Lipid encapsulated interfering RNA

• US 7,799,565 B2
• Filed: 06/07/2005
• Issued: 09/21/2010
• Est. Priority Date: 06/07/2004
• Status: Expired due to Fees

• Current Assignee: Arbutus Biopharma Corporation
• Original Assignee: Protiva Biotherapeutics Inc (Arbutus Biopharma Corporation)
• Inventors: Heyes, James, MacLachlan, Ian, Palmer, Lorne R.
• Primary Examiner(s): Kelly; Robert M
• Assistant Examiner(s): HIRIYANNA, KELAGINAMANE T
• Application Number:
  US11/148,152
• Publication Number:
  US 20060008910A1
• Time in Patent Office: 1,932 Days
• Field of Search: None
• 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. 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;

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 (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 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;

19. The nucleic acid-lipid particle in accordance with claim 18, wherein at least one of R³ and R⁴ is selected from the group consisting of:

linoleyl and linolenyl.

20. The nucleic acid-lipid particle in accordance with claim 18, wherein R¹ and R² are methyl.

21. The nucleic acid-lipid particle in accordance with claim 20, wherein R³ and R⁴ are both linoleyl.

22. The compound in accordance with claim 20, wherein R³ and R⁴ 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 (C₁₂), a PEG-dimyristyloxypropyl (C₁₄), a PEG-dipalmityloxypropyl (C₁₆), and a PEG-distearyloxypropyl (C₁₈).