Methods and compositions for inactivating glutamine synthetase gene expression

Abstract

Disclosed herein are methods and compositions for inactivating a glutamine synthetase (GS) gene, using fusion proteins comprising a zinc finger protein and a cleavage domain or cleavage half-domain. Polynucleotides encoding said fusion proteins are also provided, as are cells comprising said polynucleotides and fusion proteins.

1 . A method of inactivating an endogenous cellular glutamine synthetase (GS) gene in a cell, the method comprising:
 introducing, into a cell, a homing endonuclease having a target site in the GS gene, wherein the homing endonuclease binds to the target site and cleaves the GS gene.   
     
     
         2 . The method of  claim 1 , wherein the homing endonuclease is selected from the group consisting of I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-Pan I, I-SceII, I-PpoI, I-SceIII, 1-CreI, I-TevI, I-TevII and I-TevIII. 
     
     
         3 . The method of  claim 2 , wherein the homing endonuclease is engineered to bind to the target site. 
     
     
         4 . The method of  claim 1 , wherein the homing endonuclease is introduced into the cell as a polynucleotide. 
     
     
         5 . The method of  claim 1 , further comprising inactivating a DHFR gene in the cell using a nuclease. 
     
     
         6 . The method of  claim 1 , further comprising inactivating a FUT8 gene in the cell using a nuclease. 
     
     
         7 . The method of  claim 5 , further comprising inactivating a FUT8 gene in the cell. 
     
     
         8 . A method of producing a recombinant protein of interest in a host cell, the method comprising the steps of:
 (a) providing a host cell comprising an endogenous GS gene;   (b) inactivating the endogenous GS gene of the host cell by the method of  claim 1 ; and   (c) introducing an expression vector comprising a transgene, the transgene comprising a sequence encoding a protein of interest into the host cell, thereby producing the recombinant protein.   
     
     
         9 . The method of  claim 8 , wherein the protein of interest comprises α1-antitrypsin. 
     
     
         10 . The method of  claim 8 , wherein the protein of interest is a monoclonal antibody. 
     
     
         11 . A cell line in which a GS gene is partially or fully inactivated, wherein the cell line is produced by
 (a) inactivating the GS gene in a cell according to the method of  claim 1 ; and   (b) culturing the cell under conditions suitable for generating a cell line in which the GS gene is partially or fully inactivated.   
     
     
         12 . The cell line of  claim 11 , wherein the cell is a mammalian cell selected from the group consisting of a COS cell, a CHO cell, a VERO cell, a MDCK cell, a W138 cell, a V79 cell, a B14AF28-G3 cell, a BHK cell, a HaK cell, a NSO cell, a SP2/0-Ag14 cell, a HeLa cell, an HEK293 cell, and a perC6 cell. 
     
     
         13 . The cell line of  claim 11 , further comprising a nuclease-inactivated FUT8 gene. 
     
     
         14 . The cell line of  claim 11 , further comprising a nuclease-inactivated DHFR gene. 
     
     
         15 . The cell line of  claim 11 , further comprising nuclease inactivated FUT8 and DHFR genes.