Optimized engineered nucleases having specificity for the human t cell receptor alpha constant region gene

Abstract

The present invention encompasses engineered nucleases which recognize and cleave a recognition sequence within the first exon of the human T cell receptor (TCR) alpha constant region gene. The engineered meganucleases can exhibit at least one optimized characteristic, such as enhanced (i.e., increased) specificity or efficiency of cleavage, when compared to the first-generation meganuclease TRC 1-2x.87EE. The present invention also encompasses methods of using such engineered nucleases to make genetically-modified cells, and the use of such cells in a pharmaceutical composition and in methods for treating diseases, such as cancer.

1 . An engineered meganuclease that recognizes and cleaves a recognition sequence comprising SEQ ID NO: 5 within a human T cell receptor (TCR) alpha constant region gene, wherein said engineered meganuclease comprises a first subunit and a second subunit, wherein said first subunit binds to a first recognition half-site of said recognition sequence and comprises a first hypervariable (HVR1) region, wherein said second subunit binds to a second recognition half-site of said recognition sequence and comprises a second hypervariable (HVR2) region, and wherein said HVR2 has:
 (a) at least 81% sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 7; or   (b) at least 86% sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 8.   
     
     
         2 . The engineered meganuclease of  claim 1 , wherein said HVR2 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, and 77 of SEQ ID NO: 7 or SEQ ID NO: 8. 
     
     
         3 . The engineered meganuclease of  claim 1  or  claim 2 , wherein said HVR2 region comprises residues corresponding to residues 48, 50, 71, 72, and 73 of SEQ ID NO: 7. 
     
     
         4 . The engineered meganuclease of any one of  claims 1 - 3 , wherein said HVR2 region comprises residues corresponding to residues 48 and 50 of SEQ ID NO: 8. 
     
     
         5 . The engineered meganuclease of any one of  claims 1 - 4 , wherein said HVR2 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 48, 50, 68, 70, 71, 72, 73, 75, and 77 of SEQ ID NO: 7 or 8. 
     
     
         6 . The engineered meganuclease of any one of  claims 1 - 5 , wherein said HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 7 or 8. 
     
     
         7 . The engineered meganuclease of any one of  claims 1 - 6 , wherein said HVR2 region comprises residues 24-79 of SEQ ID NO: 7 or 8. 
     
     
         8 . The engineered meganuclease of any one of  claims 1 - 7 , wherein said second subunit comprises an amino acid sequence having at least 80% sequence identity to residues 7-153 of SEQ ID NO: 7 or 8. 
     
     
         9 . The engineered meganuclease of any one of  claims 1 - 8 , wherein said second subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 7 or 8. 
     
     
         10 . The engineered meganuclease of any one of  claims 1 - 9 , wherein said second subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 7 or 8. 
     
     
         11 . The engineered meganuclease of any one of  claims 1 - 10 , wherein said second subunit comprises an amino acid sequence having at least 80% sequence identity to residues 7-153 of SEQ ID NO: 7 or 8. 
     
     
         12 . The engineered meganuclease of any one of  claims 1 - 11 , wherein said first subunit comprises an amino acid sequence having at least 80% sequence identity to residues 198-344 of SEQ ID NO: 7 or 8, and wherein said second subunit comprises an amino acid sequence having at least 80% sequence identity to residues 7-153 of SEQ ID NO: 7 or 8. 
     
     
         13 . The engineered meganuclease of any one of  claims 1 - 12 , wherein said second subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 7 or 8. 
     
     
         14 . The engineered meganuclease of any one of  claims 1 - 13 , wherein said second subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 7 or 8. 
     
     
         15 . The engineered meganuclease of any one of  claims 1 - 14 , wherein said second subunit comprises a residue corresponding to residue 80 of SEQ ID NO: 7 or 8. 
     
     
         16 . The engineered meganuclease of any one of  claims 1 - 15 , wherein said second subunit comprises a residue corresponding to residue 139 of SEQ ID NO: 7 or 8. 
     
     
         17 . The engineered meganuclease of any one of  claims 1 - 16 , wherein said second subunit comprises residues 7-153 of SEQ ID NO: 7 or 8. 
     
     
         18 . The engineered meganuclease of any one of  claims 1 - 17 , wherein said HVR1 region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 7 or 8. 
     
     
         19 . The engineered meganuclease of any one of  claims 1 - 18 , wherein said HVR1 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 7 or 8. 
     
     
         20 . The engineered meganuclease of any one of  claims 1 - 19 , wherein said HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 7 or 8. 
     
     
         21 . The engineered meganuclease of any one of  claims 1 - 20 , wherein said HVR1 region comprises residues 215-270 of SEQ ID NO: 7 or 8. 
     
     
         22 . The engineered meganuclease of any one of  claims 1 - 21 , wherein said first subunit comprises an amino acid sequence having at least 80% sequence identity to residues 198-344 of SEQ ID NO: 7 or 8. 
     
     
         23 . The engineered meganuclease of any one of  claims 1 - 22 , wherein said first subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 7 or 8. 
     
     
         24 . The engineered meganuclease of any one of  claims 1 - 23 , wherein said first subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 7 or 8. 
     
     
         25 . The engineered meganuclease of any one of  claims 1 - 24 , wherein said first subunit comprises a residue corresponding to residue 271 of SEQ ID NO: 7 or 8. 
     
     
         26 . The engineered meganuclease of any one of  claims 1 - 25 , wherein said first subunit comprises residues 198-344 of SEQ ID NO: 7 or 8. 
     
     
         27 . The engineered meganuclease of any one of  claims 1 - 26 , wherein said engineered meganuclease comprises a linker, wherein said linker covalently joins said first subunit and said second subunit. 
     
     
         28 . The engineered meganuclease of any one of  claims 1 - 27 , wherein said engineered meganuclease comprises the amino acid sequence of SEQ ID NO: 7 or 8. 
     
     
         29 . The engineered meganuclease of any one of  claims 1 - 28 , wherein said engineered meganuclease exhibits at least one of the following optimized characteristics as compared to TRC 1-2x.87EE meganuclease set forth as SEQ ID NO: 9: improved specificity, reduced persistence time in cells, and enhanced efficiency of modification of the human TCR alpha constant region gene. 
     
     
         30 . A polynucleotide comprising a nucleic acid sequence encoding said engineered meganuclease of any one of  claims 1 - 29 . 
     
     
         31 . The polynucleotide of  claim 30 , wherein said polynucleotide is an mRNA. 
     
     
         32 . The polynucleotide of  claim 31 , wherein said mRNA is a polycistronic mRNA encoding said engineered meganuclease of any one of  claims 1 - 29  and at least one additional polypeptide or nucleic acid. 
     
     
         33 . A recombinant DNA construct comprising said polynucleotide of  claim 30 . 
     
     
         34 . The recombinant DNA construct of  claim 33 , wherein said recombinant DNA construct encodes a viral vector. 
     
     
         35 . The recombinant DNA construct of  claim 34 , wherein said viral vector is an adenoviral vector, a lentiviral vector, a retroviral vector, or an adeno-associated viral (AAV) vector. 
     
     
         36 . The recombinant DNA construct of  claim 34  or  claim 35 , wherein said viral vector is a recombinant AAV vector. 
     
     
         37 . A viral vector comprising said polynucleotide of  claim 30 . 
     
     
         38 . The viral vector of  claim 37 , wherein said viral vector is an adenoviral vector, a lentiviral vector, a retroviral vector, or an AAV vector. 
     
     
         39 . The viral vector of  claim 37  or  claim 38 , wherein said viral vector is a recombinant AAV vector. 
     
     
         40 . A method for producing a genetically-modified eukaryotic cell comprising an exogenous sequence of interest inserted into a chromosome of said eukaryotic cell, said method comprising introducing into a eukaryotic cell one or more nucleic acids including:
 (a) a first nucleic acid encoding said engineered meganuclease of any one of  claims 1 - 29 , wherein said engineered meganuclease is expressed in said eukaryotic cell; and   (b) a second nucleic acid including said sequence of interest;   wherein said engineered meganuclease produces a cleavage site in said chromosome at a recognition sequence comprising SEQ ID NO: 5;   and wherein said sequence of interest is inserted into said chromosome at said cleavage site.   
     
     
         41 . The method of  claim 40 , wherein said second nucleic acid further comprises sequences homologous to sequences flanking said cleavage site and said sequence of interest is inserted at said cleavage site by homologous recombination. 
     
     
         42 . The method of  claim 40  or  claim 41 , wherein cell surface expression of an endogenous T cell receptor is reduced on said genetically-modified cell when compared to an unmodified control cell. 
     
     
         43 . The method of any one of  claims 40 - 42 , wherein said eukaryotic cell is a human T cell, or a cell derived therefrom, or a human NK cell, or a cell derived therefrom. 
     
     
         44 . The method of any one of  claims 40 - 43 , wherein said sequence of interest comprises a coding sequence for a chimeric antigen receptor or an exogenous T cell receptor. 
     
     
         45 . The method of  claim 44 , wherein said chimeric antigen receptor or said exogenous T cell receptor comprises an extracellular ligand-binding domain having specificity for a tumor-specific antigen. 
     
     
         46 . The method of any one of  claims 40 - 45 , wherein at least said first nucleic acid is introduced into said eukaryotic cell by an mRNA. 
     
     
         47 . The method of any one of  claims 40 - 46 , wherein at least said second nucleic acid is introduced into said eukaryotic cell by a viral vector. 
     
     
         48 . The method of  claim 47 , wherein said viral vector is an adenoviral vector, a lentiviral vector, a retroviral vector, or an AAV vector. 
     
     
         49 . The method of  claim 47  or  claim 48 , wherein said viral vector is a recombinant AAV vector. 
     
     
         50 . A method for producing a genetically-modified eukaryotic cell comprising an exogenous sequence of interest inserted into a chromosome of said eukaryotic cell, said method comprising:
 (a) introducing said engineered meganuclease of any one of  claims 1 - 29  into a eukaryotic cell; and   (b) introducing a nucleic acid including said sequence of interest into said eukaryotic cell;   wherein said engineered meganuclease produces a cleavage site in said chromosome at a recognition sequence comprising SEQ ID NO: 5;   and wherein said sequence of interest is inserted into said chromosome at said cleavage site.   
     
     
         51 . The method of  claim 50 , wherein said nucleic acid further comprises sequences homologous to sequences flanking said cleavage site and said sequence of interest is inserted at said cleavage site by homologous recombination. 
     
     
         52 . The method of  claim 50  or  claim 51 , wherein cell surface expression of an endogenous T cell receptor is reduced on said genetically-modified cell when compared to an unmodified control cell. 
     
     
         53 . The method of any one of  claims 50 - 52 , wherein said eukaryotic cell is a human T cell, or a cell derived therefrom, or a human NK cell, or a cell derived therefrom. 
     
     
         54 . The method of any one of  claims 50 - 53 , wherein said sequence of interest comprises a coding sequence for a chimeric antigen receptor or an exogenous T cell receptor. 
     
     
         55 . The method of  claim 54 , wherein said chimeric antigen receptor or said exogenous T cell receptor comprises an extracellular ligand-binding domain having specificity for a tumor-specific antigen. 
     
     
         56 . The method of any one of  claims 50 - 55 , wherein said nucleic acid is introduced into said eukaryotic cell by a viral vector. 
     
     
         57 . The method of  claim 56 , wherein said viral vector is an adenoviral vector, a lentiviral vector, a retroviral vector, or an AAV vector. 
     
     
         58 . The method of  claim 55  or  claim 56 , wherein said viral vector is a recombinant AAV vector. 
     
     
         59 . A method for producing a genetically-modified eukaryotic cell by disrupting a target sequence in a chromosome of said eukaryotic cell, said method comprising:
 introducing into a eukaryotic cell a nucleic acid encoding said engineered meganuclease of any one of  claims 1 - 29 , wherein said engineered meganuclease is expressed in said eukaryotic cell;   wherein said engineered meganuclease produces a cleavage site in said chromosome at a recognition sequence comprising SEQ ID NO: 5, and wherein said target sequence is disrupted by non-homologous end-joining at said cleavage site.   
     
     
         60 . The method of  claim 59 , wherein cell surface expression of an endogenous T cell receptor is reduced on said genetically-modified cell when compared to an unmodified control cell. 
     
     
         61 . The method of  claim 59  or  claim 60 , wherein said eukaryotic cell is a human T cell, or a cell derived therefrom, or a human NK cell, or a cell derived therefrom. 
     
     
         62 . The method of any one of  claims 59 - 61 , wherein said nucleic acid is introduced into said eukaryotic cell by an mRNA. 
     
     
         63 . A method for producing a genetically-modified eukaryotic cell by disrupting a target sequence in a chromosome of said eukaryotic cell, said method comprising:
 introducing into a eukaryotic cell said engineered meganuclease of any one of  claims 1 - 29 ;   wherein said engineered meganuclease produces a cleavage site in said chromosome at a recognition sequence comprising SEQ ID NO: 5, and wherein said target sequence is disrupted by non-homologous end-joining at said cleavage site.   
     
     
         64 . The method of  claim 63 , wherein cell surface expression of an endogenous T cell receptor is reduced on said genetically-modified cell when compared to an unmodified control cell. 
     
     
         65 . The method of  claim 63  or  claim 64 , wherein said eukaryotic cell is a human T cell, or a cell derived therefrom, or a human NK cell, or a cell derived therefrom. 
     
     
         66 . A genetically-modified eukaryotic cell prepared by the method of any one of  claims 40 - 58 . 
     
     
         67 . The genetically-modified eukaryotic cell of  claim 66 , wherein said cell comprises reduced off-target effects by said engineered meganuclease, reduced persistence time of said engineered meganucleases in said eukaryotic cell, or both, as compared to the TRC 1-2x.87EE meganuclease set forth as SEQ ID NO: 9. 
     
     
         68 . A genetically-modified eukaryotic cell prepared by the method of any one of  claims 59 - 65 . 
     
     
         69 . The genetically-modified eukaryotic cell of  claim 68 , wherein said cell comprises reduced off-target effects by said engineered meganuclease, reduced persistence time of said engineered meganuclease in said eukaryotic cell, or both, as compared to the TRC 1-2x.87EE meganuclease set forth as SEQ ID NO: 9. 
     
     
         70 . A population of genetically-modified eukaryotic cells comprising a plurality of said genetically-modified eukaryotic cell of  claim 66  or  claim 67 . 
     
     
         71 . The population of  claim 70 , wherein 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 75%, at least 80%, at least 85%, at least 90%, at least 95%, or up to 100%, of cells in said population are said genetically-modified eukaryotic cell of  claim 66  or  claim 67 . 
     
     
         72 . The population of  claim 70  or  claim 71 , wherein said genetically-modified eukaryotic cell is a genetically-modified human T cell, or cell derived therefrom, or a genetically-modified NK cell, or cell derived therefrom. 
     
     
         73 . The population of any one of  claims 70 - 72 , wherein said sequence of interest comprises a coding sequence for a chimeric antigen receptor or an exogenous T cell receptor. 
     
     
         74 . The population of  claim 73 , wherein said chimeric antigen receptor or said exogenous T cell receptor comprises an extracellular ligand-binding domain having specificity for a tumor-specific antigen. 
     
     
         75 . The population of any one of  claims 70 - 74 , wherein cell surface expression of an endogenous T cell receptor is reduced on said genetically-modified eukaryotic cell when compared to an unmodified control cell. 
     
     
         76 . A population of genetically-modified eukaryotic cells comprising a plurality of said genetically-modified eukaryotic cell of  claim 68  or  claim 69 . 
     
     
         77 . The population of  claim 76 , wherein 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 75%, at least 80%, at least 85%, at least 90%, at least 95%, or up to 100%, of cells in said population are said genetically-modified eukaryotic cell of  claim 68  or  claim 69 . 
     
     
         78 . The population of  claim 76  or  claim 77 , wherein said genetically-modified eukaryotic cell is a genetically-modified human T cell, or cell derived therefrom, or a genetically-modified NK cell, or cell derived therefrom. 
     
     
         79 . The population of any one of  claims 76 - 78 , wherein said genetically-modified eukaryotic cell comprises a cell surface chimeric antigen receptor or exogenous T cell receptor. 
     
     
         80 . The population of  claim 79 , wherein said chimeric antigen receptor or said exogenous T cell receptor comprises an extracellular ligand-binding domain having specificity for a tumor-specific antigen. 
     
     
         81 . The population of any one of  claims 76 - 80 , wherein cell surface expression of an endogenous T cell receptor is reduced on said genetically-modified eukaryotic cell when compared to an unmodified control cell. 
     
     
         82 . A pharmaceutical composition useful for the treatment of a disease in a subject in need thereof, wherein said pharmaceutical composition comprises a pharmaceutically-acceptable carrier and a therapeutically-effective amount of said genetically-modified eukaryotic cell of  claim 66  or  claim 67  or said population of genetically-modified eukaryotic cells of any one of  claims 70 - 75 . 
     
     
         83 . The pharmaceutical composition of  claim 82 , wherein said genetically-modified eukaryotic cell or said population is comprised of a genetically-modified human T cell, or a cell derived therefrom, or a genetically-modified NK cell, or a cell derived therefrom. 
     
     
         84 . The pharmaceutical composition of  claim 82  or  claim 83 , wherein said sequence of interest comprises a coding sequence for a chimeric antigen receptor or an exogenous T cell receptor. 
     
     
         85 . The pharmaceutical composition of  claim 84 , wherein said chimeric antigen receptor or said exogenous T cell receptor comprises an extracellular ligand-binding domain having specificity for a tumor-specific antigen. 
     
     
         86 . The pharmaceutical composition of any one of  claims 82 - 85 , wherein cell surface expression of an endogenous T cell receptor is reduced on said genetically-modified eukaryotic cell when compared to an unmodified control cell. 
     
     
         87 . A pharmaceutical composition useful for the treatment of a disease in a subject in need thereof, wherein said pharmaceutical composition comprises a pharmaceutically-acceptable carrier and a therapeutically-effective amount of said genetically-modified eukaryotic cell of  claim 68  or  claim 69  or said population of genetically-modified eukaryotic cells of any one of  claims 76 - 81 . 
     
     
         88 . The pharmaceutical composition of  claim 87 , wherein said genetically-modified eukaryotic cell is or said population is comprised of a genetically-modified human T cell, or a cell derived therefrom, or a genetically-modified NK cell, or a cell derived therefrom. 
     
     
         89 . The pharmaceutical composition of  claim 87  or  claim 88 , wherein said genetically-modified eukaryotic cell comprises a cell surface chimeric antigen receptor or exogenous T cell receptor. 
     
     
         90 . The pharmaceutical composition of  claim 89 , wherein said chimeric antigen receptor or said exogenous T cell receptor comprises an extracellular ligand-binding domain having specificity for a tumor-specific antigen. 
     
     
         91 . The pharmaceutical composition of any one of  claims 87 - 90 , wherein cell surface expression of an endogenous T cell receptor is reduced on said genetically-modified eukaryotic cell when compared to an unmodified control cell. 
     
     
         92 . A method of treating a disease in a subject in need thereof, said method comprising administering to said subject a therapeutically-effective amount of said genetically-modified eukaryotic cell of any one of  claims 66 - 69  or said population of genetically-modified eukaryotic cells of any one of  claims 70 - 81 . 
     
     
         93 . The method of  claim 80 , wherein said method comprises administering to said subject said pharmaceutical composition of any one of  claims 82 - 91 . 
     
     
         94 . The method of  claim 92  or  claim 93 , wherein said method is an immunotherapy for the treatment of a cancer in a subject in need thereof, and wherein said genetically-modified eukaryotic cell is a genetically-modified human T cell, or a cell derived therefrom, or a genetically-modified NK cell, or a cell derived therefrom, and wherein said genetically-modified eukaryotic cell comprises a cell surface chimeric antigen receptor or exogenous T cell receptor comprising an extracellular ligand-binding domain having specificity for a tumor-specific antigen, and wherein cell surface expression of an endogenous T cell receptor is reduced on said genetically-modified eukaryotic cell when compared to an unmodified control cell. 
     
     
         95 . The method of  claim 94 , wherein said cancer is selected from the group consisting of a cancer of carcinoma, lymphoma, sarcoma, blastomas, and leukemia. 
     
     
         96 . The method of  claim 94  or  claim 95 , wherein said 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. 
     
     
         97 . The method of  claim 96 , wherein said cancer of B-cell origin is selected from the group consisting of B-lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, B-cell non-Hodgkin's lymphoma, and multiple myeloma.