Methods and compositions for inactivating glutamine synthetase gene expression

• US 9,388,426 B2
• Filed: 11/24/2014
• Issued: 07/12/2016
• 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:
  US14/552,174
• Publication Number:
  US 20150087067A1
• Time in Patent Office: 596 Days
• US Class Current: 1/1
• 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 method of producing a cell or cell line in which a glutamine synthetase (GS) gene is inactivated, the method comprising introducing into the cell or cell line one or more nucleic acids encoding a pair of zinc finger nucleases, each zinc finger nuclease comprising a zinc finger DNA-binding domain and a cleavage domain or cleavage half-domain, wherein the pair of zinc finger nucleases dimerize to cleave the GS gene and further wherein the zinc finger DNA-binding domain comprises four or six zinc finger recognition regions designated F1 to F4 or F1 to F6 comprising recognition helix regions selected from the group consisting of:

2. The method of claim 1, wherein the cleavage half-domain is a wild-type FokI cleavage half-domain.

3. The method of claim 1, wherein the cleavage half-domain is an engineered FokI cleavage half-domain.

4. A cell or cell line produced by the method of claim 1, in which glutamine synthetase (GS) is partially or fully inactivated following cleavage by the pair of zinc finger nucleases and by non-homologous end joining (NHEJ) of the cleaved GS sequence, which NHEJ causes mutations in GS that eliminate amino acids critical for GS catalytic activity.

5. The method of claim 1, wherein the zinc finger nucleases of the pair of zinc finger nucleases are encoded by the same nucleic acid.

6. The method of claim 1, wherein the zinc finger nucleases of the pair of zinc finger nucleases are encoded by different nucleic acids.

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

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

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

10. The method of claim 1, wherein the cell or cell line is a mammalian cell or cell line selected from the group consisting of a COS cell or cell line, a CHO cell or cell line, a VERO cell or cell line, a MDCK cell or cell line, a WI38 cell or cell line, a V79 cell or cell line, a B14AF28-G3 cell or cell line, a BHK cell or cell line, a HaK cell or cell line, a NS0 cell or cell line, a SP2/0-Ag14 cell or cell line, a HeLa cell or cell line, an HEK293 cell or cell line, and a perC6 cell or cell line.