Composition and method for inhibiting expression of a target gene

• US 20040038921A1
• Filed: 08/11/2003
• Published: 02/26/2004
• Est. Priority Date: 10/26/2001
• Status: Abandoned Application

• Current Assignee: Alnylam Pharmaceuticals Incorporated
• Original Assignee: Ribopharma AG (Alnylam Pharmaceuticals Incorporated)
• Inventors: Limmer, Sylvia, Limmer, Stefan, Hadwiger, Philipp, Kreutzer, Roland
• Application Number:
  US10/382,634
• Publication Number:
  US 20040038921A1
• Time in Patent Office: Days
• US Class Current: 514/44
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• 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 2310/53 partially self-complementary or closed
• CPC Class Codes: C12N 2320/50 Methods for regulating/modulating their activity

Abstract

The present invention relates to pharmaceutical compositions comprising a double-stranded oligoribonucleic acid (dsRNA) having a nucleotide sequence which is substantially identical to at least a part of a target gene in a mammalian cell and which is less than 25 nucleotides in length, together with a pharmaceutically acceptable carrier. The pharmaceutical compositions are useful for inhibiting the expression of a target gene, as well as for treating diseases caused by expression of the target gene, in a mammal at very low dosages (i.e., less than 5 milligrams, preferably less than 25 micrograms, per kg body weight per day). The invention also relates to methods for inhibiting the expression of a target gene in a mammal, as well as methods for treating diseases caused by expression of the gene.

Claims

1. A pharmaceutical composition for inhibiting the expression of a target gene in a mammal, comprising a double-stranded ribonucleic acid (dsRNA) and a pharmaceutically acceptable carrier, wherein the dsRNA comprises a nucleotide sequence which is substantially identical to at least a part of the target gene and which is less than 25 nucleotides in length, and wherein the pharmaceutical composition is in a unit dosage amount of less than 5 milligram (mg) of dsRNA per kg body weight of the mammal.

2. The pharmaceutical composition of claim 1, wherein the dosage unit of dsRNA is in a range of 0.01 to 2.5 milligrams, 0.1 to 200 micrograms, 0.1 to 100 micrograms, 1.0 to 50 micrograms, or 1.0 to 25 micrograms per kilogram body weight.

3. The pharmaceutical composition of claim 1, wherein the dosage unit of dsRNA is less than 25 micrograms per kilogram body weight.

4. The pharmaceutical composition of claim 1, wherein the dsRNA further comprises a complementary RNA strand, wherein the complementary RNA strand comprises a complementary nucleotide sequence which is complementary to an mRNA transcript of a portion of the target gene, and wherein the complementary nucleotide sequence is 19 to 24 nucleotides in length, 20 to 24 nucleotides in length, or 21 to 23 nucleotides in length.

5. The pharmaceutical composition of claim 4, wherein the complementary nucleotide sequence is 22 or 23 nucleotides in length.

6. The pharmaceutical composition of claim 4, wherein the complementary RNA strand is 21 to 30 nucleotides in length, 21 to 25 nucleotides in length, or 21 to 24 nucleotides in length.

7. The pharmaceutical composition of claim 4, wherein the complementary RNA strand is 23 nucleotides in length.

8. The pharmaceutical composition of claim 1, wherein the dsRNA comprises a first complementary RNA strand and a second RNA strand, wherein the first complementary RNA strand comprises a complementary nucleotide sequence which is complementary to an RNA transcript of a portion of the target gene, and wherein the first complementary RNA strand and the second RNA strand comprise a 3′

-terminus and a 5′

-terminus, and wherein at least one of the first RNA and second RNA strands comprise a nucleotide overhang of 1 to 4 nucleotides in length.

9. The pharmaceutical composition of claim 8, wherein the nucleotide overhang is one or two nucleotides in length.

10. The pharmaceutical composition of claim 8, wherein the nucleotide overhang is on the 3′

-terminus of the first complementary RNA strand.

11. The pharmaceutical composition of claim 8, wherein the dsRNA further comprises a first end and a second end, wherein the first end comprises the 3′

-terminus of the first complementary RNA strand and the 5′

-terminus of the second RNA strand, and wherein the second end comprises the 5′

-terminus of the first complementary RNA strand and the 3′

-terminus of the second RNA strand, wherein the first end comprises a nucleotide overhang on the 3′

-terminus of the first complementary RNA strand, and wherein the second end is blunt.

12. The pharmaceutical composition of claim 11, wherein the first complementary RNA strand is 23 nucleotides in length and comprises a 2-nucleotide overhang at the 3′

-terminus, wherein the second RNA strand is 21 nucleotides in length, the wherein the second end of the dsRNA is blunt.

13. The pharmaceutical composition of claim 1, wherein the pharmaceutically acceptable carrier is an aqueous solution.

14. The pharmaceutical composition of claim 13, wherein the aqueous solution is phosphate buffered saline.

15. The pharmaceutical composition of claim 1, wherein the pharmaceutically acceptable carrier comprises a micellar structure selected from the group consisting of a liposome, capsid, capsoid, polymeric nanocapsule, and polymeric microcapsule.

16. The pharmaceutical composition of claim 15, wherein the polymeric nanocapsule and polymeric microcapsule comprise polybutylcyanoacrylate.

17. The pharmaceutical composition of claim 1, which is formulated to be administered by inhalation, infusion, injection, or orally.

18. The pharmaceutical composition of claim 1, which is formulated to be administered by intravenous or intraperitoneal injection.

19. A method for inhibiting the expression of a target gene in a mammal, which comprises administering a pharmaceutical composition comprising a double-stranded ribonucleic acid (dsRNA) and a pharmaceutically acceptable carrier, wherein the dsRNA comprises a nucleotide sequence which is substantially identical to at least a part of the target gene and which is less than 25 nucleotides in length, and wherein the pharmaceutical composition is in a unit dosage amount of less than 5 milligram (mg) of dsRNA per kg body weight of the mammal.

20. The method of claim 19, wherein the dosage unit of dsRNA is in a range of 0.01 to 2.5 milligrams, 0.1 to 200 micrograms, 0.1 to 100 micrograms, 1.0 to 50 micrograms, or 1.0 to 25 micrograms per kilogram body weight.

21. The method of claim 20, wherein the dosage unit of dsRNA is less than 25 g per kilogram body weight.

22. The method of claim 20, wherein the dsRNA further comprises a complementary RNA strand, wherein the complementary RNA strand comprises a complementary nucleotide sequence which is complementary to an mRNA transcript of a portion of the target gene, and wherein the complementary nucleotide sequence is 19 to 24 nucleotides in length, 20 to 24 nucleotides in length, or 21 to 23 nucleotides in length.

23. The method of claim 22, wherein the complementary nucleotide sequence is 22 or 23 nucleotides in length.

24. The method of claim 22, wherein the complementary RNA strand is 21 to 30 nucleotides in length, 21 to 25 nucleotides in length, or 21 to 24 nucleotides in length.

25. The method of claim 22, wherein the complementary RNA strand is 23 nucleotides in length.

26. The method of claim 19, wherein the dsRNA comprises a first complementary RNA strand and a second RNA strand, wherein the first complementary RNA strand comprises a complementary nucleotide sequence which is complementary to an RNA transcript of a portion of the target gene, and wherein the first complementary RNA strand and the second RNA strand comprise a 3′

-terminus and a 5′

-terminus, and wherein at least one of the first RNA and second RNA strands comprise a nucleotide overhang of 1 to 4 nucleotides in length.

27. The method of claim 26, wherein the nucleotide overhang is one or two nucleotides in length.

28. The method of claim 26, wherein the nucleotide overhang is on the 3′

-terminus of the first complementary RNA strand.

29. The method of claim 26, wherein the dsRNA further comprises a first end and a second end, wherein the first end comprises the 3′

-terminus of the first complementary RNA strand and the 5′

-terminus of the second RNA strand, and wherein the second end comprises the 5′

-terminus of the first complementary RNA strand and the 3′

-terminus of the second RNA strand, wherein the first end comprises a nucleotide overhang on the 3′

-terminus of the first complementary RNA strand, and wherein the second end is blunt.

30. The method of claim 29, wherein the first complementary RNA strand is 23 nucleotides in length and comprises a 2-nucleotide overhang at the 3′

-terminus, wherein the second RNA strand is 21 nucleotides in length, the wherein the second end of the dsRNA is blunt.

31. The method of claim 29, wherein the pharmaceutically acceptable carrier is an aqueous solution.

32. The method of claim 31, wherein the aqueous solution is phosphate buffered saline.

33. The method of claim 29, wherein the pharmaceutically acceptable carrier comprises a micellar structure selected from the group consisting of a liposome, capsid, capsoid, polymeric nanocapsule, and polymeric microcapsule.

34. The method of claim 33, wherein the polymeric nanocapsule and polymeric microcapsule comprise polybutylcyanoacrylate.

35. The method of claim 29, which is formulated to be administered by inhalation, infusion, injection, or orally.

36. The method of claim 29, which is formulated to be administered by intravenous or intraperitoneal injection.

37. A method for treating a disease caused by the expression of a target gene in a mammal, which comprises administering a pharmaceutical composition comprising a double-stranded ribonucleic acid (dsRNA) and a pharmaceutically acceptable carrier, wherein the dsRNA comprises a nucleotide sequence which is substantially identical to at least a part of the target gene and which is less than 25 nucleotides in length, and wherein the pharmaceutical composition is in a unit dosage amount of less than 5 milligram (mg) of dsRNA per kg body weight of the mammal.

38. The method of claim 37, wherein the unit dosage amount of dsRNA is in a range of 0.01 to 2.5 milligrams, 0.1 to 200 micrograms, 0.1 to 100 micrograms, 1.0 to 50 micrograms, or 1.0 to 25 micrograms per kilogram body weight.

39. The method of claim 37, wherein the unit dosage amount of dsRNA is less than 25 micrograms per kilogram body weight.

40. The method of claim 37, wherein the dsRNA further comprises a complementary RNA strand, wherein the complementary RNA strand comprises a complementary nucleotide sequence which is complementary to an mRNA transcript of a portion of the target gene, and wherein the complementary nucleotide sequence is 19 to 24 nucleotides in length, 20 to 24 nucleotides in length, or 21 to 23 nucleotides in length.

41. The method of claim 40, wherein the complementary nucleotide sequence is 22 or 23 nucleotides in length.

42. The method of claim 40, wherein the complementary RNA strand is 21 to 30 nucleotides in length, 21 to 25 nucleotides in length, or 21 to 24 nucleotides in length.

43. The method of claim 40, wherein the complementary RNA strand is 23 nucleotides in length.

44. The method of claim 37, wherein the dsRNA comprises a first complementary RNA strand and a second RNA strand, wherein the first complementary RNA strand comprises a complementary nucleotide sequence which is complementary to an RNA transcript of a portion of the target gene, and wherein the first complementary RNA strand and the second RNA strand comprise a 3′

-terminus and a 5′

-terminus, and wherein at least one of the first RNA and second RNA strands comprise a nucleotide overhang of 1 to 4 nucleotides in length.

45. The method of claim 44, wherein the nucleotide overhang is one or two nucleotides in length.

46. The method of claim 44, wherein the nucleotide overhang is on the 3′

-terminus of the first complementary RNA strand.

47. The method of claim 44, wherein the dsRNA further comprises a first end and a second end, wherein the first end comprises the 3′

-terminus of the first complementary RNA strand and the 5′

-terminus of the second RNA strand, and wherein the second end comprises the 5′

-terminus of the first complementary RNA strand and the 3′

-terminus of the second RNA strand, wherein the first end comprises a nucleotide overhang on the 3′

-terminus of the first complementary RNA strand, and wherein the second end is blunt.

48. The method of claim 47, wherein the first complementary RNA strand is 23 nucleotides in length and comprises a 2-nucleotide overhang at the 3′

-terminus, wherein the second RNA strand is 21 nucleotides in length, the wherein the second end of the dsRNA is blunt.

49. The method of claim 37, wherein the pharmaceutically acceptable carrier is an aqueous solution.

50. The method of claim 49, wherein the aqueous solution is phosphate buffered saline.

51. The method of claim 37, wherein the pharmaceutically acceptable carrier comprises a micellar structure selected from the group consisting of a liposome, capsid, capsoid, polymeric nanocapsule, and polymeric microcapsule.

52. The method of claim 51, wherein the polymeric nanocapsule and polymeric microcapsule comprise polybutylcyanoacrylate.

53. The method of claim 37, which is formulated to be administered by inhalation, infusion, injection, or orally.

54. The method of claim 37, which is formulated to be administered by intravenous or intraperitoneal injection.