METHODS AND COMPOSITIONS FOR MODULATING NUCLEASE-MEDIATED GENOME ENGINEERING IN HEMATOPOIETIC STEM CELLS

• US 20160024474A1
• Filed: 07/23/2015
• Published: 01/28/2016
• Est. Priority Date: 07/25/2014
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Biosciences, Inc. (Sangamo Therapeutics, Inc.)
• Inventors: Conway, Anthony, Cost, Gregory J., Gregory, Philip D.
• Granted Patent: US 9,816,074 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 35/28 Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2501/065 Modulators of histone acetylation
• CPC Class Codes: C12N 2501/2306 Interleukin-6 (IL-6)
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 5/0647 Haematopoietic stem cells; Uncommitted or multipotent progenitors
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

The present disclosure is in the field of genome engineering, particularly targeted modification of the genome of a hematopoietic stem cell.

Claims

1. A method for increasing nuclease-mediated gene modification in a population of stem cells, wherein the stem cells comprise exogenous nucleases that cleave the genomes of the stem cells, the method comprising

culturing the stem cells in the presence of a histone deactylase inhibitor (HDACI).

2. The method of claim 1, wherein the HDACI comprises valproic acid (VPA).

3. The method of claim 1, wherein the cells are cultured in the presence of the HDACI before, during and/or after nuclease-mediated cleavage of the genome.

4. The method of claim 1, further comprising culturing the cells in the presence of lithium chloride (Li).

5. The method of claim 1, further comprising introducing one or more exogenous donor sequences into the stem cells such that the donor molecule is introduced into the genomes of the cells following cleavage by the nuclease.

6. The method of claim 1, wherein the exogenous nucleases are delivered to the stem cells as a nucleic acid encoding the nuclease.

7. The method of claim 6, wherein the nucleic acid is mRNA.

8. The method of claim 1, wherein the exogenous nuclease is selected from the group consisting of a zinc finger nuclease (ZFN), a TALE-nuclease (TALEN), a CRISPR/Cas nuclease system or combinations thereof.

9. A method for increasing targeted integration of a donor sequence following nuclease-mediated cleavage in a population of stem cells, the method comprising:

increasing the percent of overall gene modification the stem cells according to the method of claim 5.

10. The method of claim 9, wherein the donor sequences are delivered prior to, after, or along with the nucleases.

11. The method of claim 9, wherein the donor sequences comprise a sequence selected from the group consisting of sequences encoding a protein, regulatory sequences, sequence that encode a structural nucleic acid such as a microRNA or siRNA.

12. The method of claim 11, wherein the protein is selected from the group consisting of a regulatory protein that modulates expression of a gene;

an antibody, an antigen, an enzyme, a growth factor, a receptor, a hormone, a lymphokine, a cytokine, a reporter and combinations thereof.

13. A cell culture comprising:

a stem cell comprising an exogenous nuclease; and

a histone deactylase inhibitor (HDACI).

14. The cell culture of claim 13, wherein the stem cell further comprises a donor sequence.

15. A genetically modified cell descended from the cell culture of claim 13.

17. An ex vivo method of providing a genetically modified cell to a subject, the method comprising administering a stem cell according to claim 15 to the subject in a bone marrow transplant such that stem cell differentiates and matures in the subject.

18. The method of claim 17, wherein the genetically modified cell expresses a protein.

19. The method of claim 17, wherein the subject is subjected to myeloablative pre-conditioning.

16. A kit comprising an exogenous nuclease and a HDACI.