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

• US 20110159541A1
• Filed: 01/27/2011
• Published: 06/30/2011
• Est. Priority Date: 07/12/2007
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Biosciences, Inc. (Sangamo Therapeutics, Inc.)
• Inventors: Collingwood, Trevor, Cost, Gregory J.
• Granted Patent: US 8,772,025 B2
• Time in Patent Office: Days
• US Class Current: 435/69.6
• 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:

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

2. 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:

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

5. 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.

6. The method of claim 5, 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.

8. The method of claim 6, wherein the first and second polypeptides are encoded by different nucleic acids.

11. 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 5;

and

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

12. The cell line of claim 11, 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.

7. The method of 6, wherein the first and second polypeptides are encoded by the same nucleic acid.

9. 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.

10. The method of claim 9, wherein the protein of interest comprises an antibody.