Methods and compositions concerning siRNA's as mediators of RNA interference

• US 20060142228A1
• Filed: 12/23/2004
• Published: 06/29/2006
• Est. Priority Date: 12/23/2004
• Status: Abandoned Application

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Ambion Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Ford, Lance P., Krebs, Joseph
• Application Number:
  US11/020,560
• Publication Number:
  US 20060142228A1
• Time in Patent Office: Days
• US Class Current: 514/44.A
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2330/30 chemically synthesised
• CPC Class Codes: C12Y 102/01012 Glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) (1.2.1.12)
• CPC Class Codes: C12Y 502/01008 Peptidylprolyl isomerase (5.2.1.8), i.e. cyclophilin

Abstract

The present invention concerns an isolated siRNA of from about 5 to about 20 nucleotides that mediates RNA interference. Also disclosed are methods of reducing expression of a target gene in a cell comprising obtaining at least one siRNA of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 basepairs in length; and delivering the siRNA into the cell. The siRNAs can be chemically synthesized RNA or an analog of a naturally occurring RNA.

Claims

1. An isolated RNA of from about 5 to about 20 nucleotides that mediates RNA interference of a target mRNA.

2. The isolated RNA of claim 1 further comprising a terminal 3′

hydroxyl group.

3. The isolated RNA of claim 1 which is chemically synthesized RNA or an analog of a naturally occurring RNA.

4. The isolated RNA of claim 1, wherein the RNA is from about 12 to about 18 nucleotides in length.

5. The isolated RNA of claim 1, wherein the RNA is from about 14 to about 16 nucleotides in length.

6. The isolated RNA of claim 1, wherein the RNA is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 nucleotides in length.

7. The isolated RNA of claim 1, wherein the RNA is an siRNA.

8. The isolated RNA of claim 1, wherein the RNA is a single-stranded.

9. The isolated RNA of claim 1, wherein the RNA is a double-stranded.

10. The isolated RNA of claim 9, wherein the 3′

ends of the double stranded RNA comprises a 2, 3, 4, or 5 nucleotide overhang.

11. The isolated RNA of claim 10, wherein the nucleotide overhang is a 2 nucleotide overhang.

12. The isolated RNA of claim 11, wherein the 2 nucleotides are thymine.

13. The isolated RNA of claim 9, wherein the 5′

ends of the double stranded RNA comprises a 2, 3, 4, or 5 nucleotide overhang.

14. The isolated RNA of claim 13, wherein the nucleotide overhang is a 2 nucleotide overhang.

15. The isolated RNA of claim 14, wherein the 2 nucleotides are thymine.

16. The isolated RNA of claim 1, wherein the RNA is formulated into a pharmaceutically acceptable composition.

17. The isolated RNA of claim 1, wherein the RNA associates with a protein complex.

18. The isolated RNA of claim 17, wherein the protein complex is RNA-induced silencing complex (RISC).

19. The isolated RNA of claim 1, wherein the isolated RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

13;

SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, SEQ ID NO;

17;

SEQ ID NO;

18, SEQ ID NO;

19, SEQ ID NO;

20, SEQ ID NO;

21, SEQ ID NO;

22, SEQ ID NO;

23, SEQ ID NO;

24, SEQ ID NO;

25, SEQ ID NO;

26, SEQ ID NO;

27, SEQ ID NO;

28, SEQ ID NO;

29, SEQ ID NO;

30;

SEQ ID NO;

31, SEQ ID NO;

32, SEQ ID NO;

33, SEQ ID NO;

34, SEQ ID NO 35, SEQ ID NO;

36;

SEQ ID NO;

37, SEQ ID NO;

38, SEQ ID NO;

39, SEQ ID NO;

40, SEQ ID NO;

41, SEQ ID NO;

42, SEQ ID NO;

43, SEQ ID NO;

44, SEQ ID NO;

45, SEQ ID NO;

46, SEQ ID NO;

47, SEQ ID NO;

48, SEQ ID NO;

49, SEQ ID NO;

50, SEQ ID NO;

51, SEQ ID NO;

52, SEQ ID NO;

53, and SEQ ID NO;

54.

20. A method of reducing expression of a target gene in a cell comprising:

a) obtaining at least one siRNA of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length; and

b) delivering the siRNA into the cell.

21. The method of claim 20, wherein the siRNA is 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides in length.

22. The method of claim 21, wherein the siRNA is 12, 13, 14, or 15 nucleotides in length.

23. The method of claim 20, wherein the siRNA is chemically synthesized siRNA or an analog of a naturally occurring siRNA.

24. The method of claim 20, further comprising isolating the siRNA prior to delivery.

25. The method of claim 20, further comprising obtaining at least two siRNAs and delivering them into the cell.

26. The method of claim 20, further comprising obtaining a pool of siRNAs and delivering the pool into the cell.

27. The method of claim 20, wherein the cell is comprised in an organism.

28. The method of claim 20, wherein the cell is a human cell.

29. The method of claim 20, wherein multiple siRNA molecules are delivered into the cell.

30. The method of claim 20, wherein the siRNA comprises a terminal 3′

hydroxyl group.

31. The method of claim 20, wherein the siRNA is double-stranded.

32. The method of claim 31, wherein the 3′

ends of the double stranded siRNA comprise a 2, 3, 4, or 5 nucleotide overhang.

33. The method claim 32, wherein the nucleotide overhang is a 2 nucleotide overhang.

34. The method of claim 33, wherein the 2 nucleotides are thymine.

35. The method of claim 31, wherein the 5′

ends of the double stranded siRNA comprise a 2, 3, 4, or 5 nucleotide overhang.

36. The method of claim 35, wherein the nucleotide overhang is a 2 nucleotide overhang.

37. The method of claim 36, wherein the two nucleotides are thymine.

38. The method of claim 20, wherein the siRNA is formulated into a pharmaceutically acceptable composition.

39. The method of claim 20, wherein the siRNA associates with a protein complex.

40. The method of claim 39, wherein the protein complex is RNA-induced silencing complex (RISC).

41. The method of claim 20, wherein the siRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

13;

SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, SEQ ID NO;

17;

SEQ ID NO;

18, SEQ ID NO;

19, SEQ ID NO;

20, SEQ ID NO;

21, SEQ ID NO;

22, SEQ ID NO;

23, SEQ ID NO;

24, SEQ ID NO;

25, SEQ ID NO;

26, SEQ ID NO;

27, SEQ ID NO;

28, SEQ ID NO;

29, SEQ ID NO;

30;

SEQ ID NO;

31, SEQ ID NO;

32, SEQ ID NO;

33, SEQ ID NO;

34, SEQ ID NO 35, SEQ ID NO;

36;

SEQ ID NO;

37, SEQ ID NO;

38, SEQ ID NO;

39, SEQ ID NO;

40, SEQ ID NO;

41, SEQ ID NO;

42, SEQ ID NO;

43, SEQ ID NO;

44, SEQ ID NO;

45, SEQ ID NO;

46, SEQ ID NO;

47, SEQ ID NO;

48, SEQ ID NO;

49, SEQ ID NO;

50, SEQ ID NO;

51, SEQ ID NO;

52, SEQ ID NO;

53, and SEQ ID NO;

54.