METHODS AND COMPOSITIONS FOR ENHANCING NUCLEASE-MEDIATED GENE DISRUPTION

• US 20140242702A1
• Filed: 02/24/2014
• Published: 08/28/2014
• Est. Priority Date: 02/25/2013
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc., Sigma-Aldrich Company (Sigma-Aldrich Corporation)
• Original Assignee: Sangamo Biosciences, Inc., Sigma-Aldrich Company (Sigma-Aldrich Corporation)
• Inventors: Chen, Fuqiang, Kang, Qiaohua, Wechsler, Thomas
• Granted Patent: US 10,227,610 B2
• Time in Patent Office: Days
• US Class Current: 435/462
• CPC Class Codes: A61K 2300/00 Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• CPC Class Codes: A61K 31/502 ortho- or peri-condensed with carbocyclic ring systems, e.g. cinnoline, phthalazine
• CPC Class Codes: A61K 31/5377 not condensed and containing further heterocyclic rings, e.g. timolol
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 2319/80 containing a DNA binding domain, e.g. Lacl or Tet-repressor
• CPC Class Codes: C07K 2319/81 containing a Zn-finger domain for DNA binding
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

Methods and compositions for increasing nuclease-mediated genomic modification using DNA repair inhibitors are provided.

Claims

1. A method for targeted genomic disruption in a cell, the method comprising:

administering at least one nuclease to the cell, wherein the nuclease cleaves endogenous genomic sequences in the cell;

administering at least one inhibitor of classic or alternative non-homologous end joining (NHEJ) to the cell.

2. The method of claim 1, wherein the inhibitor inhibits a protein selected from the group consisting of a DNA-dependent-protein kinase catalytic subunit (DNA-PKcs), a Poly-(ADP-ribose) polymerase 1/2 (PARP1/2) and combinations thereof.

3. The method of claim 2, wherein the protein is selected from the group consisting of PARP1, Ku70/80, DNA-PKcs, XRCC4/XLF, Ligase IV, Ligase III, XRCC1, Artemis Polynucleotide Kinase (PNK) and combinations thereof.

4. The method of claim 1, wherein the targeted genomic disruption comprises a deletion.

5. The method of claim 1, wherein the targeted genomic disruption comprises an insertion.

6. The method of claim 5, further comprising administering an exogenous sequence to the cell, wherein the exogenous sequence is integrated into the genome via homology directed repair (HDR) mechanisms following cleavage by the nuclease.

7. The method of claim 6, wherein the exogenous sequence is selected from the group consisting of a sequence encoding a protein, a regulatory sequence, a sequence that encodes a structural RNA and combinations thereof.

8. The method of claim 1, further comprising administering a small molecule or a polypeptide that activates HDR or microhomology-mediated end joining (MMEH).

9. The method of claim 8, wherein the activator activates a protein selected from the group consisting of Mre11, Rad50, NSB1, Rad52, and combinations thereof.

10. The method of claim 1, wherein the nuclease is selected from the group consisting of a nuclease comprising a zinc finger protein, a nuclease comprising a TALE effector domain DNA-binding protein, a CRISPR/Cas system and combinations thereof.

11. The method of claim 10, wherein the nuclease is administered using an expression vector or as mRNA.

12. A cell made by the method of claim 1.

13. The cell of claim 12, wherein the cell is a eukaryotic cell.

14. The cell of claim 13, wherein the eukaryotic cell is a mammalian or plant cell.

15. A kit for producing a cell according to claim 12, the kit comprising at least one nuclease that bind to a target site in the cell and one or more inhibitors of proteins involved in classic or alternative NHEJ.

16. The kit of claim 15, further comprising an activator of HDR or MMEJ repair pathways.

17. The kit of claim 15, further comprising an exogenous sequence.