Targeted disruption of t cell receptor genes using engineered zinc finger protein nucleases

Abstract

Disclosed herein are methods and compositions for inactivating TCR genes, using zinc finger nucleases (ZFNs) comprising a zinc finger protein and a cleavage domain or cleavage half-domain in conditions able to preserve cell viability. Polynucleotides encoding ZFNs, vectors comprising polynucleotides encoding ZFNs and cells comprising polynucleotides encoding ZFNs and/or cells comprising ZFNs 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.

1 - 11 . (canceled) 
     
     
         12 . A method of inactivating a TCR gene in a cell, the method comprising integrating a transgene into the TCR gene by a zinc finger nuclease comprising a zinc finger protein that binds to a target site in the TCR gene, wherein the target site is within any of SEQ ID NO: 1, 7, 13, 18, 23, 29, 34, 37, 41, 45, 49, 54, 58 or 63, and further wherein the binding of the zinc finger protein at the target site in the TCR gene results in cleavage of the TCR gene such that the TCR gene is inactivated by integration of the transgene. 
     
     
         13 . The method of  claim 12 , wherein the transgene encodes a chimeric antigen receptor (CAR). 
     
     
         14 . The method of  claim 12 , wherein the TCR gene is an endogenous TCRα or TCRβ gene. 
     
     
         15 . The method of  claim 14 , wherein the transgene is integrated into exon 1 or exon 3 of the endogenous TCRα gene. 
     
     
         16 . The method of  claim 12 , wherein the cell is a T-Cell. 
     
     
         17 . The method of  claim 12 , wherein the cell is a stem cell. 
     
     
         18 . The method of  claim 17 , wherein the cell is a hematopoietic stem cell. 
     
     
         19 . A method of treating cancer, infections, autoimmune disorders or graft-versus-host disease (GVHD) in a subject in need thereof, the method comprising introducing into the subject an isolated cell or a cell line comprising an inactivated endogenous T-cell receptor (TCR) gene, wherein the TCR gene is inactivated by integration of a transgene into the TCR gene by a zinc finger nuclease comprising a zinc finger protein that binds to a target site in the TCR gene, wherein the target site is within any of SEQ ID NO: 1, 7, 13, 18, 23, 29, 34, 37, 41, 45, 49, 54, 58 or 63, and further wherein the binding of the zinc finger protein at the target site in the TCR gene results in cleavage of the TCR gene such that the TCR gene is inactivated by integration of the transgene. 
     
     
         20 . The method of  claim 19 , wherein the transgene encodes a chimeric antigen receptor (CAR). 
     
     
         21 . The method of  claim 19 , wherein the TCR gene is an endogenous TCRα or TCRβ gene. 
     
     
         22 . The method of  claim 21 , wherein the transgene is integrated into exon 1 or exon 3 of the endogenous TCRα gene. 
     
     
         23 . The method of  claim 19 , wherein the cell is a T-Cell. 
     
     
         24 . The method of  claim 19 , wherein the cell is a stem cell. 
     
     
         25 . The method of  claim 24 , wherein the cell is a hematopoietic stem cell.