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.

What is claimed is: 
     
         1 . A zinc finger DNA-binding domain comprising four or six zinc finger recognition regions designated F1 to F4 or F1 to F6, wherein the zinc finger DNA-binding domains comprise recognition helix regions selected from the group consisting of: 
       
         
           
                 
                 
                 
                 
               
                     
                   (i) 
                   F1: QSSDLSR 
                   (SEQ ID NO: 2) 
                 
                     
                     
                 
                     
                     
                   F2: RSDNLRE 
                   (SEQ ID NO: 3) 
                 
                     
                     
                 
                     
                     
                   F3: RSDTLSN 
                   (SEQ ID NO: 4) 
                 
                     
                     
                 
                     
                     
                   F4: RKDVRIT; 
                   (SEQ ID NO: 5) 
                 
                     
                     
                 
                     
                   (ii) 
                   F1: RSDHLST 
                   (SEQ ID NO: 7) 
                 
                     
                     
                 
                     
                     
                   F2: QSSDLRR 
                   (SEQ ID NO: 8) 
                 
                     
                     
                 
                     
                     
                   F3: RSDHLSQ 
                   (SEQ ID NO: 9) 
                 
                     
                     
                 
                     
                     
                   F4: QSANRTT 
                   (SEQ ID NO: 10) 
                 
                     
                     
                 
                     
                     
                   F5: RSDNLSQ 
                   (SEQ ID NO: 11) 
                 
                     
                     
                 
                     
                     
                   F6: ASNDRKK; 
                   (SEQ ID NO: 12) 
                 
                     
                     
                 
                     
                   (iii) 
                   F1: QSGALAR 
                   (SEQ ID NO: 14) 
                 
                     
                     
                 
                     
                     
                   F2: RSDALTQ 
                   (SEQ ID NO: 15) 
                 
                     
                     
                 
                     
                     
                   F3: RSDSLSA 
                   (SEQ ID NO: 16) 
                 
                     
                     
                 
                     
                     
                   F4: RSAHLSR 
                   (SEQ ID NO: 17) 
                 
                     
                     
                 
                     
                   (iv) 
                   F1: RSDHLST 
                   (SEQ ID NO: 7) 
                 
                     
                     
                 
                     
                     
                   F2: QSSDLRR 
                   (SEQ ID NO: 8) 
                 
                     
                     
                 
                     
                     
                   F3: RSDSLSV 
                   (SEQ ID NO: 19) 
                 
                     
                     
                 
                     
                     
                   F4: DNANRTK; 
                   (SEQ ID NO: 20) 
                 
                     
                     
                 
                     
                   (v) 
                   F1: QSSDLSR 
                   (SEQ ID NO: 2) 
                 
                     
                     
                 
                     
                     
                   F2: RSDNLRE 
                   (SEQ ID NO: 3) 
                 
                     
                     
                 
                     
                     
                   F3: RSSALTR 
                   (SEQ ID NO: 22) 
                 
                     
                     
                 
                     
                     
                   F4: RSDALTQ; 
                   (SEQ ID NO: 15) 
                 
                     
                     
                 
                     
                   (vi) 
                   F1: QSSDLSR 
                   (SEQ ID NO: 2) 
                 
                     
                     
                 
                     
                     
                   F2: RSDNLRE 
                   (SEQ ID NO: 3) 
                 
                     
                     
                 
                     
                     
                   F3: QSSHLTR 
                   (SEQ ID NO: 24) 
                 
                     
                     
                 
                     
                     
                   F4: TSSNRKT; 
                   (SEQ ID NO: 25) 
                 
                     
                     
                 
                     
                   (vii) 
                   F1: QSSDLSR 
                   (SEQ ID NO: 2) 
                 
                     
                     
                 
                     
                     
                   F2: RSDNLRE 
                   (SEQ ID NO: 3) 
                 
                     
                     
                 
                     
                     
                   F3: RSDSLLR 
                   (SEQ ID NO: 26) 
                 
                     
                     
                 
                     
                     
                   F4: RSDALTQ; 
                   (SEQ ID NO: 15) 
                 
                     
                     
                 
                     
                   (viii) 
                   F1: RSDHLSQ 
                   (SEQ ID NO: 9) 
                 
                     
                     
                 
                     
                     
                   F2: QSANRTT 
                   (SEQ ID NO: 10) 
                 
                     
                     
                 
                     
                     
                   F3: RSDNLSQ 
                   (SEQ ID NO: 11) 
                 
                     
                     
                 
                     
                     
                   F4: ASNDRKK; 
                   (SEQ ID NO: 12) 
                 
                     
                   and 
                     
                     
                 
                     
                     
                 
                     
                   (ix) 
                   F1: RSDHLSQ 
                   (SEQ ID NO: 9) 
                 
                     
                     
                 
                     
                     
                   F2: RNADRIT 
                   (SEQ ID NO: 29) 
                 
                     
                     
                 
                     
                     
                   F3: RSDHLSQ 
                   (SEQ ID NO: 9) 
                 
                     
                     
                 
                     
                     
                   F4: QSANRTT 
                   (SEQ ID NO: 10) 
                 
                     
                     
                 
                     
                     
                   F5: RSDNLSQ 
                   (SEQ ID NO: 11) 
                 
                     
                     
                 
                     
                     
                   F6: ASNDRKK. 
                   (SEQ ID NO: 12) 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         2 . A fusion protein comprising a zinc finger DNA-binding domain according to  claim 1  and at least one cleavage domain or at least one cleavage half-domain. 
     
     
         3 . The fusion protein of  claim 2 , wherein the cleavage half-domain is a wild-type FokI cleavage half-domain. 
     
     
         4 . The fusion protein of  claim 2 , wherein the cleavage half-domain is an engineered FokI cleavage half-domain. 
     
     
         5 . A polynucleotide encoding the zinc finger DNA-binding domain according to  claim 1 . 
     
     
         6 . An isolated cell comprising a protein according to  claim 1 . 
     
     
         7 . A cell line in which glutamine synthetase (GS) is partially or fully inactivated by a fusion protein according to claim. 
     
     
         8 . A method of inactivating an endogenous cellular GS gene in a cell, the method comprising:
 introducing, into a cell, a first nucleic acid encoding a first polypeptide, wherein the first polypeptide comprises a fusion protein according to  claim 2 :   
       such that the polypeptide is expressed in the cell, whereby the polypeptide binds to its target site and cleaves the GS gene. 
     
     
         9 . The method of  claim 8 , further comprising introducing a nucleic acid encoding a second polypeptide, wherein the second polypeptide comprises:
 (i) a zinc finger DNA-binding domain that is engineered to bind to a second target site in the GS gene; and   (ii) a cleavage domain; such that the second polypeptide is expressed in the cell, whereby the first and second polypeptides bind to their respective target sites and cleave the GS gene.   
     
     
         10 . The method of  claim 9 , wherein the first and second polypeptides are encoded by the same nucleic acid. 
     
     
         11 . The method of  claim 10 , wherein the first and second polypeptides are encoded by different nucleic acids. 
     
     
         12 . The method of  claim 8 , further comprising inactivating a DHFR gene in the cell. 
     
     
         13 . The method of  claim 10 , further comprising inactivating a FUT8 gene in the cell. 
     
     
         14 . 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 8 ; 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.   
     
     
         15 . The method of  claim 14 , wherein the protein of interest comprises al-antitrypsin. 
     
     
         16 . The method of  claim 14 , wherein the protein of interest is a monoclonal antibody. 
     
     
         17 . 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 8 ; and   (b) culturing the cell under conditions suitable for generating a cell line in which the GS gene is partially or fully inactivated.   
     
     
         18 . The cell line of  claim 15 , 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 WI38 cell, a V79 cell, a B14AF28-G3 cell, a BHK cell, a HaK cell, a NS0 cell, a SP2/0-Ag14 cell, a HeLa cell, an HEK293 cell, and a perC6 cell.