Targeted disruption of the t cell receptor

Abstract

Disclosed herein are methods and compositions for inactivating TCR genes, using engineered nucleases comprising at least one DNA binding domain and a cleavage domain or cleavage half-domain in conditions able to preserve cell viability. Polynucleotides encoding nucleases, vectors comprising polynucleotides encoding nucleases and cells comprising polynucleotides encoding nucleases and/or cells comprising nucleases are also provided. Disclosed herein are also methods and compositions for expressing a functional exogenous TCR in the absence of endogenous TCR expression in T lymphocytes, including lymphocytes with a central memory phenotype. Polynucleotides encoding exogenous TCR, vectors comprising polynucleotides encoding exogenous TCR and cells comprising polynucleotides encoding exogenous TCR and/or cells comprising exogenous TCR are also provided.

What is claimed is: 
     
         1 . A composition comprising isolated cells, the isolated cells comprising:
 a pair of zinc finger nucleases (ZFNs), each ZFN of the pair comprising a cleavage domain and a zinc finger DNA-binding domain that binds to a target site within exon c1 of an endogenous T-cell receptor alpha (TCRA) gene, wherein the zinc finger DNA-binding domains of the pair of ZFNs bind to target sites as shown in SEQ ID NO:17 and SEQ ID NO:18 or to target sites as shown in SEQ ID NO:18 and SEQ ID NO:21; and   an insertion and/or deletion within TGGACTT within exon c1 the TCRA gene.   
     
     
         2 . The composition of  claim 1 , further comprising genetically modified cells descended from the isolated cells. 
     
     
         3 . The composition of  claim 1 , further comprising cells comprising an insertion and/or a deletion within one or more of SEQ ID NO:8-21, 92-103, AACAGT, AGTGCT, TTGAAA, AATCCTC, and/or CTCCT of the TCRA gene. 
     
     
         4 . The composition of  claim 1 , further comprising cells comprising an inactivated beta 2 microglobulin (B2M), PD1 and/or CTLA4 gene. 
     
     
         5 . The composition of  claim 1 , further comprising genetically modified cells comprising a transgene encoding a chimeric antigen receptor (CAR), a transgene encoding an Antibody-coupled T-cell Receptor (ACTR) and/or a transgene encoding an engineered TCR. 
     
     
         6 . The composition of  claim 1 , wherein the isolated cells are lymphoid cells, stem cells, or progenitor cells. 
     
     
         7 . The composition of  claim 5 , wherein the genetically modified cells are T-cells, induced pluripotent stem cells (iPSCs), embryonic stem cells, mesenchymal stem cells (MSCs), or hematopoietic stem cells (HSCs).