the endpoints of a range. It is equal to any real number in the range. For example, it may be described as having a value between 0 and 2. The variables can take on values ​​of 0, 1, or 2 if the variables are discrete in nature. , 0.0, 0.1, 0.01, 0.001, or if the variable is continuous in nature, It can take on any other real value >= 0 and <= 2.

[0186] 2.1 Principles of the invention The present invention provides improved (i.e., increased) specificity and reduced off-target cleavage, mR Decreased retention time in cells after expression from NA, when used in human T cells and in vitro Improved cell characteristics and cell viability when used in a full-scale CAR T cell manufacturing process It has improved characteristics compared to its parent first-generation meganucleases, including improved cellular properties. This is based in part on the discovery of optimized second-generation meganucleases that

[0187] Similar to the previously described TRC1-2x.87EE meganuclease, these optimized , second-generation meganucleases target the TR1 region within exon 1 of the TCR alpha constant region gene. C1-2 recognition sequence (SEQ ID NO: 5). Cleavage at this recognition sequence results in NHEJ is possible at the position and the expression of the human T cell receptor alpha chain subunit is This can disrupt the expression and / or function of T cell receptors on the cell surface. Additionally, cleavage at this recognition sequence directly targets the TCR alpha constant region gene. Homologous recombination of exogenous nucleic acid sequences can also be performed. The antibody can encode a telomerase antigen receptor, an exogenous TCR receptor, or any other polypeptide of interest. The compositions and methods of the present disclosure can include sequences of interest, such as sequences that encode knockout of endogenous T cell receptors (e.g., alpha / beta T cell receptors) by and exogenous nucleic acid sequences (e.g., chimeric antigen receptors or exogenous TCRs). Such cells, when administered to allogeneic subjects, can prevent graft-versus-host disease (GVHD). This may indicate a state in which the immune system is reduced or no induction of graft-versus-host disease (GVHD) is observed.

[0188] 2.2 Recognizes and cleaves the TRC1-2 recognition sequence in the T cell receptor alpha constant region gene Optimized meganucleases Uses site-specific nucleases to make DNA cuts in the genome of living cells It is possible that such DNA breaks occur in the same or highly homologous DNA fragments within the genome. Generating permanent genome alterations using homologous recombination of cleavage target sites with A sequences It is known in the art that it is possible to The present invention can be practiced using engineered recombinant meganucleases.

[0189] In certain embodiments, the nucleases used to practice the invention are single-stranded membrane Single-chain meganucleases are linked by a linker peptide. Each of the two domains contains an N-terminal subunit and a C-terminal subunit. It recognizes half of the recognition sequence (i.e., recognition half-site), and the DNA cleavage site is the site of two sequences. The recognition sequence is located in the middle of the subunit near the surface. DNA cleavage by the cleavage enzyme generates a pair of four-base-pair, 3' single-stranded overhangs. , offset by 4 base pairs.

[0190] The recombinant meganuclease of the present invention comprises the sequence of the TCR alpha constant region gene (SEQ ID NO: 3) It is engineered to recognize and cleave the TRC1-2 recognition sequence (SEQ ID NO: 5) within exon 1. The engineered meganucleases of the present invention comprise a first hypervariable (HVR1) region. a first subunit and a second subunit comprising a second hypervariable (HVR2) region; Furthermore, the first subunit contains a first recognition half-site in the recognition sequence (i.e. The second subunit binds to the second recognition half site in the recognition sequence. The recombinant meganuclease binds to the single-stranded TRC2 half-site. In embodiments where the meganuclease comprises an HVR1 region and a first half site The first subunit to bind is positioned as the N-terminal subunit and binds to the HVR2 region. The second subunit that binds to the second half-site is located as the C-terminal subunit. The first subunit and the second subunit can be oriented so that they can be attached to each other. In an alternative embodiment, a first sequence comprising the HVR1 region and binding to the first half-site is The subunit is positioned as the C-terminal subunit and contains the HVR2 region. The second subunit that binds to the fusiform site appears to be positioned as the N-terminal subunit. The first and second subunits can be directed to the TRC1. Exemplary engineered meganucleases that recognize and cleave the -2 recognition sequence are provided in Table 1. are.

[0191] [Table 1]

[0192] In some embodiments, the engineered meganucleases of the present disclosure are first generation meganucleases. At least one optimized feature compared to the TRC1-2x.87EE enzyme Such optimized features include reduced off-target cleavage and increased fragment size from mRNA. Decreased retention time in cells after expression, enhanced (i.e., increased) efficiency of cleavage, and TCR The alpha constant region gene is modified to cause improved (i.e., increased) specificity. Thus, in certain embodiments, the operation of the present disclosure when delivered to a population of eukaryotic cells is The engineered meganucleases cleave and / or modify the TCR alpha constant region gene. In one of these embodiments, a greater proportion of cells can be produced that are in an altered state. In one embodiment, a population of eukaryotic cells is infected with a TCR alpha constant region gene that has been truncated and / or inserted. / Contains deletions ("indels"), at least 40%, at least 45%, at least 5 0%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, At least 96%, at least 97%, at least 98%, at least 99%, or Cleavage of the TCR alpha constant region gene by meganuclease and / or modifications include T7 endonuclease I assay, digital PCR, mismatch detection, and detection assay, mismatch cleavage assay, high-resolution melting curve analysis (HRMA), heterodimerization assay, Double-stranded mobility analysis, sequencing, and fluorescent PCR capillary gel electrophoresis (e.g. For example, Zischewski et al. (2017) Biotechnology A Advances 35(1):95-104, which is incorporated by reference in its entirety. The method may be measured using any method known in the art, including methods incorporating the method described above. It is possible.

[0193] In certain embodiments, the engineered meganucleases of the present disclosure are first generation TRCs. Compared to the 1-2x.87EE meganuclease, The persistence of mRNA or protein in cells is reduced when RT is increased. -PCR, Northern blot analysis, nuclease protection assay, in situ hybridization These include but are not limited to immunoblotting, immunocytochemistry, immunoblotting, and immunoprecipitation. The amount of blood flow can be measured using any method known in the art, including but not limited to:

[0194] 2.3 Methods for Delivery and Expression of Optimized Meganucleases The present invention utilizes a recognition sequence found within the human TCR alpha constant region gene (SEQ ID NO: 3). Genetically modified T cells and The present invention provides a method for producing the T cells. The T cells can be obtained from peripheral blood mononuclear cells, bone marrow, lymph nodes, or other organs. tissue, umbilical cord blood, thymocytes, cells from the site of infection, ascites, pleural effusion, spleen tissue, and tumors In certain embodiments of the present disclosure, the present invention provides a method for the production of steroid hormones, which can be obtained from a wide variety of sources, including tumors. Any number of T cell lines available in the art may be used. In some embodiments of the present disclosure, T cell The cells may be extracted from a unit of blood collected from a subject using any number of techniques known to those of skill in the art. In one embodiment, cells are obtained from the circulating blood of an individual by apheresis.

[0195] The modified T cell receptor alpha gene is a double-stranded gene expressed by the engineered meganuclease of the present disclosure. The strand breaks lead to the first exon of the TCR alpha constant region gene (i.e., the targeted exon). The exogenous sequence of interest is inserted into the target gene (the target gene), and the target gene is then synthesized by such a meganuclease. The resulting cleavage site allows direct insertion of the exogenous sequence homologous to the desired exon into the targeted exon. It may be possible to perform recombination.

[0196] As used herein, the terms "exogenous" or "heterologous" in reference to a nucleotide sequence The term refers to sequences that are purely synthetic, that originate from a foreign species, or that originate from the same species. In this case, the composition and / or the planned human therapeutic intervention may be altered from its natural form in the genomic locus. "Sequences that are substantially modified from the original" is intended to mean sequences that are substantially modified from the original.

[0197] In various embodiments, the exogenous sequence of interest comprises a coding sequence for a protein of interest. The coding sequence may be for any protein of interest. .

[0198] In certain embodiments, the exogenous sequence of interest encodes a chimeric antigen receptor (CAR). Generally, the CAR of the present disclosure comprises at least an extracellular domain and a nucleic acid sequence encoding the In some embodiments, the extracellular domain is also known as the ligand binding domain. In some embodiments, the target-specific binding element is referred to as the main or portion. The intracellular domain, i.e., the cytoplasmic domain, contains at least one costimulatory domain and For example, it contains one or more signaling domains such as CD3ζ.

[0199] In some embodiments, a CAR useful in the present invention is also known as a ligand binding domain or moiety. The ligand-binding domain contains an extracellular target-specific binding element called the The choice depends on the type and number of ligands that define the surface of the target cell. For example, the ligand-binding domain may bind to a target cell that is associated with a particular disease state. They can be selected to recognize ligands that act as cell surface markers. Examples of cell surface markers that can act as ligands for the ligand-binding domain within the AR include It is involved in viral, bacterial and parasitic infections, autoimmune diseases, and cancer cells. In some embodiments, the CAR specifically binds to an antigen on a tumor cell. The methodology involves engineering desired ligand-binding moieties that bind to target tumor-specific antigens of interest. In the context of this disclosure, "tumor antigen" or "tumor-specific antigen" " refers to an antigen common to a specific hyperproliferative disorder such as cancer.

[0200] In some embodiments, the extracellular ligand binding domain of the CAR binds any antigen or ligand of interest. is epitope specific, particularly for any tumor antigen or epitope of interest. As a non-limiting example, in some embodiments, the target antigen is ErbB2 (HER2 / neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpC AM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvII) I), CD19, CD20, CD22, CD30, CD40, CLL-1, disialogan Glioside GD2, ductal epithelial mucin, gp36, TAG-72, glycosphingolipids, Glioma-associated antigen, B-human chorionic gonadotropin, alpha-fetoprotein (AF) P), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostase-specific antigen (PSA) , PAP, NY-ESO-1, LAGA-la, p53, prostein, PSMA, residual and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, Neutrophil elastase, ephrin B2, insulin growth factor I (IGF I), IGF I Major histocompatibility complex I, IGFI receptor, mesothelin, and tumor-specific peptide epitopes Tumor-associated surface antigens such as MHC molecules, 5T4, ROR1, NKp30, and NK G2D, a tumor stromal antigen, and the extra domain A (EDA) and extra domain B of fibronectin Streptavidin domain B (EDB) and Al domain of tenascin-C (TnC Al) and fibroblast-associated protein (fap), CD3, CD4, CD8, CD24, CD2 5, CD33, CD34, CD38, CD123, CD133, CD138, CTLA- 4. B7-1 (CD80), B7-2 (CD86), endoglin, major histocompatibility genes Lineage of complex (MHC) molecules, BCMA (CD269, TNFRSF17), CS1, etc. specific or cell-specific antigens, or HIV-specific antigens (e.g., HIV gpl20) virus-specific surface antigen, EBV-specific antigen, CMV-specific antigen, E6 or E7 tumor antigen HPV-specific antigens such as proteins, Lassa virus-specific antigens, influenza virus-specific antigens antigens, as well as any derivatives or variants of these surface markers. In certain embodiments, the ligand binding domain is specific for CD19.

[0201] In some embodiments, the extracellular domain of the chimeric antigen receptor is an antigen specific for an autoantigen (Payne et al., 2002). t al.(2016)Science,Vol.353(6295):179-184 (See, e.g., J. Immunol. 2004, 103:131-132, 2004), which are activated by autoantigen-specific B cell receptors on B lymphocytes. can be recognized by specific T cells, preventing antibody-mediated autoimmunity. These CARs induce the killing of diseased autoreactive B lymphocytes. This condition may be referred to as CAAR.

[0202] In some embodiments, the extracellular domain of the chimeric antigen receptor is a chimeric antigen receptor that is naturally occurring against an antigen of interest. The original ligand, or fragments of the naturally occurring ligand that retain the ability to bind the antigen of interest. It may contain segments.

[0203] In some embodiments, the CAR comprises an extracellular ligand binding domain or a hinge or a segment. Using the sequence of the enzyme, autoantigens with intracellular signaling domains and costimulatory domains can be identified. A transmembrane domain is any membrane-bound or transmembrane domain. For example, transmembrane polypeptides are derived from T cell receptors. subunits (i.e., α, β, γ, or ζ, polypeptides that make up the CD3 complex) ), IL2 receptor p55 (chain), p75 (β chain or γ chain), Fc receptor subunit CD proteins such as the CD8 alpha chain (e.g., Fcy receptor III) or CD8 alpha chain Alternatively, the transmembrane domain may be synthetic, containing amino acids such as leucine and valine. In certain instances, the transmembrane domain may comprise a CD8α transmembrane polypeptide. It's Chido.

[0204] The hinge region functions to link the transmembrane domain to the extracellular ligand-binding domain. For example, the hinge region may be up to 300 amino acids long. Preferably, the amino acid sequence contains 10 to 100 amino acids, and most preferably 25 to 50 amino acids. The hinge region can be made of all or part of the extracellular domain of CD8, CD4, or CD28. all or part of a naturally occurring molecule, such as a portion of a polypeptide chain, or all or part of an antibody constant region Alternatively, the hinge region may be derived from a synthetic sequence corresponding to a naturally occurring hinge sequence. The hinge domain may be a synthetic sequence or may be a completely synthetic hinge sequence. The antibody may contain a portion of the human CD8 alpha chain, the FcγR111 receptor, or IgG1. This can be done.

[0205] The intracellular signaling domain of the CAR is expressed in at least one of the cells in which the CAR is placed. Activation of two normal effector functions and / or activation of proliferative and cell survival pathways The term "effector function" refers to a specialized function of a cell. Effector functions include, for example, cytolytic or helper activity, including cytokine secretion. Intracellular signaling domains such as CD3ζ may respond to binding of the extracellular domain. As discussed, activation signals can be provided to cells that Signals induce cellular effector functions, such as cytolytic activity or cytokine secretion. It can be emitted.

[0206] The intracellular domain of CAR mediates cell proliferation, cell survival, and / or cell proliferation. One or more intracellular co-stimulatory signals that promote cytokine secretion after fusion Such intracellular costimulatory domains include N1, N6, C D27, CD28, CD8, 4-1BB(CD137), OX40, CD30, CD40 , PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, L Ligands that specifically bind to IGHT, NKG2C, B7-H3, and CD83 These include, but are not limited to, those known in the art.

[0207] CARs can be specific for any type of cancer cell. Such cancers include carcinomas, lymphomas, and the like. Pancreatic cancer, sarcoma, 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 In certain embodiments, the tumors may include, but are not limited to, B-cell lymphoma. The cancer of origin is B-lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, B-cell non-Hodgkin lymphoma, This includes, but is not limited to, lymphoma, and multiple myeloma.

[0208] The sequence of interest may further encode an exogenous T cell receptor (TCR). The exogenous T cell receptor may comprise an alpha chain and a beta chain, or alternatively, a gas Exogenous TCRs useful in the present invention may comprise any TCR of interest. It may have specificity for an antigen or epitope.

[0209] In other embodiments, the sequence of interest is a wild-type or modified version of a target sequence. It can encode an endogenous gene.

[0210] The sequence of interest is a T2A element, a P2A element, an E2A element, and an F2 including, but not limited to, IRES elements such as A elements and 2A elements It is not possible to translate more than two genes from the same promoter, as is possible with the present technology. It may include elements or peptides known in the art. In specific embodiments, Such elements in the exogenous sequence of interest encode a protein of interest (e.g., CAR). It can be located 5' upstream or 3' downstream of the nucleic acid sequence it is targeting.

[0211] The exogenous sequences of interest described herein may further comprise additional regulatory sequences. For example, the sequence of interest may be a homologous recombination transcription enhancer sequence, a Kozak sequence, a polyadenylation sequence, a transcription termination sequence, a selectable marker sequence (e.g., an antibiotic resistance gene), an origin of replication, and The sequences of interest described herein may include at least one Examples of nuclear localization signals are known in the art. knowledge (e.g., Lange et al., J. Biol. Chem., 2007, 282:5101-5105).

[0212] The engineered meganucleases of the present invention can be delivered to cells in the form of a protein. Preferably, it may be delivered in the form of a nucleic acid encoding the engineered meganuclease. Such nucleic acids may be DNA (e.g., circular or linear plasmid DNA or PCR products) or or RNA (e.g., mRNA). For embodiments delivered in DNA form, this sequence is used to regulate transcription of the meganuclease gene. The mammalian host must be operably linked to a promoter that promotes the expression of the mammalian host. Mammalian promoters include the cytomegalovirus early (CMV) promoter (Thomson et al., 2001). n et al. (1984), Proc Natl Acad Sci USA.81 (3):659-63) or the SV40 early promoter (Benoist and Ch Ambon (1981), Nature. 290 (5804): 304-10) The promoters are time-expressed and tetracycline-inducible (Dingermann et al. (1992), Mol Cell Biol. 12(9):4038-45) The engineered meganucleases of the present invention include inducible promoters such as synthetic promoters. The synthetic promoter may be operably linked to a JeT promoter (International Publication No. 2002 / 012514).

[0213] In some embodiments, the mRNA encoding the engineered meganuclease is expressed in the cell. The reason for this is that this mRNA encodes the engineered meganuclease. This is because it reduces the likelihood that the engineered gene will be integrated into the cell's genome. Such mRNA encoding a nuclease can be prepared by techniques known in the art, such as in vitro transcription. In some embodiments, the mRNA can be produced using methods known in the art. Guanosine, anti-reverse cap analog (ARCA) (U.S. Patent No. 7,074,596 ), CleanCap® analogs such as Cap1 analogs (Trilink, San Diego, CA) or enzymes using vaccinia cap enzyme or analogs In some embodiments, the mRNA is 5' capped using a selective capping method. mRNA, expression of the encoded engineered meganuclease and Various 5' and 3' untranslated sequences can be used to enhance the stability of the mRNA and / or the mRNA itself. Such elements may include, for example, post-translational regulatory factors of woodchuck hepatitis virus. mRNA can contain post-translational regulatory elements such as transcription factors. Lysine, 5-methylcytidine, N6-methyladenosine, 5-methyluridine, or 2 -thiouridine, or other nucleoside analogs or naturally occurring nucleosides. Additional nucleoside analogs include those described in, for example, U.S. Pat. No. 8,278,036. include.

[0214] In certain embodiments, the mRNA encoding the engineered meganuclease of the present invention The present invention relates to a policy that encodes two or more meganucleases that are co-expressed in a cell. Polycistronic mRNAs can be polycistronic mRNAs that share the same target gene. It is possible to encode two or more meganucleases that target different recognition sequences within a gene. Alternatively, polycistronic mRNAs can be produced in which cleavage sites are located within both genes. As described herein, at least one meganuclease and similar genes or a second recognition sequence located in a second gene. At least one additional nuclease can be encoded that targets the recognition sequence. Polycistronic mRNA can be expressed by IRES elements, T2A elements, and P2A elements, E2A elements, and F2A elements, The ability to translate three or more genes (i.e., cistrons) from the same mRNA molecule The peptides may include any element or peptide known in the art. do.

[0215] In another specific embodiment, a nucleic acid encoding an engineered meganuclease of the invention can be introduced into cells using a single-stranded DNA template. 5' and / or downstream of the ganucleases-encoding sequence. or a 3' AAV inverted terminal repeat (ITR). In other embodiments, the single-stranded DNA 5' and / or 3' homology arms of the engineered meganuclease-encoding sequence It can further include upstream and / or downstream.

[0216] In another specific embodiment, the gene encoding the meganuclease of the present invention is linear. In some instances, the meganuclease can be introduced into cells using a DNA template. The circular plasmid DNA is linearized before being introduced into the cells. The fragment may be digested with one or more restriction enzymes to be purified.

[0217] The purified meganuclease protein is then introduced into cells to cleave genomic DNA. This can be accomplished using methods known in the art, including those described in more detail herein below. By various different mechanisms known in the art, the target sequence is cleaved by homologous recombination or non-homologous recombination. End ligation can be performed.

[0218] In some embodiments, a meganuclease protein or a molecule encoding a meganuclease The DNA / mRNA containing the targeting ligand is conjugated to a cell-penetrating peptide or Facilitates cellular uptake. Examples of cell-penetrating peptides known in the art include poly- Arginine (Jearawiriyapaisarn, et al. (2008) Mol Ther. 16:1624-9), TAT peptide derived from HIV virus (Hudec z,et al.(2005),Med.Res.Rev.25:679-736),M PG (Simeoni, et al. (2003) Nucleic Acids Res .31:2717-2724), Pep-1 (Deshayes et al. (200 4)Biochemistry43:7698-7706), and HSV-1 VP- 22(Deshayes et al.(2005)Cell Mol Life Sc i. 62:1839-49). In an alternative embodiment, the meganuclease protein The DNA / mRNA encoding the protein or meganuclease is called the meganuclease transcript. Protein / DNA / mRNA binds and is internalized by the target cell. The antibody is then covalently linked to an antibody that recognizes a specific cell surface receptor expressed on the target cell. Alternatively, the meganuclease protein / DNA / mRNA is a natural ligand (or part of a natural ligand) for such a cell surface receptor. ) can be covalently or non-covalently bound to the 014)Tissue Barriers.2(4):e944449;Dinda,e t al.(2013)Curr Pharm Biotechnol.14:1264 -74;Kang,et al.(2014)Curr Pharm Biotechn ol.15(3):220-30;Qian,et al.(2014)Expert Opin Drug Metab Toxicol.10(11):1491-508) .

[0219] In some embodiments, a meganuclease protein or a molecule encoding a meganuclease The DNA / mRNA carrying the nanoparticles is covalently or non-covalently bound or It has been encapsulated within such nanoparticles using methods known in the art (Sharma , et al. (2014) Biomed Res Int. 2014). Nanoparticles are, Nanoscale, whose length scale is less than 1 Dm, preferably less than 100 nm Such nanoparticles may be metal, lipid, polymer, or biological macromolecules. Using a core composed of multiple copies of the recombinant meganuclease protein, mRNA The DNA can be designed in a nanoparticle core or attached to or encapsulated in the nanoparticle core. Increases the number of copies of meganuclease protein / mRNA / DNA transported into the cells, To achieve this, we increased the intracellular expression of each engineered meganuclease and enhanced its target recognition sequence. The surface of such nanoparticles can be coated with polymers or lipids (e.g. chitosan, cationic polymers, or cationic lipids) to The surface can be modified to provide additional functionality to enhance transport and uptake of small molecule drugs. Forming core-shell nanoparticles such as (Jian, et al. (2012) Biomat 33(30):7621-30). The nanoparticles can additionally target molecules. to target the nanoparticles to appropriate cell types and / or for cellular uptake. Examples of such target molecules include those specific to cell surface receptors. Natural ligands (or parts of natural ligands) for heterologous antibodies and cell surface receptors Includes.

[0220] In some embodiments, a protein encoding a meganuclease or a meganuclease is The DNA / mRNA is encapsulated in liposomes or transported using cationic lipids. (e.g., Lipofectamine™, Life Technologies, Logies Corp., Carlsbad, CA; Zuris et al. (20 15) Nat Biotechnol.33:73-80;Mishra et al. (2011) J Drug Deliv. 2011:863734). Liposome and lipoplex formulations can protect small molecule drugs from degradation. cellular uptake and proliferation through fusion with and / or disruption of the target cell membrane. This can promote transportation efficiency.

[0221] In some embodiments, a meganuclease protein or a molecule encoding a meganuclease The DNA / mRNA is encapsulated within a polymeric scaffold (e.g., PLGA). or in combination with cationic polymers (e.g., PEI, PLL) (Tamboli et al. (2011) Ther Deliv. 2 (4 ):523-536). Polymeric carriers can be prepared through the control of polymer erosion and drug diffusion. The drug can be designed to provide a tunable drug release rate, and the high drug encapsulation efficiency allows for the desired target Can provide protection for small molecule drugs until they are delivered intracellularly to target cell populations .

[0222] In some embodiments, the meganuclease protein, or recombinant meganuclease, The encoding DNA / mRNA 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, shield charge interactions, and promote nonspecific Hydrophilic polymers (e.g., polyethylene glycol) that can reduce unwanted interactions The micellar shell may include a micellar shell formed using

[0223] In some embodiments, the meganuclease protein, or recombinant meganuclease, The encoding DNA / mRNA is suitable for administration and / or delivery to target cells. emulsions or nanoemulsions (i.e., with an average particle size of less than 1 nm) for The term "emulsion" refers to a water-immiscible phase mixed with an aqueous phase. If the water is in a polar state, the nonpolar residues (e.g., long-chain hydrocarbons) that are away from water and the polar residues that are towards water Any water-in-oil type, including lipid structures that can be formed as a result of hydrophobic forces driving the lipid head groups. refers to, but is not limited to, oil-in-water, water-in-oil-in-water, or oil-in-water-in-oil dispersions or droplets These other lipid structures may be unilamellar, paucilamellar, or Emulsions include, but are not limited to, multilamellar lipid vesicles, micelles, and lamellar phases. The solution is composed of an aqueous phase and a lipophilic phase (typically containing oil and an organic solvent). Emulsions often also contain one or more surfactants. The agents are described, for example, in U.S. Patent Application Nos. 2002 / 0045667 and 2004 / 00 43041, and U.S. Patent Nos. 6,015,832, 6,506,803, Those described in US Pat. Nos. 6,635,676 and 6,559,189 are well known, and each of which is incorporated herein by reference in its entirety.

[0224] In some embodiments, the meganuclease protein, or recombinant meganuclease, The encoding DNA / mRNA is then transported via multifunctional polymer conjugates, DNA dendrimers, and and covalently or non-covalently attached to polymeric dendrimers. (Mastorakos et al. (2015) Nanoscale.7( 9):3845-56;Cheng et al. (2008) J Pharm Sci 97(1):123-43). Dendrimer generation controls maximum loading capacity and size. In addition, the display of multiple surface groups can provide stable Improved quality, reduced non-specific interactions, and cell-specific targeting and and can be utilized to enhance drug release.

[0225] In some embodiments, the gene encoding the meganuclease is delivered via a viral vector. Such vectors are known in the art and are known as retroviral vectors. vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viruses (AAV) vectors (Vannucci, et al. (2013 New Millimeter Wave) (Reviewed in J. Clin. Biol. 36:1-22). Recombinant AAV useful in the present invention. The vectors are used for viral transduction into cells and for the insertion of nuclease genes into the cell genome. In certain embodiments, the recombinant AAV may have any serotype that allows for the insertion of The vectors have the AAV2 or AAV6 serotype. They may also be self-complementary so as not to require the synthesis of double-stranded DNA in the host cell (Mc Carty, et al. (2001) Gene Ther. 8:1248-54).

[0226] If the meganuclease gene is in DNA form (e.g., a plasmid) and / or virus, When delivered using a vector (e.g., AAV), this gene is operably linked to the promoter. In some embodiments, the viral vector-derived endogenous Viral promoters such as promoters (e.g., LTRs in lentiviral vectors) or can be the well-known cytomegalovirus promoter or the SV40 early promoter. In a preferred embodiment, the meganuclease gene is transfected into a target cell (e.g., a T cell). Preferably, the gene is operably linked to a promoter that drives expression of the gene.

[0227] The present invention provides a method for the identification of exogenous sequences of interest in T cell receptor alpha constant region sequences at the TRC1-2 recognition sequence. In some embodiments, the exogenous sequence of interest is introduced into the 5' region of the gene. The homologous arms include a 3' homologous arm adjacent to the sequence to be inserted. Corresponding nuclease recognition 5' upstream and 3' downstream of the sequence where the cleavage site is created Generally, the homology arms are at least 50 base pairs long, preferably 100 base pairs long. or at least 100 base pairs in length and up to 2000 base pairs or more in length. At least 90%, preferably at least 95%, or more, of the genome The sequence may have sequence homology to the corresponding sequence of

[0228] The exogenous sequences of interest of the present invention may be introduced into cells by any of the methods described above. In certain embodiments, the exogenous sequence of interest is a lentivirus, retrovirus, adenovirus, or The vector may be introduced using a viral vector such as a human avian virus or, preferably, a recombinant AAV vector. Recombinant AAV vectors useful for introducing exogenous nucleic acids are used to express the virus in cells. Any cell that is capable of introducing an exogenous nucleic acid sequence into the cell genome and inserting an exogenous nucleic acid sequence into the cell genome. In certain embodiments, the recombinant AAV vector may have an AAV2 or AAV3 lotype. The recombinant AAV vectors have the AV6 serotype. They may also be self-complementary so that DNA synthesis is not required.

[0229] In another specific embodiment, the exogenous sequence of interest is introduced into the cell using a single-stranded DNA template. The single-stranded DNA may contain an exogenous sequence of interest, and in a preferred embodiment, 5' phase to facilitate insertion of nucleic acid sequences into meganuclease cleavage sites by co-recombination The single-stranded DNA may contain a 5' homologous arm and a 3' homologous arm. The 5' AAV inverted terminal repeat (ITR) and the 3' AAV I, which is 3' downstream of the 3' homology arm. It may further comprise a TR.

[0230] In another specific embodiment, the genes encoding the engineered nucleases of the invention and and / or the exogenous sequence of interest of the present invention can be transfected using a linear DNA template. In some cases, plasmid DNA can be introduced into cells by gene transfer. The circular plasmid DNA is linearized before insertion into one or more restriction enzymes. So it can be digested.

[0231] T cells modified according to the present invention can be engineered to express a meganuclease and / or an exogenous sequence of interest. Activation is required before the introduction of the cells. For example, T cells require sufficient Anti-CD3 and anti-CD3 antibodies, either soluble or conjugated to a support (i.e., beads), can be administered over a period of time. and an anti-CD28 antibody.

[0232] The genetically modified cells of the present invention express one or more inducible suicide genes. These cells can be further modified to induce cell death, This allows for the selective destruction of cells in vitro or in vivo. The gene can convert a cytotoxic polypeptide, a non-toxic prodrug, into a cytotoxic drug. polypeptides that have the ability to activate cytotoxic gene pathways in cells, and / or That is, a suicide gene can encode a polypeptide that or other compounds, the nucleic acid encoding a product that causes cell death. A typical example of a suicide gene is the one encoding the thymidine kinase of herpes simplex virus. Additional examples include varicella-zoster virus thymidine kinase and 5-fluorouracil. It can convert rosytosine into 5-fluorouracil, a highly toxic compound. The bacterial gene is a gene encoding cytosine deaminase. As a non-limiting example, caspase-9, caspase-8, or cytosine deaminase In some cases, caspase-9 induces specific protein dimers. The suicide gene can be activated using a therapeutic antibody and / or Cell surface expressed proteins that make cells sensitive to cytotoxic monoclonal antibodies In a further example, the suicide gene may encode a polypeptide that is capable of inhibiting the growth of an anti-C D20 expresses antigenic motifs and suicide genes recognized by mAb rituximab A recombinant antigen polypeptide containing an epitope that allows selection of cells that are For example, two rituximab-binding epitopes and QBEnd1 RQR8 polypeptides described in WO 2013153391, including 0-binding epitopes. For such genes, rituximab can be used to target the cellular deoxyribonucleotides when needed. In a further example, the suicide gene can be administered to a subject to induce cleavage. a QBEnd10-binding epitope expressed in combination with a type EGFR polypeptide; It may contain taupe.

[0233] The eukaryotic cells and compositions modified by the methods described herein may be modified to express endogenous T cell receptors. (i.e., alpha / beta T cell receptors) and can reduce the expression of proteins of interest (e.g., Therefore, the present invention provides a method for producing a protein of interest. Further, a population of eukaryotic cells expressing endogenous T cell receptors (e.g., alpha / beta T cells) is provided. For example, a population may express CAR (i.e., CAR+) or expressing exogenous T cell receptors (i.e., exogenous TCR+), and eukaryotic cells that have been modified to reduce expression of endogenous T cell receptors (i.e., TCR- In various embodiments of the invention, at least 10%, at least 15%, at least at least 20%, at least 25%, at least 30%, at least 35%, at least 4 0%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, At least 98%, at least 99%, or up to 100% of the cells are In certain embodiments, the population is at least At least 10%, at least 15%, at least 20%, at least 25%, at least 30 %, at least 35%, at least 40%, at least 45%, at least 50%, at least at least 55%, at least 60%, at least 65%, at least 70%, at least 7 5%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to It can include 100% cells that are both TCR- and CAR+.

[0234] In some embodiments, when introduced into a population of cells, the engineered meganuclei of the present disclosure The enzyme delivers the first generation TCR1-2x.87EE meganuclease into a cell population. A cell population that is both TCR- and CAR+ at a higher percentage than when arise.

[0235] Furthermore, cells genetically modified with the engineered meganucleases of the present disclosure exhibit improved characteristics. This characteristic reduces off-target cleavage and its effects, and enhances the ability of meganucleases in cells. This reduces the maintenance time of the enzyme and enhances (i.e., increases) CAR T proliferation. Furthermore, the cells are genetically modified with the TRC1-2x.87EE meganuclease. In addition, the meganucleases of the present disclosure (or The cell population into which the nucleic acid encoding the TRC1-2x.87EE meganucleic acid has been introduced is compared to a cell population into which the enzyme (or a nucleic acid encoding it) has been introduced. have a large proportion of modified cells and a larger proportion of less differentiated cells In certain embodiments, the population of cells into which the engineered meganucleases of the present disclosure are introduced is Further, a population of cells into which the first generation TRC1-2x.87EE meganuclease is introduced is A significantly higher proportion of central memory T cells (expressing CD45RO, CCR7, and CD62L) Indicates the type of product that is being sold.

[0236] 2.4 Pharmaceutical Compositions In some embodiments, the present invention provides a genetically modified eukaryotic cell of the present invention, ... A pharmaceutical composition comprising a population of altered eukaryotic cells and a pharmaceutically acceptable carrier is provided. Such pharmaceutical compositions can be prepared according to known techniques. n,The Science And Practice of Pharmacy(2 1st ed.,Philadelphia,Lippincott,Williams In preparing pharmaceutical formulations according to the present invention, Thus, the cells are typically mixed with a pharmaceutically acceptable carrier and the resulting composition is administered to a subject. Of course, the carrier must be acceptable in the sense of being compatible with any other ingredients in the formulation. In some embodiments, the present invention provides a method for treating a subject's The pharmaceutical composition may contain one or more additional agents useful in treating a disease in a subject. In additional embodiments, the pharmaceutical compositions of the present invention may further comprise a cytokine (e.g., I and / or IL-2, IL-7, IL-15, and / or IL-21. This promotes in vivo cell expansion and engraftment of genetically modified T cells. Pharmaceutical compositions comprising the genetically modified eukaryotic cells of the present invention may be administered with additional drugs or biological agents. Alternatively, they may be co-administered in separate compositions. can be given.

[0237] The present disclosure also provides a method for producing a genetically modified cell line as described herein for use as a pharmaceutical. The present disclosure provides a method for treating a disease in a subject in need thereof. The genetically modified cells, or populations thereof, described herein, may be used in the manufacture of a pharmaceutical for the treatment of a disease. In one such aspect, the medicament is a cancer immunotherapy in a subject in need thereof. It is useful for therapy.

[0238] Cells into which the meganucleases of the present disclosure have reduced off-target cleavage; This reduces the time that meganucleases remain in cells, allowing for more efficient translation of the TCR alpha constant region gene. The cells destroy CAR T cells with high efficiency and enhance (i.e., increase) CAR T proliferation. TRC1-2 previously genetically modified with RC1-2x.87EE meganuclease Compared to cells previously genetically modified with x.87EE meganuclease, In some embodiments, the present invention provides a method for producing a cell comprising: The pharmaceutical compositions disclosed in the invention also contain TRC1-2x.87EE meganuclease. Therefore, when compared to the administration of a pharmaceutical composition containing genetically modified cells, When administered to a subject, the compound improves efficacy in treating a disease (e.g., cancer).

[0239] In some embodiments, when introduced into a population of cells, the engineered meganuclei of the present disclosure The enzyme delivers the first generation TCR1-2x.87EE meganuclease into a cell population. A cell population that is both TCR- and CAR+ at a higher percentage than when arise.

[0240] Furthermore, cells genetically modified with the engineered meganucleases of the present disclosure exhibit improved characteristics. This characteristic reduces off-target cleavage and its effects, and enhances the ability of meganucleases in cells. This reduces the maintenance time of the enzyme and enhances (i.e., increases) CAR T proliferation. Furthermore, the cells are genetically modified with the TRC1-2x.87EE meganuclease. In addition, the meganucleases of the present disclosure (or The cell population into which the nucleic acid encoding the TRC1-2x.87EE meganucleic acid has been introduced is compared to a cell population into which the enzyme (or a nucleic acid encoding it) has been introduced. have a large proportion of modified cells and a larger proportion of less differentiated cells In certain embodiments, the population of cells into which the engineered meganucleases of the present disclosure are introduced is , further from a population of cells pre-transfected with the parental TRC1-2x.87EE meganuclease. A significantly higher proportion of central memory T cells (expressing CD45RO, CCR7, and CD62L) Indicates the type of product that is being sold.

[0241] The pharmaceutical compositions of the present invention can be used to treat any tumor that can be targeted by T cell adoptive immunotherapy. In certain embodiments, the pharmaceutical compositions of the present invention may be useful for treating disease conditions such as The pharmaceutical compositions and medicaments of the present disclosure are useful in the treatment of cancer. Non-limiting examples of cancers that can be treated include cancers of B-cell origin, neuroblastoma, osteosarcoma, pre-cancerous carcinoma, and ovarian cancer. prostate cancer, renal cell carcinoma, rhabdomyosarcoma, liver cancer, stomach cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer Cancer, breast cancer, lung cancer, cutaneous or intraocular malignant melanoma, ovarian cancer, kidney cancer, uterine cancer, ovarian cancer, colon cancer , colon cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, fallopian tube cell tumor, uterine Endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, non-Hodgkin's lymphoma, Esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, Stem cancer, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvic cell tumor, Central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brainstem tumors Rheoma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, asbestos-induced Environmentally induced cancers, including those caused by: multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myeloma, Myeloid lymphoma, chronic myeloid leukemia, chronic lymphocytic leukemia, large cell immunoblastic lymphoma , acute lymphoblastic leukemia, mycosis fungoides, anaplastic large cell lymphoma, and T-cell lymphoma Cancers including, but not limited to, carcinomas, lymphomas, sarcomas, and any combination of such cancers. In certain embodiments, the tumors are of B-cell origin. The cancers are B-cell acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, and B-cell lymphoma. , diffuse large B-cell lymphoma, Pre-B ALL (precursor B-cell lymphocytic leukemia, Pediatric indications), mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, Burkitt's lymphoma lymphoma, multiple myeloma, and B-cell non-Hodgkin's lymphoma.

[0242] In some of these embodiments, cancer is treated with the genetically modified cells or populations thereof of the present disclosure. In some embodiments, the subject receiving the genetically modified cells or population thereof may undergo radiation therapy, external cytotoxicity therapy, or other treatments. The patient may then be given additional therapeutic agents such as chemotherapy or chemotherapy drugs.

[0243] The present invention also provides genetically modified cells, including a plurality of genetically modified cells described herein. A population of cells is further provided, which is capable of expressing an exogenous nucleic acid molecule encoding a sequence of interest in the genome. The exogenous nucleic acid molecule is inserted into a T cell receptor alpha constant region gene. Thus, in various embodiments of the present invention, cell surface expression of endogenous TCR is reduced. A population of genetically modified cells is provided, wherein at least 10%, at least At least 15%, at least 20%, at least 25%, at least 30%, at least 35% , at least 40%, at least 45%, at least 50%, at least 55%, less At least 60%, at least 65%, at least 70%, at least 75%, at least 80% %, at least 85%, at least 90%, at least 95%, at least 96%, at least At least 97%, at least 98%, at least 99%, or up to 100% of the cells In a further embodiment of the invention, the genetically modified cell is A population of cells is provided, wherein at least 10%, at least 15%, At least 20%, at least 25%, at least 30%, at least 35%, at least At least 40%, at least 45%, at least 50%, at least 55%, at least 60% , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 95%, at least 96%, at least 97% %, at least 98%, at least 99%, or up to 100% of the cells are chimeric antigen-receptor cells. The genetically modified cells described herein further express a receptor.

[0244] 2.5. Methods of Administration of Genetically Modified Cells Another aspect described herein is the administration of an effective amount of the genetic modification of the present disclosure to a subject in need of treatment. In certain embodiments, the administration of the modified eukaryotic cells or populations thereof is The pharmaceutical composition is administered to a subject in need of treatment. For example, a therapeutically effective amount of a population of cells In certain embodiments, the disease may be cancer, and the The administration of the genetically modified eukaryotic cells of the invention constitutes immunotherapy. The administered cells exhibit reduced proliferation. It can reduce, decrease the number of, or kill target cells in the recipient. Unlike conventional eukaryotic cells, the genetically modified eukaryotic cells of the present disclosure are capable of replicating and multiplying in vivo. and be long-lasting so as to result in sustained disease control.

[0245] Examples of possible routes of administration include parenteral (e.g., intravenous (IV), intramuscular (IM), intradermal, In addition, the administration may be by continuous infusion, or This may be by single bolus or multiple boluses. In specific embodiments, The drug lasts for less than about 12 hours, less than 6 hours, less than 4 hours, less than 3 hours, less than 2 hours, or less than 1 hour. In yet other embodiments, the infusion is initially slow and may be performed and then increased over time.

[0246] In some embodiments, the genetically modified eukaryotic cells or populations thereof of the present disclosure are used to treat cancer. Such cancers include carcinomas, lymphomas, sarcomas, and blastoma, leukemia, cancer of B-cell origin, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, melanoma, Cancers include prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, and Hodgkin's lymphoma. In specific embodiments, the cancer and disorder can include, but is not limited to, pr eB ALL (pediatric indication), adult ALL, mantle cell lymphoma, diffuse large cell type Including, but not limited to, B-cell lymphoma, salvage after allogeneic bone marrow transplant These cancers do not express, for example, CD19, CD20, CD22, and / or ROR1. Some non-limiting examples include the use of a combination of targeting CARs in the treatment of cancer. Genetically modified eukaryotic cells or populations thereof are useful in the treatment of cancers of B-cell origin, such as neuroblastoma, osteosarcoma, and premature eukaryotic cancer. prostate cancer, renal cell carcinoma, rhabdomyosarcoma, liver cancer, stomach cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer Cancer, breast cancer, lung cancer, cutaneous or intraocular malignant melanoma, ovarian cancer, kidney cancer, uterine cancer, ovarian cancer, colon cancer , colon cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, fallopian tube cell tumor, uterine Endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, non-Hodgkin's lymphoma, Esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, Stem cancer, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvic cell tumor, Central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brainstem tumors Rheoma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, asbestos-induced Environmentally induced cancers, including those caused by: multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myeloma, Myeloid lymphoma, chronic myeloid leukemia, chronic lymphocytic leukemia, large cell immunoblastic lymphoma , acute lymphoblastic leukemia, mycosis fungoides, anaplastic large cell lymphoma, and T-cell lymphoma Cancers including, but not limited to, carcinomas, lymphomas, sarcomas, and any combination of such cancers. In certain embodiments, the target is a tumor, such as a B-cell tumor, a melanoma, a blastoma, a leukemia, or a germ cell tumor. Cancers of cellular origin include B-cell acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, and B-cell lymphoma. Diffuse large B-cell lymphoma, Pre-B ALL (precursor B-cell lymphocytic leukemia) Hematologic malignancies, pediatric indications), mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, Burkina Fasciitis lymphoma, multiple myeloma, and B-cell non-Hodgkin's lymphoma do not have.

[0247] When an "effective dose" or "therapeutic dose" is indicated, age, weight, tumor size (if present), (if applicable), taking into account the degree of infection or metastasis and individual differences in the condition of the patient (subject). The exact amount to be administered can then be determined by a physician. Pharmaceutical compositions comprising the genetically modified cells or populations thereof described in the document are within the scope of Administered at a dosage of 104 to 109 cells / kg body weight, including whole integers. In this case, the dosage is 105 to 106 cells / kg body weight, including all integer values ​​within that range. In some embodiments, the cell compositions are administered multiple times at these dosages. It can be administered by using infusion techniques well known in immunotherapy (e.g., Rosen berg et al.,New Eng.J.of Med.319:1676,19 88). The optimal dosage and treatment regimen for a particular patient depends on the nature of the disease. Pharmacist-directed drug distribution by monitoring patients for symptoms and adjusting treatment accordingly. This can be readily determined by one of ordinary skill in the art.

[0248] In some embodiments, the genetically modified eukaryotic cells or populations thereof of the present disclosure comprise a target For example, the genetic modification of the present disclosure reduces at least one symptom of the disease or condition. Administration of the T cells or population thereof may reduce at least one symptom of cancer. Symptoms are well known in the art and can be determined by known techniques.

[0249] 2.6 Methods for Producing Recombinant Viral Vectors In some embodiments, the present invention provides viral vectors (e.g., vectors) for use in the methods of the present invention. The recombinant AAV vector is a vector derived from a host cell, such as HEK-293. The viral cap and rep genes are typically produced in mammalian cell lines. , creating space for one or more therapeutic genes (e.g., meganuclease genes) to be delivered. To prevent self-replication, the vector is removed from the packaging cell line. In this case, it is necessary to provide these genes in trans. It is necessary to provide the necessary "helper" (e.g., adenovirus) components to support (Cots et al. (2013), Curr. Gene Ther. 13 (5 ):370-81). Recombinant AAV vectors often encode "helper" components. a first plasmid containing the cap and rep genes, a second plasmid containing the cap and rep genes, and A third region containing the viral ITRs containing the intervening DNA sequence that is packaged into the virus. Cell lines are transfected with plasmids and generated using triple transfection. The genome (ITRs of interest and one or more intervening genes) is enclosed in the capsid. The viral particles containing the virions are subsequently purified by freeze-thaw cycles, sonication, detergents, or other methods known in the art. The particles are then isolated from the cells by other means known in the art. Purified using density gradient centrifugation or affinity chromatography, and then delivering one or more genes of interest to a cell, tissue, or organism, such as a human patient. will be done.

[0250] Recombinant AAV particles are typically produced (manufactured) in cells, ensuring that the engineered mediator Care should be taken when practicing the present invention so that the ganucleases are not expressed in the packaging cells. It should be noted that the viral genome of the present invention contains the recognition sequence for the meganuclease. Any meganuclease expressed in the packaging cell line may contain a sequence The meganuclease cleaves the viral genome before it can be packaged into viral particles. This may allow for improved packaging efficiency and / or fragmented genomes. This results in a decrease in the packaging of the meganuclease in the packaging cells. To prevent expression, several approaches can be used, including the following.

[0251] The meganuclease controls a tissue-specific promoter that is not active in packaging cells. For example, muscle tissue can be (a) placed with one or more meganuclease genes. When generating viral vectors for delivery, muscle-specific promoters can be used. An example of a muscle-specific promoter is C5-12 (Liu, et al. (2004) Hum Gene Ther.15:783-92), muscle-specific creatine kinase (MCK) protein promoter (Yuasa, et al. (2002) Gene Ther. 9:1576- 88), or the smooth muscle 22 (SM22) promoter (Haase, et al. (201 3) BMC Biotechnol.13:49-54) including CNS (neurons) Examples of specific promoters are 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-amino acid Trypsins (e.g., Pa1AT), and hemopexins (e.g., Phpx) (Kramer et al., (2003) Mol. Therapy 7:375-85), Hybrid A rat liver-specific promoter (hepatic locus control region and liver-specific promoter from the ApoE gene) Human thyroxine-binding globulin (TBG) promoter Examples of eye-specific promoters include the ocular promoter, the apolipoprotein A-II promoter, and the apolipoprotein A-II promoter. opsin, and the corneal epithelial-specific K12 promoter (Martin et al. (2 002)Methods(28):267-75)(Tong et al.,(200 7) J Gene Med, 9:956-66). Other tissue-specific promoters known in the art do not exhibit high activity in HEK-293. Therefore, when introduced into the viral vector of the present invention, It is not expected to result in significant levels of meganuclease gene expression. In addition, the viral vectors of the present invention may be used in combination with other cell types, including the use of incompatible tissue-specific promoters. Use of strains (i.e., the well-known HeLa cell line (human epithelial cells) and liver-specific Hemoperoxidase Other examples of tissue-specific promoters are PDZD4 (cerebellum), C6 (liver), ASB5 (muscle), PPP1R12B (heart) ), SLC5A12 (kidney), cholesterol-regulating APOM (liver), ADPRHL1 ( heart) and the monogenic malformation syndrome TP73L (muscle). (Jacox et al. l.,(2010),PLoS One v.5(8):e12274).

[0252] Alternatively, the vector may be derived from a different species in which the meganuclease may not be expressed. For example, viral particles can be packaged in non-mammalian packaging cells. The well-known cytomegalovirus early promoter, which is not active in cells, or SV40 Microbial cells, insect cells, or plant cells using mammalian promoters such as early promoters In a preferred embodiment, the viral particles can be produced in the cells described in Gao, et al. ao et al. (2007), J.Biotechnol.131(2):138- 43) and produced in insect cells using the baculovirus system. Meganucleases under the control of promoters cannot be expressed in insect cells (Airenne et al.(2013),Mol.Ther.21(4):739 -49) In addition, insect cells have different mRNA splicing patterns than mammalian cells. Therefore, the coding sequence of the meganuclease is linked to the human growth hormone ( mammalian introns, such as the HGH intron or the SV40 large T antigen intron Introns can be efficiently integrated from pre-mRNA transcripts in insect cells. is not spliced, so insect cells do not express a functional meganuclease. In contrast, the resulting recombinant AA Mammalian cells into which V particles are transported reliably splice pre-mRNA transcripts and produce functional Haifeng Chen is developing a new method to express meganuclease proteins in insect packages. Expression of the toxic proteins barnase and diphtheria toxic fragment A in Zing cells reported the use of HGH and the SV40 large T antigen intron to attenuate This allows the production of recombinant AAV vectors carrying these toxic genes (Ch en(2012),Mol Ther Nucleic Acids.1(11):e5 7).

[0253] The meganuclease gene requires a small molecule inducer for meganuclease expression. The inducible promoter may be operably linked to an inducible promoter such that the promoter is et-On system (Clontech; Chen et al. (2015), BMC Biotechnol.15(1):4)) and the RheoSwitch system (I ntrexon;Sowa et al.(2011),Spine,36(10):E Both systems, and similar systems known in the art, include small Activates transcription in response to molecular activators (doxycycline or ecdysone, respectively) The ligand-induced transcription factors (Tet repressor and Ecdysone receptor, respectively) The latest inventions using such ligand-inducible transcriptional activators are The implementation includes: 1) generating a meganucleotide under the control of a promoter that responds to the corresponding transcription factor; The meganuclease gene is arranged in a manner that the meganuclease factor has one or more binding sites for a transcription factor. and 2) encoding a transcription factor in the packaged viral genome. The latter step involves providing a transcriptional activator to the same cell. If not, the meganuclease is expressed in the target cells or tissues after delivery of the recombinant AAV. The transcriptional activator is then treated with its cognate small molecule activator. This approach induces the expression of the meganuclease gene only in cells or tissues where the gene is present. By selecting when and to which tissues small molecule inducers are transported, This allows for the control of meganuclease gene expression in a spatiotemporal manner. However, inducers have a significantly limited transport capacity and can transport large amounts of the viral genome. The requirement to include this in makes this approach flawed.

[0254] In another preferred embodiment, the recombinant AAV particle comprises a transcription factor that prevents expression of the meganuclease. The transcriptional repressor is produced in a mammalian cell line that expresses a transcriptional repressor. The Tet repressor, Lac repressor, Cro repressor, and lambda repressor are known. Many nuclear hormone receptors, such as the ecdysone receptor, also contain cognate hormone receptors. In the absence of a mon ligand, it functions as a transcriptional repressor. Packaging cells are transfected / transformed using a vector encoding a transcriptional repressor. The meganuclease gene is transduced into the viral genome (packaging vector). The molecule contains a binding site for a repressor so that the repressor silences the promoter. The promoter is operably linked to a gene that has been modified to encode a transcriptional repressor. The genes involved can be located in a variety of locations. The genes can be encoded on separate vectors. , can be integrated into a packaging vector outside the ITR sequence, cap / rep vector Alternatively, the gene may be incorporated into an adenovirus helper vector, or may 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 promoter sites are known in the art. For example, Chang and Roninson have reported that the Lac repressor The strong, constitutive CMV and RSV promoters containing the .alpha.-transducer were engineered. , expression of the gene from this modified promoter is suppressed in cells expressing the repressor. showed that the effect was significantly attenuated (Chang and Roninson (1996), G ene 183:137-42). The use of non-human transcriptional repressors allows for the creation of meganuclear Transcription of the enzyme gene is inhibited only in packaging cells expressing the repressor, resulting in In target cells or tissues transduced with recombinant AAV vectors, It is certain that this is not the case.

[0255] 2.7 Engineered Nuclease Variants Embodiments of the present invention include the engineered nucleases described herein, and variants thereof. Further embodiments of the present invention include vectors encoding the nucleases described herein. and variants of such polynucleotides. Includes.

[0256] As used herein, "variant" is intended to mean substantially the same sequence. "Variant" polypeptides are polypeptides that contain one or more internal regions of the native protein. Deletion or addition of one or more amino acids at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 A polypeptide derived from a "natural" polypeptide by substitution of one or more amino acids in As used herein, "natural" polynucleotides are intended to mean peptides. The nucleotide or polypeptide comprises the parent sequence from which the variant is derived. The variant polypeptides encompassed by are biologically active. The variant polypeptide possesses the desired biological activity of the native protein. That is, it is the TRC found in the human T cell receptor alpha constant region (SEQ ID NO: 3). 1-2 recognition sequence (SEQ ID NO: 5). This results in improved (i.e., increased) specificity and reduced off-target cleavage and intracellular The maintenance time and enhanced (i.e., increased) TCR alpha constant region gene modification efficacy A small number of studies on first generation TRC1-2 meganucleases are selected from the group consisting of Such variants may be produced, for example, by human manipulation, and may exhibit at least one improved property. Biologically active variants of the native polypeptides of the embodiments (e.g., variants of the sequence SEQ ID NO: 7 and SEQ ID NO: 8), or a recognition half-site binding subunit as described herein. Biologically active variants of the polypeptides may be amino acids of the native polypeptides or native subunits. At least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75 ... 5%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 91%, approximately 92%, approximately 9 3%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity This is due to the sequence alignment programs and parameters described elsewhere herein. The biological activity of the polypeptide or subunit of the embodiment is determined by the Active variants may contain as few as about 1-40 amino acid residues, or as few as about 1-20. As few as about 1–10 amino acid residues As few as about 5 amino acid residues As few as 4, 3, 2, or even 1 amino acid residue It may differ from variants of polypeptides or subunits in similarly small amounts. stomach.

[0257] The polypeptides of this embodiment may be modified in various ways, including amino acid substitutions, deletions, truncations, and insertions. The procedure for such manipulations is generally known in the art. For example, amino acid sequence variants can be prepared by mutations in the DNA. Methods for inducing and altering polynucleotides are well known in the art. , Kunkel (1985) Proc. Natl. Acad. Sci. USA82:48 8-492, Kunkel et al. (1987) Methods in Enzy mol.154:367-382, U.S. Patent No. 4,873,192, Walker and Gaastra, eds. (1983) Techniques in Mol ecular Biology (MacMillan Publishing Comp See any of the publications cited therein. Derivation of appropriate amino acid substitutions that do not affect the biological activity of the target protein is performed by Dayton et al. Atlas of Protein Sequence by hoff et al. (1978) nce and Structure(Natl.Biomed.Res.Found. , Washington, DC) model, which serves as a reference. The replacement of amino acids with other amino acids having similar properties is incorporated herein by reference. Conservative substitutions such as:

[0258] In some embodiments, the engineered meganucleases of the invention comprise the HVs described herein. The parent HVR regions may include variants of the R1 and HVR2 regions. It may comprise residues 24-79 or residues 215-270 of the engineered meganuclease. Thus, variant HVRs are residues of the engineered meganucleases exemplified herein. At least 80 for the amino acid sequence corresponding to residues 24-79 or residues 215-270 %, at least 85%, at least 90%, at least 91%, at least 92%, at least at least 93%, at least 94%, at least 95%, at least 96%, at least 9 7%, at least 98%, at least 99% or more sequence homology The variant HVR regions contain amino acid sequences that allow the production of engineered meganucleases. Furthermore, the present invention maintains its biological activity (i.e., binding and cleaving the recognition sequence). In some embodiments, the variant HVR1 region or variant HVR2 region is a variant of the parent HVR1 region or variant HVR2 region. It may contain residues corresponding to amino acid residues found at specific positions within the HVR. In this context, "corresponding" means that the amino acid residues in the variant HVRs are in the same relative positions. position (i.e., for amino acids maintained in the parent sequence) present in the parent HVR sequence. This means that the amino acid residues are the same (i.e., separate identical residues). and the parent HVR sequence contains a serine residue at position 26, The variant HVR also contains a serine at a position relative to (i.e., corresponding to) parent position 26. .

[0259] In certain embodiments, the engineered meganuclease of the invention comprises residue 21 of SEQ ID NO:7. For the amino acid sequence corresponding to 5-270, at least 90%, at least 91%, or at least at least 92%, at least 93%, at least 94%, at least 95%, at least 9 6%, at least 97%, at least 98%, at least 99% or more sequence identity It contains the HVR1 region with isoforms.

[0260] In certain embodiments, the engineered meganuclease of the invention comprises residues of SEQ ID NO:7. For the amino acid sequence corresponding to 24-79, at least 81%, at least 82%, or at least at least 83%, at least 84%, at least 85%, at least 86%, at least 8 7%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of the sequence Contains the homologous HVR2 region.

[0261] In some embodiments, the engineered meganuclease of the invention comprises residue 24 of SEQ ID NO:8. For the amino acid sequence corresponding to ~79, at least 85%, at least 86%, at least At least 87%, at least 88%, at least 89%, at least 90%, at least 91% , at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher Contains the HVR2 region with sequence homology.

[0262] In certain embodiments, the engineered meganuclease of the invention comprises sequences 215 to 216 of SEQ ID NO:7. HVR1 with at least 97% sequence identity to the amino acid sequence corresponding to 270 region, and the amino acid sequence corresponding to residues 24 to 79 of SEQ ID NO: 7, It contains the HVR2 region with 1% sequence homology.

[0263] In other specific embodiments, the engineered meganuclease of the present invention comprises the sequence 21 of SEQ ID NO:8. an HVR1 region having an amino acid sequence corresponding to residues 5-270 of SEQ ID NO:8; HVR2 with at least 86% sequence identity to the amino acid sequence corresponding to ~79 Includes the area.

[0264] Amino acid modifications to the DNA recognition domain of wild-type I-Crel meganuclease A substantial number have already been identified (e.g., U.S. Pat. No. 8,021,867) and are single The specificity of the individual bases in the DNA recognition sequence half-sites can be varied individually or in combination. The resulting rationally designed meganucleases are The nucleases have half-site specificities that differ from the wild-type enzyme. Table 2 shows the recognition half-sites. Increased specificity based on the base present at each half-site position (-1 to -9) of the half-site. These can be made into engineered meganuclease monomers or subunits to enhance provides possible substitutions.

[0265] [Table 2] JPEG2025131624000003.jpg50159

[0266] Engineered meganucleases to modulate DNA binding affinity and / or activity Certain modifications can be made in the monomers or subunits. For example, The engineered meganuclease monomer or subunit may be I-Crel or SEQ ID NO: 7 or the residue corresponding to position 19 of SEQ ID NO: 8 (WO 2009001159) and can contain G, S, or A, and can be I-Crel or SEQ ID NO: 7 or sequence may contain Y, R, K, or D at the residue corresponding to position 66 of number 8; and and / or I-Crel or SEQ ID NO:7 or SEQ ID NO:8 (U.S. Pat. No. 8,021,867 The residue corresponding to position 80 of (numbered) may contain E, Q, or K.

[0267] With respect to polynucleotides, a "variant" is one or more of the naturally occurring polynucleotides. including deleting and / or adding one or more nucleotides at multiple sites Those skilled in the art will readily appreciate that the nucleic acids of this embodiment may be reversed so that the open reading frame is maintained. Conserved variants may be constructed for polynucleotides. The amino acid sequence of one of the polypeptides of the embodiment may be obtained by degeneracy of the genetic code. The variant polynucleotides include those encoding the sequence. including synthetically derived polynucleotides, such as those produced using genetic mutagenesis. However, this further encodes the recombinant nuclease of the embodiment. A variant of a particular polynucleotide in a form that is less specific to that particular polynucleotide. At least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, approximately 80%, approximately 85%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, about 96%, about 97%, about 98%, or about 99% sequence identity, which is Determined by the sequence alignment program and parameters described elsewhere in the specification Variants of the particular polynucleotides of the embodiments (i.e., the reference polynucleotide) The polypeptide encoded by the variant polynucleotide is Comparing the percentage of sequence homology of polypeptides encoded by polynucleotides It can also be evaluated by

[0268] Deletions, insertions, and substitutions of the variant protein sequences encompassed herein are not intended to be limiting unless otherwise specified. It is not expected to cause drastic changes in the characteristics of the petites, but Human T cells, where it is difficult to predict the effects of substitutions, deletions, or insertions before making them. The TRC1-2 receptor is found within exon 1 of the receptor alpha constant region gene (SEQ ID NO: 3). The polypeptide was screened for its ability to preferentially recognize and cleave the recognition sequence (SEQ ID NO: 5). Those skilled in the art will understand that this effect may be assessed by performing a lean test. cormorant. [Example]

[0269] This invention is further illustrated by the following examples, which should not be construed as limiting. Those skilled in the art will be able to use the methods described herein through routine experimentation and numerous methods for specific substances. It may be possible to recognize or ascertain without sufficient equivalents and procedures. Such equivalents are intended to be encompassed by the claims following the examples below.

[0270] Example 1 Characterization of meganucleases with specificity for the TRC1-2 recognition sequence 1.1. Meganuclease that recognizes and cleaves the TRC1-2 recognition sequence

[0271] TRC1-2L.1592 (SEQ ID NO: 7) and TRC1-2L.1775 (SEQ ID NO: 8) is a second-generation TRC1-2 meganuclease that recognizes the TRC1-2 sequence. (SEQ ID NO: 5). This sequence is expressed in human T cell receptor agonists. These second-generation meganucleases are based on SV40, First meganuclease subunits, linker sequences, and second meganucleases Each TRC1- contains an N-terminal nuclease localization signal derived from a subunit of The first subunit of the meganuclease binds to the TRC1 recognition half-site of SEQ ID NO: 5. The second subunit binds to the TRC2 recognition half-site. (See Figure 1). The TRC1-binding subunit and the TRC2-binding subunit are Each contains a 56 base pair hypervariable region, designated HVR1 and HVR2, respectively. It is being done.

[0272] The HVR1 region of each TRC1 binding subunit is represented by residue 2 of SEQ ID NO:7 and SEQ ID NO:8. Consists of 15-270. TRC1-2L.1592 and TRC1-2L.1775 The RC1-binding subunits are identical to each other outside of the HVR1 region. The HVR1 region of the ganucleases is located at positions 215, 217, 219, 221, 223, and 22 4, 229, 231, 233, 235, 237, 259, 266, and 268 wild type Contains modifications to the I-Crel sequence (SEQ ID NO: 1). Modified relative to wild-type I-Crel Even if not present, the arginine residue at position 261 of SEQ ID NO: 7 and SEQ ID NO: 8 This group, along with the modified HVR1 residues, is believed to be responsible for the specificity of the nuclease. The HVR1 region of RC1-2L.1592 is the HVR1 region of the TRC1-2x.87EE meganuclease. It shares 96.43% sequence identity with the HVR1 region of TRC1-2L.177 The HVR1 region of 5 is similar to the HVR1 region of TRC1-2x.87EE meganuclease. , sharing 100% sequence homology.

[0273] The HVR2 region of each TRC2 binding subunit is represented by residues 2 and 3 of SEQ ID NO:7 and SEQ ID NO:8. Consists of 4-79. TRC1-2L.1592 and TRC1-2L.1775 The two binding subunits are identical to each other outside the HVR2 region, but E(TRC1-2L SEQ ID NO: 7 and sequence Except for position 80 of number 8. The HVR2 region of each TRC1-2 meganuclease is , positions 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 48, 5 0, 68, 70, 75, and 77 relative to the wild-type I-Crel sequence (SEQ ID NO: 1) The TRC1-2L.1592 meganuclease contains modifications to the wild-type I-Crel. and further contains modifications at positions 71, 72, and 73. The arginine residue at position 139 was modified relative to the wild-type I-Crel sequence, It is also noteworthy that, together with the HVR2 residues, this is thought to be responsible for the specificity of the nuclease. The HVR2 region of TRC1-2L.1592 is the same as that of the first-generation TRC1-2x.8 It shares only 80.36% sequence identity with the HVR2 region of 7EE meganuclease. The HVR2 region of TRC1-2L.1775 is located at the TRC1-2x.87EE meganucleotide site. It shares only 85.71% sequence homology with the HVR2 region of ase.

[0274] 2. Optimization of first-generation TRC1-2 nuclease The TRC1-2x.87EE meganuclease reported so far is GUIDE-s eq(Tsai et al. (2015), Nat Biotechnology 3 3:187-197), but has potential drawbacks for meganucleases. Evaluate recognition site specificity using a method designed to find target sites Generally, potential off-target sites are probed in double-stranded DNA breaks. The TRC1-2 meganuclei are identified by capturing certain oligonucleotides. The enzyme generates a 4-base pair 3' overhang, so the probe oligo Also, ligation efficiency is improved at sites more suitable for nuclease cleavage. It contains randomized 4-base pair overhangs, which improves the

[0275] Specificity analysis of TRC1-2x.87EE revealed various potential off-target genes in human T cells. These off-target sites can be classified into two related categories: This is due to the unique targets that are hit frequently and the repetition of targets that are hit less frequently. The important amino acids involved in the sequences of these off-targets were The acid was again randomized, and then simultaneous selection was performed to cleave the target site. Counterselection was performed to avoid cleavage at off-target sites. The target site is a discriminatory response to both off-targets (i.e., nuclease The two off-target genes used were alternated between successive rounds of selection to isolate the off-target gene. The fragment is 5'-TGGCCTGGAGaAACAgtgtaaa-3' (SEQ ID NO: 16). Off1, which is a low frequency cut but at a highly repeated site in the genome. Off2 was 5'-cGGCCTGtAGtAcaggAcCTGA-3' (SEQ ID NO: No. 17), which was a frequently hit unique off-target (lowercase indicates target). (The mismatches represent the mismatches with the corresponding positions.) A variety of nuclease libraries were used.

[0276] After selection, 96 clones from each successive library were isolated. Well plates were prepared for isolating plasmid DNA. Each plasmid DNA was , the target region integrated at the break between the three direct repeat sequences in the GFP gene. These were individually transfected into CHO cells containing the target site. The GFP gene is repaired by double-strand annealing. This can be measured by counting the number of GFP-positive cells on a flow cytometer. They used the target site and off-target sites to target nuclease promoters to cells. In this way, the inventors have the most discriminatory effect on off-targets. It is possible to evaluate which nucleases further cleave the target site, excluding those that differentiate. The inventors identified five candidates. Three of the candidates were original to TRC1-2. L.1462, L.1466, and L.1469 were reisolated from the library. All three answers were unique but related to each other. These were isolated from RC-library 2 and named L.1108 and L.1118. Each of these candidates is an intermediate nuclease with respect to the development of second generation nucleases of the present invention. Represents ze.

[0277] To further improve the nuclease, we randomly selected important amino acids involved in the recognition sequence specificity. L.1462, L.1466 and L.1469 are stored in a specific library. L.1108 and L.1118 were collected in a second library. De novo randomization by CR was introduced into both groups. Similar selection strategies were used for de novo randomization. This was carried out by the lab. This was done to select the target site and to select Off1 or Off2. Off-targets were rotated between rounds of selection. The 96-well plates of the answer were generated from the selection and tested in the CHO iGFFP assay. The target site and both Off1 and Off2 cleavage were measured. The L.1462, L.1466 and L.1467 were identified through this additional round of optimization. Responses from libraries based on L.1469 include L.1775 and L.1843 One answer from the library based on L.1108 and L.1118 is L. All of the novel nucleases exhibited strong activity against their intended targets and both The novel nuclease showed strong discrimination against off-targets (described further below). The enzyme was used in an oligo capture assay (further below) to determine potential off-target sites. described above), which generally reduces the number of potential off-targets, especially L.1 592 has few potential legitimate off-target sites. L.1469, L.1592, L.1775, and L.1843 are for a period of seven days. This was further evaluated in a long-term iGFFP assay to assess the stability of the GFP signal over time. This is a common measure of toxicity. 1775 and L.1843 were further tested in primary T cells for function.

[0278] 3. Evaluation of TRC1-2 recognition sequence cleavage and off-target cleavage Which TRC1-2 meganuclease recognizes the TRC1-2 recognition sequence (SEQ ID NO: 5) and To determine whether each TRC1-2 meganuclease can cleave the CHO A cell reporter assay (see WO 2012 / 167192, Figure 3) To perform the assay, non-functional genomic DNA was integrated into the genome of the cells. A pair of CHO cell receptors carrying an expression cassette for the green fluorescent protein (GFP) gene The GFP gene in each cell line was transfected with either meganuclease or so that intracellular cleavage of the recognition sequence stimulates a homologous recombination event that generates a functional GFP gene. In both cell lines, one of the recognition sequences was a TR The second recognition sequence is derived from the C1-2 gene and is designated "CHO23 / 24". Specifically recognized by the meganuclease: TRC1-2 recognition sequence (SEQ ID NO: 5) CHO reporter cells containing the CHO23 / 24 recognition sequence are referred to as "TRC1-2 cells." are referred to herein.

[0279] TRC1-2 cells were cultured using TRC1-2 meganuclease (e.g., TRC1-2x.87 EE, TRC1-2L.1592, TRC1-2L.1775 or TRC1-2L. 1843) or encodes the CHO23 / 34 meganuclease. 4e5 CHO cells were transfected with plasmid DNA according to the manufacturer's instructions. Following the instructions, 96-well plates were transfected using Lipofectamine 2000 (ThermoFisher). The plate was transfected with 50 ng of plasmid DNA. At 8 hours, cells were assessed by flow cytometry to determine whether non-transfected neurons were present. Determine the ratio of GFP-positive cells compared to the positive control (1–2 bs). All TRC1-2 meganucleases significantly outperformed the negative control. One of the CHO23 / 24 positive controls was used. GFP-polysaccharide was detected in cell lines containing the TRC1-2 recognition sequence at frequencies above those of the reactive control. Each TRC1-2 meganuclease was found to produce a potent inhibitor of the cell This demonstrates that the target TRC1-2 recognition sequence can be efficiently recognized and cleaved. (Figures 4A to 4C).

[0280] Alternatively, this TRC1-2 meganuclease may also be used to counterselect off-targets. The GFP sequence was then inherited into TRC-off 1 and TRC-off 2 cells, which contained GFP direct repeats. Unlike the target site TRC1-2 in CHO cells, the target site TRC-off1 and The desired nuclease activity in ON and OFF2 CHO cells was compared with baseline levels of GFP-positive cells. This is because it is possible to discriminate against cleavage of off-target sequences. The CHO23-24 target site was used as a positive control in these experiments. When the target site is cleaved by CHO23-24 nuclease, G This indicates that FP can be produced unchanged. TRC1-2x.87EE and TRC1-2x.87EE showed comparable %GFP levels to the control. Improved (i.e., increased) discrimination against Off1 and Off2 target sites compared to This is shown in Figures 5A to 5C.

[0281] TRC1-2.L1469, L.1592, L.1775 and L.1843 The efficacy of the engineered meganuclease was confirmed by transfecting meganuclease mRNA into TRC1-2 cells. The time-dependent changes were also measured 2, 5, and 7 days after transfection into the cells. In the study, TRC1-2 cells (1.0 × 10 6) was prepared by BioRad according to the manufacturer's instructions. Using Gene Pulser Xcell, 1 x 10 cells per 6 Copy Meganuk At 48 hours after transfection, the cells were electroporated with GFAP mRNA. The percentage of P-positive cells was assessed by flow cytometry. CHO23 / 24 meganuclease was also included at each time point as a positive control. Each meganuclease produced significant GFP-positive CHO23-24. The ratio of GFP-positive cells was shown in Fig. 6 and Fig. 7. Only L.1469 increased the number of GFP-positive cells over time. There is some residual toxicity that shows a tendency to decrease and is improved with subsequent optimization. This shows that the remaining nuclease is levels equivalent to or higher than those of the steroid class, and are stable or increasing over time GFP-positive cells were observed.

[0282] The extended iGFFP assay also included two on-off series, Off 1 and Off 2, over a 7-day period. It was used to evaluate a similar group of meganucleases for discrimination against the target. In this case, cells containing either Off1 or Off2 and CHO23-24 used a BioRad Gene Pulser Xcell according to the manufacturer's instructions. , 1 x 10 cells per 6 1 copy of meganuclease mRNA. At 2, 5, and 7 days after gene transfer, cells were evaluated by flow cytometry. The percentage of GFP-positive cells was determined. The HO23-24 meganuclease was also included. Each nuclease was a TRC1- Compared to 2x.87EE, it showed improved discrimination against off-targets ( (Figures 8A and 8B). The L.1592 is designed to minimize disconnection of either Off1 or Off2. L.1469 showed a similar effect to that of mock control cells. Although dramatically lower than that observed by 2x.87EE, The results showed that some cleavage of L.1775 and L. 1843 improves off-target discrimination over its parent, L.1469 Shows.

[0283] 4. Oligo Capture Assay and Analysis of Off-target Cleavage In these studies, oligo capture assays were used to identify TRC1-2 meganucleases. Similar to GUIDE-seq, we identified off-target cleavage induced by nucleotides. The oligonucleotide capture assay captures oligonucleotides at break sites within the genomic DNA of cells. and recognize potential off-target sites created by TRC1-2 meganuclease. GUIDE-seq was developed for CRISPR-Cas9, which generates DNA breaks. To apply this technology to the nucleases of the present invention, chemical and analytical approaches were developed. Unlike CRISPR-cas9, the manipulations described herein do not result in significant modifications. The modified meganuclease generates a 4-base pair 3' overhang. To achieve this, the oligonucleotides used for oligo capture were TRC1-2 meganuclear Randomize 4 base pair overhangs that can be compatible with the overhangs generated by the enzyme. High frequency insertions are observed due to the greater efficiency of cohesive ends joining than smooth ends. It is believed that cells encode nucleases and double-stranded DNA oligonucleotides. After two days, genomic DNA from these cells was isolated and The DNA was sheared to small sizes by sonication. The fragments are then ligated into the sheared DNA and PCR is used to create fragments containing adapters at certain ends and those containing adapters at other ends. At the end of the PCR product, any DNA fragment containing the captured oligonucleotide was amplified. The purified DNA was then used to generate a sequencing library using standard commercial kits. was prepared.

[0284] The sequencing library was a V2 2x1 sequence using an Illumina MiSeq. This data was used to identify the appropriate sites for capturing oligonucleotides. The proteins were selected and analyzed to predict potential off-target sites. From the PAM search using CRISPR-cas9, the TRC1-2 meganuclease sequence was identified. The software developed examines each sequence and detects the presence or absence of adapters. Verify that the sequence is present and that the capture oligos flanking this sequence are present, indicating that this is a valid read. The software also checks for PCR overlap and reduces PCR bias. Identical reads are removed to help reduce the number of reads. Sequence reads are aligned to a reference genome. The classified sequences within the 1000 base pair window were identified as potential TRC1-2 meganuclei. The DNA is scanned for enzyme sites.

[0285] Each TRC1-2 meganuclease is a linked dimer. Each monomer consists of two half-sides. The software recognizes 9 base pair half sites with a 4 base pair spacer in the middle between the bases. Search for each half-site with a disallowed gap and the sequence that shows the closest match. The central four base pairs are not considered for off-target selection. , the TRC1-2 meganucleases account for the large amount of degeneracy at these positions within the target site. The software considers salts in the binding half-sites as generally acceptable. Outputs a list of potential off-target sites along with the number of base mismatches. does not include the central 4 base pair mismatch. The software considers 7 or more base pairs as mismatches. CRISPR-Cas9 excluding any off-targets identified as matched Unlike the conventional method, it does not filter out any off-targets based on any mismatch filter. Instead, oligos are randomly captured at weak or hot spots in the genome. The background noise generated by capturing the image can be removed in two ways. First, we ran untreated mock samples through the oligo capture to identify the constructs that lacked any nucleases present. A window of binding sites can be drawn from the nuclease containing sample. They performed the assay in triplicate and repeated at least two of the three times. The removal of arbitrary regions that do not have a specific shape is a good way to empirically remove random embedded noise. We also found that this is a viable method.

[0286] Although read counts do not correlate with the frequency of breaks at specific sites, they occur more frequently. This data allows us to identify potentially more relevant or plausible off-targets. Based on the number of potentially plausible off-target sites, One way to graphically visualize oligo capture data is shown in Figure 9. Each off-target generated by the enzyme is captured by the probe oligo at that site. The target site is plotted based on the number of unique sequence reads for each target site. This is the case for all TRC1-2 meganucleases tested. Better nucleases remove higher count sites, resulting in positions further to the left of the plot. In this plot, there are very few dots higher than the background noise. This means that, for example, the TRC1-2L.1592 is now much faster than the first generation TRC1-2x.87EE. It is clear that this also excludes many of the high read count sites.

[0287] Additional visualization methods allow the inventors to visualize the results not only in terms of the number of reads recovered at a particular site, but also in terms of the number of reads recovered at a particular site. The oligonucleotides are also determined by the number of mismatches between the putative off-target site and the target site. You can view the captured data, which allows you to see whether the random embedded noise or the sequencing The actual oligo integration site can be determined more accurately when compared with the genomic noise. In Figure 10, off-target sites are plotted according to the number of reads arranged on the x-axis. The number of mismatched base pairs compared to the target site is indicated by color. Darker areas indicate a higher overall match between the off-target and target binding sites. The boxes indicate the areas of highest confidence. Targets are those with high aligned read counts to the target site or Either there is a very high similarity between the two, and one of these is the background Reduces the likelihood of a region being noisy compared to regions in the confidence region. Figure 10 shows the difference between the TRC1-2L.1592 and the TRC1-2x.87EE. , demonstrating the increased specificity of the optimized meganuclease. 2 shows a decrease in the number of higher read count sites and more similarity to target sites The decrease in the area is shown.

[0288] Example 2 In vitro analysis of optimized TRC1-2 meganuclease Assessment of gene editing efficiency, post-editing proliferation, and differentiation

[0289] In the first series of experiments, four optimized second-generation TRC1-2 meganuclei were The enzymes were screened for their gene editing efficiency and post-editing proliferation and differentiation potential. Three different operators used T cells obtained from different healthy human T cell donors. All nuclease variants were evaluated in T cells. donors K708, K799 from ey Biologics (Memphis, TN); K708 and K6784T cells were procured using the following protocol. The cells were treated according to the following procedure: human CD3 positive selection reagent (Stem T cells were enriched using ImmunoCult Anti-CD2 / CD3 / CD28 (StemCell Technologies) was used. and stimulated with 4D NucleoFEctor (Lonza). RNA was delivered to K799-derived T cells, which were treated according to the following protocol. Namely, CD4 and CD8 MicroBeads and CliniMACS Cell Isolator T cells were enriched using a transfection system (Miltenyi Biotec) and transfected with TransAct (M iltenyi) and nuclease-free RNA was extracted using MaxCyte-GT. Transported.

[0290] Four optimized nuclease variants (TRC1-2, L.1496, L.159 The editing efficiency, proliferation, and differentiation of the precursor nuclei (L.2, L.1775, and L.1843) were evaluated. ase, TRC1-2x.87EE, and electroporated mock T cells. Three days after the initial stimulation with ImmunoCult / TransAct, T cells were The cells are collected, electroporated with RNA encoding one of the nucleases, and T Direct insertion of the AAV6 vector encoding the CAR gene into the RC1-2 cleavage site Control cultures that did not receive AAV were set up in parallel. It was.

[0291] On the 4th and 8th days after editing, the NucleoCounter NC-200 (Ch The overall cellularity of the cultures was measured using a cellularity test (Cell Signaling Technology). D3-PE (BioLegend clone UCHT1) and anti-FMC63sc for Fv-AlexaFluor647 (a novel clone generated and conjugated in-house) Differentiation was measured by staining culture samples with the antibody of interest. 86 (clone OKT4 BioLegend), CD8-BV711 (clone RPA -T8, BioLegend), CD62L-BB515 (clone SK11 BD B Biosciences), and CD45RO-PE / Cy7 (clone UCHL1, B Using ioLegend, we analyzed central memory cells for both the CD4 and CD8 compartments. By comparing the frequencies of cytoplasmic cells, transitional memory cells, and effector memory cells, It was appreciated.

[0292] The results of these experiments are summarized in Figure 11. The CD3-positive phenotype was measured by TRC cells transforming The knockout frequency (constant) was determined for each nuclease in three separate donors. For all three donors tested (and using both cell preparation methods), TRC Both the 1-2L.1592 and L.1775 were similar to the TRC1-2x.87EE. In comparison, knockout cells were generated with L.1469 and L.2169 at a higher efficiency than those of L.1469. and L.1843 showed lower knockout frequencies than the three donors tested. This was true for all. L.1775 showed slightly higher editing efficiency than L.1592. Increased editing of the TRC1-2 recognition sequence was associated with an increased rate of CAR gene insertion. In all three donors, L.1592 and L.1775 showed comparable or superior Editing and insertion frequencies were established.

[0293] At day 8 after editing, cell count data was used to calculate fold expansion of CAR T cells. Across all three donors, L.1592, L.1775 and L.1843 L.1469 promoted higher proliferation after electroporation than x.87EE. In two of the three donors, L.1843 promoted lower proliferation than 7EE. Of the three optimized nucleases, L.1 was the most widely propagated. 775 demonstrated variable proliferation rates depending on the donor.

[0294] CD4:CD8 ratio and memory subset data were also captured on day 8 post-editing. Compared to x.87EE, both optimized nucleases produced significantly less CD4: No major disturbances in the CD8 ratio were observed, except for L.1592, L.17 75, and L.1843 often resulted in a large frequency of CD4+ cells. Compared with x.87EE, the number of cells distant from the central memory cells and in the transition and ef Differentiation into vector memory cell populations was observed to a greater extent in L.1469-edited cells. In contrast, if it was compiled in L.1592, L.1775, or L.1843, In this case, a similar or greater frequency of cells maintained the central memory phenotype.

[0295] These studies demonstrated that three of the four optimized nucleases significantly improved editing efficiency, cell proliferation, and It was superior to TRC1-2x.87EE in terms of characteristics such as differentiation and differentiation. Our nuclease, L.1469, did not function as well as x.87EE. Among the three variants with improved in vitro function, variant L.17 75 demonstrates the highest frequency of edited cells in culture. The variants show minimal post-editing proliferation and accelerate T cell differentiation in culture. The L.1843 gene can maximize post-edit growth and is a preferred central memory However, in terms of knockout frequency, the frequency of L.1775 or It was less effective than either of the L.1592. Surprisingly, L. 1592 improves upon the first generation x.87EE by using all three of these standards. This indicates that

[0296] 2. Oligo Capture Assay and Analysis of Off-target Cleavage Oligo capture was performed using the method previously described in Example 1 from each of the three donors. Oligo capture was performed on three replicates of isolated T cells. The results are shown in Figure 12. The dots represent the sequences recovered at each putative off-target site and at the intended target site. This represents the number of sequencing reads. In previous studies, sites with eight or more mismatches were Since it was not shown to be cleaved by TRC1-2L.1592, Putative sites with 8 or more mismatches to the target were excluded. All intended target sites are highlighted with circles. Mismatches compared to intended targets The number of mismatches is indicated by the darkness of each circle, which is darker when there are few mismatches. The plots do not have the false background removed and are specific to each sample. Figure 1 represents oligo capture data with read counts normalized to the number of reads recovered This explains the difference in the total number of reads. 2 has high lead-chain activity when used for editing and targeted insertion in CAR T cell populations. This indicates that there are fewer targets and fewer targets that are more similar to the target site.

[0297] 3. In vitro investigation of editing efficiency, proliferation and cytokine secretion A second series of in vitro studies demonstrated that editing T cells and the proliferation of edited T cells after editing were The ability of cells and nuclease barriers to respond to encounter antigen-associated target cells In terms of the potency of CAR T cells generated using this agent, the efficiency of second-generation The optimized TRC1-2 meganuclease was evaluated.

[0298] Apheresis material was obtained from Key Biologics (Memphis, TN). T cells were procured from donor K708 and transfected with human CD3 positive selection reagent (Ste mCell Technologies) and ImmunoCult Antibody -CD2 / CD3 / CD28 (StemCell Technologies) Stimulation and nuclease analysis using 4D NucleoFector (Lonza) RNA was delivered in triplicate. Triplicate samples were run in parallel.

[0299] Three optimized meganucleases (TRC1-2, L.1592, L.1775 and The editing efficiency, proliferation, and differentiation of the nucleases TRC1 and L.1843 were investigated using the precursor nuclease TRC1. -2x.87EE and electroporated mock T cells. Three days after the first stimulation with t / TransAct, T cells were harvested and used in the nuclei. Electroporation with RNA encoding one of the TRC1-2 cleavage enzymes The cells were immediately transduced with an AAV6 vector encoding the inserted CAR gene. At 4 and 8 days after collection, cultures were harvested and analyzed by Beckman-Coulter Editing efficiency and proliferation were measured using a CytoFLEX-LX flow cytometer. Endogenous T cell receptor knockout efficiency was assessed using anti-CD3-PE (BioLegend cl). one UCHT1) and CAR knock-in was performed using anti-FMC62scFv- Evaluated using AlexaFluor647 (a novel clone generated and conjugated in-house) was done.

[0300] Proliferation, cytotoxicity, and cytokine production were measured using the CD19+ tumor line, Raji or Nalm6 was evaluated using co-cultured CAR T cells with E:T ratios of 1:1 and 1:2. CD19-negative K562 myeloid leukemia cells were used as a control. Culture supernatants were collected and analyzed using a Luminex MAGPIX instrument and MilliPl Using ex MAP15-plex beads set (Millipore), secreted Cytokines were analyzed. Proliferation and target killing were measured by dividing the culture cell samples into Anti-CD4-APC (BioLegend clone OKT4), anti-CD8-FI TC (BioLegend clone RPA-T8), and anti-CD19-PE ( Staining was performed using BioLegend clone HIB19, and the staining was performed using CytoFLEX-L X was used to obtain fluorescence data along with cell counts.

[0301] Compared to T cells electroporated without RNA (sham control), at day 8 The total cellularity of the cultures was determined by the TRC1-2 meganucleases x.87EE and L. The L.1592 expression level was reduced by approximately 50% in T cells edited with L.1775 (Figure 13A). or L.1843-edited cultures to the same extent in total cellularity of the cultures. Considering the editing efficiency and the edited fragments produced by the process, no degradation of the When the total number of cells was counted, L.1592 produced the most TCR knockout cells ( Figure 13B). Variants x.87EE and L.1775 produced the lowest amount of L.1843. While producing similar numbers of edited cells, the same pattern was observed in cultures containing C This was also determined when the number of AR+ / TCR- cells was measured (Fig. 13C).

[0302] When CAR T cells were co-cultured with antigen-relevant target cells, TRC1-2x.87 CAR T cells generated using EE expanded approximately three-fold over the number transduced (shown by the horizontal dotted line). (As defined in Figure 14). Surprisingly, using the optimized nuclease CAR T cells generated with x.87EE proliferated even more rapidly than x.87EE, proliferating after 5 days. When the E:T ratio increased to 1:2, the x.87EE and L.184 CAR T cell proliferation after editing with 3 was reduced by approximately half compared to a 1:1 ratio. This was observed in CAR T cells generated with L.1775 or L.1592. and significantly better than other TRC1-2 nucleases (p<0.0001, Figure 15A ) was found to be effective in reducing the residual number of CD19+ Raji cells (at an E:T ratio of 1:2). As measured by the CAR T cell receptor activator (Figure 15B), all four CAR T products were showed a 90% or greater reduction in Raji numbers when compared to untreated control cultures. CAR T cells generated using the optimized nuclease expressed x.87EE. The results showed that the Raji cells were significantly better removed than cells produced using

[0303] Analysis of co-culture supernatants demonstrated that CAR T cells were expressed in the optimized nucleoside analogue rather than x.87EE. High levels of effector cytokines were produced when the antibody was made with ribosomal enzymes. L.1592-edited CAR T cells induced the highest levels of IL-2, TNF-α, and IL-1. α, IFNγ, and granzyme B (Figures 16A-16D), and then maximal levels In the case of IL-2 and TNFα, x.87 Cytokine production of EE-edited CAR T cells and L.1592-edited CAR The differences between T cells were 2-3 fold, while all other differences were minor.

[0304] Overall, the optimized TRC1-2 meganuclease, L.1775, L The L.1592 and L.1843 were superior in functionality to the X.87EE. The relative ability of the nucleases to demonstrate the production of AR T cells (Figure 13), and the The ability of CAR T cells to respond to encounter the target antigen (Figures 14-16) From multiple experiments, L.1775 showed the highest editing efficiency (knockout frequency). ) generally support the L.1843 gene, which can maximize the proliferation of T cells after editing, while the L.1 592 simultaneously allows for the second highest editing efficiency and the highest or second highest proliferation. was found to produce the greatest overall number of CAR T cells. CAR T cells generated with L.1592 were significantly more potent than other optimized meganucleases. demonstrated functional advantages (proliferation, target cell killing, and cytokine production) over .

[0305] 4. In vitro maintenance time of the optimized TRC1-2 meganuclease The study hypothetically demonstrated that the second-generation optimized TRC1-2 meganuclease could be more effective than the first-generation T To determine whether RC1-2x.87EE has a shorter in vitro retention time than RC1-2x.87EE, Further investigation was carried out to determine whether shorter maintenance times could be beneficial for gene editing and off-target cleavage. This can be advantageous in the context of potential reductions in

[0306] In these studies, T cells were isolated using CD4 and CD8 microbeads and LD columns ( Apheresis was performed by magnetically enriching CD4+ and CD8+ cells using a Miltenyi (Miltenyi) Cells were cultured in 5% FBS (GE Healthcare). Hyclone), 10 ng / ml IL-2 (Cellgenix), and 1% Anti-CD3 in Xuri medium (GE) containing antibiotic / antimycotic solution (Gibco) / Activated with anti-CD28 TransAct reagent (Miltenyi) for 3 days The cells were then transfected with in vitro transfected TRC1-2x.87EE or TRC1-2 Using mRNA encoding L.1592 nuclease (Trilink), Electroporate 1 μg of mRNA per 6 cells using the MaxCyte electroporation system. The cells were then incubated in a 1% antibiotic / antimycotic solution containing 30 ng / ml IL-2. The nucleotide sequence of the ... It encodes an anti-CD19 chimeric antigen receptor (SAB Tech) designed to be After electroporation, the cells were transduced with a recombinant AAV6 vector carrying the donor template. At 6 hours, samples were quantified and supplemented with 5% FBS, 30 ng / ml IL-2, and 1 % antibiotic / antimycotic solution. In this study, the remaining unedited CD3+ T cells were extracted from the electroporated population of TRCs into LD columns, C Magnetic isolation was performed using liniMACS buffer and CD3 microbeads (Miltenyi). The particles were removed by magnetic depletion. The cells were then incubated at 37°C in +5% FBS / 1% anti-antibody +10 ng / ml IL-15 and I They were cultured in Xuri medium supplemented with L-21 for the remainder of the experiment.

[0307] At 6 hours, 24 hours, 48 ​​hours, 96 hours, and 168 hours after electroporation, T cell samples were quenched, and an equal amount of viable cells was pelleted and diluted with RIPA buffer (EM). The cells were resuspended in 1000 mL of PBS (Millipore) and protease inhibitors (Roche) were added. Add 100 ml of PBS, mix thoroughly, and process further as described below for Western blotting. They were either frozen or incubated on ice for 30 minutes before use.

[0308] Mock cells from the same donor were activated as the nuclease-treated group and cultured in the same medium. The sham cells were also electroporated for 24 hours after the nuclease treatment group. collected at the time.

[0309] For Western blot analysis, the lysate was centrifuged and the supernatant was transferred to a new tube. The protein concentration was determined by BCA assay (Pierce). Determine and add 15 μg of total protein for each sample to sample buffer + DTT (NuP The gel was incubated at 90°C for 10 minutes. 5 μg of each sample was added to each A single mock sample at 24 hours post-electroporation served as a control. After electroporation, the samples were transferred to a PVDF membrane (Novex) (NuPage Electroporation system and reagents). The membrane was blocked with 5% non-fat dry milk in TBS-T and then Staining was performed with the secondary antibody.

[0310] Blot Primary Antibody

[0311] A Rabbit polyclonal anti-nuclease (Precision BioScience) ES in-house development, used at 1:6500)

[0312] B Mouse anti-B-actin (Sigma, used at 1:15000)

[0313] The membranes were washed six times and then incubated with the appropriate secondary antibodies.

[0314] Blot Secondary Antibody

[0315] A Goat anti-rabbit HRP (Invitrogen, used at 1:50,000)

[0316] B Goat anti-mouse HRP (Invitrogen, used at 1:75000)

[0317] After a washing step, the membrane was exposed to ECL Prime (Amersham) and wrapped in Saran Wrap ( and imaged using a UVP ChemiDoc-IT 815 Imager. Obtained.

[0318] As shown in Figure 17, the absence of nuclease expression was detected in the mock samples, as expected. TRC1-2x.87EE or TRC1-2L.1592 nuclear In samples electroporated with mRNA encoding the nuclease, the nuclease protein , was highly expressed at 6 hours after electroporation, the earliest time point analyzed. At 24 hours post-transfection, the protein remains detectable, but expression is reduced by either nuclear factor. The TRC1-2x expression level also decreased substantially from 6 hours after electroporation. A significant decrease was observed for TRC1-2L 0.1592 compared to 0.87EE. In the mRNA-treated sample of RC1-2L.1592, the nuclease protein was electropositive. It was not detectable at 48 hours after air perforation or at any later time point. Expression of the RC1-2x.87EE protein is still detectable at this time point. Expression was stable across all samples and time points, and the same amount of protein was present in each sample. It was shown that it is added to the

[0319] These studies demonstrated that the TRC1-2x.87EE and TRC1-2L.1592 nuclei The enzyme was expressed at high levels 6 hours after electroporation of mRNA. Expression of TRC1-2L.1592 declined more rapidly than TRC1-2x.87EE in the As shown in Figure 11, the TRC1-2L.1592 is The gene editing efficiency was not reduced compared to the control group. Reducing the duration of expression while maintaining high gene editing activity is a key step in TRC 1-2L.1592 are desirable characteristics, and these properties may be enhanced (i.e., increased). The high proliferation capacity of T cells and TRC1 compared with TRC1-2x.87EE This correlates with the low off-target activity of TRC1-2L.1592. Compared to the 2x.87EE, it showed an unexpectedly favorable improvement.

[0320] Example 3 Evaluation of optimized TRC1-2 meganuclease in CAR T production

[0321] TRC1-2L.1592 meganuclease will be further evaluated for large-scale processing steps The first generation TRC1-2x.87EE meganuclear cell line was developed and demonstrated successful production of CAR T cells at scale. It was determined whether the enzyme could be improved against creatine.

[0322] used to generate allogeneic CAR T cells harboring TRC1-2x.87EE The large-scale process involves the collection of fresh leukoplakia from healthy, pre-qualified donors. The Leukopak product was washed and immunomagnetically enriched for target T cells. Platelets were removed before the procedure. The enriched T cells were then washed and placed in growth medium. After a 3-day activation period, the cells were washed and electrophoresed. Concentrated in pore buffer. Addition of mRNA encoding TRC1-2x.87EE. The mixture of cells and mRNA was then treated using an electroporation device. Dilution with growth medium containing the AAV vector encoding the CAR insert gene After the expansion period, cells were harvested on day 8 and immunomagnetically isolated from the CD3-positive population. After depletion, target CD3-negative cells were added. Finally, cells were harvested on day 13, washed, and frozen. The allogeneic CAR T carrying TRC1-2L.1592 was concentrated into a protective solution and frozen. The large-scale process used to generate cells for TRC1-2x.87EE The procedure was carried out essentially as described. Growth of TRC1-2L.1592 Except for the culture medium formulation, it was animal origin free (AOF).

[0323] The total number of viable cells was determined at key time points during the production process (Figure 18). The same procedure is followed from day 0 to the depletion step up to day 8. However, TRC1-2L .1592 has a significantly higher T cell receptor knockout efficiency, which is why TRC1-2L The depletion step in the .1592 process is the same as the TRC1-2x.87EE process. More than twice the number of cells recovered at the time of processing were recovered. Growth between days 8 and 13 The kinetics were similar, resulting in approximately 2x more total viable cells at day 13.

[0324] CD3 knockout efficiency (i.e., a measure of endogenous T cell receptor knockout) was measured in 8 The results were determined by flow cytometry at each production step on day 1 (Figure 19). The proportion of gene-edited cells (out of total viable cells) was 100%. The process of TRC1-2L.1592 is better than that of C1-2x.87EE. At times it was nearly 20% higher.

[0325] Finally, CAR knock-in efficiency was assessed by flow cytometry at three key time points during each production process. Unexpectedly, the CAR-positive The proportion of transduced cells (CD3-negative cells) in TRC1-2x.87EE Approximately 25% higher during the TRC1-2L.1592 process than during the CAR process. The quine ratio remained stable for both processes between day 8 and the end of the process on day 13. At the end of the process, TRC1-2L.1592 had a similarly high percentage of CAR. -Generates positive cells.

[0326] Finally, these studies surprisingly demonstrated that the TRC1-2L.1592 nuclease It has been shown to significantly improve the quality and quantity of the final allogeneic cell therapy product. -2L.1592 more effectively knocks out endogenous T cell receptors, thereby This results in a larger population of gene-edited CD3-negative cells, improving the overall production process. In addition, TRC1-2L.1592 showed improved CA The CAR gene was transduced into the targeted double-strand break, as evidenced by the knock-in efficiency. The insertion potentially improves the environment for homologous recombination. The increased ratio resulted in significantly higher purity with almost no CAR-negative cells. This results in a drug product.

Claims

1. A recognition sequence comprising SEQ ID NO:5 within the human T cell receptor (TCR) alpha constant region gene an engineered meganuclease that recognizes and cleaves The engineered meganuclease comprises a first subunit and a second subunit. the first subunit binds to a first recognition half-site of the recognition sequence, and the second subunit comprises a second hypervariable region (HVR1) of the recognition sequence; binding to the recognition half-site and comprising a second hypervariable (HVR2) region; Furthermore, the HVR2 (a) at least 81% to the amino acid sequence corresponding to residues 24 to 79 of SEQ ID NO:7 sequence homology of, or (b) at least 86% to the amino acid sequence corresponding to residues 24 to 79 of SEQ ID NO:8 An engineered meganuclease having sequence homology to:

2. the HVR2 region is selected from residues 24, 26, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, The residues corresponding to 2, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 2. The engineered meganuclease of claim 1, comprising a group.

3. The HVR2 region corresponds to residues 48, 50, 71, 72, and 73 of SEQ ID NO:

7.

3. The engineered meganuclease of claim 1 or 2, comprising a residue selected from the group consisting of:

4. the HVR2 region comprises residues corresponding to residues 48 and 50 of SEQ ID NO:

8.

4. The engineered meganuclease of any one of 1 to 3.

5. the HVR2 region is selected from residues 24, 26, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, ２、３３、３８、４０、４２、４４、４６、４８、５０、６８、７０、７１、７２、７３ 5. The engineered protein of claim 1, comprising residues corresponding to 75, 76, and 77. Meganucleases.

6. the HVR2 region comprises a Y at a residue corresponding to residue 66 of SEQ ID NO:7 or SEQ ID NO:8 6. The engineered meganucleic acid of any one of claims 1 to 5, comprising: Aze.

7. 1. The method of claim 1, wherein the HVR2 region comprises residues 24 to 79 of SEQ ID NO: 7 or SEQ ID NO:

8.

7. The engineered meganuclease of any one of claims 1 to 6.

8. the second subunit has at least one amino acid sequence similar to residues 7-153 of SEQ ID NO:7 or SEQ ID NO:8; Any one of claims 1 to 7, comprising an amino acid sequence with at least 80% sequence identity.

2. An engineered meganuclease according to claim 1.

9. The second subunit has a residue corresponding to residue 19 of SEQ ID NO:7 or SEQ ID NO:

8. The engineered meganucleotide of any one of claims 1 to 8, wherein the amino acid sequence comprises G, S, or A. Rease.

10. the second subunit has a residue corresponding to residue 80 of SEQ ID NO:7 or SEQ ID NO:8 The engineered meganucleotide of any one of claims 1 to 9, wherein the amino acid sequence is E, Q, or K. Rease.

11. the second subunit has at least one amino acid sequence similar to residues 7-153 of SEQ ID NO:7 or SEQ ID NO:8; Any one of claims 1 to 10, comprising an amino acid sequence having at least 80% sequence homology.

2. The engineered meganuclease according to paragraph 1.

12. the first subunit is a sequence corresponding to residues 198 to 344 of SEQ ID NO: 7 or SEQ ID NO: 8 , comprising an amino acid sequence having at least 80% sequence identity, and has at least 80% sequence homology to residues 7-153 of SEQ ID NO:7 or SEQ ID NO:8 The engineered megaprotein of any one of claims 1 to 11, comprising an amino acid sequence having the following properties: Nuclease.

13. The second subunit has a residue corresponding to residue 19 of SEQ ID NO:7 or SEQ ID NO:

8.

13. The engineered megagene of any one of claims 1 to 12, wherein the amino acid sequence comprises G, S, or A. Crease.

14. the second subunit has a residue corresponding to residue 80 of SEQ ID NO:7 or SEQ ID NO:8 The engineered megagene of any one of claims 1 to 13, wherein the amino acid sequence comprises E, Q, or K. Crease.

15. the second subunit contains a residue corresponding to residue 80 of SEQ ID NO:7 or SEQ ID NO:8 15. The engineered meganuclease of any one of claims 1 to 14, comprising:

16. the second subunit has a residue corresponding to residue 139 of SEQ ID NO:7 or SEQ ID NO:8 16. The engineered meganuclease of any one of claims 1 to 15, comprising:

17. the second subunit comprises residues 7 to 153 of SEQ ID NO:7 or SEQ ID NO:

8.

17. The engineered meganuclease of any one of claims 1 to 16.

18. the HVR1 region is an amino acid sequence corresponding to residues 215 to 270 of SEQ ID NO:7 or SEQ ID NO:8 The amino acid sequence of claim 1 has at least 80% sequence identity to the amino acid sequence of claim 1.

18. The engineered meganuclease of any one of 1 to 17.

19. the HVR1 region is selected from residues 215, 217, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 2 ２１、２２３、２２４、２２９、２３１、２３３、２３５、２３７、２５９、２６１、２ 19. The engineered polypeptide of claim 1, comprising residues corresponding to 66 and 268. Meganucleases.

20. the HVR1 region at a residue corresponding to residue 257 of SEQ ID NO:7 or SEQ ID NO:8 20. The engineered megagene of any one of claims 1 to 19, comprising Y, R, K, or D. Crease.

21. the HVR1 region comprises residues 215-270 of SEQ ID NO:7 or SEQ ID NO:

8.

21. The engineered meganuclease of any one of paragraphs 1 to 20.

22. the first subunit is a sequence corresponding to residues 198 to 344 of SEQ ID NO: 7 or SEQ ID NO: 8 22. Any of claims 1 to 21, comprising an amino acid sequence having at least 80% sequence homology. The engineered meganuclease of any one of claims 1 to 4.

23. The first subunit has a residue corresponding to residue 210 of SEQ ID NO:7 or SEQ ID NO:

8. The engineered molecule of any one of claims 1 to 22, wherein Nuclease.

24. The first subunit has a residue corresponding to residue 271 of SEQ ID NO:7 or SEQ ID NO:

8. The engineered megakaryon of any one of claims 1 to 23, wherein Nuclease.

25. The first subunit has a residue corresponding to residue 271 of SEQ ID NO:7 or SEQ ID NO:

8.

25. The engineered meganuclease of any one of claims 1 to 24, comprising:

26. the first subunit comprises residues 198 to 344 of SEQ ID NO:7 or SEQ ID NO:8 26. The engineered meganuclease of any one of claims 1 to 25.

27. the engineered meganuclease comprises a linker, the linker being connected to the first subunit any one of claims 1 to 26, wherein the unit and the second subunit are covalently linked 2. The engineered meganuclease according to paragraph 1.

28. the engineered meganuclease has the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8 28. The engineered meganuclease of any one of claims 1 to 27, comprising:

29. Compared to the TRC1-2x.87EE meganuclease set forth as SEQ ID NO: 9 When the engineered meganuclease is expressed as a nuclease with improved specificity, reduced retention time in cells, and enhancing the efficiency of modification of the human TCR alpha constant region gene.

29. The manipulated device according to any one of claims 1 to 28, exhibiting at least one of the following characteristics: Meganuclease.

30. A gene encoding the engineered meganuclease of any one of claims 1 to 29. A polynucleotide comprising a nucleic acid sequence comprising:

31. 31. The polynucleotide of claim 30, wherein the polynucleotide is mRNA.

32. The mRNA is the engineered meganuclear vector of any one of claims 1 to 29. a polypeptide encoding an enzyme and at least one additional polypeptide or nucleic acid; 32. The polynucleotide of claim 31, which is a ronic mRNA.

33. A recombinant DNA construct comprising the polynucleotide of claim 30.

34. 34. The recombinant DNA construct of claim 33, wherein the recombinant DNA construct encodes a viral vector. DNA constructs.

35. The viral vector is an adenoviral vector, a lentiviral vector, a retroviral vector, or a 35. The vector of claim 34, which is a viral vector or an adeno-associated viral (AAV) vector. The recombinant DNA constructs described above.

36. 36. The method of claim 34 or 35, wherein the viral vector is a recombinant AAV vector. A recombinant DNA construct as described.

37. A viral vector comprising the polynucleotide of claim 30.

38. The viral vector is an adenoviral vector, a lentiviral vector, a retroviral vector, or a The viral vector of claim 37, which is a viral vector or an AAV vector. 。

39. 39. The method of claim 37 or 38, wherein the viral vector is a recombinant AAV vector. The viral vector described.

40. A pre-transfected vector comprising an exogenous sequence of interest to be inserted into the chromosome of a genetically modified eukaryotic cell.

1. A method for producing the genetically modified eukaryotic cell, said method comprising: (a) encoding the engineered meganuclease of any one of claims 1 to 29; and wherein the engineered meganuclease is a first gene expressed in the eukaryotic cell. and (b) a second nucleic acid comprising said sequence of interest; and and inserting into a eukaryotic cell one or more nucleic acids comprising the engineered The meganuclease creates a cleavage site in the chromosome at a recognition sequence containing SEQ ID NO:

5. death, The method, wherein the sequence of interest is inserted into the chromosome at the break site.

41. the second nucleic acid further comprises a sequence homologous to a sequence adjacent to the cleavage site, 41. The method of claim 40, wherein the sequence is inserted into the cleavage site by homologous recombination. Law.

42. Cell surface expression of endogenous T cell receptors was compared to unmodified control cells.

42. The method of claim 40 or claim 41, wherein the expression of the How to do it.

43. The eukaryotic cells are human T cells, cells derived from human T cells, or human NK cells.

43. The method according to claim 40, wherein the cells are human NK cells or cells derived from human NK cells. The method described.

44. The sequence of interest may be a coding sequence for a chimeric antigen receptor or an exogenous T cell receptor.

44. The method of any one of claims 40 to 43, comprising a column.

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

45. The method of claim 44, comprising an extracellular ligand-binding domain having the following structure:

46. At least the first nucleic acid is introduced into the eukaryotic cell by mRNA. Item 46. The method according to any one of items 40 to 45.

47. At least the second nucleic acid is introduced into the eukaryotic cell by a viral vector. The method according to any one of claims 40 to 46,

48. The viral vector is an adenoviral vector, a lentiviral vector, a retroviral vector, or a 48. The method of claim 47, wherein the vector is a viral vector or an AAV vector.

49. 49. The method of claim 47 or 48, wherein the viral vector is a recombinant AAV vector. The method described.

50. A genetically modified eukaryotic cell comprising an exogenous sequence of interest that is inserted into the chromosome of the cell. A method for producing the genetically modified eukaryotic cell, said method comprising: (a) the engineered meganuclease of any one of claims 1 to 29 introducing it into a nuclear cell; (b) introducing into the eukaryotic cell a nucleic acid comprising the sequence of interest; The engineered meganuclease is inserted into the chromosome at a recognition sequence comprising SEQ ID NO:

5. Creating a cleavage site, The method, wherein the sequence of interest is inserted into the chromosome at the break site.

51. the nucleic acid further comprises a sequence homologous to a sequence adjacent to the cleavage site, 51. The method of claim 50, wherein the sequence is inserted at the cleavage site by homologous recombination.

52. Cell surface expression of endogenous T cell receptors was compared to unmodified control cells.

52. The method of claim 50 or claim 51, wherein the expression of How to do it.

53. The eukaryotic cells are human T cells, cells derived from human T cells, or human NK cells.

53. The method according to any one of claims 50 to 52, wherein the cells are human NK cells or cells derived from human NK cells. The method described.

54. The sequence of interest may be a coding sequence for a chimeric antigen receptor or an exogenous T cell receptor.

54. The method of any one of claims 50 to 53, comprising a column.

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

55. The method of claim 54, comprising an extracellular ligand-binding domain having the following structure:

56. 51. The method of claim 50, wherein the nucleic acid is introduced into the eukaryotic cell by a viral vector.

56. The method of any one of claims 1 to 55.

57. The viral vector is an adenoviral vector, a lentiviral vector, a retroviral vector, or a 57. The method of claim 56, wherein the vector is a viral vector or an AAV vector.

58. Claim 55 or claim 56, wherein the viral vector is a recombinant AAV vector. The method described below.

59. Genetic modification by disrupting a target sequence in the chromosome of a genetically modified eukaryotic cell. A method for producing the modified eukaryotic cell, said method comprising: A gene encoding the engineered meganuclease of any one of claims 1 to 29. and introducing a nucleic acid sequence comprising the engineered meganuclease into a eukaryotic cell, It is expressed in eukaryotic cells and The engineered meganuclease cleaves into the chromosome at a recognition sequence comprising SEQ ID NO:

5. A cleavage site is created, and the target sequence is cleaved by non-homologous end joining at the cleavage site. It's broken, way.

60. Cell surface expression of endogenous T cell receptors was compared to unmodified control cells.

60. The method of claim 59, wherein the expression level of the gene is reduced in the genetically modified cell.

61. The eukaryotic cells are human T cells, cells derived from human T cells, or human NK cells.

61. The method according to claim 59 or 60, wherein the cell is a human NK cell or a cell derived from a human NK cell. How to post.

62. 62. Any of claims 59 to 61, wherein the nucleic acid is introduced into the eukaryotic cell by mRNA. The method according to any one of claims 1 to 4.

63. Genetic modification by disrupting a target sequence in the chromosome of a genetically modified eukaryotic cell. A method for producing the modified eukaryotic cell, said method comprising:

30. The engineered meganuclease of any one of claims 1 to 29, wherein the engineered meganuclease is introduced into a eukaryotic cell. Including introducing The engineered meganuclease cleaves into the chromosome at a recognition sequence comprising SEQ ID NO:

5. A cleavage site is created, and the target sequence is cleaved by non-homologous end joining at the cleavage site. It's broken, way.

64. Cell surface expression of endogenous T cell receptors was compared to unmodified control cells.

64. The method of claim 63, wherein the expression level of the gene is reduced in the genetically modified cell.

65. The eukaryotic cells are human T cells, cells derived from human T cells, or human NK cells.

65. The method according to claim 63 or claim 64, wherein the cell is a human NK cell or a cell derived from a human NK cell. How to post.

66. A genetically modified organism prepared by the method of any one of claims 40 to 58. Modified eukaryotic cells.

67. The cells are TRC1-2x.87EE meganuclear cells, which are set forth as SEQ ID NO:

9. Reduced off-target effects with the engineered meganucleases compared to non-targeted meganucleases , a decrease in the maintenance time of the engineered meganuclease in the eukaryotic cell, or both.

67. The genetically modified eukaryotic cell of claim 66, comprising:

68. A genetically modified plant prepared by the method of any one of claims 59 to 65. A eukaryotic cell.

69. The cells are TRC1-2x.87EE meganuclear cells, which are set forth as SEQ ID NO:

9. Reduced off-target effects with the engineered meganucleases compared to non-targeted meganucleases , a decrease in the maintenance time of the engineered meganuclease in the eukaryotic cell, or both.

69. The genetically modified eukaryotic cell of claim 68, comprising:

70. 68. A method for producing a recombinant eukaryotic cell comprising the steps of: A population of genetically modified eukaryotic cells.

71. At least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% %, at least 55%, at least 60%, at least 65%, at least 70%, at least at least 75%, at least 80%, at least 85%, at least 90%, at least 9 5%, or up to 100% of the cells in the population are of the type described in claim 66 or claim 67 71. The population of genetically modified eukaryotic cells of claim 70.

72. The genetically modified eukaryotic cell may be a genetically modified human T cell or a genetically modified human T cell. Cells derived from human T cells, or genetically modified NK cells, or genetically modified 72. The population of claim 70 or claim 71, which is a cell derived from a modified NK cell. 。

73. The sequence of interest may be a coding sequence for a chimeric antigen receptor or an exogenous T cell receptor.

73. The collection of any one of claims 70 to 72, comprising a row.

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

74. The population of claim 73, comprising an extracellular ligand-binding domain having the following structure:

75. Cell surface expression of endogenous T cell receptors was compared to unmodified control cells.

75. The method of claim 70, wherein the gene expression level is reduced in the genetically modified eukaryotic cell. The population described in section .

76. 70. A method for producing a recombinant eukaryotic cell comprising the steps of: A population of genetically modified eukaryotic cells.

77. At least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% %, at least 55%, at least 60%, at least 65%, at least 70%, at least at least 75%, at least 80%, at least 85%, at least 90%, at least 9 5%, or up to 100% of the cells in the population are of the type described in claim 68 or claim 69.

77. The population of genetically modified eukaryotic cells of claim 76.

78. The genetically modified eukaryotic cell may be a genetically modified human T cell or a genetically modified human T cell. Cells derived from human T cells, or genetically modified NK cells, or genetically modified 78. The population of claim 76 or claim 77, which is a cell derived from a modified NK cell. 。

79. The genetically modified eukaryotic cells express a cell surface chimeric antigen receptor or an exogenous T cell receptor.

79. The population of any one of claims 76 to 78, comprising a body.

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

80. The population of claim 79, comprising an extracellular ligand-binding domain having the following structure:

81. Cell surface expression of endogenous T cell receptors was compared to unmodified control cells.

81. The method of claim 76, wherein the method is reduced in the genetically modified eukaryotic cell. The population described in section .

82. 1. A pharmaceutical composition useful for treating a disease in a subject in need thereof, comprising: The pharmaceutical composition comprises a pharmaceutically acceptable carrier and a therapeutically effective amount of claim 66 or claim 67. 67, or the genetically modified eukaryotic cell of any one of claims 70 to 75. A pharmaceutical composition comprising the population of genetically modified eukaryotic cells described above.

83. the genetically modified eukaryotic cells or population are genetically modified human T cells; are genetically modified human T cell-derived cells or genetically modified NK cells, or cells derived from genetically modified NK cells. composition.

84. The sequence of interest may be a coding sequence for a chimeric antigen receptor or an exogenous T cell receptor.

84. The pharmaceutical composition of claim 82 or claim 83, comprising a sequence.

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

85. The pharmaceutical composition of claim 84, comprising an extracellular ligand-binding domain having the following structure:

86. Cell surface expression of endogenous T cell receptors was compared to unmodified control cells.

86. The method of claim 82, wherein the gene expression level is reduced in the genetically modified eukaryotic cell. The pharmaceutical composition according to claim 1.

87. 1. A pharmaceutical composition useful for treating a disease in a subject in need thereof, comprising: The pharmaceutical composition comprises a pharmaceutically acceptable carrier and a therapeutically effective amount of claim 68 or claim 69. 69, or the genetically modified eukaryotic cell of any one of claims 76 to 81. A pharmaceutical composition comprising said population of genetically modified eukaryotic cells as described above.

88. the genetically modified eukaryotic cells or population are genetically modified human T cells; are genetically modified human T cell-derived cells or genetically modified NK cells, or cells derived from genetically modified NK cells. composition.

89. The genetically modified eukaryotic cells express a cell surface chimeric antigen receptor or an exogenous T cell receptor.

89. The pharmaceutical composition of claim 87 or claim 88, comprising the body.

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

90. The pharmaceutical composition of claim 89, comprising an extracellular ligand-binding domain having the following structure:

91. Cell surface expression of endogenous T cell receptors was compared to unmodified control cells.

91. The method of claim 87, wherein the gene expression level is reduced in the genetically modified eukaryotic cell. The pharmaceutical composition according to claim 1.

92. 1. A method of treating a disease in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of the genetically modified eukaryotic cell according to any one of claims 66 to 69. or the population of genetically modified eukaryotic cells according to any one of claims 70 to 81. The method comprising administering.

93. The method comprises administering to the subject the pharmaceutical composition of any one of claims 82 to 91.

81. The method of claim 80, comprising administering

94. the method is an immunotherapy for treating cancer in a subject in need thereof, The genetically modified eukaryotic cells may be genetically modified human T cells or genetically modified human T cells. cells derived from human T cells, or genetically modified NK cells, or genetically modified and a cell derived from a natural killer (NK) cell, wherein the genetically modified eukaryotic cell is capable of expressing a tumor-specific antigen. Cell surface chimeric antigen receptors comprising an extracellular ligand-binding domain with specificity for or compared to unmodified control cells containing an exogenous T cell receptor. cell surface expression of an endogenous T cell receptor is reduced in said genetically modified eukaryotic cell; 94. The method of claim 92 or claim 93.

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

95. The method of claim 94, wherein

96. 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.

96. The method of claim 94 or claim 95, wherein the compound is selected from the group consisting of:

97. The cancer of B-cell origin is B-cell acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, 9. The method of claim 8, wherein the cancer is selected from the group consisting of B-cell non-Hodgkin's lymphoma, B-cell non-Hodgkin's lymphoma, and multiple myeloma.

6. The method according to claim 6.

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