Methods for encapsulating nucleic acids in lipid bilayers

• US 6,734,171 B1
• Filed: 10/09/1998
• Issued: 05/11/2004
• Est. Priority Date: 10/10/1997
• Status: Expired due to Fees

• Current Assignee: Tekmira Pharmaceuticals Corporation
• Original Assignee: Inex Pharmaceuticals Corporation
• Inventors: Zhang, Yuan-Peng, Saravolac, Edward George, Kojic, Ljiljana D., Ludkovski, Olga, Scherrer, Peter, Cullis, Pieter R., Wheeler, Jeffery J.
• Primary Examiner(s): NGUYEN, DAVE TRONG
• Application Number:
  US09/169,573
• Time in Patent Office: 2,041 Days
• Field of Search: 424/450, 435/325, 435/320.1, 435/455, 435/458, 514/44
• US Class Current: 514/44.00R
• CPC Class Codes: A61K 48/0008 characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
• CPC Class Codes: A61K 48/0025 wherein the non-active part clearly interacts with the delivered nucleic acid
• CPC Class Codes: A61K 48/0041 the non-active part being polymeric

Abstract

The present invention relates to lipid-based formulations for nucleic acid delivery to cells, methods for the preparation of such formulations and, in particular, to lipid encapsulated plasmids. The compositions are safe and practical for clinical use. In addition, the present invention provides methods for introducing nucleic acids into cells and for inhibiting tumor growth in cells using such lipid-nucleic acid formulations.

Claims

1. A method of encapsulating a nucleic acid in a lipid bilayer carrier, said method comprising:

(a) combining said nucleic acid with a lipid-detergent mixture, said lipid-detergent mixture comprising a lipid mixture of an aggregation-preventing agent in an amount of about 5 mol % to about 20 mol %, a cationic lipid in an amount of about 0.5 mol % to about 50 mol %, and a flisogenic lipid and a detergent, to provide a nucleic acid-lipid detergent mixture; and

(b) dialyzing said nucleic acid-lipid-detergent mixture against a buffered salt solution to remove said detergent and to encapsulate said nucleic acid in a lipid bilayer carrier and provide a lipid bilayer-nucleic acid composition, wherein said buffered salt solution has an ionic strength sufficient to encapsulate of from about 40% to about 80% of said nucleic acid.

2. The method in accordance with claim 1, further comprising

(c) removing substantially all of the unencapsulated nucleic acids to provide a purified lipid bilayer-nucleic acid composition having from about 20 μ g to about 400 μ

g of nucleic acid per about 1 mg of lipid.

3. The method in accordance with claim 1, wherein said nucleic acid is a plasmid.

4. The method in accordance with claim 1, wherein said detergent is octylglucoside.

5. The method in accordance with claim 1, wherein said cationic lipid is DODAC.

6. The method in accordance with claim 1, wherein said aggregation-preventing agent is a member selected from the group consisting of gangliosides, ATTA-lipids and PEG-lipids.

7. The method in accordance with claim 6, wherein said aggregation-preventing agent is a PEG-lipid.

8. The method in accordance with claim 7, wherein said PEG-lipid is a PEG-ceramide.

9. The method in accordance with claim 8, wherein said PEG-ceramide is selected from the group consisting of PEG-Cer-C8, PEG-Cer-C 14 and PEG-Cer-C20.

10. The method in accordance with claim 1, wherein said buffered salt solution is HEPES-buffered NaCl solution.

11. The method in accordance with claim 1, wherein said buffered salt solution is a citrate solution.

12. The method in accordance with claim 1, wherein said buffered salt solution contains about 150 mM NaCl.

13. The method in accordance with claim 1, wherein about 50% to about 70% of the initial concentration of said nucleic acid becomes encapsulated.

14. The method in accordance with claim 8, wherein said cationic lipid is DODAC, said PEG-ceramide is selected from the group consisting of PEG-Cer-C8, PEG-Cer-C14 and PEG-Cer-C20, said fusogenic lipid comprises DOPE and said buffered salt solution comprises NaCl and sodium phosphate.

15. The method in accordance with claim 1, wherein the lipid bilayer carrier-encapsulated nucleic acid formed has a mean particle diameter of from about 50 nm to about 150 nm in the absence of extrusion or sonication.

16. The method in accordance with claim 1, wherein the lipid bilayer carrier-encapsulated nucleic acid formed has a mean particle diameter of from about 50 nm to about 90 nm in the absence of extrusion or sonication.

17. A method for introducing a nucleic acid into a cell, said method comprising:

(a) preparing a lipid-nucleic acid composition according to claim 1; and

(b) contacting said cell with said lipid-nucleic acid composition for a period of time sufficient to introduce said nucleic acid into said cell.

18. The method in accordance with claim 17, wherein said cell is a spleen cell.

19. The method in accordance with claim 17, wherein the efficiency of transfection is not diminished by repeat doses administered within 2 weeks.