Methods and compositions for modulating nuclease-mediated genome engineering in hematopoietic stem cells

• US 9,816,074 B2
• Filed: 07/23/2015
• Issued: 11/14/2017
• Est. Priority Date: 07/25/2014
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Therapeutics, Inc.
• Inventors: Conway, Anthony, Cost, Gregory J., Gregory, Philip D.
• Primary Examiner(s): Babic, Christopher M
• Assistant Examiner(s): Leonard, Arthur S
• Application Number:
  US14/807,183
• Publication Number:
  US 20160024474A1
• Time in Patent Office: 845 Days
• Field of Search: None
• 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 cell culture comprising:

a stem cell comprising an exogenous nuclease that cleaves the genome of the stem cell;

interleukin 6 (IL-6);

lithium chlorine (LiCl); and

a histone deactylase inhibitor (HDACI), wherein the HDACI is valproic acid (VPA) at a concentration of between 1.25 and 5 mM.

2. The cell culture of claim 1, wherein the stem cell further comprises a donor sequence.

3. A method for preparing a cell culture according to claim 1, 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 the histone deactylase inhibitor (HDACI) wherein the HDACI is valproic acid (VPA) at a concentration of between 1.25 and 5 mM;

interleukin 6 (IL-6); and

lithium chlorine (LiCl).

4. The method of claim 3, 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.

5. The method of claim 3, wherein the exogenous nucleases are delivered to the stem cells as a nucleic acid encoding the nuclease.

6. The method of claim 5, wherein the nucleic acid is mRNA.

7. The method of claim 3, 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.

8. The method of claim 4, 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.

9. The method of claim 8, 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.