Methods and compositions for inactivating glutamine synthetase gene expression

• US 8,153,399 B2
• Filed: 10/29/2009
• Issued: 04/10/2012
• Est. Priority Date: 10/29/2008
• Status: Active Grant

• Current Assignee: Sangamo Biosciences, Inc. (Sangamo Therapeutics, Inc.)
• Original Assignee: Sangamo Biosciences, Inc. (Sangamo Therapeutics, Inc.)
• Inventors: Liu, Pei-Qi, Miller, Jeffrey C.
• Primary Examiner(s): Monshipouri, Maryam
• Application Number:
  US12/589,884
• Publication Number:
  US 20100129869A1
• Time in Patent Office: 894 Days
• Field of Search: 435/69.1, 435/252.3, 530/350
• US Class Current: 435/69.1
• CPC Class Codes: C07K 14/00 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12N 9/93 Ligases (6)
• CPC Class Codes: C12P 21/02 having a known sequence of two or more amino acids, e.g. glutathione
• CPC Class Codes: C12Y 603/01002 Glutamate-ammonia ligase (6.3.1.2)
• CPC Class Codes: Y02P 20/52 using catalysts, e.g. selective catalysts

Abstract

Disclosed herein are methods and compositions for inactivating a glutamine synthetase (GS) 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 comprising four, five or six zinc finger recognition regions designated F1 to F4, F1-F5 or F1 to F-6 as shown in a single row of Table 1.

2. 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.

3. The fusion protein of claim 2, wherein the cleavage half-domain is a wild-type FokI cleavage half-domain.

4. The fusion protein of claim 2, wherein the cleavage half-domain is an engineered FokI cleavage half-domain.

5. A polynucleotide encoding the zinc finger DNA-binding domain according to claim 1.

6. An isolated cell comprising a protein according to claim 1.

7. A cell line in which glutamine synthetase (GS) is partially or fully inactivated by a fusion protein according to claim 2.

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

introducing, into a cell, a first nucleic acid encoding a first polypeptide, wherein the first polypeptide comprises a fusion protein according to claim 2;

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

9. The method of claim 8, 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 GS 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 GS gene.

10. The method of claim 9, wherein the first and second polypeptides are encoded by the same nucleic acid.

11. The method of claim 10, wherein the first and second polypeptides are encoded by different nucleic acids.

12. The method of claim 8, further comprising inactivating a DHFR gene in the cell.

13. The method of claim 10, further comprising inactivating a FUT8 gene in the cell.

14. 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 GS gene;

(b) inactivating the endogenous GS gene of the host cell by the method of claim 8; 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.

15. The method of claim 14, wherein the protein of interest comprises α

1-antitrypsin.

16. The method of claim 14, wherein the protein of interest is a monoclonal antibody.

17. A cell line in which a GS gene is partially or fully inactivated, wherein the cell line is produced by

(a) inactivating the GS gene in a cell according to the method of claim 8;

and

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

18. The cell line of claim 15, 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.