Lipid encapsulated interfering RNA

• US 7,982,027 B2
• Filed: 06/27/2006
• Issued: 07/19/2011
• Est. Priority Date: 07/16/2003
• Status: Active Grant

• Current Assignee: Arbutus Biopharma Corporation
• Original Assignee: Protiva Biotherapeutics Inc (Arbutus Biopharma Corporation)
• Inventors: Heyes, James, MacLachlan, Ian, Ambegia, Ellen Grace
• Primary Examiner(s): Whiteman; Brian
• Application Number:
  US11/426,907
• Publication Number:
  US 20060240093A1
• Time in Patent Office: 1,848 Days
• Field of Search: 536/24.5, 435/458
• US Class Current: 536/24.5
• CPC Class Codes: A61K 9/1272 with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids with cholesterol as the only non-phosphatidyl surfactant A61K9/127; lipids as modifying agent A61K47/543
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 3/06 Antihyperlipidemics
• CPC Class Codes: A61P 31/14 for RNA viruses
• CPC Class Codes: A61P 31/20 for DNA viruses

Abstract

The present invention provides compositions and methods for silencing gene expression by delivering nucleic acid-lipid particles comprising a siRNA molecule to a cell.

Claims

1. A nucleic acid-lipid particle, said nucleic acid-lipid particle comprising:

a chemically synthesized siRNA;

a cationic lipid;

a non-cationic lipid; and

a conjugated lipid that inhibits aggregation of particles, wherein the conjugated lipid that inhibits aggregation of particles comprises a polyethyleneglycol-dialkyloxypropyl (PEG-DAA) conjugate having the following structure;

2. The nucleic acid-lipid particle in accordance with claim 1, wherein said siRNA component of said nucleic acid-lipid particle is resistant in aqueous solution to degradation by a nuclease.

3. The nucleic acid-lipid particle in accordance with claim 1, wherein said particle has a median diameter of less than about 150 nm.

4. The nucleic acid-lipid particle in accordance with claim 1, wherein said siRNA comprises about 15 to about 60 nucleotides.

5. The nucleic acid-lipid particle in accordance with claim 1, wherein said cationic lipid is a member selected from the group consisting of N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), and combinations thereof.

6. The nucleic acid-lipid particle in accordance with claim 1, wherein said non-cationic lipid is a member selected from the group consisting of dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), cholesterol, and combinations thereof.

7. The nucleic acid-lipid particle in accordance with claim 1, wherein said cationic lipid comprises from about 2% to about 60% of the total lipid present in said particle.

8. The nucleic acid-lipid particle in accordance with claim 1, wherein said cationic lipid comprises from about 5% to about 45% of the total lipid present in said particle.

9. The nucleic acid-lipid particle in accordance with claim 1, wherein said cationic lipid comprises from about 40% to about 50% of the total lipid present in said particle.

10. The nucleic acid-lipid particle in accordance with claim 1, wherein said non-cationic lipid comprises from about 5% to about 90% of the total lipid present in said particle.

11. The nucleic acid-lipid particle in accordance with claim 1, wherein said non-cationic lipid comprises from about 20% to about 85% of the total lipid present in said particle.

12. The nucleic acid-lipid particle in accordance with claim 1, wherein said conjugated lipid that inhibits aggregation of particles comprises from about 0.5% to about 50% of the total lipid present in said particle.

13. The nucleic acid-lipid particle in accordance with claim 1, wherein said conjugated lipid that inhibits aggregation of particles comprises from about 0.5% to about 25% of the total lipid present in said particle.

14. The nucleic acid-lipid particle in accordance with claim 1, wherein said conjugated lipid that inhibits aggregation of particles comprises from about 1% to about 20% of the total lipid present in said particle.

15. The nucleic acid-lipid particle in accordance with claim 1, wherein said conjugated lipid that inhibits aggregation of particles comprises from about 3% to about 15% of the total lipid present in said particle.

16. The nucleic acid-lipid particle in accordance with claim 1, wherein said conjugated lipid that inhibits aggregation of particles comprises from about 4% to about 10% of the total lipid present in said particle.

17. The nucleic acid-lipid particle in accordance with claim 1, wherein said non-cationic lipid comprises dipalmitoylphosphatidylcholine (DPPC).

18. The nucleic acid-lipid particle in accordance with claim 1, wherein said nucleic acid-lipid particle further comprises cholesterol.

19. The nucleic acid-lipid particle in accordance with claim 18, wherein the cholesterol comprises from about 10% to about 60% of the total lipid present in said particle.

20. The nucleic acid-lipid particle in accordance with claim 18, wherein the cholesterol comprises from about 20% to about 45% of the total lipid present in said particle.

21. The nucleic acid-lipid particle in accordance with claim 1, wherein said particle has an siRNA:

lipid ratio (mg;

mg) of from about 0.01 to about 0.08.

22. The nucleic acid-lipid particle in accordance with claim 1, wherein said particle has an siRNA:

lipid ratio (mg;

mg) of about 0.04.

23. The nucleic acid-lipid particle in accordance with claim 1, wherein said particle has a median diameter of less than about 100 nm.

24. The nucleic acid-lipid particle in accordance with claim 1, wherein said non-cationic lipid comprises a phospholipid and cholesterol, and wherein R¹ and R² are both myristyl (C14).

25. The nucleic acid-lipid particle in accordance with claim 1, wherein said siRNA is about 15 to about 30 base pairs in length.

26. The nucleic acid-lipid particle in accordance with claim 1, wherein said siRNA is about 19 to about 25 base pairs in length.

27. The nucleic acid-lipid particle in accordance with claim 1, wherein said siRNA comprises 3′

overhangs.

28. The nucleic acid-lipid particle in accordance with claim 1, wherein said siRNA comprises 2′

-O-methyl ribonucleotides.

29. A pharmaceutical composition comprising a nucleic acid-lipid particle in accordance with claim 1 and a pharmaceutically acceptable carrier.

30. The nucleic acid-lipid particle in accordance with claim 1, wherein R¹ and R² are selected from the group consisting of lauryl (C12), myristyl (C14), palmityl (C16), stearyl (C18), and icosyl (C20).

31. The nucleic acid-lipid particle in accordance with claim 1, wherein R¹ and R² are the same.

32. The nucleic acid-lipid particle in accordance with claim 31, wherein R¹ and R² are both myristyl (C14).

33. The nucleic acid-lipid particle in accordance with claim 31, wherein R¹ and R² are both stearyl (C18).

34. The nucleic acid-lipid particle in accordance with claim 1, wherein said alkyl groups are saturated.

35. The nucleic acid-lipid particle in accordance with claim 1, wherein said alkyl groups are unsaturated.

36. The nucleic acid-lipid particle in accordance with claim 1, wherein said PEG has an average molecular weight of from about 550 daltons to about 8,500 daltons.

37. The nucleic acid-lipid particle in accordance with claim 1, wherein said PEG has an average molecular weight of from about 1,000 daltons to about 5,000 daltons.

38. The nucleic acid-lipid particle in accordance with claim 1, wherein said PEG has an average molecular weight of from about 1,000 daltons to about 3,000 daltons.

39. The nucleic acid-lipid particle in accordance with claim 1, wherein said PEG has an average molecular weight of about 2,000 daltons.

40. A method of introducing an siRNA into a cell, said method comprising contacting said cell with a nucleic acid-lipid particle in accordance with claim 1.

41. The method in accordance with claim 40, wherein the presence of said nucleic acid-lipid particle in said cell is detectable at least 24 hours after administration of said particle.

42. The method in accordance with claim 40, wherein the presence of said nucleic acid-lipid particle in said cell is detectable at least 48 hours after administration of said particle.

43. The method in accordance with claim 40, wherein said cell is in a mammal.

44. The method in accordance with claim 43, wherein more than 10% of said particles are present in the plasma of said mammal 24 hours after administration.

45. The method in accordance with claim 43, wherein the presence of said siRNA at a site distal to the site of administration is detectable for at least 48 hours after administration of said particle.

46. The method in accordance with claim 43, wherein the presence of said siRNA at a site distal to the site of administration is detectable for at least 24 hours after administration of said particle.

47. The method in accordance with claim 43, wherein the mammal is a human.

48. A method for silencing the expression of a target sequence, said method comprising administering to a mammalian subject a therapeutically effective amount of a nucleic acid-lipid particle in accordance with claim 1.

49. The method in accordance with claim 48, wherein said administration is intravenous.

50. The method in accordance with claim 48, wherein said mammalian subject is a human.

51. The method in accordance with claim 50, wherein said human has a disease or disorder associated with expression or overexpression of a gene comprising a target sequence for said siRNA.

52. The method in accordance with claim 51, wherein said gene is selected from the group consisting of a gene associated with viral infection and survival, a gene associated with a metabolic disease or disorder, and a gene associated with tumorigenesis and cell transformation.

53. A method for the in vivo delivery of an siRNA, said method comprising administering to a mammalian subject a nucleic acid-lipid particle in accordance with claim 1.

54. The method in accordance with claim 53, wherein said administration is intravenous.

55. The method in accordance with claim 53, wherein said mammalian subject is a human.

56. The method in accordance with claim 55, wherein said human has a disease or disorder associated with expression or overexpression of a gene comprising a target sequence for said siRNA.

57. The method in accordance with claim 56, wherein said gene is selected from the group consisting of a gene associated with viral infection and survival, a gene associated with a metabolic disease or disorder, and a gene associated with tumorigenesis and cell transformation.

58. A method for the in vivo delivery of an siRNA to a liver cell, said method comprising administering to a mammalian subject a nucleic acid-lipid particle in accordance with claim 1.

59. The method in accordance with claim 58, wherein said administration is intravenous.

60. The method in accordance with claim 58, wherein said mammalian subject is a human.

61. The method in accordance with claim 60, wherein said human has a disease or disorder associated with expression or overexpression of a gene comprising a target sequence for said siRNA.

62. The method in accordance with claim 61, wherein said gene is associated with a metabolic disease or disorder.

63. The method in accordance with claim 62, wherein said metabolic disease or disorder is a dyslipidemia.