Lipid-encapsulated polyanionic nucleic acid
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%.
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.