Modified iRNA agents

• US 20050164235A1
• Filed: 09/21/2004
• Published: 07/28/2005
• Est. Priority Date: 04/17/2003
• Status: Abandoned Application

• Current Assignee: Alnylam Pharmaceuticals Incorporated
• Original Assignee: Alnylam Pharmaceuticals Incorporated
• Inventors: Manoharan, Muthiah, Rajeev, Kallanthottathil G.
• Application Number:
  US10/946,873
• Publication Number:
  US 20050164235A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A61K 47/55 the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
• CPC Class Codes: A61K 47/554 the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
• CPC Class Codes: A61P 31/14 for RNA viruses
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• 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 2310/11 Antisense
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/323 modified ring structure
• CPC Class Codes: C12N 2310/344 Position-specific modifications, e.g. on every purine, at the 3'-end
• CPC Class Codes: C12N 2310/3515 Lipophilic moiety, e.g. cholesterol
• CPC Class Codes: C12N 2320/32 Special delivery means, e.g. tissue-specific
• CPC Class Codes: C12N 2320/51 modulating the chemical stability, e.g. nuclease-resistance
• CPC Class Codes: C12N 2330/30 chemically synthesised

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The invention relates to iRNA agents, which preferably include a monomer in which the ribose moiety has been replaced by a moiety other than ribose. The inclusion of such a monomer can allow for modulation of a property of the iRNA agent into which it is incorporated, e.g., by using the non-ribose moiety as a point to which a ligand or other entity, e.g., a carbohydrate; or a steroid, e.g., cholesterol, which is optionally substituted with at least one carbohydrate. is directly, or indirectly, tethered. The invention also relates to methods of making and using such modified iRNA agents.

Claims

1. An iRNA agent comprising a first strand and a second strand, wherein at 2 least one subunit having a formula (I) is incorporated into at least one of said strands:

2. The compound of claim 1, wherein X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰, and Z is absent.

3. The compound of claim 2, wherein R¹ is (CH₂)_nOR^b and R³ is OR^a.

4. The compound of claim 3, wherein R¹ and R³ are cis.

5. The compound of claim 3, wherein R¹ and R³ are trans.

6. The compound of claim 3, wherein n is 1.

7. The compound of claims 3, A is 0.

8. The compound of claim 3, wherein A is S.

9. The compound of claim 2, wherein R¹ is (CH₂)_nOR^b and R³ is OR^b.

10. The compound of claim 2, wherein R¹ is (CH₂)_nOR^a and R³ is OR^b.

11. The compound of claim 3, wherein R⁷ is (CH₂)₅NHR^d or (CH₂)₅NHC(O)R^d.

12. The compound of claim 3, wherein Rd is chosen from the group of a galactose radical, an N-acetylgalactose radical, or a mannose radical.

13. The compound of claim 2, wherein R¹ is OR^b and R³ is (CH₂)_nOR^b.

14. The compound of claim 2, wherein R¹ is OR^b and R³ is (CH₂)_nOR^b.

15. The compound of claim 2, wherein R¹ is OR^a and R³ is (CH₂)OR^b.

16. The compound of claim 15, wherein R¹ and R³ are cis.

17. The compound of claim 15, wherein R¹ and R³ are trans.

18. The compound of claim 15, wherein n is 1.

19. The compound of claim 2, wherein R¹ is (CH₂)_nOR^b and R⁹ is OR^a.

20. The compound of claim 19, wherein R¹ and R⁹ are cis.

21. The compound of claim 19, wherein R¹ and R⁹ are trans.

22. The compound of claim 19, wherein n is 1.

23. The compound of claim 2, wherein R¹ is OR^a and R⁹ is (CH₂)_nOR^b.

24. The compound of claim 23, wherein R¹ and R⁹ are cis.

25. The compound of claim 23, wherein R¹ and R⁹ are trans.

26. The compound of claim 23, wherein n is 1.

27. The compound of claim 2, wherein R¹ is (CH₂)_nOR^b and R⁹ is OR^b.

28. The compound of claim 2, wherein R¹ is (CH₂)_nOR^a and R⁹ is OR^b.

29. The compound of claim 2, wherein R¹ is OR^b and R⁹ is (CH₂)_nOR^b.

30. The compound of claim 2, wherein R¹ is OR^b and R⁹ is (CH₂)_nOR^a.

31. The compound of claim 2, wherein R³ is (CH₂)_nOR^b and R⁹ is OR^a.

32. The compound of claim 2, wherein R³ is (CH₂)_nOR^b and R⁹ is OR^b.

33. The compound of claim 2, wherein R³ is (CH₂)_nOR^a and R⁹ is OR^b.

34. The compound of claim 2, wherein R³ is OR^a and R⁹ is (CH₂)_nOR^b.

35. The compound of claim 2, wherein R³ is OR^b and R⁹ is (CH₂)_nOR^b.

36. The compound of claim 2, wherein R³ is OR^b and R⁹ is (CH₂)_nOR^a.

37. The compound of claim 1, wherein one of R¹, R², R³, R⁴, R⁹, and R¹⁰ is OR^a and one of R¹, R², R³, R⁴, R⁹, and R¹⁰ is (CH₂)_nOR^b.

38. The compound of claim 1, wherein one of R¹, R², R³, R⁴, R⁹, and R¹⁰ is (CH₂)_nOR^a and one of R¹, R², R³, R⁴, R⁹, and R¹⁰ is OR^b.

39. The compound of claim 1, wherein one of R¹, R², R³, R⁴, R⁹, and R¹⁰ is OR^b and one of R¹, R², R³, R⁴, R⁹, and R¹⁰ is (CH₂)_nOR^b.

60. A method of modulating expression of a target gene in a subject, the method comprising:

administering an iRNA agent of claim 1 to a subject.

61. A pharmaceutical composition comprising an iRNA agent of claim 1 and a pharmaceutically acceptable carrier.

62. A kit comprising an iRNA agent of claim 1, a sterile container in which the iRNA agent is disclosed, and instructions for use.

63. A method of treating a human having or at risk for developing a disorder of the liver, the method comprising administering an iRNA agent of claim 1 or 40.

64. The method of claim 63, wherein the disorder of the liver is infection with a hepatitis virus.

65. The method of claim 64, wherein the virus is HCV.

66. The method of claim 64, wherein the virus is HBV.

67. The iRNA agent of claim 1, wherein the iRNA agent comprises a first subunit in which R^d is a carbohydrate radical, and a second subunit in which R^d is a steroid radical, which is optionally tethered to at least one carbohydrate radical.

68. The iRNA agent of claim 67, wherein the first and second subunits are incorporated into the same strand.

69. The iRNA agent of claim 67, wherein the first and second subunits are incorporated into different strands.

70. The iRNA of claim 67, wherein R^d in the first subunit is a galactose radical.

71. The iRNA agent of claim 67, wherein R^d in the first subunit is an N-acetylgalactoseamine radical.

72. The iRNA agent of claim 67, wherein R^d in the first subunit is mannose radical.

73. The iRNA agent of claim 67, wherein R^d in the second subunit is a steroid radical.

74. The iRNA agent of claim 73, wherein R^d in the second subunit is a cholesterol radical.

75. The iRNA agent of claim 73, wherein R^d in the second subunit is a cholanic acid radical.

76. The iRNA agent of claim 1, wherein the iRNA agent comprises a first subunit and a second subunit in which R^d is a carbohydrate radical in both the first and second subunits.

77. The iRNA agent of claim 76, wherein R^d is the same carbohydrate radical in the first and second subunit.

78. The iRNA agent of claim 76, wherein the carbohydrate radical in the first subunit is different from the carbohydrate radical in the second subunit.

40. An iRNA agent comprising a first strand and a second strand, wherein at least one subunit is derivatized with a moiety comprising a carbohydrate radical or a steroid radical, the steroid radical being optionally tethered to at least one carbohydrate radical, which enhances entrance into a cell.

41. The compound of claim 40, wherein the subunit is derivatized with a galactose radical, an N-acetylgalactose radical, or a mannose radical.

42. The compound of claim 40, wherein the subunit is derivatized with a steroid tethered to at least one carbohydrate radical.

43. The iRNA agent of claim 40, comprising at least one one subunit having a formula (I) is incorporated into at least one of said strands:

44. The compound of claim 43, wherein R^d is chosen from a galactose radical, an N-acetylgalactose radical, or a mannose radical.

45. The compound of claim 42, wherein R^d is steroid tethered to at least one carbohydrate radical.

46. The compound of claim 43, wherein X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰, and Z is absent.

47. The compound of claim 46, wherein R¹ is (CH₂)_nOR^b and R³ is OR^a.

48. The iRNA agent of claim 40, wherein the iRNA is 21 nucleotides in length and there is a duplex region of about 19 pairs.

49. The iRNA agent of claim 40, wherein the iRNA agent includes a duplex region between 17 and 23 pairs in length.

50. The compound of claim 40, wherein the first or second strand is a sense strand, and the moiety is attached to the sense strand.

51. The compound of claim 50, wherein the moiety is attached to a 3′

terminal subunit.

52. The compound of claim 50, wherein the moiety is attached to a 5′

terminal subunit.

53. The compound of claim 50, wherein the moiety is attached an internal subunit.

54. The iRNA agent of claim 40, wherein the moiety enhances entrance into a primary cell.

55. The iRNA agent of claim 40, wherein the moiety enhances entrance into a hepatocyte cell.

56. The iRNA agent of claim 40, wherein the cell comprises an exogenous gene, and the iRNA agent targets the exogenous gene.

57. The iRNA agent of claim 56, wherein the exogenous gene is a hepatitis viral gene.

58. A composition comprising an iRNA agent to which a carbohydrate radical or a steroid radical, which is optionally substiuted with at least one carbohydrate radical is conjugated, which is free of, has a reduced amount of, or is essentially free of, other reagents that facilitate or enhance delivery through the cell membrane.

59. The composition of claim 58, wherein said composition is free of, has a reduced amount of, or is essentially free of:

an additional lipophilic moiety;

a transfection agent, e.g., concentrations of an ion or other substance which substantially alters cell permeability to an iRNA agent.

79. An iRNA agent comprising a first strand and a second strand, wherein at least one subunit is derivatized with a porphyrin, which enhances entrance into a cell.

80. The iRNA agent of claim 79, wherein the porphyrin further comprises a carbohydrate radical;

or a steroid radical optionally substituted with at least one carbohydrate radical.