Lipid-encapsulated polyanionic nucleic acid

• US 8,021,686 B2
• Filed: 02/08/2008
• Issued: 09/20/2011
• Est. Priority Date: 05/14/1997
• Status: Expired due to Fees

• Current Assignee: University of British Columbia
• Original Assignee: University of British Columbia
• Inventors: Debeyer, Dan, Ansell, Steven M., Scherrer, Peter, Klimuk, Sandra K., Harasym, Troy, Semple, Sean C., Cullis, Pieter R., Hope, Michael J.
• Primary Examiner(s): NAFF, DAVID M
• Application Number:
  US12/028,696
• Publication Number:
  US 20080200417A1
• Time in Patent Office: 1,320 Days
• Field of Search: None
• US Class Current: 424/450
• 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: A61K 9/1277 Processes for preparing; Proliposomes
• 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 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: Y10T 428/2984 Microcapsule with fluid core [includes liposome]

Abstract

Methods for the preparation of a lipid-nucleic acid composition are provided. According to the methods, a mixture of lipids containing a protonatable or deprotonatable lipid, for example an amino lipid and a lipid such as a PEG- or Polyamide oligomer-modified lipid is combined with a buffered aqueous solution of a charged therapeutic agent, for example polyanionic nucleic acids, to produce particles in which the therapeutic agent is encapsulated in a lipid vesicle. Surface charges on the lipid particles are at least partially neutralized to provide surface-neutralized lipid-encapsulated compositions of the therapeutic agents. The method permits the preparation of compositions with high ratios of therapeutic agent to lipid and with encapsulation efficiencies in excess of 50%.

Claims

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

a polyanionic nucleic acid;

an amino lipid comprising an amino group having a pKa of from 4 to 11;

a neutral lipid;

a sterol; and

a polyethyleneglycol-diacylglycerol (PEG-DAG) conjugate,

wherein said nucleic acid-lipid particle comprises a lipid layer surrounding and encapsulating a central region containing the polyanionic nucleic acid, and wherein the lipid layer comprises the amino lipid, the neutral lipid, the sterol, and the PEG-DAG conjugate.

2. The nucleic acid-lipid particle in accordance with claim 1, wherein said amino lipid is a member selected from the group consisting of:

(1,2-dioleyloxy-3-dimethylamino-propane (DODAP), N,N-dimethyl-2,3-dioleyl-oxy)propylamine (DODMA), and a mixture thereof.

3. The nucleic acid-lipid particle in accordance with claim 1, wherein said neutral lipid is a member selected from the group consisting of:

dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), distearoylphosphatidylcholine (DSPC), sphingomyelin and a mixture thereof.

4. The nucleic acid-lipid particle in accordance with claim 1, wherein said sterol is cholesterol.

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

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

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

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

9. A method of introducing a nucleic acid into a cell, said method comprising contacting said cell with the nucleic acid-lipid particle in accordance with claim 1.

10. The method of claim 9, wherein said nucleic acid is DNA.

11. The method of claim 9, wherein said nucleic acid is RNA.

12. The method of claim 9, wherein said nucleic acid is an oligonucleotide.

13. A method for reducing expression of a disease-associated gene in a mammalian cell, comprising exposing the cell to the nucleic acid-lipid particle in accordance with claim 1 for a period of time sufficient for the nucleic acid to enter the cell, wherein the nucleic acid has a sequence complementary to the disease-associated gene and reduces the production of the gene product of the disease-associated gene in the cell.

14. The method of claim 13, wherein said nucleic acid is RNA.

15. The method of claim 13, wherein said nucleic acid is an oligonucleotide.