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

• US 9,322,036 B2
• Filed: 05/28/2014
• Issued: 04/26/2016
• Est. Priority Date: 07/12/2007
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Biosciences, Inc.
• Inventors: Collingwood, Trevor, Cost, Gregory J.
• Primary Examiner(s): Carlson, Karen Cochrane
• Application Number:
  US14/289,459
• Publication Number:
  US 20140273097A1
• Time in Patent Office: 699 Days
• US Class Current: 1/1
• 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 method of inactivating an endogenous cellular FUT8 gene in an isolated cell, the method comprising:

introducing, into an isolated 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;

2. The method of claim 1, wherein the first and second polypeptides are encoded by the same nucleic acid.

3. The method of claim 1, wherein the first and second polypeptides are encoded by different nucleic acids.

4. The method of claim 1, wherein the first or second cleavage domain is a wild-type FokI cleavage domain.

5. The method of claim 1, wherein the first or second cleavage domain is an engineered FokI cleavage domain.

6. The method of claim 1, further comprising introducing donor sequence into the cell such that the donor sequence is integrated into the inactivated FUT8 gene.

7. A method of producing a recombinant protein of interest in an isolated cell, the method comprising the steps of:

(a) providing an isolated cell comprising an endogenous FUT8 gene;

(b) inactivating the endogenous FUT8 gene of the isolated cell by the method of claim 1; and

(c) introducing an expression vector comprising a transgene, the transgene comprising a sequence encoding a protein of interest into the cell, thereby producing the recombinant protein.

8. The method of claim 7, wherein the protein of interest comprises an antibody.

9. A cell or cell line in which Fut8 is partially or fully inactivated, wherein the cell or cell line is produced by

(a) inactivating Fut8 in a cell according to the method of claim 1;

and

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

10. The cell or cell line of claim 9, wherein the cell is a mammalian cell or cell line 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.

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

12. The cell or cell line of claim 11, wherein dihydrofolate reducatase (DHFR) and glutamine synthetase (GS) have been inactivated.