Methods and compositions for inactivating alpha 1,6 fucosyltransferase (fut8) gene expression

A method of inactivating an endogenous cellular FUT8 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) a first zinc finger DNA-binding domain, wherein the DNA-binding domain comprises four or five zinc finger recognition regions ordered F1 to F4, F1 to F5 or F1 to F6 from N-terminus to C-terminus, and wherein F1 to F4, F1 to F5 or F1 to F6 comprise amino acid sequences selected from the group consisting of:
• (i)
• F1:
• QSSDLSR (SEQ ID NO:
• 9);
• F2:
• TSGNLTR (SEQ ID NO:
• 10);
• F3:
• RSDDLSK (SEQ ID NO:
• 11);
• and
• F4:
• DRSALAR (SEQ ID NO:
• 12);
• (ii)
• F1:
• RSDVLSA (SEQ ID NO:
• 14);
• F2:
• QNATRIN (SEQ ID NO:
• 15);
• F3:
• DRSNLSR (SEQ ID NO:
• 16);
• and
• F4:
• RLDNRTA (SEQ ID NO:
• 17);
• (iii)
• F1:
• RSDNLRE (SEQ ID NO:
• 26);
• F2:
• NNTQLIE (SEQ ID NO:
• 27);
• F3:
• TSSILSR (SEQ ID NO:
• 28);
• F4 RSDNLSA (SEQ ID NO:
• 29);
• and
• F5:
• RKDTRIT (SEQ ID NO:
• 30);
• and
• (iv)
• F1:
• RSDNLSV (SEQ ID NO:
• 19);
• F2:
• QNATRIN (SEQ ID NO:
• 15);
• F3:
• RSDNLST (SEQ ID NO:
• 20)
• or RSDHLSQ (SEQ ID NO:
• 24);
• F4:
• QSATRTK (SEQ ID NO:
• 21);
• F5 RSDNLSR (SEQ ID NO:
• 22);
• and
• F6:
• RNDNRKT (SEQ ID NO:
• 23);
• and
• (b) a first cleavage domain;
• such that the polypeptide is expressed in the cell, whereby the polypeptide binds to the target site and cleaves the FUT8 gene;
• and
• introducing a nucleic acid encoding a second polypeptide, wherein the second polypeptide comprises:
• (i) a second zinc finger DNA-binding domain that is engineered to bind to a second target site in the FUT8 gene;
• and
• (ii) a second cleavage domain;
• such that the second polypeptide is expressed in the cell,
• whereby the first and second polypeptides bind to their respective target sites, dimerize and cleave the FUT8 gene, thereby inactivating the FUT8 gene.