Methods and compositions for inactivating alpha 1,6 fucosyltransferase (FUT8) gene expression
First Claim
Patent Images
1. A method of producing an antibody in an isolated cell, the method comprising:
- inactivating an endogenous cellular FUT8 gene in an isolated cell by introducing one or more nucleic acids encoding first and second zinc finger nucleases, each zinc finger nuclease comprising a cleavage domain and a zinc finger protein, wherein the at least one of the zinc finger nucleases comprises a zinc finger DNA-binding domain comprising four, five or six 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 as follows;
(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);
wherein the first and second zinc finger nucleases cleave and inactivate the endogenous cellular FUT8 gene in the isolated cell; and
introducing an exogenous sequence encoding an antibody into the cell, wherein the antibody is produced in the isolated cell.
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Abstract
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.
44 Citations
11 Claims
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1. A method of producing an antibody in an isolated cell, the method comprising:
-
inactivating an endogenous cellular FUT8 gene in an isolated cell by introducing one or more nucleic acids encoding first and second zinc finger nucleases, each zinc finger nuclease comprising a cleavage domain and a zinc finger protein, wherein the at least one of the zinc finger nucleases comprises a zinc finger DNA-binding domain comprising four, five or six 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 as follows; (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);wherein the first and second zinc finger nucleases cleave and inactivate the endogenous cellular FUT8 gene in the isolated cell; and introducing an exogenous sequence encoding an antibody into the cell, wherein the antibody is produced in the isolated cell. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
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Specification