Primary cell gene editing

Abstract

Methods and compositions are provided for nuclease-mediated gene editing of primary cells without the use of viral mediated delivery. Methods of treatments using edited primary cells are also provided.

What is claimed is: 
     
         1 . A primary cell comprising an exogenous T cell receptor (TCR) encoded by:
 a. a polynucleotide comprising, from 5′ to 3′, a first linker coding sequence, a first P2A peptide coding sequence, a first signal peptide sequence, a TCRβ variable region, a TCRβ constant region, a protease cleavage site, a second linker coding sequence, a second P2A peptide coding sequence, a second signal peptide sequence, and a TCRα variable region inserted in an endogenous TRAC locus, 5′ to, and in frame with, the TCRα constant region coding sequence; or   b. a polynucleotide comprising, from 5′ to 3′, a first linker coding sequence, a first P2A peptide coding sequence, a first signal peptide sequence, a TCRα variable region, a TCRα constant region, a protease cleavage site, a second linker coding sequence, a second P2A peptide coding sequence, a second signal peptide sequence, and a TCRβ variable region inserted in an endogenous TRBC locus, 5′ to, and in frame with, the TCRβ constant region coding sequence.   
     
     
         2 . The primary cell of  claim 1 , wherein the TCR recognizes a cancer antigen. 
     
     
         3 . The primary cell of  claim 2 , wherein the cancer antigen is a neoantigen. 
     
     
         4 . The primary cell of  claim 1 , wherein the cell is substantially free of viral mediated delivery. 
     
     
         5 . The primary cell of  claim 1  further comprising a nuclease composition. 
     
     
         6 . The primary cell of  claim 5 , wherein the nuclease composition disrupts a TCRβ locus and/or a TCRα locus. 
     
     
         7 . The primary cell of  claim 1 , wherein the primary cell is a T cell or an NK cell. 
     
     
         8 . The primary cell of  claim 7 , wherein the T cell is selected from the group consisting of a cytotoxic T lymphocyte (CTL), a CD8 +  T cell, a CD4 +  T cell, a tumor infiltrating T cell, an engineered T cell, a regulatory T cell (Treg), a helper T cell, a Th1 cell, a Th2 cell, a Th17 cell, an alpha-beta T cell, and a gamma-delta T cell. 
     
     
         9 . The primary cell of  claim 1 , wherein the primary cell is a stem cell. 
     
     
         10 . The primary cell of  claim 9 , wherein the primary cell is a hematopoietic stem cell. 
     
     
         11 . The primary cell of  claim 1 , wherein the cell is isolated from a subject known or suspected to have cancer. 
     
     
         12 . A population of cells comprising the primary cell of  claim 1 . 
     
     
         13 . The population of cells of  claim 12  comprising at least about 1×10 6  cells. 
     
     
         14 . The population of cells of  claim 12 , wherein the primary cell is not sorted. 
     
     
         15 . A composition comprising the primary cell of  claim 1 . 
     
     
         16 . The composition of  claim 15  further comprising a pharmaceutically acceptable excipient.