Lipid-based formulations

• US 20030077829A1
• Filed: 04/30/2002
• Published: 04/24/2003
• Est. Priority Date: 04/30/2001
• Status: Abandoned Application

• Current Assignee: Protiva Biotherapeutics Inc (Arbutus Biopharma Corporation)
• Original Assignee: Protiva Biotherapeutics Inc (Arbutus Biopharma Corporation)
• Inventors: MacLachlan, Ian
• Application Number:
  US10/136,707
• Publication Number:
  US 20030077829A1
• Time in Patent Office: Days
• US Class Current: 435/458
• CPC Class Codes: A61K 9/1271 Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers liposome as conjugate A61K47/6911
• 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 15/88 using microencapsulation, e.g. using amphiphile liposome vesicle

Abstract

The present invention provides lipid-based formulations for delivering nucleic acids to a cell, and assays for optimizing the transfection efficiency of such lipid-based formulations.

Claims

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

a nucleic acid;

a cationic lipid;

a non-cationic lipid; and

a polyethyleneglycol-diacylglycerol (PEG-DAG) conjugate.

2. 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), and a mixture thereof.

3. 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.

4. The nucleic acid-lipid particle in accordance with claim 1, wherein said PEG-DAG conjugate is a member selected from the group consisting of PEG-dilaurylglycerol (C12), a PEG-dimyristylglycerol (C14), a PEG-dipalmitoylglycerol (C16), and a PEG-disterylglycerol (C18).

5. 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.

6. The nucleic acid-lipid particle in accordance with claim 1, wherein said cationic lipid comprises from about 5% to about 45% of the total lipid present in said particle.

7. The nucleic acid-lipid particle in accordance with claim 1, wherein said cationic lipid comprises from about 5% to about 15% of the total lipid present in said particle.

8. 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.

9. 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.

10. 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.

11. The nucleic acid-lipid particle in accordance with claim 1, wherein said PEG-DAG conjugate comprises from 1% to about 20% of the total lipid present in said particle.

12. The nucleic acid-lipid particle in accordance with claim 1, wherein said PEG-DAG conjugate comprises from 4% to about 15% of the total lipid present in said particle.

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

14. The nucleic acid-lipid particle in accordance with claim 13, further comprising cholesterol.

15. The nucleic acid-lipid particle in accordance with claim 14, wherein the cholesterol comprises from about 10% to about 60% of the total lipid present in said particle.

16. 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.

17. The nucleic acid-lipid particle in accordance with claim 1, wherein the cationic lipid comprises 7.5% of the total lipid present in said particle;

the non-cationic lipid comprises 82.5% of the total lipid present in said particle; and

the PEG-DAG conjugate comprises 10% of the total lipid present in said particle.

18. The nucleic acid-lipid particle in accordance with claim 1, wherein the nucleic acid-lipid particle comprises:

DODMA;

DSPC; and

a PEG-DAG conjugate.

19. The nucleic acid-lipid particle in accordance with claim 18, wherein the PEG-DAG conjugate is PEG-dilaurylglycerol (C12).

20. The nucleic acid-lipid particle in accordance with claim 19, further comprising cholesterol.

21. The nucleic acid-lipid particle in accordance with claim 18, wherein the PEG-DAG conjugate is PEG-dimyristylglycerol (C14).

22. The nucleic acid-lipid particle in accordance with claim 21, further comprising cholesterol.

23. The nucleic acid-lipid particle in accordance with claim 18, wherein the PEG-DAG conjugate is PEG-dipalmitoylglycerol (C16).

24. The nucleic acid-lipid particle in accordance with claim 23, further comprising cholesterol.

25. The nucleic acid-lipid particle in accordance with claim 18, wherein the PEG-DAG conjugate is PEG-disterylglycerol (C18).

26. The nucleic acid-lipid particle in accordance with claim 25, further comprising cholesterol.

27. The nucleic acid-lipid particle in accordance with claim 1, wherein said nucleic acid is DNA.

28. The nucleic acid-lipid particle in accordance with claim 1, wherein said nucleic acid is a plasmid.

29. The nucleic acid-lipid particle in accordance with claim 1, wherein said nucleic acid is an antisense oligonucleotide.

30. The nucleic acid-lipid particle in accordance with claim 1, wherein said nucleic acid is a ribozyme.

31. The nucleic acid-lipid particle in accordance with claim 1, wherein said nucleic acid encodes a therapeutic product of interest.

32. The nucleic acid-lipid particle in accordance with claim 31, wherein said therapeutic product of interest is a peptide or protein.

33. 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.

34. 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.

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

36. A pharmaceutical composition comprising a nucleic acid-lipid particle in accordance with claim 1 and a pharmaceutically acceptable carrier.

37. A pharmaceutical composition in accordance with claim 36 comprising a nucleic acid-lipid particle, wherein the nucleic acid-lipid particle comprises:

DODMA, DSPC, and a PEG-DAG conjugate;

and a pharmaceutically acceptable carrier.

38. A pharmaceutical composition in accordance with claim 37, wherein the PEG-DAG conjugate is PEG-dilaurylglycerol (C12).

39. A pharmaceutical composition in accordance with claim 37, wherein the PEG-DAG conjugate is PEG-dimyristylglycerol (C14).

40. A pharmaceutical composition in accordance with claim 37, wherein the PEG-DAG conjugate is PEG-dipalmitoylglycerol (C16).

41. A pharmaceutical composition in accordance with claim 37, wherein the PEG-DAG conjugate is PEG-disterylglycerol (C18).

42. A method of introducing a nucleic acid into a cell;

said method comprising contacting said cell with a nucleic acid-lipid particle comprising a cationic lipid, a non-cationic lipid, a PEG-DAG conjugate, and a nucleic acid.