Compositions and methods for inhibiting expression of a target gene

• US 20040175703A1
• Filed: 03/07/2003
• Published: 09/09/2004
• Est. Priority Date: 11/24/1999
• Status: Active Grant

• Current Assignee: Alnylam Pharmaceuticals Incorporated
• Original Assignee: Ribopharma AG (Alnylam Pharmaceuticals Incorporated)
• Inventors: Limmer, Stefan, Hadwiger, Philipp, Kreutzer, Roland, Limmer, Sylvia
• Granted Patent: US 7,829,693 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 33/06 Antimalarials
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/04 specific for metastasis
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 5/00 Drugs for disorders of the endocrine system
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/1131 against viruses
• CPC Class Codes: C12N 15/1138 against receptors or cell surface proteins
• 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 a double-stranded ribonucleic acid (dsRNA) having a nucleotide sequence which is substantially identical to at least a part of a target gene and which is no more than 49, preferably less than 25, nucleotides in length, and which comprises a complementary (antisense) RNA strand having a 1 to 4 nucleotide overhang at the 3′-end and a blunt 5′-end. The invention further relates to a pharmaceutical composition comprising the dsRNA and 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, at 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, as well as methods for treating diseases caused by the expression of the gene.

Claims

1. A double-stranded ribonucleic acid (dsRNA), comprising a complementary RNA strand and a sense RNA strand, wherein the sense RNA strand comprises a nucleotide sequence which is substantially identical to at least a part of a target gene, wherein the complementary RNA strand comprises a complementary nucleotide sequence which is complementary to an mRNA transcript of a portion of the target gene, wherein the complementary RNA strand comprises a 3′

-end and a 5′

-end, wherein the 3′

-end has a nucleotide overhang of 1 to 4 nucleotides and wherein the 5′

-end is blunt, and wherein the dsRNA is no more than 49 nucleotides in length.

2. The dsRNA of claim 1, wherein the dsRNA is less than 25 nucleotides in length.

3. The dsRNA of claim 1, wherein the dsRNA is 19 to 23 nucleotides in length.

4. The dsRNA of claim 1, wherein the nucleotide overhang is 1 or 2 nucleotides in length.

5. The dsRNA of claim 1, wherein the nucleotides of the nucleotide overhang are replaced with nucleoside thiophosphates.

6. The dsRNA of claim 1, further comprising a linker between the complementary RNA strand and the sense RNA strand.

7. The dsRNA of claim 6, wherein the linker is a chemical linker.

8. The dsRNA of claim 7, wherein the chemical linker is selected from the group consisting of a hexaethylene glycol linker, apoly-(oxyphosphinico-oxy-1,3-propandiol) linker, and an oligoethyleneglycol linker.

9. The dsRNA of claim 7, wherein the chemical linker is a hexaethylene glycol linker.

10. The dsRNA of claim 6, wherein the linker is between the 5′

-end of the complementary RNA strand and the 3′

-end of the sense RNA strand.

11. The dsRNA of claim 1, wherein the target gene is selected from the group of genes consisting of an oncogene, a cytokine gene, an idiotype protein gene, a prion gene, a gene that encodes a protein that induces angiogenesis, a gene that encodes an adhesion protein, a gene that encodes a cell surface receptor, a gene that encodes a protein involved in a metastasizing and/or invasive process, a gene that encodes a proteinase, a gene that encodes a protein that regulates apoptosis, a gene that encodes a EGF receptor, or a MDR1 gene.

12. The dsRNA of claim 1, wherein the target gene is an MDR1 gene.

13. The dsRNA of claim 1, wherein the target gene is a gene of a human papilloma virus, a hepatitis C virus, or a human immunodeficiency virus.

14. The dsRNA of claim 1, where the target gene comprises a sequence of SEQ ID NO:

1-140.

40. The pharmaceutical composition of claim 3, wherein the linker is between the 5′

-end of the complementary RNA strand and the 3′

-end of the sense RNA strand.

15. A method of inhibiting the expression of a target gene in a cell, the method comprising:

(a) introducing into the cell a double-stranded ribonucleic acid (dsRNA), wherein the dsRNA comprises a complementary RNA strand and a sense RNA strand, wherein the sense RNA strand comprises a nucleotide sequence which is substantially identical to at least a part of the target gene, wherein the complementary RNA strand comprises a complementary nucleotide sequence which is complementary to an mRNA transcript of a portion of the target gene, wherein the complementary RNA strand comprises a 3′

-end and a 5′

-end, wherein the 3′

-end has a nucleotide overhang of 1 to 4 nucleotides and wherein the 5′

-end is blunt, and wherein the dsRNA is no more than 49 nucleotides in length; and

(b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the target gene, thereby inhibiting expression of the target gene in the cell.

16. The method of claim 15, wherein the dsRNA is less than 25 nucleotides in length.

17. The method of claim 15, wherein the dsRNA is 19 to 23 nucleotides in length.

18. The method of claim 15, wherein the nucleotide overhang is 1 or 2 nucleotides in length.

19. The method of claim 15, wherein the nucleotides of the nucleotide overhang are replaced with nucleoside thiophosphates.

20. The method of claim 15, further comprising a linker between the complementary RNA strand and the sense RNA strand.

21. The method of claim 20, wherein the linker is a chemical linker.

22. The method of claim 21, wherein the chemical linker is selected from the group consisting of a hexaethylene glycol linker, apoly-(oxyphosphinico-oxy-1,3-propandiol) linker, and an oligoethyleneglycol linker.

23. The method of claim 21, wherein the chemical linker is a hexaethylene glycol linker.

24. The method of claim 20, wherein the linker is between the 5′

-end of the complementary RNA strand and the 3′

-end of the sense RNA strand.

25. The method of claim 15, wherein the target gene is selected from the group of genes consisting of an oncogene, a cytokine gene, an idiotype protein gene, a prion gene, a gene that encodes a protein that induces angiogenesis, a gene that encodes an adhesion protein, a gene that encodes a cell surface receptor, a gene that encodes a protein involved in a metastasizing and/or invasive process, a gene that encodes a proteinase, a gene that encodes a protein that regulates apoptosis, a gene that encodes a EGF receptor, or a MDR1 gene.

26. The method of claim 15, wherein the target gene is an MDR1 gene.

27. The method of claim 15, wherein the target gene is a gene of a human papilloma virus, a hepatitis C virus, or a human immunodeficiency virus.

28. The method of claim 15, where the target gene comprises a sequence of SEQ ID NO:

1-140.

29. A pharmaceutical composition for inhibiting the expression of a target gene in a mammal, comprising:

(a) a double-stranded ribonucleic acid (dsRNA), wherein the dsRNA comprises a complementary RNA strand and a sense RNA strand, wherein the sense RNA strand comprises a nucleotide sequence which is substantially identical to at least a part of the target gene, wherein the complementary RNA strand comprises a complementary nucleotide sequence which is complementary to an mRNA transcript of a portion of the target gene, wherein the complementary RNA strand comprises a 3′

-end and a 5′

-end, wherein the 3′

-end has a nucleotide overhang of 1 to 4 nucleotides and wherein the 5′

-end is blunt, and wherein the dsRNA is no more than 49 nucleotides in length; and

(b) a pharmaceutically acceptable carrier.

30. The pharmaceutical composition of claim 29, wherein the dosage unit of dsRNA is in a range of 0.01 to 5.0 milligrams (mg), 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 of the mammal.

31. The pharmaceutical composition of claim 29, wherein the dosage unit of dsRNA is less than 25 micrograms per kilogram body weight of the mammal.

32. The pharmaceutical composition of claim 29, wherein the dsRNA is less than 25 nucleotides in length.

33. The pharmaceutical composition of claim 29, wherein the dsRNA is 19 to 23 nucleotides in length.

34. The pharmaceutical composition of claim 29, wherein the nucleotide overhang is 1 or 2 nucleotides in length.

35. The pharmaceutical composition of claim 29, wherein the nucleotides of the nucleotide overhang are replaced with nucleoside thiophosphates.

36. The pharmaceutical composition of claim 29, further comprising a linker between the complementary RNA strand and the sense RNA strand.

37. The pharmaceutical composition of claim 36, wherein the linker is a chemical linker.

38. The pharmaceutical composition of claim 37, wherein the chemical linker is selected from the group consisting of a hexaethylene glycol linker, apoly-(oxyphosphinico-oxy-1,3-propandiol) linker, and an oligoethyleneglycol linker.

39. The pharmaceutical composition of claim 37, wherein the chemical linker is a hexaethylene glycol linker.

41. The pharmaceutical composition claim 29, wherein the target gene is selected from the group of genes consisting of an oncogene, a cytokine gene, an idiotype protein gene, a prion gene, a gene that encodes a protein that induces angiogenesis, a gene that encodes an adhesion protein, a gene that encodes a cell surface receptor, a gene that encodes a protein involved in a metastasizing and/or invasive process, a gene that encodes a proteinase, a gene that encodes a protein that regulates apoptosis, a gene that encodes a EGF receptor, or a MDR1 gene.

42. The pharmaceutical composition of claim 29, wherein the target gene is an MDR1 gene.

43. The pharmaceutical composition of claim 29, wherein the target gene is a gene of a human papilloma virus, a hepatitis C virus, or a human immunodeficiency virus.

44. The pharmaceutical composition of claim 29, where the target gene comprises a sequence of SEQ ID NO:

1-140.

45. The pharmaceutical composition of claim 29, wherein the pharmaceutically acceptable carrier is an aqueous solution.

46. The pharmaceutical composition of claim 45, wherein the aqueous solution is phosphate buffered saline.

47. The pharmaceutical composition 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.

48. The pharmaceutical composition of claim 47, wherein the polymeric nanocapsule and polymeric microcapsule comprise polybutylcyanoacrylate.

49. The pharmaceutical composition of claim 29, which is formulated to be administered by inhalation, infusion, injection, or orally.

50. The pharmaceutical composition of claim 29, which is formulated to be administered by intravenous or intraperitoneal injection.

51. 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 complementary RNA strand and a sense RNA strand, wherein the sense RNA strand comprises a nucleotide sequence which is substantially identical to at least a part of the target gene, wherein the complementary RNA strand comprises a complementary nucleotide sequence which is complementary to an mRNA transcript of a portion of the target gene, wherein the complementary RNA strand comprises a 3′

-end and a 5′

-end, wherein the 3′

-end has a nucleotide overhang of 1 to 4 nucleotides and wherein the 5′

-end is blunt, and wherein the dsRNA is no more than 49 nucleotides in length.

52. The method of claim 51, wherein the dosage unit of dsRNA is in a range of 0.01 to 5.0 milligrams (mg), 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 of the mammal.

53. The method of claim 51, wherein the dosage unit of dsRNA is less than 25 micrograms per kilogram body weight of the mammal.

54. The method of claim 51, wherein the dsRNA is less than 25 nucleotides in length.

55. The method of claim 51, wherein the dsRNA is 19 to 23 nucleotides in length.

56. The method of claim 51, wherein the nucleotide overhang is 1 or 2 nucleotides in length.

57. The method of claim 51, wherein the nucleotides of the nucleotide overhang are replaced with nucleoside thiophosphates.

58. The method of claim 51, further comprising a linker between the complementary RNA strand and the sense RNA strand.

59. The method of claim 58, wherein the linker is a chemical linker.

60. The method of claim 59, wherein the chemical linker is selected from the group consisting of a hexaethylene glycol linker, apoly-(oxyphosphinico-oxy-1,3-propandiol) linker, and an oligoethyleneglycol linker.

61. The method of claim 59, wherein the chemical linker is a hexaethylene glycol linker.

62. The method of claim 58, wherein the linker is between the 5′

-end of the complementary RNA strand and the 3′

-end of the sense RNA strand.

63. The method of claim 51, wherein the target gene is selected from the group of genes consisting of an oncogene, a cytokine gene, an idiotype protein gene, a prion gene, a gene that encodes a protein that induces angiogenesis, a gene that encodes an adhesion protein, a gene that encodes a cell surface receptor, a gene that encodes a protein involved in a metastasizing and/or invasive process, a gene that encodes a proteinase, a gene that encodes a protein that regulates apoptosis, a gene that encodes a EGF receptor, or a MDR1 gene.

64. The method of claim 51, wherein the target gene is an MDR 1 gene.

65. The method of claim 51, wherein the target gene is a gene of a human papilloma virus, a hepatitis C virus, or a human immunodeficiency virus.

66. The method of claim 51, where the target gene comprises a sequence of SEQ ID NO:

1-140.

67. The method of claim 51, wherein the pharmaceutically acceptable carrier is an aqueous solution.

68. The method of claim 67, wherein the aqueous solution is phosphate buffered saline.

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

70. The method of claim 69, wherein the polymeric nanocapsule and polymeric microcapsule comprise polybutylcyanoacrylate.

71. The method of claim 51, which is formulated to be administered by inhalation, infusion, injection, or orally.

72. The method of claim 51, which is formulated to be administered by intravenous or intraperitoneal injection.