Lipid-nucleic acid particles prepared via a hydrophobic lipid-nucleic acid complex intermediate and use for gene transfer

• US 6,586,410 B1
• Filed: 05/08/2000
• Issued: 07/01/2003
• Est. Priority Date: 06/07/1995
• Status: Expired due to Term

• Current Assignee: Tekmira Pharmaceuticals Corporation
• Original Assignee: Inex Pharmaceuticals Corporation
• Inventors: Scherrer, Peter, Cullis, Pieter R., Wheeler, Jeffery J., Bally, Marcel B., Zhang, Yuan-Peng, Reimer, Dorothy L., Hope, Michael
• Primary Examiner(s): McGarry, Sean
• Assistant Examiner(s): EPPS -SMITH, JANET L
• Application Number:
  US09/566,700
• Time in Patent Office: 1,149 Days
• Field of Search: 424/450, 435/320.1, 435/375, 435/458, 514/44, 536/23.1, 536/24.5
• US Class Current: 514/44.00R
• CPC Class Codes: A61K 47/6911 the form being a liposome
• 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
• CPC Class Codes: Y10S 436/829 Liposomes, e.g. encapsulation

Abstract

Novel lipid-nucleic acid particulate complexes which are useful for in vitro or in vivo gene transfer are described. The particles can be formed using either detergent dialysis methods or methods which utilize organic solvents. Upon removal of a solubilizing component (i.e., detergent or an organic solvent) the lipid-nucleic acid complexes form particles wherein the nucleic acid is serum-stable and is protected from degradation. The particles thus formed have access to extravascular sites and target cell populations and are suitable for the therapeutic delivery of nucleic acids.

Claims

1. 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 conjugated lipid that inhibits aggregation of particles, and a nucleic acid, wherein said nucleic acid in said nucleic acid-lipid particle is resistant in aqueous solution to degradation with a nuclease.

2. The method of claim 1, wherein said particle is substantially non-toxic.

3. The method of claim 1, wherein said particle has a median diameter of less than about 150 nm.

4. The method of 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), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), and a mixture of two or more of the above.

5. The method of claim 1, wherein said particle further comprises a non-cationic lipid.

6. The method of claim 5, wherein said non-cationic lipid is selected from the group consisting of DOPE, POPC and EPC.

14. The method of claim 1, wherein the nucleic acid:

lipid ratio within said particle is at least 5 mg nucleic acid per mmol lipid.

15. The method of claim 1, wherein the nucleic acid:

lipid ratio within said particle is at least 20 mg nucleic acid per mmol lipid.

16. The method of claim 1, wherein the nucleic acid:

lipid ratio within said particle is at least 40 mg nucleic acid per mmol lipid.

17. The method of claim 1, wherein said nucleic acid is DNA.

18. The method of claim 1, wherein said nucleic acid is a plasmid.

19. The method of claim 1, wherein said nucleic acid is an antisense oligonucleotide.

20. The method of claim 1, wherein said nucleic acid is a ribozyme.

21. The method of claim 1, wherein said cationic lipid comprises 50% or less of the lipid present in said particle.

22. The method of claim 1, wherein said cationic lipid comprises from an amount greater than 0% to about 20% of the lipid present in said nucleic acid-lipid particle.

23. The method of claim 1, wherein the nucleic acid component of said particle is substantially not degraded after exposure of said particle to a nuclease at 37°

C. for 20 minutes.

24. The method of claim 1, wherein the nucleic acid component of said particle is substantially not degraded after incubation of said particle in serum at 37°

C. for 30 minutes.

25. The method of claim 1, wherein more than 10% of a plurality of such particles are present in plasma one hour after intravenous administration.

26. The method of claim 1, wherein said cell is present inside of a mammal, and wherein said transformation of said cell by said particle at a site distal to the site of administration is detectable for at least four days after intravenous injection.

27. The method of claim 1, wherein said cell is present inside of a mammal, and wherein said nucleic acid-lipid particle is administered parenterally to said mammal.

28. The method of claim 27, wherein said particle is administered to said mammal by intravenous injection.

29. The method of claim 27, wherein said particle is administered to said mammal by intraperitoneal delivery.

7. The method of claimed 1, wherein said conjugated lipid is a PEG-lipid.

8. The method of claim 7, wherein said PEG-lipid comprises from 1% to about 15% of the total lipid present in said particle.

9. The method of claim 7, wherein said PEG-lipid is PEG-ceramide.

10. The method of claim 9, wherein the ceramide of said PEG-ceramide comprises a fatty acid group having 8 carbon atoms.

11. The method of claim 9, wherein the ceramide of said PEG-ceramide comprises a fatty acid group having 14 carbon atoms.

12. The method of claim 9, wherein the ceramide of said PEG-ceramide comprises a fatty acid group having 20 carbon atoms.

13. The method of claim 7, wherein said PEG-lipid is PEG-phosphatidylethanolamine.