Liposomal transfection of nucleic acids into animal cells

• US 5,279,833 A
• Filed: 04/04/1990
• Issued: 01/18/1994
• Est. Priority Date: 04/04/1990
• Status: Expired due to Term

• Current Assignee: Yale University
• Original Assignee: Yale University
• Inventors: Rose, John K.
• Primary Examiner(s): Griffin, Ronald W.
• Assistant Examiner(s): Kunz, Gary L.
• Application Number:
  US07/504,498
• Time in Patent Office: 1,385 Days
• Field of Search: 424/450, 536/27, 435/6, 435/240.2, 436/71, 558/169, 558/172, 564/282, 564/291, 564/463
• 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: C12N 15/88 using microencapsulation, e.g. using amphiphile liposome vesicle

Abstract

A reagent for introducing a nucleic acid into an animal cell. The reagent comprises (a) a neutral lipid such as dioleoylphosphatidylethanolamine and (b) a cationic lipid selected from the group consisting of an ammonium salt such as dimethyldioctadecylammonium bromide or cetyldimethylethylammonium bromide, an amine such as stearylamine, and a benzethonium salt such as methylbenzethonium chloride

Claims

1. A liposome for introducing a nucleic acid into an animal cell comprising

(a) a neutral lipid selected from the group consisting of lecithin, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphinogomyelin, cephalin, cardiolipin, phosphatidic acid, cerebrosides, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, palmitoyloleoylphosphatidylcholine, palmitoyloleoylphosphatidylethanolamine, diheptadecanoylphosphatidylethanolamine, dilauroylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, distearoylphosphatidylethanolamine, beta-linoleoyl-gamma-palmitoylphosphatidylethanolamine and beta-oleoyl-gamma-palmitoylphosphatidylethanolamine, and

(b) a cationic lipid selection from the group consisting of dimethyldioctadecylammonium bromide, cetylidimethylethylammonium bromide, stearylamine and methylbenzethonium chloride, wherein the weight ratio of the cationic lipid to the neutral lipid is 6:

30 to 12;

30.

2. The liposome of claim 1, wherein the neutral lipid is dioleoylphosphatidylethanolamine.

3. The liposome of claim 1, wherein the cationic lipid is cetyldimethylethylammonium bromide.

4. The liposome of claim 1, wherein the cationic lipid is dimethyldioctadecylammonium bromide.

5. The liposome of claim 1, wherein the cationic lipid is stearylamine.

6. The liposome of claim 1, wherein the cationic lipid is methylbenzothonium chloride.

7. A composition comprising the liposome of claim 1, a nucleic acid, and an animal cell selected from the group consisting of arythrocytes, baby hamster kidney cells, AtI 20 cells, RK-13 cells, HeLa cells, Madin-Darby canine kidney cells and monkey COS cells.

8. A method for introducing RNA or DNA into an animal cell comprising (a) contacting said RNA or DNA with the liposome of claim 1 and then (b) contacting said liposome-RNA or liposome-DNA complex with an animal cell.

9. The method according to claim 8, wherein the method is carried out at a temperature of 37°

C. for a period of three hours.

10. The method according to claim 8, wherein the animal cell is selected from the group consisting of arythrocytes, baby hamster kidney cells, AtI 20 cells, RK-13 cells, Heln cells, Madin-Darby canine kidney cells and monkey COS cells.

11. The method according to claim 9, wherein the animal cell is selected from the group consisting of erythrocytes, baby hamster kidney cells, AtI 20 cells, RK-13 cells, Heln cells, Madin-Darby canine kidney cells and monkey COS cells.