Methods and compositions for inactivating alpha 1,6 fucosyltransferase (FUT8) gene expression

• US 7,919,313 B2
• Filed: 07/10/2008
• Issued: 04/05/2011
• Est. Priority Date: 07/12/2007
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Biosciences, Inc.
• Inventors: Cost, Gregory J., Collingwood, Trevor
• Primary Examiner(s): CARLSON, KAREN C
• Application Number:
  US12/218,035
• Publication Number:
  US 20090042250A1
• Time in Patent Office: 999 Days
• Field of Search: None
• US Class Current: 435/325
• CPC Class Codes: C07K 14/4703 Inhibitors; Suppressors
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/87 Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2800/80 Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
• CPC Class Codes: C12N 9/1051 Hexosyltransferases (2.4.1)
• CPC Class Codes: C12P 21/00 Preparation of peptides or proteins single cell protein C12N1/00

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.

Claims

1. A zinc finger DNA-binding domain, wherein the DNA-binding domain comprises six zinc finger recognition regions ordered F1 to F6 from N-terminus to C-terminus, and wherein F1, F2, F4, F5 and F6 comprise the following amino acid sequences:

F1;

RSDNLSV (SEQ ID NO;

19);

F2;

QNATRIN (SEQ ID NO;

15);

F4;

QSATRTK (SEQ ID NO;

21);

F5 RSDNLSR (SEQ ID NO;

22); and

F6;

RNDNRKT (SEQ ID NO;

23).

2. The zinc finger DNA-binding domain of claim 1, wherein F3 comprises RSDNLST (SEQ ID NO:

20).

3. The zinc finger DNA-binding domain of claim 1, wherein F3 comprises RSDHLSQ (SEQ ID NO:

24).

4. 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.

5. The fusion protein of claim 4, wherein the cleavage half-domain is a wild-type FokI cleavage half-domain.

6. The fusion protein of claim 5, wherein the cleavage half-domain is an engineered FokI cleavage half-domain.

7. An isolated polynucleotide encoding the zinc finger DNA-binding domain of claim 1.

8. A polynucleotide encoding the protein according to claim 4.

9. An isolated cell comprising a protein according to claim 4.

10. The cell of claim 9, wherein Fut8 is partially inactivated.

11. The cell of claim 9, wherein Fut8 is fully inactivated.

12. The cell of claim 9, wherein one or more additional genes are partially or fully inactivated.

13. The cell of claim 12, wherein dihydrofolate reductase (DHFR) and glutamine synthetase (GS) have been inactivated.

14. A zinc finger DNA-binding domain, wherein the DNA-binding domain comprises four zinc finger recognition regions ordered F1 to F4 from N-terminus to C-terminus, and wherein F1, F2, F3, and F4 comprise the following amino acid sequences:

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).

15. A fusion protein comprising a zinc finger DNA-binding domain according to claim 14 and at least one cleavage domain or at least one cleavage half-domain.

16. The fusion protein of claim 15, wherein the cleavage half-domain is a wild-type FokI cleavage half-domain.

17. The fusion protein of claim 16, wherein the cleavage half-domain is an engineered FokI cleavage half-domain.

18. An isolated polynucleotide encoding the protein according to claim 14.

19. A polynucleotide encoding the protein according to claim 15.

20. An isolated cell comprising a protein according to claim 15.

21. The cell of claim 20, wherein Fut8 is partially inactivated.

22. The cell of claim 20, wherein Fut8 is fully inactivated.

23. The cell of claim 20, wherein one or more additional genes are partially or fully inactivated.

24. The cell of claim 23, wherein dihydrofolate reductase (DHFR) and glutamine synthetase (GS) have been inactivated.