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 composition comprising a modified cell wherein an endogenous TCR locus of the modified cell comprises an exogenous nucleic acid sequence encoding:
 a. a TCR gene;   b. a first P2A-coding sequence positioned upstream of the TCR gene and a second P2A-coding sequence positioned downstream of the TCR gene sequence, wherein the first and second P2A-coding sequences are codon-diverged relative to each other;   c. a sequence coding for the amino acid sequence Gly Ser Gly positioned immediately upstream of the P2A-coding sequences; and   d. a sequence coding for a Furin cleavage site positioned upstream of the second P2A-coding sequence, and   wherein expression of the exogenous nucleic acid results in a TCR polypeptide free of non-TCR amino acid sequences.   
     
     
         2 . The composition of  claim 1 , wherein the exogenous nucleic acid sequence comprises a human growth hormone signal peptide positioned between the first P2A-coding sequence and the TCR gene. 
     
     
         3 . The composition of  claim 1 , wherein the TCR polypeptide is a TCR alpha or TCR beta polypeptide. 
     
     
         4 . The composition of  claim 1 , wherein the exogenous nucleic acid further comprises a second TCR gene that results in a second TCR polypeptide free of non-TCR amino acid sequences. 
     
     
         5 . The composition of  claim 4 , wherein the exogenous nucleic acid comprises:
 a. a first human growth hormone signal sequence positioned between the first P2A-coding sequence and the first TCR gene; and   b. a second human growth hormone signal sequence positioned between the second P2A-coding sequence and the second TCR gene,   wherein the first and the second human growth hormone signal sequences are codon diverged relative to each other.   
     
     
         6 . The composition of  claim 4 , wherein the first and second TCR polypeptides are capable of associating together to form a functional TCR. 
     
     
         7 . The composition of  claim 1 , wherein the cell is a primary cell. 
     
     
         8 . The composition of  claim 1 , wherein the cell is a patient-derived cell. 
     
     
         9 . The composition of  claim 1 , wherein the cell is a lymphocyte. 
     
     
         10 . The composition of  claim 1 , wherein the cell is a T cell. 
     
     
         11 . The composition of  claim 1 , wherein the cell is substantially free of viral-mediated delivery components. 
     
     
         12 . The composition of  claim 1 , wherein the TCR gene sequence encodes for a TCR that recognizes a cancer antigen. 
     
     
         13 . The composition of  claim 12 , wherein the cancer antigen is a neoantigen. 
     
     
         14 . The composition of  claim 12 , wherein the cancer antigen is a patient specific neoantigen. 
     
     
         15 . The composition of  claim 1 , wherein the TCR gene sequence is a patient specific TCR gene sequence. 
     
     
         16 . The composition of  claim 1 , wherein at least one endogenous TCR locus is disrupted. 
     
     
         17 . The composition of  claim 1  further comprising a pharmaceutically acceptable excipient. 
     
     
         18 . The composition of  claim 6  further comprising a pharmaceutically acceptable excipient. 
     
     
         19 . A composition comprising a modified cell comprising an exogenous nucleic acid sequence integrated into an endogenous TCR locus, wherein the exogenous nucleic acid sequence comprises:
 a. a first P2A-coding sequence and second P2A-coding sequences that are codon-diverged relative to each other;   b. a TCR gene sequence positioned between the first and second P2A-coding sequences;   c. a sequence coding for the amino acid sequence Gly Ser Gly positioned immediately upstream of the P2A-coding sequences; and   d. a sequence coding for a Furin cleavage site positioned upstream of the second P2A-coding sequence.   
     
     
         20 . The composition of  claim 19 , wherein the exogenous nucleic acid sequence further comprises a human growth hormone signal sequence positioned between the first P2A-coding sequence and the TCR gene sequence. 
     
     
         21 . The composition of  claim 19 , wherein the exogenous nucleic acid sequence comprises a second TCR sequence positioned downstream the second P2A-coding sequence. 
     
     
         22 . The composition of  claim 21 , wherein the exogenous nucleic acid sequence comprises:
 a. a first human growth hormone signal sequence positioned between the first P2A-coding sequence and the first TCR gene sequence; and   b. a second human growth hormone signal sequence positioned between the second P2A-coding sequence and the second TCR gene sequence;
 wherein the first and the second human growth hormone signal sequences are codon diverged relative to each other. 
   
     
     
         23 . The composition of  claim 19 , wherein the cell further comprises a disruption of at least one endogenous TCR gene locus. 
     
     
         24 . The composition of  claim 19 , wherein the cell is a primary cell, a patient-derived cell, a lymphocyte, or a T cell. 
     
     
         25 . The composition of  claim 19 , wherein the cell is substantially free of viral-mediated delivery components. 
     
     
         26 . The composition of  claim 19 , wherein the TCR gene sequence encodes for a TCR that recognizes a cancer antigen. 
     
     
         27 . The composition of  claim 26 , wherein the cancer antigen is a neoantigen, a patient specific neoantigen. 
     
     
         28 . The composition of  claim 19 , wherein the TCR gene sequence is a patient specific TCR gene sequence. 
     
     
         29 . The compositions of  claim 19  further comprising a pharmaceutically acceptable excipient.