Methods for producing interfering RNA molecules in mammalian cells and therapeutic uses for such molecules

• US 20040096843A1
• Filed: 02/13/2003
• Published: 05/20/2004
• Est. Priority Date: 02/14/2002
• Status: Active Grant

• Current Assignee: City of Hope
• Original Assignee: City of Hope
• Inventors: Lee, Nan-Sook, Rossi, John J.
• Granted Patent: US 7,820,632 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/1132 against retroviridae, e.g. HIV
• CPC Class Codes: C12N 2310/111 spanning the whole gene, or a large part of it
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/53 partially self-complementary or closed
• CPC Class Codes: C12N 2330/30 chemically synthesised
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2600/178 miRNA, siRNA or ncRNA

Abstract

Methods for producing interfering RNA molecules in mammalian cells are provided. Therapeutic uses for the expressed molecules, including inhibiting expression of HIV, are also provided.

Claims

1. A method for producing a double stranded interfering RNA molecule in a mammalian cell, comprising:

a) inserting DNA sequences encoding a sense strand and an antisense strand of the RNA molecule into a vector comprising an RNA pol III promoter; and

b) introducing the vector into a mammalian cell so that the RNA molecule can be expressed.

2. The method of claim 1, wherein the RNA molecule is a small interfering RNA (siRNA) molecule.

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

4. The method of claim 1, wherein the DNA sequences are inserted into separate vectors.

5. The method of claim 1, wherein the DNA sequences are inserted into the same vector.

6. The method of claim 1, wherein the vector is a plasmid vector.

7. The method of claim 1, wherein the vector is a viral vector.

8. The method of claim 1, wherein the RNA pol III promoter is a mammalian U6 RNA Pol III promoter.

9. The method of claim 1, wherein the vector is introduced into the mammalian cell in vitro.

10. The method of claim 1, wherein the vector is introduced into the mammalian cell in vivo.

12. The method of claim 9, wherein the mammalian cell is a human cell.

13. The method of claim 9, wherein the DNA sequences are in separate vectors.

14. The method of claim 9, wherein the DNA sequences are in the same vector.

11. A method for inhibiting the expression of a target gene, comprising:

introducing one or more vectors into a mammalian cell, wherein the one or more vectors comprise a suitable promoter and DNA sequences encoding a sense strand and an antisense strand of a double stranded, small interfering RNA (siRNA) molecule, so that the siRNA molecule can be expressed and initiate RNA interference of expression of a target gene in the mammalian cell, thereby inhibiting expression of the target gene.

15. The method of claim 11, wherein the vector is a plasmid vector.

16. The method of claim 11, wherein the vector is a viral vector.

17. The method-of claim 11, wherein the promoter is an RNA pol III promoter.

18. The method of claim 17, wherein the RNA pol III promoter is a mammalian U6 RNA Pol III promoter.

19. The method of claim 11, wherein the target gene is an HIV target gene.

20. The method of claim 19, wherein the HIV is HIV-1.

21. The method of claim 20, wherein the HIV target gene is HIV-1 rev.

22. The method of claim 20, wherein the HIV target gene is HIV-1 tat.

23. The method of claim 20, wherein the HIV target gene is both HIV-1 rev and HIV-1 tat.

24. The method of claim 19, wherein the mammalian cell is an HIV-infected human cell.

25. A method for testing the expression and function of small interfering RNA (siRNA) molecules, comprising:

co-introducing into a mammalian cell i) one or more vectors comprising a suitable first promoter and DNA sequences encoding a sense strand and an antisense strand of an siRNA molecule, and ii) a vector comprising a target gene and a suitable second promoter, so that the siRNA molecule can be expressed and initiate RNA interference of expression of the target gene, thereby inhibiting expression of the target gene.

26. The method of claim 25, wherein the mammalian cell is a human cell.

27. The method of claim 25, wherein the DNA sequences are in separate vectors.

28. The method of claim 25, wherein the DNA sequences are in the same vector.

29. The method of claim 25, wherein the first promoter is an RNA pol III promoter.

30. The method of claim 29, wherein the RNA pol III promoter is a mammalian U6 RNA Pol III promoter.