Targeted integration into stem cells

Patent metadata

Field	Value
Publication number	US-9834787-B2
Application number	US-79874910-A
Country	US
Kind code	B2
Filing date	Apr 8, 2010
Priority date	Apr 9, 2009
Publication date	Dec 5, 2017
Grant date	Dec 5, 2017

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.

First claim

What is claimed is:

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.

Assignees

• Gregory Philip D
• Hockemeyer Dirk
• Holmes Michael C
• Jaenisch Rudolf
• Soldner Frank
• Urnov Fyodor
• Yao Shuyuan
• Sangamo Therapeutics Inc

Inventors

• Gregory Philip D
• Hockemeyer Dirk
• Holmes Michael C
• Jaenisch Rudolf
• Soldner Frank
• Urnov Fyodor
• Yao Shuyuan

Classifications

• A61P7/04

  Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents · CPC title

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N2740/15043

  viral genome or elements thereof as genetic vector · CPC title

• C12N2830/008

  cell type or tissue specific enhancer/promoter combination · CPC title

• C12N2800/30

  Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT · CPC title