Lipid encapsulated interfering RNA
View Patent ↗The present invention provides lipid-based formulations for delivering, e.g., introducing, nucleic acid-lipid particles comprising an interference RNA molecule to a cell, and assays for optimizing the delivery efficiency of such lipid-based formulations.
1. A nucleic acid-lipid particle, said nucleic acid-lipid particle comprising:
(a) an interfering RNA;
(b) a cationic lipid having one of the following structures:
wherein:
R 1 and R 2 are independently selected from the group consisting of: H and C 1 -C 3 alkyl; and
R 3 and R 4 are both linoleyl or independently selected from the group consisting of alkyl groups having from about 10 to about 20 carbon atoms, wherein both R 3 and R 4 comprise at least three sites of unsaturation;
(c) a non-cationic lipid; and
(d) a conjugated lipid that inhibits aggregation of particles.
2. The nucleic acid-lipid particle of claim 1 , wherein said cationic lipid is selected from the group consisting of: 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA) and 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA).
3. The nucleic acid-lipid particle of claim 1 , wherein said interfering RNA in said nucleic acid-lipid particle is resistant in aqueous solution to degradation by a nuclease.
4. The nucleic acid-lipid particle of claim 1 , wherein said particle has a median diameter of less than about 150 nm.
5. The nucleic acid-lipid particle of claim 1 , wherein said interfering RNA comprises a small interfering RNA (siRNA).
6. The nucleic acid-lipid particle of claim 5 , wherein said siRNA comprises 15-60 (duplex) nucleotides.
7. The nucleic acid-lipid particle of claim 1 , wherein said interfering RNA is transcribed from a plasmid.
8. The nucleic acid-lipid particle of claim 1 , wherein said interfering RNA comprises double-stranded RNA (dsRNA).
9. 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 a mixture thereof.
10. 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 a mixture thereof.
11. 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, a PEG-dialkyloxypropyl, a PEG-phospholipid, a PEG-ceramide, and a mixture thereof.
12. The nucleic acid-lipid particle in accordance with claim 11 , wherein the conjugated lipid that inhibits aggregation of particles comprises a polyethyleneglycol (PEG)-dialkyloxypropyl conjugate.
13. The nucleic acid-lipid particle in accordance with claim 12 , wherein the PEG-dialkyloxypropyl 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 ).
14. The nucleic acid-lipid particle in accordance with claim 1 , wherein the conjugated lipid that inhibits aggregation of particles has the Formula:
A-W—Y (Formula VII), wherein A is a lipid moiety; W is a hydrophilic polymer; and Y is a polycationic moiety.
15. The nucleic acid-lipid particle composition of claim 14 , wherein W is a polymer selected from the group consisting of polyethyleneglycol (PEG), polyamide, polylactic acid, polyglycolic acid, polylactic acid/polyglycolic acid copolymers and combinations thereof, said polymer having a molecular weight of about 250 to about 7000 daltons.
16. The nucleic acid-lipid particle composition of claim 14 , wherein Y has at least 4 positive charges at a selected pH.
17. The nucleic acid-lipid particle composition of claim 14 , wherein Y is a member selected from the group consisting of lysine, arginine, asparagine, glutamine, derivatives thereof and combinations thereof.
18. A nucleic acid-lipid particle, said nucleic acid-lipid particle comprising:
(a) an interfering RNA;
(b) a cationic lipid having one of the following structures:
wherein:
R 1 and R 2 are independently selected from the group consisting of: H and C 1 -C 3 alkyls; and
R 3 and R 4 are both linoleyl or are independently selected from the group consisting of alkyl groups having from about 10 to about 20 carbon atoms, wherein R 3 and R 4 are independently selected from the group consisting of: dodecatrienyl, tetradectrienyl, hexadecatrienyl, linolenyl, and icosatrienyl;
(c) a non-cationic lipid; and
(d) a polyethyleneglycol (PEG)-lipid conjugate.
19. The nucleic acid-lipid particle in accordance with claim 18 , wherein at least one of R 3 and R 4 is selected from the group consisting of: linoleyl and linolenyl.
20. The nucleic acid-lipid particle in accordance with claim 18 , wherein R 1 and R 2 are methyl.
21. The nucleic acid-lipid particle in accordance with claim 20 , wherein R 3 and R 4 are both linoleyl.
22. The compound in accordance with claim 20 , wherein R 3 and R 4 are both linolenyl.
23. The nucleic acid-lipid particle in accordance with claim 18 , 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 a mixture thereof.
24. The nucleic acid-lipid particle in accordance with claim 18 , wherein the polyethyleneglycol (PEG)-lipid conjugate is member selected from the group consisting of a PEG-diacylglycerol, a PEG-dialkyloxypropyl, a PEG-phospholipid, a PEG-ceramide, and a mixture thereof.
25. The nucleic acid-lipid particle in accordance with claim 24 , wherein the polyethyleneglycol (PEG)-lipid conjugate comprises a polyethyleneglycol (PEG)-dialkyloxypropyl conjugate.
26. The nucleic acid-lipid particle in accordance with claim 25 , wherein the PEG-dialkyloxypropyl 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 ).