siRNA Kinase and Methods of Use

• US 20100154072A1
• Filed: 05/29/2007
• Published: 06/17/2010
• Est. Priority Date: 05/29/2006
• Status: Abandoned Application

• Current Assignee: IMBA, INSTITUT FUR MOLEKULARE BIOTECHNOLOGIE GMBH
• Original Assignee: IMBA, INSTITUT FUR MOLEKULARE BIOTECHNOLOGIE GMBH
• Inventors: Martinez, Javier, Weitzer, Stefan
• Application Number:
  US12/302,583
• Publication Number:
  US 20100154072A1
• Time in Patent Office: Days
• US Class Current: 800/18
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2320/31 Combination therapy
• CPC Class Codes: C12N 2320/50 Methods for regulating/modulating their activity
• CPC Class Codes: C12N 9/1205 Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
• CPC Class Codes: C12Y 207/01078 Polynucleotide 5'-hydroxyl-kinase (2.7.1.78)

Abstract

The invention is based on the finding that Clp1 has the activity of an RNA kinase. Clp1 is therefore useful as an enhancer of siRNA activity. It can be used per se, in kits or expressed in cell lines. Clp1 transgenic and Clp1 knock-out non-human animals are useful for studying the function of siRNAs. Clp1 is also useful in gene therapy to enhance the efficacy of therapeutic siRNAs.

Claims

1. A method for phosphorylating RNA comprising using a Clp1 molecule as an RNA kinase.

2. The method of claim 1, wherein the Clp1 molecule for the transfers of the γ

-phosphate of ATP to the 5′

end of ssRNA, dsRNA or the RNA strand of RNA/DNA hybrids.

3. The method of claim 1, wherein said Clp1 is recombinant.

4. The method of claim 1, wherein said Clp1 is human Clp1 (HsClp1) with the amino acid sequence as set forth in SEQ ID NO:

2, or with the amino acid sequence encoded by a polynucleotide which hybridizes under stringent conditions to a polynucleotide having a nucleotide sequence as set forth in SEQ ID NO;

1 or to the region encoding HsClp1 contained therein, or a variant or fragment thereof with said RNA kinase activity.

5. The method of claim 1, wherein said Clp1 molecule is a component of a kit.

10. The kit of claim 6, wherein said Clp1 is recombinant human Clp1 as defined in claim 4.

11. The method of claim 1, wherein said Clp1 molecule is exogenously expressed in an animal cell to exhibit its kinase activity in said cell.

12. The method of claim 11, wherein said animal cell is a mammalian cell.

13. The method of claim 12, wherein said cell is derived from a cell line.

15. The cell line of claim 13 which does not endogenously express Clp1.

6. A kit containing

a. a recombinant Clp1 with said RNA kinase activity;

b. γ

-ATP; and

c. a reaction buffer containing buffer components and one or more metal ions selected from Mg²⁺, Mn²⁺, Ni² or mixtures thereof, for use in a final concentration range of ca. 1-10 mM.

7. The kit of claim 6, wherein said concentration range of the metal ion 2-5 mM.

8. The kit of claim 6, wherein γ

-ATP is present in radioactively labeled form.

9. The kit of claim 8, wherein γ

-ATP is present as [γ

-³²P]ATP, [γ

-³³P]ATP, [γ

-¹⁸O]ATP or [γ

-³⁵S]ATP.

14. An animal cell line genetically engineered to express Clp1.

16. The cell line of claim 14 or 15, which, in addition, expresses an siRNA molecule.

17. The cell line of claim 16, wherein said siRNA molecule is controlled by the same promoter as said Clp1 molecule.

18. A Clp1 transgenic non-human animal.

19. The transgenic animal of claim 18 which is a mouse.

20. A Clp1 knock-out mouse.

21. A pharmaceutical composition comprising a DNA encoding a therapeutic siRNA directed against a disease-associated gene of interest and a DNA encoding a human Clp1 molecule with RNA kinase activity.