Targeted integration into the PPP1R12C locus

• US 9,267,154 B2
• Filed: 07/30/2014
• Issued: 02/23/2016
• 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:
  US14/447,378
• Publication Number:
  US 20140349394A1
• Time in Patent Office: 573 Days
• Field of Search: None
• US Class Current: 1/1
• 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. An isolated genetically modified cell comprising an exogenous polynucleotide integrated into an endogenous PPP1R12C (AAVS1) gene using at least one nuclease, wherein the exogenous polynucleotide is integrated into intron 1 of the endogenous AAVS1 gene and further wherein the exogenous polynucleotide does not comprise a wild-type AAV gene.

2. The cell of claim 1, wherein the wherein the one or more nucleases target SEQ ID NO:

1 or SEQ ID NO;

2.

3. The cell of claim 1, wherein the exogenous polynucleotide encodes a polypeptide.

4. The cell of claim 1, wherein the exogenous polynucleotide comprises an exogenous promoter.

5. The cell of claim 1, wherein expression of the exogenous polynucleotide is driven by an endogenous promoter.

6. The cell of 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.

7. The cell of claim 6, wherein the reporter comprises GFP.

8. The cell of claim 1, where the exogenous polynucleotide encodes a RNA, and further wherein the RNA is selected from the group consisting of one or more shRNAs, one or more RNAi molecules, one or more miRNAs, one or more mRNAs, and combinations thereof.

9. The cell of claim 1, wherein the exogenous polynucleotide further comprises a first nucleotide sequence that is homologous to a first sequence in the AAVS1 gene.

10. The cell of claim 9, wherein the exogenous polynucleotide further comprises a second nucleotide sequence that is homologous to a second sequence in the AAVS1 gene.

11. The cell of claim 9, wherein the exogenous polynucleotide comprises a tandem cassette.

12. The cell of claim 1, wherein the exogenous polynucleotide is a plasmid.

13. The cell of claim 1, wherein the polynucleotide is a linear DNA molecule.

14. The cell of claim 1, wherein the at least one nuclease comprises one or more cleavage half-domains are from a Type IIS restriction endonuclease.

15. The cell of claim 14, wherein the Type IIS restriction endonuclease is selected from the group consisting of FokI and StsI.

16. The cell of claim 15, wherein at least one of the cleavage half-domains comprises an alteration in the amino acid sequence of the dimerization interface.

17. The cell of claim 1, wherein the cell is a stem cell.

18. The cell of claim 17, wherein the cell is a hematopoietic stem cell.

19. The cell of claim 1, where the cell is a cell line.