siRNA silencing of apolipoprotein B
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.
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 (C 12 ), a PEG-dimyristyloxypropyl (C 14 ), a PEG-dipalmityloxypropyl (C 16 ), and a PEG-distearyloxypropyl (C 18 ).
17 . The nucleic acid-lipid particle in accordance with claim 15 , wherein said PEG-DAA conjugate is a PEG-dimyristyloxypropyl (C 14 ).
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.