Targeted integration into stem cells

• US 9,834,787 B2
• Filed: 04/08/2010
• Issued: 12/05/2017
• Est. Priority Date: 04/09/2009
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc., Whitehead Institute for Biomedical Research
• Original Assignee: Sangamo Therapeutics, Inc.
• Inventors: Gregory, Philip D., Hockemeyer, Dirk, Holmes, Michael C., Jaenisch, Rudolf, Soldner, Frank, Urnov, Fyodor, Yao, Shuyuan
• Primary Examiner(s): Ton, Thaian N
• Application Number:
  US12/798,749
• Publication Number:
  US 20110027235A1
• Time in Patent Office: 2,798 Days
• Field of Search: 424 937, 435 29, 435325, 435435, 435366, 435 691
• CPC Class Codes: A61P 7/04 Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2710/10343 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2740/15043 viral genome or elements thereof as genetic vector
• 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 2830/008 cell type or tissue specific enhancer/promoter combination
• 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 sequences of interest such as lineage-specific or cell fate reporter constructs or protein encoding sequences into stem cells.

Claims

1. An isolated pluripotent or multipotent stem cell comprising a transgene, wherein the transgene is integrated into an endogenous PITX3 or Factor IX gene of the stem cell using a pair of zinc finger nucleases that bind to and cleave the endogenous PITX3 or Factor IX gene, wherein the pair of zinc finger nucleases bind to paired target sites of SEQ ID NO:

47 and SEQ ID NO;

48 of the PITX3 gene, or paired target sites of SEQ ID NO;

49 and SEQ ID NO;

50 of the PITX3 gene, or to paired target sites of SEQ ID NO;

51 and SEQ ID NO;

56 of the Factor IX gene, and further wherein the pair of zinc finger nucleases cleaves the PITX3 or Factor IX gene within or between the paired target sites such that the transgene is integrated into the PITX3 or Factor IX gene.

2. The isolated stem cell of claim 1, wherein the stem cell is selected from the group consisting of a hematopoietic stem cell, a mesenchymal stem cell, an embryonic stem cell, a neuronal stem cell, a muscle stem cell, a liver stem cell, a skin stem cell, and an induced pluripotent stem cell.

3. The isolated stem cell of claim 1, wherein the stem cell is a mammalian stem cell.

4. The isolated stem cell of claim 3, wherein the stem cell is a human induced pluripotent stem cell (hiPSC).

5. The isolated stem cell of claim 1, wherein the transgene is flanked by recombinase sites.

6. The isolated stem cell of claim 1, further comprising a sequence encoding a reporter construct.

7. A stem cell according to claim 1, wherein the isolated transgene comprises a sequence encoding a lineage-specific or cell fate marker as shown in Table 1.