Coupling endonucleases with end-processing enzymes drives high efficiency gene disruption

Patent metadata

Field	Value
Publication number	US-12378546-B2
Application number	US-202318526930-A
Country	US
Kind code	B2
Filing date	Dec 1, 2023
Priority date	Feb 28, 2011
Publication date	Aug 5, 2025
Grant date	Aug 5, 2025

Abstract

The present disclosure relates to the co-expression of an endonuclease with an end-processing enzyme for the purpose of enhanced processing of the polynucleotide ends generated by endonuclease cleavage.

First claim

What is claimed is:

1. A method of increasing mutagenesis at a double-strand DNA (dsDNA) break at a selected dsDNA target site in a eukaryotic cell comprising:
a) selecting a dsDNA target site for mutagenesis; and
b) introducing into the eukaryotic cell a polynucleotide sequence encoding:
(i) an endonuclease having a >14 base pair cleavage site, wherein said endonuclease binds and cleaves the selected dsDNA target site; and
(ii) an exonuclease;

wherein the exonuclease exhibits exonuclease activity at the cleaved dsDNA target site, resulting in increased mutagenesis at the selected dsDNA target site as compared to mutagenesis that occurs in the absence of exonuclease activity.

2. The method of claim 1 , wherein the endonuclease is engineered to bind and cleave the selected dsDNA target site.

3. The method of claim 1 , wherein the eukaryotic cell is a human cell.

4. The method of claim 1 , wherein the mutagenesis is an insertion or deletion at the selected dsDNA target site.

5. The method of claim 1 , wherein the exonuclease exhibits 3′ to 5′ exonuclease activity.

6. The method of claim 1 , wherein the exonuclease is Trex2 or a biologically active fragment thereof.

7. The method of claim 1 , wherein the exonuclease is Trex2.

8. The method of claim 1 , wherein the endonuclease and the exonuclease are encoded by a single polynucleotide.

9. The method of claim 8 , wherein the endonuclease is coupled to the exonuclease by a linker domain.

10. The method of claim 9 , wherein the linker domain is a peptide linker comprising 4 to 30 amino acids.

11. The method of claim 10 , wherein the linker domain is a G4S linker.

12. The method of claim 10 , wherein the linker domain is a T2A linker.

13. The method of claim 1 , wherein the dsDNA target site is within a non-coding sequence of a gene.

14. The method of claim 13 , wherein the non-coding sequence is a regulatory sequence.

15. The method of claim 14 , wherein the regulatory sequence is a promoter, enhancer, or splice site.

16. The method of claim 1 , wherein the dsDNA target site is within a coding sequence of a gene.

17. The method of claim 9 , wherein the linker domain is a chemical linker.

Assignees

• Seattle Childrens Hospital
• Seattle Childrens Res Institute

Inventors

• Scharenberg Andrew M
• Certo Michael T
• Gwiazda Kamila Sabina

Classifications

• A61K2035/124

  the cells being hematopoietic, bone marrow derived or blood cells · CPC title

• C12N2840/203

  having an IRES · CPC title

• C12N2800/80

  Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites · CPC title

• A61K35/28

  Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells · CPC title

• A61K9/0019

  Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner (non-active ingredients are additionally classified in A61K47/00) · CPC title