METHODS AND COMPOSITIONS FOR INACTIVATING ALPHA 1,6 FUCOSYLTRANSFERASE (FUT8) GENE EXPRESSION

摘要

Disclosed herein are methods and compositions for inactivating a FUT8 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.

主权项

1. 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);andF4: DRSALAR (SEQ ID NO: 12);(ii) F1: RSDVLSA (SEQ ID NO: 14);F2: QNATRIN (SEQ ID NO: 15);F3: DRSNLSR (SEQ ID NO: 16);andF4: 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);andF5: 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);andF6: 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.