Methods and compositions for enhancing targeted transgene integration

Patent metadata

Field	Value
Publication number	US-2016168593-A1
Application number	US-201514967885-A
Country	US
Kind code	A1
Filing date	Dec 14, 2015
Priority date	Dec 15, 2014
Publication date	Jun 16, 2016
Grant date	—

Abstract

Disclosed herein are methods and compositions for enhancing insertion of transgene sequences encoding proteins that is aberrantly expressed in disease or disorder such as a lysosomal storage disease or a hemophilia by administering one or more topoisomerases inhibitors, one or more stabilizers of R loop formation or inhibitors of R-loop repair and/or one or more up-regulators of the TC-NER pathway to the target cell.

First claim

What is claimed is:

1 . A method of integrating a transgene into a cell's genome, the method comprising introducing the transgene into the cell wherein the cell is grown in the presence of one or more topoisomerase inhibitors, one or more stabilizers of R loop formation or inhibitors of R-loop repair and/or one or more up-regulators of the TC-NER pathway, such that the transgene is integrated into the genome.

2 . The method of claim 1 , wherein the topoisomerase inhibitors inhibit topoisomerase I and/or II.

3 . The method of claim 2 , wherein the inhibitor is camptothecin, etoposide, doxorubicin, cisplatin, topotecan, irinotecan, tenipopside, mitoxantrone, etoposide phosphate, and/or topotecan hydrochloride.

4 . The method of claim 1 , wherein the inhibitor of R-loop formation or persistence is RNAases H1/H2, AQR, Sentataxin, XAB2, PRP19, CCDC16, PPIE and hISY1.

5 . The method of claim 1 , wherein the TC-NER pathway is XPF, XPG, XPA, XPB, XPD and CSB.

6 . The method of claim 1 , further comprising introducing one or more nucleases targeted to an endogenous locus into the cell.

7 . The method of claim 6 wherein the endogenous locus is a gene.

8 . The method of claim 7 , wherein the endogenous gene is a safe harbor gene.

9 . The method of claim 8 , wherein the safe harbor gene is an albumin gene.

10 . The method of claim 1 , wherein expression of the transgene is controlled by an endogenous albumin promoter.

11 . The method of claim 1 , wherein the transgene encodes a protein and/or a structural nucleic acid.

12 . The method of claim 11 , wherein the protein is deficient or lacking in a lysosomal storage disease, hemophilia, epidermolysis bullosa, diabetes, cancer, clotting disorders or AAT deficient emphysema.

13 . The method of claim 11 , wherein the protein is a Factor VII protein, a Factor VIII (F8) protein, a Factor IX (F.IX) protein, a Factor X protein, a glucocerebrosidase protein, an α galactosidase protein, an iduronate-2-sulfatase protein, an alpha-L iduronidase protein and/or a sphingomyelin phosphodiesterase protein.

14 . The method of claim 6 , wherein the nuclease comprises a zinc finger nuclease (ZFN), a TALEN or a CRISPR/Cas nuclease system.

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

16 . The method of claim 15 , wherein the cell is a hepatic cell or a stem cell.

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

18 . A kit comprising the compositions one or more transgene donors, one or more topoisomerase inhibitor(s), one or more R-loop stabilizers and/or inhibitors of R-loop repair.

19 . The kit of claim 18 , further comprising one or more nucleases.

Assignees

• Sangamo Biosciences Inc

Inventors

• Cost Gregory J
• Wechsler Thomas

Classifications

• C12N2750/14143

  viral genome or elements thereof as genetic vector · CPC title

• C12N15/907 Primary

  in mammalian cells · CPC title

• A61K31/7048

  having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin {, digitoxin or digoxin} · CPC title

• A61K48/00

  Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy · CPC title

• A61K31/4745

  condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines (yohimbine derivatives, vinblastine A61K31/475; ergoline derivatives A61K31/48) · CPC title