Use of pre t alpha or functional variant thereof for expanding tcr alpha deficient t cells

Abstract

A method of expanding TCRalpha deficient T-cells by expressing pTalpha or functional variants thereof into said cells, thereby restoring a functional CD3 complex. This method is particularly useful to enhance the efficiency of immunotherapy using primary T-cells from donors. This method involves the use of pTalpha or functional variants thereof and polynucleotides encoding such polypeptides to expand TCRalpha deficient T-cells. Such engineered cells can be obtained by using specific rare-cutting endonuclease, preferably TALE-nucleases. The use of Chimeric Antigen Receptor (CAR), especially multi-chain CAR, in such engineered cells to target malignant or infected cells. The invention opens the way to standard and affordable adoptive immunotherapy strategies for treating cancer and viral infections.

1 - 40 . (canceled) 
     
     
         41 . A method for genetic modification of a cell comprising:
 providing human primary T cells comprising a target DNA gene sequence;   culturing the T cells;   electroporating mRNAs encoding two half-TALE-nucleases that together target said target DNA gene sequence into the T cells;   wherein one of said mRNAs encodes a half-TALE-nuclease targeting said target DNA gene sequence,   wherein another of said mRNAs encodes the other half-TALE-nuclease targeting said target DNA gene sequence; and   introducing into the T cells an exogeneous nucleic acid comprising at least a sequence homologous to a portion of the target nucleic acid sequence; and   transiently expressing the half-TALE-nucleases in the cells to generate a double-strand break at the DNA target gene sequence such that homologous recombination occurs between the target nucleic acid sequence and the exogeneous nucleic acid to generate a sufficient number of T cells comprising the exogeneous nucleic acid integrated into the target gene for immunotherapy.   
     
     
         42 . The method of  claim 41 , wherein the exogenous nucleic acid comprises first and second portions which are homologous to region 5′ and 3′ of the target nucleic acid sequence. 
     
     
         43 . The method of  claim 42 , wherein the exogenous nucleic acid further comprises a third portion positioned between the first and the second portion which comprises no homology with the regions 5′ and 3′ of the target nucleic acid sequence. 
     
     
         44 . The method of  claim 42 , wherein the first and second portion each comprise homologous sequences of at least 50 bp. 
     
     
         45 . The method of  claim 42 , wherein the first and second portion each comprise homologous sequences of more than 100 bp. 
     
     
         46 . The method of  claim 42 , wherein the first and second portion each comprise homologous sequences of more than 200 bp. 
     
     
         47 . The method of  claim 42 , wherein the exogenous nucleic acid is from 200 base pairs to 6000 base pairs. 
     
     
         48 . The method of  claim 42 , wherein the exogenous nucleic acid is from 1000 base pairs to 2000 base pairs. 
     
     
         49 . The method of  claim 42 , wherein the exogenous nucleic acid is inserted within the open reading frame of the target gene. 
     
     
         50 . The method of  claim 42 , wherein the integration of the exogenous nucleic acid inactivates the target gene. 
     
     
         51 . The method of  claim 42 , wherein the exogenous polynucleotide sequence encodes a recombinant protein. 
     
     
         52 . The method of  claim 51 , wherein the recombinant protein comprises a polypeptide selected from:
 a tail-less version of pTalpha (SEQ ID NO: 121);   a pTalpha extracellular domain fused to the transmembrane (TM) and the IC domains from TCRα (SEQ ID NO: 122);   a human pTalpha protein (SEQ ID NO: 107);   a pTalpha deletion mutants (SEQ ID NO: 108 to SEQ ID NO: 114); and   a pTalpha mutant containing intracellular activation domains (SEQ ID NO: 115 to SEQ ID NO: 120).   
     
     
         53 . A cell prepared by the method of  claim 41 .