Methods and compositions for generation of Bax-and Bak-deficient cell lines

• US 20100003756A1
• Filed: 06/10/2009
• Published: 01/07/2010
• Est. Priority Date: 06/10/2008
• Status: Active Grant

• Current Assignee: Sangamo Biosciences, Inc. (Sangamo Therapeutics, Inc.)
• Original Assignee: Sangamo Biosciences, Inc. (Sangamo Therapeutics, Inc.)
• Inventors: Gregory, Philip D., Collingwood, Trevor
• Granted Patent: US 8,597,912 B2
• Time in Patent Office: Days
• US Class Current: 435/455
• CPC Class Codes: C07K 2319/81 containing a Zn-finger domain for DNA binding
• CPC Class Codes: C12N 2501/48 Regulators of apoptosis
• CPC Class Codes: C12N 2510/02 Cells for production
• CPC Class Codes: C12N 5/0018 Culture media for cell or tissue culture media for specific animal cell type C12N5/06
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

Disclosed herein are methods and compositions for generation of Bak- and/or Bax-deficient cell lines using engineered nucleases.

Claims

1. A zinc finger DNA-binding domain comprising four, five or six zinc finger recognition regions as shown in Table 1 or Table 2.

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 Bak is partially or fully inactivated.

8. A cell line in which Bax is partially or fully inactivated.

9. A cell line in which Bak and Bax are partially or fully inactivated.

10. A method of inactivating an endogenous cellular BAK or BAX 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 BAK or BAX 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 at least one of the BAK and BAX genes.

11. The method of claim 10, 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 BAK or BAX 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 at least one of the BAK or BAX genes.

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

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

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 endogenous BAX and BAK genes;

(b) inactivating one or both of the endogenous BAX and BAK genes of the host cell by the method of claim 10; 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 an antigen.

16. The method of claim 15, wherein the antigen comprises-an influenza antigen.

17. A cell line in which at least one of Bak and Bax are partially or fully inactivated, wherein the cell line is produced by

(a) inactivating at least one of Bak and Bax in a cell according to the method of claim 10;

and

(b) culturing the cell under conditions suitable for generating a cell line in which at least one of Bak and Bax is partially or fully inactivated.

18. The cell line of claim 17, 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.