Targeted integration into stem cells

• US 20110027235A1
• Filed: 04/08/2010
• Published: 02/03/2011
• Est. Priority Date: 04/09/2009
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc., Whitehead Institute for Biomedical Research
• Original Assignee: Sangamo Biosciences, Inc. (Sangamo Therapeutics, Inc.), Whitehead Institute for Biomedical Research
• Inventors: Urnov, Fyodor, Hockemeyer, Dirk, Soldner, Frank, Yao, Shuyuan, Jaenisch, Rudolf, Holmes, Michael C., Gregory, Philip D.
• Granted Patent: US 9,834,787 B2
• Time in Patent Office: Days
• US Class Current: 424/93.7
• 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. A stem cell comprising a transgene, wherein the transgene comprises a gene product, wherein the transgene is integrated into a specified region of the genome of the stem, cell such that gene product is transcribed or translated upon or during differentiation of the stem cell into a lineage-specific or mature cell type.

2. The stem cell of claim 1, wherein the transgene further comprises a lineage-specific or cell fate promoter operably linked to the gene product.

3. The stem cell of claim 1, wherein the gene product comprises a lineage-specific or cell fate gene that is integrated into the genome such that expression of the lineage-specific or cell fate gene is driven by an endogenous promoter.

4. The 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, an induced pluripotent stem cell and combinations thereof.

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

6. The stem cell of claim 5, wherein the stem cell is a human induced pluripotent stem cell (hiPSC).

7. The stem cell of claim 1, wherein the gene product comprises a protein.

8. The stem cell of claim 1, wherein the gene product comprises sequences that are transcribed into an RNA product.

9. The stem cell of claim 8, wherein the RNA product is selected from the group consisting of micro RNAs (miRNAs), shRNAs, RNAis and combinations thereof.

10. The stem cell of claim 7, wherein the sequence encoding the protein is expressed constitutively, inducibely or tissue-specifically.

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

12. The stem cell of claim 1 further comprising an additional reporter construct.

13. A method of isolating cells of a selected cell type, the method comprising:

culturing a population of stem cells according to claim 1, wherein the gene product is expressed in cells of the selected cell type and isolating the cells that express the gene product, thereby isolating cells of the selected type.

14. A method of determining the effect of a compound on stem cell differentiation, the method comprising:

culturing first and second populations of stem cells according to claim 1 in the presence and absence of the compound, and

evaluating expression of the gene product in the first and second populations, wherein a difference in the expression of the gene product in the presence of the compound indicates an effect of the compound on stem cell differentiation.

15. A method of producing a gene product in a stem cell, the method comprising:

providing a population of stem cells according to claim 1, wherein the sequence is integrated into a non-essential site in the stem cells, and

culturing the population of stem cells, wherein the population of cultured stem cells uniformly expresses the gene product.

16. A method of treating a disease characterized by reduced expression of a functional gene product in a subject in need of treatment, the method comprising:

administering a population of stem cells according to claim 15 to the subject, wherein the stem cells express the functional gene product.

17. The method of claim 16, wherein the disease is hemophilia B, and the protein is Factor IX.

18. The method of claim 13, wherein the transgene is introduced into the stem cell using a plasmid, linear DNA, an adenovirus vector or a retroviral vector.

19. A method of reducing or eliminating positional variation effects associated with transgene insertion into a specified region of a stem cell, the method comprising:

providing a stem cell in which the transgene inserted at one allele of the specified region comprises a gene expression construct comprising a regulatable construct and in which the other allele of the specified region comprises a transactivator for the regulatable promoter.