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

Patent metadata

Field	Value
Publication number	US-11873479-B2
Application number	US-202117244190-A
Country	US
Kind code	B2
Filing date	Apr 29, 2021
Priority date	Feb 28, 2011
Publication date	Jan 16, 2024
Grant date	Jan 16, 2024

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 polynucleotide encoding a fusion polypeptide, wherein said polypeptide comprises a homing endonuclease that (i) binds and cleaves a selected dsDNA target site in a mammalian cell, (ii) is selected from the group consisting of: I-LtrI, I-GpiI, I-GzeI, I-MpeMI, I-PanMI, I-CreI, I-OnuI, and I-HjeMI, and (iii) is linked by a linker domain to Trex2 or a biologically active fragment thereof, wherein said linker comprises from about 4 to 30 amino acids and is flexible such that said homing endonuclease and said Trex2 or biologically active fragment thereof retain their respective biological activities.

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

3. The polynucleotide of claim 2 , wherein the non-coding sequence is a regulatory sequence.

4. The polynucleotide of claim 3 , wherein the regulatory sequence is a promoter, an enhancer, or a splice site.

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

6. The polynucleotide of claim 5 , wherein the gene is CCR-5.

7. The polynucleotide of claim 5 , wherein the gene is Stat3.

8. The polynucleotide of claim 1 , wherein the mammalian cell is a human cell.

9. The polynucleotide of claim 1 , wherein the homing endonuclease is engineered from I-CreI.

10. The polynucleotide of claim 1 , wherein the homing endonuclease is engineered from I-OnuI.

11. The polynucleotide of claim 1 , wherein Trex2 is linked by a linker domain to said homing endonuclease.

12. The polynucleotide of claim 1 , wherein the biologically active fragment of Trex2 is linked by a linker domain to said homing endonuclease.

13. The polynucleotide of claim 1 , wherein the linker domain is a G4S linker.

14. The polynucleotide of claim 1 , wherein the linker domain is a T2A linker.

15. A polynucleotide encoding a fusion polypeptide, wherein said polypeptide comprises an I-OnuI homing endonuclease that (i) binds and cleaves a selected dsDNA target site in a mammalian cell, and (ii) is linked by a linker domain comprising 4 to 30 amino acids to a Trex2 or a biologically active fragment thereof.

16. A method of increasing mutagenesis at a doublestrand DNA (dsDNA) break at a selected dsDNA target site in a eukaryotic cell comprising introducing into said cell the polynucleotide of claim 1 .

Assignees

• Seattle Childrens Hospital
• Seattle Childrens Res Institute

Inventors

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

Classifications

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N15/102 Primary

  Mutagenizing nucleic acids · 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

• A61K35/28

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

• A61K38/45 Primary

  Transferases (2) · CPC title