GENETICALLY-MODIFIED CELLS COMPRISING A MODIFIED HUMAN T CELL RECEPTOR ALPHA CONSTANT REGION GENE
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: 8-27.
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 1 , 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 1 ;
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 .