Genetically-modified cells comprising a modified human t cell receptor alpha constant region gene

Abstract

Disclosed herein is a genetically-modified cell comprising in its genome a modified human T cell receptor alpha constant region gene, wherein the cell has reduced cell-surface expression of the endogenous T cell receptor. The present disclosure further relates to methods for producing such a genetically-modified cell, and to methods of using such a cell for treating a disease in a subject.

1 .- 69 . (canceled) 
     
     
         70 . A recombinant meganuclease comprising an amino acid sequence set forth in any one of SEQ ID NOs: 9-27. 
     
     
         71 . A polynucleotide comprising a nucleic acid sequence encoding said recombinant meganuclease of  claim 70 . 
     
     
         72 . The polynucleotide of  claim 71 , wherein said polynucleotide is an mRNA. 
     
     
         73 . A recombinant DNA construct comprising said polynucleotide of  claim 71 . 
     
     
         74 . A method for producing a genetically-modified human T cell comprising a modified human TCR alpha constant region gene, said method comprising introducing into a human T cell:
 (a) a first nucleic acid comprising a sequence encoding said recombinant meganuclease of  claim 70 , wherein said recombinant meganuclease is expressed in said human T cell and generates a cleavage site in the T cell receptor (TCR) alpha constant region gene which is endogenous to said human T cell at a recognition sequence consisting of SEQ ID NO: 3; and   (b) a second nucleic acid comprising an exogenous polynucleotide, wherein said exogenous polynucleotide comprises a nucleic acid sequence encoding a chimeric antigen receptor;   wherein said exogenous polynucleotide encoding said chimeric antigen receptor is inserted into said TCR alpha constant region gene at said cleavage site to generate said genetically-modified human T cell; and   wherein said genetically-modified human T cell expresses said chimeric antigen receptor on the cell-surface; and   wherein said genetically-modified human T cell does not express an endogenous TCR on the cell-surface.   
     
     
         75 . The method of  claim 74 , wherein said first nucleic acid is an mRNA. 
     
     
         76 . The method of  claim 74 , wherein said second nucleic acid comprises from 5′ to 3′:
 (a) a 5′ homology arm that is homologous to the 5′ upstream sequence flanking said cleavage site; 
 (b) said exogenous polynucleotide; and 
 (c) a 3′ homology arm that is homologous to the 3′ downstream sequence flanking said cleavage site; 
 wherein said exogenous sequence is inserted by homologous recombination into said TCR alpha constant region gene at said cleavage site. 
 
     
     
         77 . The method of  claim 74 , wherein said exogenous polynucleotide comprises a promoter that drives expression of said chimeric antigen receptor. 
     
     
         78 . The method of  claim 74 , wherein said chimeric antigen receptor comprises an extracellular ligand-binding domain specific for CD19. 
     
     
         79 . The method of  claim 74 , wherein said second nucleic acid is introduced into said human T cell by contacting said human T cell with a recombinant adeno-associated viral (AAV) vector comprising said second nucleic acid sequence. 
     
     
         80 . The method of  claim 79 , wherein said recombinant AAV vector has a serotype of AAV6. 
     
     
         81 . A genetically-modified human T cell prepared by the method of  claim 74 . 
     
     
         82 . A method for producing a genetically-modified human T cell, said method comprising introducing into a human T cell a nucleic acid comprising a sequence encoding said recombinant meganuclease of  claim 70 ;
 wherein said recombinant meganuclease is expressed in said human T cell and generates a cleavage site in the T cell receptor (TCR) alpha constant region gene which is endogenous to said human T cell at a recognition sequence consisting of SEQ ID NO: 3; and   wherein said genetically-modified human T cell does not express an endogenous TCR on the cell-surface.   
     
     
         83 . The method of  claim 82 , wherein said nucleic acid is an mRNA. 
     
     
         84 . The method of  claim 82 , wherein said genetically-modified human T cell expresses a cell-surface chimeric antigen receptor. 
     
     
         85 . The method of  claim 84 , wherein said chimeric antigen receptor comprises an extracellular ligand-binding domain specific for CD19. 
     
     
         86 . A genetically-modified human T cell prepared by the method of  claim 82 .