Targeted integration into the PPP1R12C locus

• US 8,110,379 B2
• Filed: 04/24/2008
• Issued: 02/07/2012
• Est. Priority Date: 04/26/2007
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Biosciences, Inc.
• Inventors: DeKelver, Russell, Gregory, Philip D., Paschon, David, Tam, Phillip, Urnov, Fyodor
• Primary Examiner(s): QIAN, CELINE X
• Application Number:
  US12/150,103
• Publication Number:
  US 20080299580A1
• Time in Patent Office: 1,384 Days
• Field of Search: None
• US Class Current: 435/70.1
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/435 from animals; from humans
• CPC Class Codes: C07K 14/43595 from coelenteratae, e.g. medusae
• CPC Class Codes: C07K 2319/81 containing a Zn-finger domain for DNA binding
• CPC Class Codes: C12N 15/1082 Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2800/10 Plasmid DNA
• CPC Class Codes: C12N 2800/30 Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Y 301/21004 Type II site-specific deoxyribonuclease (3.1.21.4)

Abstract

Disclosed herein are methods and compositions for targeted integration of an exogenous sequence into the human PPP1R12C locus, for example, for expression of a polypeptide of interest.

Claims

1. A method for expressing at least one product of an exogenous nucleic acid sequence in an isolated cell, the method comprising:

(a) expressing a first fusion protein in the cell, the first fusion protein comprising a first DNA-binding domain and a first cleavage domain or cleavage half-domain, wherein the first DNA-binding domain has been engineered to bind to a first target site in the PPP1R12C gene in the genome of the cell, wherein the DNA-binding domain is a zinc finger binding domain; and

(b) contacting the cell with a polynucleotide comprising an exogenous nucleic acid sequence under conditions such that the first fusion protein cleaves the genome of the cell in the PPP1R12C gene and the exogenous nucleic acid sequence is inserted in a homology dependent manner into the cleavage site and expressed.

2. The method of claim 1, further comprising:

expressing a second fusion protein in the cell, the second fusion protein comprising a second zinc finger binding domain and a second cleavage half domain, wherein the second zinc finger binding domain binds to a second target site in the PPP1R12C gene in the genome of the cell, wherein the second target site is different from the first target site;

wherein binding of the first fusion protein to the first target site, and binding of the second fusion protein to the second target site, positions the cleavage half-domains such that the genome of the cell is cleaved in the PPP1R12C gene, thereby resulting in integration of the exogenous sequence into the genome of the cell in the PPP1R12C gene and expression of the product of the exogenous sequence.

3. The method according to claim 1, wherein the exogenous nucleic acid sequence encodes a polypeptide.

4. The method according to claim 1, wherein the exogenous nucleic acid sequence produces a polynucleotide.

5. The method according to claim 3, wherein the polypeptide is selected from the group consisting of an antibody, an antigen, an enzyme, a growth factor, a cell surface receptor, a nuclear receptor, a hormone, a lymphokine, a cytokine, a reporter, functional fragments thereof and combinations thereof.

6. The method of claim 5, wherein the reporter comprises GFP.

7. The method of claim 4 wherein the polynucleotide is selected from the group consisting of one or more shRNAs, one or more RNAi molecules, one or more miRNAs and combinations thereof.

8. The method according to claim 1, wherein the exogenous sequence further comprises a promoter.

9. The method according to claim 1, wherein the exogenous sequence does not comprise a promoter.

10. The method according to claim 1, wherein the exogenous sequence further comprises a first nucleotide sequence that is homologous but non-identical to a first sequence in the PPP1R12C gene.

11. The method according to claim 10, wherein the exogenous sequence further comprises a second nucleotide sequence that is homologous but non-identical to a second sequence in the PPP1R12C gene.

12. The method according to claim 11, wherein the first and second nucleotide sequences flank the exogenous sequence.

13. The method of claim 1, wherein the exogenous sequence is a plasmid.

14. The method of claim 1, wherein the polynucleotide is a linear DNA molecule.

15. A method for identifying integration of an exogenous sequence into the genome of a cell, the method comprising:

expressing an exogenous sequence in the cell according to claim 1, wherein the exogenous nucleic acid sequence comprises a marker gene; and

identifying cells expressing the marker gene, thereby identifying cells into which the exogenous sequence has been integrated.

16. The method of claim 15, wherein the exogenous sequence is operably linked to a promoter.

17. The method of claim 15, wherein the marker gene comprises a screening marker.

18. The method of claim 17, wherein the screening marker comprises GFP.

19. The method according to claim 1, wherein the first and second cleavage half-domains are from a Type IIS restriction endonuclease.

20. The method according to claim 19, wherein the Type IIS restriction endonuclease is selected from the group consisting of FokI and StsI.

21. The method according to claim 1, wherein the cell is arrested in the G2 phase of the cell cycle.

22. The method according to claim 2, wherein at least one of the fusion proteins comprises an alteration in the amino acid sequence of the dimerization interface of the cleavage half-domain.

23. The method according to claim 1, wherein the cell is a mammalian cell.

24. The method according to claim 23, wherein the cell is a human cell.

25. The method of according to claim 1, wherein the zinc finger binding domain comprises four zinc fingers, ordered F1 to F4 from N-terminal to C-terminal and further wherein:

26. The method of according to claim 1, wherein the zinc finger binding domain comprises four zinc fingers, ordered F1 to F4 from N-terminal to C-terminal and further wherein: