IRNA agents with biocleavable tethers

• US 7,723,509 B2
• Filed: 11/09/2004
• Issued: 05/25/2010
• Est. Priority Date: 04/17/2003
• Status: Active Grant

• Current Assignee: Alnylam Pharmaceuticals Incorporated
• Original Assignee: Alnylam Pharmaceuticals Incorporated
• Inventors: Manoharan, Muthiah, Rajeev, Kallanthottathil G.
• Primary Examiner(s): VIVLEMORE, TRACY ANN
• Application Number:
  US10/985,426
• Publication Number:
  US 20050256069A1
• Time in Patent Office: 2,023 Days
• Field of Search: None
• US Class Current: 536/24.5
• 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 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/323 modified ring structure
• CPC Class Codes: C12N 2310/3511 intercalating or cleaving agent
• CPC Class Codes: C12N 2320/51 modulating the chemical stability, e.g. nuclease-resistance

Abstract

The invention relates to iRNA agents, which preferably include a monomer in which the ribose moiety has been replaced by a moiety other than ribose that further includes a tether having one or more linking groups, in which at least one of the linking groups is a cleavable linking group. The tether in turn can be connected to a selected moiety, e.g., a ligand, e.g., a targeting or delivery moiety, or a moiety which alters a physical property. The cleavable linking group is one which is sufficiently stable outside the cell such that it allows targeting of a therapeutically beneficial amount of an iRNA agent (e.g., a single stranded or double stranded iRNA agent), coupled by way of the cleavable linking group to a targeting agent—to targets cells, but which upon entry into a target cell is cleaved to release the iRNA agent from the targeting agent.

Claims

1. An iRNA agent comprising a first strand and optionally a second strand, wherein at least one subunit having a formula (I) is incorporated into at least one of the strands:

2. The agent of claim 1, wherein at least one of the linking groups is cleaved at least about 100 times faster under intracellular conditions than under extracellular conditions.

3. The agent of claim 1, wherein at least one of the linking groups is cleaved at least about 50 times faster under intracellular conditions than under extracellular conditions.

4. The agent of claim 1, wherein at least one of the linking groups is cleaved at least about 10 times faster under intracellular conditions than under extracellular conditions.

5. The agent of claim 1, wherein R¹ is CH₂OR^b and R⁹ is OR^a or OH.

6. The agent of claim 5, wherein R¹ and R⁹ are cis.

7. The agent of claim 5, wherein R¹ and R⁹ are trans.

8. The agent of claim 1, wherein R¹ is OR^a and R⁹ is CH₂OR^b.

9. The agent of claim 8, wherein R¹ and R⁹ are cis.

10. The agent of claim 8, wherein R¹ and R⁹ are trans.

11. The agent of claim 1, wherein R¹ is CH₂OR^b and R⁹ is OR^b.

12. The agent of claim 1, wherein R¹ is CH₂OR^a and R⁹ is OR^b.

13. The agent of claim 1, wherein R¹ is OR^b and R⁹ is CH₂OR^b.

14. The agent of claim 1, wherein R¹ is OR^b and R⁹ is CH₂OR^a.

15. The agent of claim 1, wherein the R^d steroid radical is a cholesterol radical.

16. The agent of claim 1, wherein the iRNA agent is a single-stranded iRNA agent.

17. The agent of claim 16, wherein the single-stranded iRNA agent is an siRNA agent.

18. The agent of claim 17, wherein R^d is a cholesterol radical.

19. The agent of claim 16, wherein the iRNA agent is less than 30 nucleotides in length.

20. The agent of claim 19, wherein the iRNA agent is 21-23 nucleotides in length.

21. The agent of claim 1, wherein the iRNA agent is a double-stranded iRNA agent comprising a first strand and a second strand.

22. The agent of claim 21, wherein the double-stranded iRNA agent is an siRNA agent.

23. The agent of claim 22, wherein R^d is a cholesterol radical.

24. The agent of claim 21, wherein the iRNA agent is less than 30 nucleotides in length.

25. The agent of claim 24, wherein the iRNA agent is 21-23 nucleotides in length.

26. The agent of claim 21, wherein the at least one subunit of formula (I) is incorporated into both strands.

27. The agent of claim 1, wherein one of R³ or R⁹ is OH.

28. The agent of claim 1, wherein X is N(CO)R⁷.

29. The agent of claim 1, wherein X is NR⁷.

30. The agent of claim 1, wherein R^d is uvaol.

31. The agent of claim 1, wherein R^d is hecigenin.

32. The agent of claim 1, wherein R^d is diosgenin.

33. The agent of claim 1, wherein R^d is cholic acid.

34. The agent of claim 1, wherein R^d is bile acid.

35. The agent of claim 1, wherein R^d is dihydrotestosterone.

36. The agent of claim 1, wherein R^d is a cholesteryl residue.

37. The agent of claim 1, wherein R^d is O3-(oleoyl)lithocholic acid radical.

38. The agent of claim 1, wherein R^d is an O3-(oleoyl) cholenic acid radical.

39. The agent of claim 1, wherein A is O in each occurrence.

40. The agent of claim 1, wherein A is S in at least one occurrence.

41. The agent of claim 5, wherein R¹ is CH₂OR^b and R⁹ is OH.