Methods and compositions for inactivating glutamine synthetase gene expression

• US 20100311124A1
• Filed: 07/09/2010
• Published: 12/09/2010
• Est. Priority Date: 10/29/2008
• Status: Abandoned Application

• Current Assignee: Sangamo Biosciences, Inc.
• Original Assignee: Sangamo Biosciences, Inc.
• Inventors: Miller, Jeffrey C., Liu, Pei-Qi
• Application Number:
  US12/803,928
• Publication Number:
  US 20100311124A1
• Time in Patent Office: Days
• US Class Current: 435/69.6
• 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 method of inactivating an endogenous cellular glutamine synthetase (GS) gene in a cell, the method comprising:

introducing, into a cell, a homing endonuclease having a target site in the GS gene, wherein the homing endonuclease binds to the target site and cleaves the GS gene.

2. The method of claim 1, wherein the homing endonuclease is selected from the group consisting of I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-Pan I, I-SceII, I-PpoI, I-SceIII, 1-CreI, I-TevI, I-TevII and I-TevIII.

3. The method of claim 2, wherein the homing endonuclease is engineered to bind to the target site.

4. The method of claim 1, wherein the homing endonuclease is introduced into the cell as a polynucleotide.

5. The method of claim 1, further comprising inactivating a DHFR gene in the cell using a nuclease.

6. The method of claim 1, further comprising inactivating a FUT8 gene in the cell using a nuclease.

7. The method of claim 5, further comprising inactivating a FUT8 gene in the cell.

8. 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 1; 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.

9. The method of claim 8, wherein the protein of interest comprises α

1-antitrypsin.

10. The method of claim 8, wherein the protein of interest is a monoclonal antibody.

11. 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 1;

and

(b) culturing the cell under conditions suitable for generating a cell line in which the GS gene 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 W138 cell, a V79 cell, a B14AF28-G3 cell, a BHK cell, a HaK cell, a NSO cell, a SP2/0-Ag14 cell, a HeLa cell, an HEK293 cell, and a perC6 cell.

13. The cell line of claim 11, further comprising a nuclease-inactivated FUT8 gene.

14. The cell line of claim 11, further comprising a nuclease-inactivated DHFR gene.

15. The cell line of claim 11, further comprising nuclease inactivated FUT8 and DHFR genes.