LIPID ENCAPSULATED INTERFERING RNA

• US 20060240093A1
• Filed: 06/27/2006
• Published: 10/26/2006
• 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
• Granted Patent: US 7,982,027 B2
• Time in Patent Office: Days
• US Class Current: 424/450
• 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. An nucleic acid-lipid particle, said nucleic acid-lipid particle comprising:

a siRNA;

a cationic lipid;

a non-cationic lipid; and

a conjugated lipid that inhibits aggregation of particles.

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

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

5. The nucleic acid-lipid particle of claim 1, wherein said siRNA is transcribed from a plasmid encapsulated in said particle.

6. 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), and N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), and combinations thereof.

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

8. The nucleic acid-lipid particle in accordance with claim 1, wherein the conjugated lipid that inhibits aggregation of particles is a member selected from the group consisting of a polyethyleneglycol (PEG)-lipid conjugate, a polyamide (ATTA)-lipid conjugate, and combinations thereof.

9. The nucleic acid-lipid particle in accordance with claim 1, wherein the conjugated lipid that inhibits aggregation of particles comprises a polyethyleneglycol-lipid and the PEG-lipid is member selected from the group consisting of a PEG-diacylglycerol (DAG), a PEG dialkyloxypropyl, a PEG-phospholipid, a PEG-ceramide, and combinations thereof.

10. The nucleic acid-lipid particle in accordance with claim 9, wherein the conjugated lipid that inhibits aggregation of particles comprises a polyethyleneglycol (PEG)-dialkyloxypropyl conjugate.

11. The nucleic acid-lipid particle in accordance with claim 10, wherein the PEG-dialkyloxypropyl conjugate is a member selected from the group consisting of a PEG-dilauryloxypropyl (C₁₂), a PEG-dimyristyloxypropyl (C₁₄), a PEG-dipalmityloxypropyl (C₁₆), and a PEG-distearyloxypropyl (C₁₈), and combinations thereof.

12. A method of introducing an siRNA into a cell, said method comprising contacting said cell with a nucleic acid-lipid particle comprising

a siRNA;

a cationic lipid;

a non-cationic lipid; and

a conjugated lipid that inhibits aggregation of particles.

13. The method of claim 12, wherein said siRNA component of said nucleic acid-lipid particle is resistant in aqueous solution to degradation with a nuclease.

14. The method of claim 12, wherein said particle has a median diameter of less than about 150 nm.

15. The method of claim 12, wherein said siRNA is transcribed from a plasmid encapsulated in said particle.

16. The method of claim 12, 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), and N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), and combinations thereof.

17. The method of claim 12, wherein said non-cationic lipid is a member selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), cholesterol, and combinations thereof.

18. The method of claim 12, wherein the conjugated lipid that inhibits aggregation of particles is a member selected from the group consisting of a polyethyleneglycol (PEG)-lipid conjugate, a polyamide (ATTA)-lipid conjugate, and combinations thereof.

19. The method of claim 12, wherein the conjugated lipid that inhibits aggregation of particles comprises a polyethyleneglycol (PEG)-lipid and the PEG-lipid is member selected from the group consisting of a PEG-diacylglycerol, a PEG dialkyloxypropyl, a PEG-phospholipid, a PEG-ceramide, and combinations thereof.

20. The method of claim 12, wherein the conjugated lipid that inhibits aggregation of particles comprises a polyethyleneglycol (PEG)-dialkyloxypropyl conjugate.

21. The method of claim 20, wherein the PEG-dialkyloxypropyl conjugate is a member selected from the group consisting of a PEG-dilauryloxypropyl (C₁₂), a PEG-dimyristyloxypropyl (C₁₄), a PEG-dipalmityloxypropyl (C₁₆), and a PEG-distearyloxypropyl (C₁₈).

22. The method of claim 12, wherein the presence of said nucleic acid-lipid particle in target cells is detectable at least 48 hours after administration of said particle.

23. The method of claim 12, wherein the presence of said nucleic acid-lipid particle is detectable at least 24 hours after administration of said particle.

24. The method of claim 12, wherein said cell is in a mammal.

25. The method of claim 24, wherein more than 10% of such particles are present in plasma of said mammal 24 hours after administration.

26. The method of claim 24, wherein presence of a siRNA at a site distal to the site of administration is detectable for at least 48 hours after administration of said particle.

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

28. The method of claim 27, wherein the mammal is a human.

29. A method for silencing expression of a target sequence, said method comprising administering to a mammalian subject an effect amount of a a therapeutically effective amount of a nucleic acid-lipid particle comprising:

a siRNA;

a cationic lipid;

a non-cationic lipid; and

a conjugated lipid that inhibits aggregation of particles.

30. The method of claim 29, wherein said administration is intravenous.

31. The method of claim 29, wherein said mammal is a human.

32. The method of claim 31, wherein said human has a disease or disorder associated with expression of a gene comprising a target sequence for said siRNA.

33. The method of claim 32, wherein said disease or disorder is hepatitis.

34. A method for in vivo delivery of siRNA, said method comprising administering to a mammalian subject a nucleic acid-lipid particle comprising:

a siRNA;

a cationic lipid;

a non-cationic lipid; and

a conjugated lipid that inhibits aggregation of particles.

35. The method of claim 34, wherein said administration is intravenous.

36. The method of claim 34, wherein said mammal is a human.

37. The method of claim 36, wherein said human has a disease or disorder associated with expression of a gene comprising a target sequence for said siRNA.

38. The method of claim 37, wherein said disease or disorder is associated with overexpression of said gene.

39. The method of claim 37, wherein said disease or disorder is hepatitis.

40. A method for in vivo delivery of siRNA to a liver cell, said method comprising administering to a mammalian, subject a nucleic acid-lipid particle comprising:

a siRNA;

a cationic lipid;

a non-cationic lipid; and

a conjugated lipid that inhibits aggregation of particles.

41. The method of claim 40, wherein said mammal is a human.

42. The method of claim 41, wherein said human has a disease or disorder associated with expression of a gene comprising a target sequence for said siRNA.

43. The method of claim 42, wherein said disease or disorder is associated with overexpression of said gene.

44. The method of claim 42, wherein said disease or disorder is hepatitis.

45. The method of claim 40, wherein said administration is intravenous.

46. A method of treating a disease in a mammalian subject, said method comprising administering to said subject a therapeutically effective amount of a nucleic acid-lipid particle comprising:

a siRNA;

a cationic lipid;

a non-cationic lipid; and

a conjugated lipid that inhibits aggregation of particles,

wherein said disease is associated with expression of a gene comprising a target sequence for said siRNA.

47. The method of claim 46, wherein said mammal is a human.

48. The method of claim 46, wherein said disease is hepatitis.

49. The method of claim 48, wherein said hepatitis is Hepatitis B.

50. The method of claim 48, wherein said hepatitis is Hepatitis C.

51. The method of claim 46, wherein said administration is intravenous.