siRNA silencing of apolipoprotein B

• US 20060134189A1
• Filed: 11/17/2005
• Published: 06/22/2006
• Est. Priority Date: 11/17/2004
• Status: Abandoned Application

• Current Assignee: Protiva Biotherapeutics Inc (Arbutus Biopharma Corporation)
• Original Assignee: Protiva Biotherapeutics Inc (Arbutus Biopharma Corporation)
• Inventors: Judge, Adam, MacLachlan, Ian, Palmer, Lorne R., Sood, Vandana, Lee, Amy C.H., Jeffs, Lloyd B.
• Application Number:
  US11/283,550
• Publication Number:
  US 20060134189A1
• Time in Patent Office: Days
• US Class Current: 424/450
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61K 9/0019 Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner non-active ingredients are additionally classified in A61K47/00
• CPC Class Codes: A61K 9/127 Liposomes
• CPC Class Codes: A61P 3/06 Antihyperlipidemics
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 5/14 of the thyroid hormones, e.g. T3, T4
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• 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 15/88 using microencapsulation, e.g. using amphiphile liposome vesicle
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2320/32 Special delivery means, e.g. tissue-specific

Abstract

The present invention provides nucleic acid-lipid particles comprising siRNA molecules that silence ApoB expression and methods of using such nucleic acid-lipid particles to silence ApoB expression.

Claims

1. A nucleic acid lipid particle comprising:

an siRNA molecule that silences Apolipoprotein B (ApoB) expression;

a cationic lipid; and

a non-cationic lipid.

2. The nucleic acid-lipid particle in accordance with claim 1, wherein said nucleic acid-lipid particle comprises an siRNA molecule comprising a sequence set forth in Table 1, rows A-F of Table 2, and Tables 3-7.

3. The nucleic acid-lipid particle in accordance with claim 1, wherein said nucleic acid-particle comprises at least two siRNA molecules, wherein each siRNA molecule comprises a sequence independently selected from the sequences set forth in Table 1, rows A-F of Table 2, and Tables 3-7.

4. The nucleic acid-lipid particle in accordance with claim 1, wherein the siRNA silences ApoB expression by at least about 2 fold more than an siRNA that is not in a nucleic acid-lipid particle.

5. The nucleic acid-lipid particle in accordance with claim 1, wherein the siRNA silences ApoB expression by at least about 5 fold more than an siRNA that is not in a nucleic acid-lipid 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), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLendMA), and a mixture thereof.

7. The nucleic acid-lipid particle in accordance with claim 1, wherein said cationic lipid is DLinDMA.

8. The nucleic acid-lipid particle in accordance with claim 1, wherein said non-cationic lipid is an anionic lipid.

9. The nucleic acid-lipid particle in accordance with claim 1, wherein said non-cationic lipid is a neutral lipid.

10. 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), palmitoyloleyolphosphatidylglycerol (POPG), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, palmitoyloleoylphosphatidylethanolamine (POPE), 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, and a mixture thereof.

11. The nucleic acid-lipid particle in accordance with claim 1, wherein said non-cationic lipid is DSPC.

12. The nucleic acid-lipid particle in accordance with claim 1, further comprising a conjugated lipid that inhibits aggregation of particles

13. The nucleic acid-lipid particle in accordance with 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 a mixture thereof.

14. The nucleic acid-lipid particle in accordance with claim 13, wherein the PEG-lipid conjugate is member selected from the group consisting of:

a PEG-diacylglycerol (PEG-DAG), a PEG dialkyloxypropyl (PEG-DAA), a PEG-phospholipid, a PEG-ceramide (PEG-Cer), and a mixture thereof.

15. The nucleic acid-lipid particle in accordance with claim 13, wherein the conjugated lipid that inhibits aggregation of particles comprises a PEG-DAA conjugate.

16. The nucleic acid-lipid particle in accordance with claim 15, wherein the PEG-DAA 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).

17. The nucleic acid-lipid particle in accordance with claim 15, wherein said PEG-DAA conjugate is a PEG-dimyristyloxypropyl (C₁₄).

18. The nucleic acid-lipid particle in accordance with claim 1, wherein said cationic lipid comprises from about 5 mol % to about 15 mol % of the total lipid present in said particle.

19. The nucleic acid-lipid particle in accordance with claim 1, wherein said cationic lipid comprises from about 30 mol % to about 50 mol % of the total lipid present in said particle.

20. The nucleic acid-lipid particle in accordance with claim 1, wherein said cationic lipid comprises from about 30 mol % to about 50 mol % of the total lipid present in said particle.

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

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

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

24. The nucleic acid-lipid particle in accordance with claim 15, wherein said PEG-DAA conjugate comprises from 0.5 mol % to about 20 mol % of the total lipid present in said particle.

25. The nucleic acid-lipid particle in accordance with claim 15, wherein said PEG-DAA conjugate comprises from 2 mol % to about 15 mol % of the total lipid present in said particle.

26. The nucleic acid-lipid particle in accordance with claim 15, wherein said PEG-DAA conjugate comprises about 2 mol % of the total lipid present in said particle.

27. The nucleic acid-lipid particle in accordance with claim 1, further comprising cholesterol.

28. The nucleic acid-lipid particle in accordance with claim 27, wherein the cholesterol comprises from about 0 mol % to about 10 mol % of the total lipid present in said particle.

29. The nucleic acid-lipid particle in accordance with claim 27, wherein the cholesterol comprises from about 10 mol % to about 60 mol % of the total lipid present in said particle.

30. The nucleic acid-lipid particle in accordance with claim 27, wherein the cholesterol comprises from about 20 mol % to about 45 mol % of the total lipid present in said particle.

31. The nucleic acid-lipid particle in accordance with claim 1, wherein the nucleic acid in said nucleic acid-lipid particle is not substantially degraded after exposure of said particle to a nuclease at 37°

C. for 20 minutes.

32. The nucleic acid-lipid particle in accordance with claim 1, wherein the nucleic acid in said nucleic acid-lipid particle is not substantially degraded after incubation of said particle in serum at 37°

C. for 30 minutes.

33. The nucleic acid-lipid particle in accordance with claim 1, wherein the nucleic acid is fully encapsulated in said nucleic acid-lipid particle.

34. The nucleic acid-lipid particle in accordance with claim 1, wherein said particle has a nucleic acid:

lipid ratio (mg;

mg) of from about 0.01 to about 0.2.

35. The nucleic acid-lipid particle in accordance with claim 1, wherein said particle has a nucleic acid:

lipid ratio (mg;

mg) of from about 0.02 to about 0.1.

36. The nucleic acid-lipid particle in accordance with claim 1, wherein said particle has a nucleic acid:

lipid ratio (mg;

mg) of about 0.04.

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

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

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

40. A method of introducing an siRNA that silences ApoB expression into a cell, said method comprising contacting said cell with a nucleic acid-lipid particle comprising a cationic lipid, a non-cationic lipid, and said siRNA.

41. The method of claim 40, wherein said nucleic acid-lipid particle further comprises a conjugated lipid that inhibits aggregation of particles.

42. The method of claim 40, wherein said nucleic acid-lipid particle comprises an siRNA molecule comprising any one of the sequences set forth in Table 1, rows A-F of Table 2, and Tables 3-7.

43. The method of claim 40, wherein said nucleic acid-lipid particle comprises at least two siRNA molecules, wherein each siRNA molecule comprises a sequence independently selected from the sequences set forth in Table 1, rows A-F of Table 2, and Tables 3-7.

44. The method of claim 40, wherein said siRNA in said nucleic acid-lipid particle is resistant in aqueous solution to degradation with a nuclease.

45. The method of claim 40, wherein said cationic lipid is a member selected from the group consisting of DODAC, DDAB, DOTAP, DOTMA, DODMA, DLinDMA, DLenDMA, and a mixture thereof.

46. The method of claim 40, wherein said non-cationic lipid is a member selected from the group consisting of DOPE, POPC, EPC, DSPC, POPG, DPPE, DMPE, DSPE, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, POPE, SOPE, cholesterol, and a mixture thereof.

47. The method of claim 40, wherein the conjugated lipid that inhibits aggregation of particles comprises a PEG-lipid conjugate selected from the group consisting of:

a PEG-DAG, a PEG-DAA, a PEG-phospholipid, a PEG-Cer, and a mixture thereof.

48. The method of claim 40, wherein said cell is in a mammal.

49. The method of claim 48, wherein said contacting comprises administering said nucleic acid-lipid particle via a route selected from the group consisting of:

intravenous, subcutaneous, and intraperitoneal.

50. The method of claim 48, wherein the mammal is a human.

51. The method of claim 50, wherein said human has a disease or disorder associated with expression of a ApoB and wherein expression of ApoB is silenced by said siRNA.

52. The method of claim 50, wherein said disease or disorder is associated with overexpression of ApoB and wherein expression of ApoB is silenced by said siRNA.

53. The method of claim 50, wherein said human has a disease or disorder selected from the group consisting of:

atherosclerosis, angina pectoris, high blood pressure, diabetes, and hypothyroidism.

54. The method of claim 50, wherein said human has a disease or disorder involving hypercholesterolemia and wherein serum cholesterol levels are lowered when expression of ApoB is silenced by said siRNA.

55. The method of claim 54, wherein said disease is a member selected from the group consisting of:

atherosclerosis, angina pectoris, and high blood pressure.

56. An isolated nucleic acid comprising a sequence set forth in in Table 1, rows A-F of Table 2, and Tables 3-7.