siRNA silencing of filovirus gene expression
View Patent ↗The present invention provides siRNA molecules that target Filovirus gene expression and methods of using such siRNA molecules to silence Filovirus gene expression. The present invention also provides nucleic acid-lipid particles that target Filovirus gene expression comprising an siRNA that silences Filovirus gene expression, a cationic lipid, and a non-cationic lipid.
1. An siRNA molecule for silencing Ebola virus L-polymerase (L-pol) expression comprising a double-stranded sequence consisting of SEQ ID NO:23 and SEQ ID NO:24.
2. The siRNA molecule in accordance with claim 1 , wherein said double-stranded sequence comprises a hairpin loop structure.
3. A pharmaceutical composition comprising an siRNA molecule in accordance with claim 1 and a phamaceutically acceptable carrier.
4. A nucleic acid-lipid particle comprising: an siRNA molecule in accordance with claim 1 ; a cationic lipid; and a non-cationic lipid.
5. The nucleic acid-lipid particle in accordance with claim 4 , 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), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLendMA), and a mixture thereof.
6. The nucleic acid-lipid particle in accordance with claim 4 , wherein said cationic lipid is DLinDMA.
7. The nucleic acid-lipid particle in accordance with claim 4 , wherein said non-cationic lipid is an anionic lipid.
8. The nucleic acid-lipid particle in accordance with claim 4 , wherein said non-cationic lipid is a neutral lipid.
9. The nucleic acid-lipid particle in accordance with claim 4 , wherein said non-cationic lipid is a member selected from the group consisting of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), egg phosphatidylcholine (EPC), cholesterol, and a mixture thereof.
10. The nucleic acid-lipid particle in accordance with claim 4 , wherein said non-cationic lipid is DSPC.
11. The nucleic acid-lipid particle in accordance with claim 4 , further comprising a conjugated lipid that inhibits aggregation of particles.
12. The nucleic acid-lipid particle in accordance with claim 11 , wherein said 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.
13. The nucleic acid-lipid particle in accordance with claim 12 , wherein said PEG-lipid is a member selected from the group consisting of a PEG-diacylglycerol, a PEG dialkyloxypropyl, a PEG-phospholipid, a PEG-ceramide, and a mixture thereof.
14. The nucleic acid-lipid particle in accordance with claim 12 , wherein said conjugated lipid that inhibits aggregation of particles comprises a polyethyleneglycol (PEG)-dialkyloxypropyl (PEG-DAA) conjugate.
15. The nucleic acid-lipid particle in accordance with claim 14 , wherein said PEG-DAA conjugate is a member selected from the group consisting of a PEG-dilauryloxypropyl (C 12 ), a PEG-dimyristyloxypropyl (C 14 ), a PEG-dipalmityloxypropyl (C 16 ), and a PEG-distearyloxypropyl (C 18 ).
16. The nucleic acid-lipid particle in accordance with claim 14 , wherein said PEG-DAA conjugate is a PEG-dimyristyloxypropyl (C 14 ).
17. The nucleic acid-lipid particle in accordance with claim 4 , wherein said cationic lipid comprises from about 20 mol % to about 50 mol % of the total lipid present in said particle.
18. The nucleic acid-lipid particle in accordance with claim 4 , wherein said cationic lipid comprises about 40 mol % of the total lipid present in said particle.
19. The nucleic acid-lipid particle in accordance with claim 4 , wherein said non-cationic lipid comprises from about 5 mol % to about 90 mol % of the total lipid present in said particle.
20. The nucleic acid-lipid particle in accordance with claim 4 , wherein said non-cationic lipid comprises about 20 mol % of the total lipid present in said particle.
21. The nucleic acid-lipid particle in accordance with claim 14 , wherein said PEG-DAA conjugate comprises from 0 mol % to about 20 mol % of the total lipid present in said particle.
22. The nucleic acid-lipid particle in accordance with claim 14 , wherein said PEG-DAA conjugate comprises about 2 mol % of the total lipid present in said particle.
23. The nucleic acid-lipid particle in accordance with claim 4 , further comprising cholesterol.
24. The nucleic acid-lipid particle in accordance with claim 23 , wherein the cholesterol comprises from about 10 mol % to about 60 mol % of the total lipid present in said particle.
25. The nucleic acid-lipid particle in accordance with claim 23 , wherein the cholesterol comprises about 48 mol % of the total lipid present in said particle.
26. The nucleic acid-lipid particle in accordance with claim 4 , wherein said siRNA molecule in said nucleic acid-lipid particle is not substantially degraded after exposure of said particle to a nuclease at 37° C. for 20 minutes.
27. The nucleic acid-lipid particle in accordance with claim 4 , wherein said siRNA molecule in said nucleic acid-lipid particle is not substantially degraded after incubation of said particle in serum at 37° C. for 30 minutes.
28. The nucleic acid-lipid particle in accordance with claim 4 , wherein said siRNA molecule is fully encapsulated in said nucleic acid-lipid particle.
29. The nucleic acid-lipid particle in accordance with claim 4 , wherein said particle has a siRNA:lipid ratio of from about 0.01 to about 0.2.
30. The nucleic acid-lipid particle in accordance with claim 4 , wherein said particle has a siRNA:lipid ratio of from about 0.02 to about 0.1.
31. The nucleic acid-lipid particle in accordance with claim 4 , wherein said particle has a siRNA:lipid ratio of about 0.04.
32. The nucleic acid-lipid particle in accordance with claim 4 , wherein said particle has a median diameter of from about 50 nm to about 150 nm.
33. The nucleic acid-lipid particle in accordance with claim 4 , wherein said particle has a median diameter of from about 70 nm to about 90 nm.
34. A pharmaceutical composition comprising a nucleic acid-lipid particle in accordance with claim 4 and a pharmaceutically acceptable carrier.
35. The nucleic acid-lipid particle in accordance with claim 4 , wherein said non-cationic lipid comprises DSPC and cholesterol.
36. The nucleic acid-lipid particle in accordance with claim 10 , wherein said DSPC comprises about 10 mol % of the total lipid present in said particle.
37. The nucleic acid-lipid particle in accordance with claim 4 , wherein said cationic lipid comprises from about 2 mol % to about 60 mol % of the total lipid present in said particle.
38. The nucleic acid-lipid particle in accordance with claim 4 , wherein said non-cationic lipid comprises dipalmitoylphosphatidylcholine (DPPC).
39. The nucleic acid-lipid particle in accordance with claim 4 , wherein said non-cationic lipid comprises DPPC and cholesterol.
40. The siRNA molecule in accordance with claim 1 , wherein said siRNA molecule comprises 3′ overhangs of about 1 to about 4 nucleotides on one or both sides of the double-stranded sequence.
41. The siRNA molecule in accordance with claim 1 , wherein said siRNA molecule comprises at least one modified nucleotide.
42. The siRNA molecule in accordance with claim 1 , wherein said siRNA molecule comprises at least one modified nucleotide in one or both strands of the double-stranded sequence.
43. The siRNA molecule in accordance with claim 1 , wherein said siRNA molecule comprises at least one 2′-O-methyl (2′OMe) nucleotide.
44. The siRNA molecule in accordance with claim 1 , wherein said siRNA molecule comprises at least one 2′OMe nucleotide selected from the group consisting of a 2′OMe-guanosine nucleotide, 2′OMe-uridine nucleotide, 2′OMe-adenosine nucleotide, 2′OMe-cytosine nucleotide, and mixtures thereof.
45. The nucleic acid-lipid particle in accordance with claim 11 , wherein said conjugated lipid that inhibits aggregation of particles comprises from about 0.5 mol % to about 20 mol % of the total lipid present in said particle.