Zinc finger proteins and method for inactivating a dhfr gene in a chinese hamster ovary cell

• US 8,313,925 B2
• Filed: 03/09/2009
• Issued: 11/20/2012
• Est. Priority Date: 07/26/2005
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Biosciences, Inc.
• Inventors: Gregory, Philip D., Miller, Jeffrey C.
• Primary Examiner(s): DUNSTON, JENNIFER ANN
• Application Number:
  US12/381,217
• Publication Number:
  US 20090258363A1
• Time in Patent Office: 1,352 Days
• Field of Search: None
• US Class Current: 435/69.1
• CPC Class Codes: A61K 48/0008 characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
• CPC Class Codes: A61K 48/005 characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
• CPC Class Codes: A61K 48/0058 Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/81 containing a Zn-finger domain for DNA binding
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 15/90 Stable introduction of foreign DNA into chromosome
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

Disclosed herein are methods and compositions for targeted integration of a exogenous sequence into a predetermined target site in a genome for use, for example, in protein expression and gene inactivation.

Claims

1. A protein comprising an engineered zinc finger protein 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. The protein according to claim 1, further comprising a cleavage domain.

5. The protein of claim 3, wherein the cleavage domain is a cleavage half-domain.

7. The protein of claim 5, wherein the cleavage half-domain is a wild-type FokI cleavage half-domain or a FokI cleavage half-domain comprising an amino acid mutation selected from the group consisting of E490K, E490R, and Q486E.

9. A polynucleotide encoding the protein of claim 1.

11. An isolated cell comprising the protein of claim 1.

13. An isolated cell comprising the polynucleotide of claim 9.

2. A protein comprising an engineered zinc finger protein 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:

4. The protein according to claim 2, further comprising a cleavage domain.

6. The protein of claim 4, wherein the cleavage domain is a cleavage half-domain.

8. The protein of claim 6, wherein the cleavage half-domain is a wild-type FokI cleavage half-domain or a FokI cleavage half-domain comprising an amino acid mutation selected from the group consisting of E490K, E490R, and Q486E.

10. A polynucleotide encoding the protein of claim 2.

12. An isolated cell comprising the protein of claim 2.

14. An isolated cell comprising the polynucleotide of claim 10.

15. A method for inactivating an endogenous dhfr gene in a Chinese Hamster Ovary (CHO) cell, the method comprising:

introducing, into a CHO 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 dhfr gene, wherein the first target site comprises SEQ ID NO;

223 or SEQ ID NO;

228; and

(ii) a cleavage domain;

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

16. The method of claim 15, 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:

F1;

QSGALAR (SEQ ID NO;

229)

F2;

RSDNLRE (SEQ ID NO;

230)

F3;

QSSDLSR (SEQ ID NO;

231)

F4;

TSSNRKT (SEQ ID NO;

232).

17. The method of claim 15, 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:

F1;

RSDTLSE (SEQ ID NO;

224)

F2;

NNRDRTK (SEQ ID NO;

225)

F3;

RSDHLSA (SEQ ID NO;

226)

F4;

QSGHLSR (SEQ ID NO;

227).

18. The method of claim 15, wherein the first nucleic acid further encodes 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 dhfr 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 dhfr gene.

19. A method of producing a recombinant protein of interest in a CHO cell, the method comprising the steps of:

(a) providing a CHO cell comprising an endogenous dhfr gene;

(b) inactivating the endogenous dhfr gene of the CHO cell by the method of claim 15;

(c) introducing an expression vector comprising a transgene, the transgene comprising a dhfr gene and a sequence encoding a protein of interest into the CHO cell; and

(d) selecting cells in which the transgene is stably integrated into and expressed by the CHO cell, thereby producing the recombinant protein.

20. The method of claim 19, further comprising the step of exposing the CHO cell comprising the integrated transgene to methotrexates.

21. A dhfr-deficient Chinese hamster ovary (CHO) cell line produced by:

inactivating endogenous dhfr genes in a population of CHO cells by introducing, into the cells, 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 dhfr gene, wherein the target site comprises SEQ ID NO;

223 or SEQ ID NO;

228; and

(ii) a cleavage domain;

such that the polypeptide is expressed in the CHO cell, whereby the polypeptide binds to the target site and cleaves the dhfr gene; and

culturing the cells such that a dhfr-deficient CHO cell line is produced.

22. The dhfr-deficient cell line of claim 21, wherein the zinc finger 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:

23. The dhfr-deficient cell line of claim 21, wherein the zinc finger 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:

24. The dhfr-deficient cell line of claim 21, wherein the first nucleic acid further encodes 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 dhfr 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 dhfr gene.