Targeted disruption of the mhc cell receptor

Abstract

Disclosed herein are methods and compositions for inactivating MHC 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.

What is claimed is: 
     
         1 . An isolated lymphoid cell, induced pluripotent stem cell (iPSC), mesenchymal stem cell (MSC), hematopoietic stem cell (HSC) or progenitor cell in which expression of a beta 2 microglobulin (B2M) gene is partially or completely inactivated by an insertion and/or a deletion within TCAAAT (designated as site D in  FIG. 1B ) or TCAAATT (designated as cite C in  FIG. 1B ), of exon 2 of the B2M gene, wherein the insertion and/or deletion is made by an exogenous fusion molecule comprising a zinc finger protein (ZFP) or transcription activator-like effector (TALE) DNA-binding domain that binds to a target site as shown in SEQ ID NO: 8, 9, 10 or 11 and a cleavage domain or a cleavage half-domain;
 wherein when the cleavage site is TCAAAT, the target site is SEQ ID NO: 8 or SEQ ID NO: 9; and   wherein when the cleavage site is TCAAATT, the target site is SEQ ID NO: 9 or SEQ ID NO:10.   
     
     
         2 . The cell of  claim 1 , further comprising an inactivated T-cell receptor gene, PD1 and/or CTLA4 gene. 
     
     
         3 . The cell of  claim 1 , further comprising a transgene encoding a chimeric antigen receptor (CAR), a transgene encoding an Antibody-coupled T-cell Receptor (ACTR) and/or a transgene encoding an engineered TCR. 
     
     
         4 . The cell of  claim 1 , wherein the cell is a T-cell. 
     
     
         5 . The cell of  claim 1 , wherein the cleavage half-domain is a wild-type of engineered FokI cleavage half-domain. 
     
     
         6 . The cell of  claim 1 , wherein the exogenous fusion molecule comprises a zinc finger nuclease (ZFN). 
     
     
         7 . The cell of  claim 6 , wherein the ZFN comprises a ZFP with six zinc fingers designated F1-F6 in the order F1 to F6 for the ZFP designed as SBS57017, SBS57332, SBS57469 or BS57331 in a single row of Table 1. 
     
     
         8 . The cell of  claim 7 , wherein the ZFN is used as part of a ZFN pair, each ZFN of the pair comprising a ZFP with five or six zinc fingers designated F1-F5 or F1-F6 in the order F1-F5 or F1-F6 shown in a single row of Table 1, and wherein the ZFN is as follows:
 SBS57017 and SBS57327;   SBS57017 and SBS57328;   SBS57332 and SBS57327;   SBS57469 and SBS57327;   SBS57469 and SBS57328; or   SBS57331 and SBS57326.   
     
     
         9 . The cell of  claim 1 , wherein the exogenous fusion molecule comprises a TALE protein with the repeat variable diresidues (RVDs) for the TALE designated as 103076, 10377, 103079, 103081, 103083, 103085, or 103087 shown in a single row of Table 2B. 
     
     
         10 . A pharmaceutical composition comprising a cell according to  claim 1 . 
     
     
         11 . A fusion molecule comprising a DNA-binding domain that binds to exon 2 of a B2M gene and a cleavage domain or cleavage half-domain, wherein the DNA-binding domain comprises a ZFP with six zinc fingers designated F1-F6 in the order F1-F6 for the ZFP designated as SBS57017, SBS57332, SBS57469 or SBS57331 in a single row of Table 1;
 or a TALE protein with the repeat variable diresidues for the TALE designated as 103076, 103077, 103079, 103081, 103083, 103085, or 103087 s shown in a single row of Table 2B.   
     
     
         12 . A polynucleotide encoding the fusion molecule of  claim 11 . 
     
     
         13 . The polynucleotide of  claim 12 , wherein the polynucleotide is a viral vector, a plasmid or mRNA. 
     
     
         14 . The cell of  claim 2 , further comprising a transgene encoding a chimeric antigen receptor (CAR), a transgene encoding an Antibody-coupled T-cell Receptor (ACTR) and/or a transgene encoding an engineered TCR. 
     
     
         15 . The cell of  claim 2 , wherein the cell is a T-cell. 
     
     
         16 . The cell of  claim 2 , wherein the cleavage half-domain is a wild-type of engineered FokI cleavage half-domain. 
     
     
         17 . The cell of  claim 2 , wherein the exogenous fusion molecule comprises a zinc finger nuclease (ZFN). 
     
     
         18 . The cell of  claim 17 , wherein the ZFN comprises a ZFP with six zinc fingers designated F1-F6 in the order F1 to F6 for the ZFP designed as SBS57017, SBS57332, SBS57469 or BS57331 in a single row of Table 1. 
     
     
         19 . The cell of  claim 18 , wherein the ZFN is used as part of a ZFN pair, each ZFN of the pair comprising a ZFP with five or six zinc fingers designated F1-F5 or F1-F6 in the order F1-F5 or F1-F6 shown in a single row of Table 1, and wherein the ZFN is as follows:
 SBS57017 and SBS57327;   SBS57017 and SBS57328;   SBS57332 and SBS57327;   SBS57469 and SBS57327;   SBS57469 and SBS57328; or   SBS57331 and SBS57326.   
     
     
         20 . The cell of  claim 19 , wherein the exogenous fusion molecule comprises a TALE protein with the repeat variable diresidues (RVDs) for the TALE designated as 103076, 10377, 103079, 103081, 103083, 103085, or 103087 shown in a single row of Table 2B.