Lipid encapsulated interfering RNA

• US 20060008910A1
• Filed: 06/07/2005
• Published: 01/12/2006
• Est. Priority Date: 06/07/2004
• Status: Active Grant

• Current Assignee: Arbutus Biopharma Corporation
• Original Assignee: Protiva Biotherapeutics Inc (Arbutus Biopharma Corporation)
• Inventors: Heyes, James, MacLachlan, Ian, Palmer, Lorne R.
• Granted Patent: US 7,799,565 B2
• Time in Patent Office: Days
• US Class Current: 435/458
• CPC Class Codes: A61K 31/7088 Compounds having three or more nucleosides or nucleotides
• CPC Class Codes: A61K 31/7105 Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
• 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: 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 2310/351 Conjugate
• CPC Class Codes: C12N 2320/32 Special delivery means, e.g. tissue-specific

Abstract

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.

Claims

1. An nucleic acid-lipid particle, said nucleic acid-lipid particle comprising:

(a) an interfering RNA;

(b) a cationic lipid of Formula I and having the following structure;

wherein;

R¹ and R² are independently selected from the group consisting of;

H and C₁-C₃ alkyls; and

R³ and R⁴ are independently selected from the group consisting of alkyl groups having from about 10 to about 20 carbon atoms, wherein at least one of R³ and R⁴ comprises at least two 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 (DAG), 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 (C₁₂), a PEG-dimyristyloxypropyl (C₁₄), a PEG-dipalmityloxypropyl (C₁₆), and a PEG-distearyloxypropyl (C₁₈).

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 II)

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 method of introducing an interfering RNA into a cell, said method comprising contacting said cell with a nucleic acid-lipid particle comprising

(a) said interfering RNA;

(b) a cationic lipid of Formula I and having the following structure;

wherein;

R¹ and R² are independently selected from the group consisting of;

H and C₁-C₃ alkyls; and

R³ and R⁴ are independently selected from the group consisting of alkyl groups having from about 10 to about 20 carbon atoms, wherein at least one of R³ and R⁴ comprises at least two sites of unsaturation;

(c) a non-cationic lipid; and

(d) a conjugated lipid that inhibits aggregation of particles.

19. The method of claim 18, wherein said interfering RNA in said nucleic acid-lipid particle is resistant in aqueous solution to degradation with a nuclease.

20. The method of claim 18, wherein said particle has a median diameter of less than about 150 nm.

21. The method of claim 18, wherein said interfering RNA comprises a small interfering RNA (siRNA).

22. The method of claim 18, wherein said interfering RNA is transcribed from a plasmid.

23. The method of claim 18, wherein said non-cationic lipid is a member selected from the group consisting o 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, palmitoyloleoyl-phosphatidylethanolamine (POPE), 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, and a mixture thereof.

24. The method of claim 18, 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.

25. The method of claim 18, wherein the conjugated lipid that inhibits aggregation of particles is a polyethyleneglycol (PEG)-lipid.

26. The method of claim 25, wherein 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.

27. The method of claim 26, wherein the conjugated lipid that inhibits aggregation of particles is a polyethyleneglycol (PEG)-dialkyloxypropyl conjugate.

28. The method of claim 27, wherein the PEG-dialkyloxypropyl conjugate is PEG-dimyristyloxypropyl (C₁₄).

29. The method of claim 18, wherein said cell is in a mammal.

30. The method of claim 29, wherein the mammal is a human.

31. The method of claim 29, wherein presence of an interfering RNA at a site distal to the site of administration is detectable for at least 48 hours after administration of said particle.

32. The method of claim 29, wherein presence of an interfering RNA at a site distal to the site of administration is detectable for at least 24 hours after administration of said particle.

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

(a) said interfering RNA;

(b) a cationic lipid of Formula I and having the following structure;

wherein;

R¹ and R² are independently selected from the group consisting of;

H and C₁-C₃ alkyls; and

R³ and R⁴ are independently selected from the group consisting of alkyl groups having from about 10 to about 20 carbon atoms, wherein at least one of R³ and R⁴ comprises at least two sites of unsaturation;

(c) a non-cationic lipid; and

(d) a conjugated lipid that inhibits aggregation of particles.

34. The method of claim 33, wherein said mammal is a human.

35. The method of claim 34, wherein said human has a disease or disorder associated with expression of a gene and wherein expression of said gene is reduced by said interfering RNA.

36. The method of claim 35, wherein said disease or disorder is associated with overexpression of said gene.

37. The method of claim 33, wherein said administration is intravenous.