Sequential gene editing in primary immune cells

Abstract

The invention pertains to the field of adaptive cell immunotherapy. It aims at reducing the occurrence of translocations and cell deaths when several specific endonuclease reagents are used altogether to genetically modify primary immune cells at different genetic loci. The method of the invention allows to yield safer immune primary cells harboring several genetic modifications, such as triple or quadruple gene inactivated cells, from populations or sub-populations of cells originating from a single donor or patient, for their subsequent use in therapeutic treatments.

We claim: 
     
         1 . An mRNA molecule encoding a PD-1-targeting TALEN comprising the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 9. 
     
     
         2 . The mRNA molecule of  claim 1 , encoding the amino acid sequence of SEQ ID NO: 5. 
     
     
         3 . The mRNA molecule of  claim 1 , encoding the amino acid sequence of SEQ ID NO: 6. 
     
     
         4 . The mRNA molecule of  claim 1 , encoding the amino acid sequence of SEQ ID NO: 9. 
     
     
         5 . A population of gene-edited primary immune cells produced by transiently expressing a rare-cutting sequence-specific endonuclease reagent that is a PD-1-targeting TALEN in the primary immune cells, wherein the PD-1-targeting TALEN comprises the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 9. 
     
     
         6 . A population of gene-edited primary immune cells produced by transiently expressing a pair of PD-1-targeting TALEN monomers in the primary immune cells, wherein the PD-1-targeting TALEN monomers comprise the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 9. 
     
     
         7 . The population of gene-edited primary immune cells of  claim 6 , wherein the pair of PD-1 targeting TALEN monomers comprise a TALEN monomer having the amino acid sequence of SEQ ID NO: 5 and a TALEN monomer having the amino acid sequence of SEQ ID NO: 6. 
     
     
         8 . The population of gene-edited primary immune cells of  claim 6 , wherein the pair of PD-1 targeting TALEN monomers comprise a TALEN monomer having the amino acid sequence of SEQ ID NO: 9 and a TALEN monomer having the amino acid sequence of SEQ ID NO: 6. 
     
     
         9 . The population of gene-edited primary immune cells of  claim 5 , wherein the primary immune cells are T cells. 
     
     
         10 . The population of gene-edited primary immune cells of  claim 5 , wherein the primary immune cells are NK cells. 
     
     
         11 . The population of gene-edited primary immune cells of  claim 5 , wherein the primary immune cells are obtained from a tumor. 
     
     
         12 . The population of gene-edited primary immune cells of  claim 5 , wherein the primary immune cells are tumor infiltrating lymphocytes (TILs).