Nucleic acids encoding engineered meganucleases with recognition sequences found in the human t cell receptor alpha constant region gene

Abstract

Disclosed herein are recombinant meganucleases engineered to recognize and cleave a recognition sequence present in the human T cell receptor alpha constant region gene. The present disclosure further relates to the use of such recombinant meganucleases in methods for producing genetically-modified eukaryotic cells.

1 - 9 . (canceled) 
     
     
         10 . A method for producing a genetically-modified eukaryotic cell by disrupting a target sequence in a chromosome of said eukaryotic cell, said method comprising:
 transfecting a eukaryotic cell with a nucleic acid encoding an engineered meganuclease, wherein said engineered meganuclease binds and cleaves at a recognition sequence consisting of SEQ ID NO: 3, wherein said engineered meganuclease comprises a first subunit and a second subunit, wherein said first subunit binds to a first recognition half-site of said recognition sequence, and wherein said second subunit binds to a second recognition half-site of said recognition sequence, and wherein said engineered meganuclease comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 8;   wherein said engineered meganuclease produces a cleavage site in said chromosome at a recognition sequence consisting of SEQ ID NO: 3, and wherein said target sequence is disrupted by non-homologous end-joining at said cleavage site.   
     
     
         11 . The method of  claim 10 , wherein said eukaryotic cell is a human T cell or a cell derived therefrom. 
     
     
         12 . The method of  claim 10  further comprising transfecting said eukaryotic cell with a second nucleic acid including an exogenous sequence of interest. 
     
     
         13 . The method of  claim 12 , wherein said second nucleic acid further comprises sequences homologous to sequences flanking said cleavage site, and wherein said exogenous sequence of interest is inserted at said cleavage site by homologous recombination. 
     
     
         14 . The method of  claim 12 , wherein said sequence of interest encodes a chimeric antigen receptor. 
     
     
         15 . The method of  claim 12 , wherein at least said second nucleic acid is introduced into said eukaryotic cell by a recombinant AAV vector. 
     
     
         16 . A method for producing a genetically-modified eukaryotic cell by disrupting a target sequence in a chromosome of said eukaryotic cell, comprising:
 introducing an engineered meganuclease into a eukaryotic cell, wherein said engineered meganuclease binds and cleaves at a recognition sequence consisting of SEQ ID NO: 3, wherein said engineered meganuclease comprises a first subunit and a second subunit, wherein said first subunit binds to a first recognition half-site of said recognition sequence, wherein said second subunit binds to a second recognition half-site of said recognition sequence, and wherein said engineered meganuclease comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 8;   wherein said meganuclease produces a cleavage site in said chromosome at a recognition sequence consisting of SEQ ID NO: 3, and wherein said target sequence is disrupted by non-homologous end-joining at said cleavage site.   
     
     
         17 . The method of  claim 16 , wherein said eukaryotic cell is a human T cell or a cell derived therefrom. 
     
     
         18 . The method of  claim 16  further comprising transfecting said eukaryotic cell with a nucleic acid including an exogenous sequence of interest. 
     
     
         19 . The method of  claim 18 , wherein said nucleic acid further comprises sequences homologous to sequences flanking said cleavage site, and wherein said exogenous sequence of interest is inserted at said cleavage site by homologous recombination. 
     
     
         20 . The method of  claim 18 , wherein said sequence of interest encodes a chimeric antigen receptor. 
     
     
         21 . The method of  claim 18 , wherein said nucleic acid is introduced into said eukaryotic cell by a recombinant AAV vector. 
     
     
         22 . A method of immunotherapy for treating cancer in a subject in need thereof, said method comprising administering to said subject a pharmaceutical composition comprising said genetically-modified cell produced by the method of claim  1  and a pharmaceutically acceptable carrier. 
     
     
         23 . The method of  claim 22 , wherein said cancer is selected from the group consisting of carcinoma, lymphoma, sarcoma, blastomas, and leukemia. 
     
     
         24 . The method of  claim 22 , wherein the cancer is selected from the group consisting of a cancer of B-cell origin, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, melanoma, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, and Hodgkin's lymphoma. 
     
     
         25 . The method of  claim 24 , wherein the cancer of B-cell origin is selected from the group consisting of B-lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, and B-cell non-Hodgkin's lymphoma. 
     
     
         26 . A use of an engineered meganuclease having at least 96% identity to SEQ ID NO: 8 to treat a subject in need of treatment thereof. 
     
     
         27 . The use of  claim 26 , wherein said subject has a cancer that is selected from the group consisting of carcinoma, lymphoma, sarcoma, blastomas, and leukemia. 
     
     
         28 . The use of  claim 26 , wherein the cancer is selected from the group consisting of a cancer of B-cell origin, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, melanoma, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, and Hodgkin's lymphoma. 
     
     
         29 . The use of  claim 28 , wherein the cancer of B-cell origin is selected from the group consisting of B-lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, and B-cell non-Hodgkin's lymphoma.