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

• US 20090042250A1
• Filed: 07/10/2008
• Published: 02/12/2009
• Est. Priority Date: 07/12/2007
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Biosciences, Inc. (Sangamo Therapeutics, Inc.)
• Inventors: Cost, Gregory J., Collingwood, Trevor
• Granted Patent: US 7,919,313 B2
• Time in Patent Office: Days
• US Class Current: 435/69.100
• 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 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;

QSSDLSR (SEQ ID NO;

9);

F2;

TSGNLTR (SEQ ID NO;

10);

F3;

RSDDLSK (SEQ ID NO;

11); and

F4;

DRSALAR (SEQ ID NO;

12).

10. A polynucleotide encoding the protein according to claim 1.

2. 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).

3. 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).

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

20).

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

24).

7. A fusion protein comprising a zinc finger DNA-binding domain according to claim 3 and at least one cleavage domain or at least one cleavage half-domain, are also provided.

8. The fusion protein of claim 7, wherein the cleavage half-domain is a wild-type FokI cleavage half-domain.

9. The fusion protein of claim 8, wherein the cleavage half-domain is an engineered FokI cleavage half-domain.

11. A polynucleotide encoding the protein according to claim 7.

12. An isolated cell comprising a protein according to claim 7.

13. The cell of claim 12, wherein Fut8 is partially inactivated.

14. The cell of claim 12, wherein Fut8 is fully inactivated.

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

16. The cell of claim 15, wherein dihydrofolate reductase (DHFR) and glutamine synthetase (GS) have been inactivated.

6. A zinc finger DNA-binding domain, wherein the DNA-binding domain comprises five zinc finger recognition regions ordered F1 to F5 from N-terminus to C-terminus, and wherein F1, F2, F3, F4 and F5 comprise the following amino acid sequences:

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).

17. A cell line in which Fut8 is partially or fully inactivated.

18. A method of inactivating an endogenous cellular FUT8 gene in a cell, the method comprising:

(a) introducing, into a cell, a first nucleic acid encoding a first polypeptide, wherein the first polypeptide comprises;

(i) a zinc finger DNA-binding domain that is engineered to bind to a first target site in an endogenous FUT8 gene; and

(ii) a cleavage domain;

such that the polypeptide is expressed in the cell, whereby the polypeptide binds to the target site and cleaves the FUT8 gene.

19. The method of claim 18, 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 FUT8 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 FUT8 gene.

20. The method of claim 19, wherein the first and second polypeptides are encoded by the same nucleic acid.

21. The method of claim 19, wherein the first and second polypeptides are encoded by different nucleic acids.

22. 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 FUT8 gene;

(b) inactivating the endogenous FUT8 gene of the host cell by the method of claim 18; 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.

23. The method of claim 22, wherein the protein of interest comprises an antibody.

24. A cell line in which Fut 8 is partially or fully inactivated, wherein the cell line is produced by

(a) inactivating Fut8 in a cell according to the method of claim 18;

and

(b) culturing the cell under conditions suitable for generating a cell line in which Fut8 is partially or fully inactivated.

25. The cell line of claim 24, 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.