Compositions comprising immunostimulatory siRNA molecules and DLinDMA or DLenDMA

• US 7,807,815 B2
• Filed: 06/30/2005
• Issued: 10/05/2010
• Est. Priority Date: 07/02/2004
• Status: Expired due to Fees

• Current Assignee: Arbutus Biopharma Corporation
• Original Assignee: Protiva Biotherapeutics Inc (Arbutus Biopharma Corporation)
• Inventors: Judge, Adam, Heyes, James, MacLachlan, Ian, Palmer, Lorne
• Primary Examiner(s): ANGELL, JON E
• Application Number:
  US11/174,453
• Publication Number:
  US 20060025366A1
• Time in Patent Office: 1,923 Days
• Field of Search: 536/24.5
• US Class Current: 536/24.5
• CPC Class Codes: A01K 2267/0331 Animal model for proliferative diseases
• CPC Class Codes: A61K 2039/55561 CpG containing adjuvants; Oligonucleotide containing adjuvants
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/117 Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/17 Immunomodulatory nucleic acids
• CPC Class Codes: C12N 2320/50 Methods for regulating/modulating their activity

Abstract

The present invention provides siRNA molecules and methods of using such siRNA molecules to modulate an immune response and to silence expression of a target gene.

Claims

1. A nucleic acid-lipid particle comprising:

(a) a modified siRNA comprising a double-stranded sequence of about 15 to about 30 nucleotides in length, said sequence comprising a non-immunostimulatory mismatch motif relative to an unmodified siRNA sequence that is capable of silencing expression of a target sequence, wherein the mismatch motif consists of a 5′

-XX′

-3′

dinucleotide corresponding to a 5′

-GU-3′

dinucleotide in the sense or antisense strand of the unmodified siRNA sequence, wherein X and X′

are independently selected from the group consisting of A, U, C, and G, with the proviso that if X is G, X′

is not U and if X′

is U, X is not G,

wherein the modified siRNA is less immunogenic than the unmodified siRNA sequence, and

wherein the modified siRNA is capable of silencing expression of the target sequence;

(b) a cationic lipid, wherein said cationic lipid is selected from the group consisting of 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), and a mixture thereof;

(c) a non-cationic lipid; and

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

2. The nucleic acid-lipid particle in accordance with claim 1, wherein the modified siRNA has reduced toxicity relative to an siRNA that is not in a nucleic acid-lipid particle.

3. The nucleic acid-lipid particle in accordance with claim 1, wherein said cationic lipid is DLinDMA.

4. The nucleic acid-lipid particle in accordance with claim 1, wherein said non-cationic lipid is a member selected from the group consisting of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl- phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, and a mixture thereof.

5. The nucleic acid-lipid particle in accordance with claim 1, wherein said non-cationic lipid is DSPC.

6. The nucleic acid-lipid particle in accordance with claim 1, wherein the conjugated lipid that inhibits aggregation of particles comprises a polyethyleneglycol (PEG)-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.

7. The nucleic acid-lipid particle in accordance with claim 1, wherein the conjugated lipid that inhibits aggregation of particles comprises a PEG-dialkyloxypropyl (DAA) conjugate.

8. The nucleic acid-lipid particle in accordance with claim 7, wherein the PEG-DAA 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).

9. The nucleic acid-lipid particle in accordance with claim 7, wherein the PEG-DAA conjugate is a PEG-dimyristyloxypropyl (C₁₄).

10. The nucleic acid-lipid particle in accordance with claim 1, wherein said cationic lipid comprises from about 15% to about 35% of the total lipid present in said particle.

11. The nucleic acid-lipid particle in accordance with claim 1, wherein said non-cationic lipid comprises from about 15% to about 25% of the total lipid present in said particle.

12. The nucleic acid-lipid particle in accordance with claim 7, wherein said PEG-DAA conjugate comprises from 1% to about 10% of the total lipid present in said particle.

13. The nucleic acid-lipid particle in accordance with claim 7, wherein said PEG-DAA conjugate comprises about 2% of the total lipid present in said particle.

14. The nucleic acid-lipid particle in accordance with claim 1, further comprising cholesterol.

15. The nucleic acid-lipid particle in accordance with claim 14, wherein the cholesterol comprises from about 40% to about 60% of the total lipid present in said particle.

16. The nucleic acid-lipid particle in accordance with claim 1, wherein the modified siRNA is fully encapsulated in said nucleic acid-lipid particle.

17. A composition comprising a nucleic acid-lipid particle in accordance with claim 1 and a carrier.

18. The nucleic acid-lipid particle in accordance with claim 1, wherein said cationic lipid comprises from about 2% to about 60% 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 40% to about 50% of the total lipid present in said particle.

20. The nucleic acid-lipid particle in accordance with claim 1, wherein said non-cationic lipid comprises from about 5% to about 90% of the total lipid present in said particle.

21. The nucleic acid-lipid particle in accordance with claim 1, wherein said non-cationic lipid comprises from about 20% to about 85% of the total lipid present in said particle.

22. The nucleic acid-lipid particle in accordance with claim 14, wherein the cholesterol comprises from about 20% to about 45% of the total lipid present in said particle.

23. The nucleic acid-lipid particle in accordance with claim 1, wherein said particle has a median diameter of from about 50 nm to about 150 nm.

24. The nucleic acid-lipid particle in accordance with claim 1, wherein said non-cationic lipid comprises a phospholipid and cholesterol, and wherein said conjugated lipid that inhibits aggregation of particles comprises a PEG-DAA conjugate.

25. The nucleic acid-lipid particle in accordance with claim 1, wherein the modified siRNA further comprises a second non-immunostimulatory mismatch motif.

26. The nucleic acid-lipid particle in accordance with claim 1, wherein the modified siRNA comprises a double-stranded sequence of about 19 to about 25 nucleotides in length.

27. The nucleic acid-lipid particle in accordance with claim 1, wherein the modified siRNA comprises 3′

overhangs.

28. The nucleic acid-lipid particle in accordance with claim 1, wherein said conjugated lipid that inhibits aggregation of particles comprises from about 1% to about 15% of the total lipid present in said particle.