Expression of cloned genes in the lung by aerosol and liposome-based delivery

• US 6,468,798 B1
• Filed: 01/14/1998
• Issued: 10/22/2002
• Est. Priority Date: 12/17/1991
• Status: Expired due to Fees

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Zhu, Ning, Debs, Robert James
• Primary Examiner(s): McGarry, Sean
• Assistant Examiner(s): EPPS -SMITH, JANET L
• Application Number:
  US09/006,841
• Time in Patent Office: 1,742 Days
• Field of Search: 424/450, 435/6, 435/69.1, 435/91.1, 435/440, 435/455, 435/458, 435/325, 435/354, 435/366, 435/371, 435/375, 435/320.1, 536/23.1, 536/23.5, 536/24.31, 536/24.5
• US Class Current: 435/458
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A61K 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• CPC Class Codes: A61K 47/54 the modifying agent being an organic compound
• CPC Class Codes: A61K 47/543 Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
• CPC Class Codes: A61K 47/547 Chelates, e.g. Gd-DOTA or Zinc-amino acid chelates; Chelate-forming compounds, e.g. DOTA or ethylenediamine being covalently linked or complexed to the pharmacologically- or therapeutically-active agent
• CPC Class Codes: A61K 47/554 the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
• CPC Class Codes: A61K 47/6911 the form being a liposome
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61K 9/0078 for inhalation via a nebulizer such as a jet nebulizer, ultrasonic nebulizer, e.g. in the form of aqueous drug solutions or dispersions
• 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: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 11/06 Antiasthmatics
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 14/4712 Cystic fibrosis
• CPC Class Codes: C12N 15/88 using microencapsulation, e.g. using amphiphile liposome vesicle

Abstract

Methods and compositions for producing a mammal capable of expressing an exogenously supplied gene in cells of the airway are disclosed. Liposome-nucleic acid complexes are prepared then delivered via aerosol to the lung airway. The invention provides a direct method for transforming pulmonary cells, treat disorders of the lung, and for delivering substances systematically following expression in the lung.

Claims

1. A method of making an aerosolized lipid-nucleic acid complex composition, comprising:

mixing a cationic liposome and a selected nucleic acid to form a cationic lipid-nucleic acid complex, which complex does not substantially aggregate in vitro; and

,

aerosolizing the cationic lipid-nucleic acid complex to provide an aerosolized complex.

2. The method of claim 1, further comprising suspending the liposome and the nucleic acid, each in aqueous solutions, prior to mixing.

3. The method of claim 1, further comprising separately diluting the liposome or the nucleic acid prior to mixing in an aqueous buffer.

4. The method of claim 1, comprising diluting the cationic lipid nucleic acid complex in a physiologically acceptable diluent prior to aerosolization.

5. The method of claim 4, comprising diluting the cationic lipid nucleic acid complex in a physiologically acceptable diluent selected from deionized water, and 5% dextrose in water.

6. The method of claim 1, comprising diluting the cationic lipid nucleic acid complex in a physiologically acceptable diluent comprising a stabilizer or biocide.

7. The method of claim 1, further comprising measuring the mean particle size of the aerosolized complex.

8. The method of claim 1, wherein mean particle size of the complex is less than 15 μ

m after aerosolization.

9. The method of claim 1, wherein mean particle size of the complex is less than 5 μ

m after aerosolization.

10. The method of claim 1, wherein mean particle size of the complex is about 0.2 to 4 μ

m after aerosolization.

11. The method of claim 1, wherein mean particle size of the complex is between about 5 μ

m and about 10 μ

m after aerosolization.

12. The method of claim 1, further comprising packaging the cationic lipid-nucleic acid complex in a container prior to aerosolization.

13. The method of claim 1, further comprising packaging the cationic lipid-nucleic acid complex prior to aerosolization, wherein the cationic lipid-nucleic acid complex is packaged in a sterile container comprising one or more unit dose of the complex.

14. The method of claim 1, wherein the cationic lipid-nucleic acid complex is aerosolized by nebulizing the complex in a nebulizer.

15. The method of claim 1, further comprising nebulizing the cationic lipid-nucleic acid complex in a nebulizer and administering the aerosolized complex through a mouthpiece or facemask.

16. The method of claim 1, wherein the nucleic acid is a DNA and the concentration of DNA in the composition is less than 5 mg DNA/ 8 ml of composition.

17. The method of claim 1, wherein the composition does not comprise a chelating agent.

18. The method of claim 1, wherein the composition is a low-salt mixture.

19. The method of claim 1, wherein the liposome and nucleic acid are mixed by agitation.

20. The method of claim 1, wherein the liposome is prepared by mixing constituent lipids of the liposome in chloroform, followed by evaporation of the chloroform and resuspension of the liposome in an aqueous solution.

21. The method of claim 1, wherein the liposome is prepared by drying a composition comprising the lipid components of the liposome, adding an aqueous solution to the resulting dried lipid components, and vortexing or sonicating the aqueous solution added to the dried lipid components to produce liposomes.

22. The method of claim 19, further comprising extruding the resulting liposomes through a polycarbonate membrane.

23. The method of claim 1, wherein the cationic liposome comprises a non-cationic lipid.

24. The method of claim 1, wherein the cationic liposome comprises cholesterol.

25. The method of claim 1, wherein the cationic liposome is a small unilamellar vesicle (SUV).

26. The method of claim 1, wherein the cationic liposome is a multilamellar vesicle (MLV).

27. The method of claim 1, wherein the cationic liposome is a large unilamellar vesicle (LUV).

28. The method of claim 1, wherein the cationic liposome is mixed with the nucleic acid at a ratio of about 4:

1 to about 1;

10 micrograms nucleic acid to nanomoles cationic lipid.

29. The method of claim 1, wherein the cationic liposome is mixed with the nucleic acid at a ratio of about 1:

1 to about 1;

2 micrograms nucleic acid to nanomoles cationic lipid.

30. The method of claim 1, wherein the cationic liposome is between about 100 nm and 10 microns in diameter.

31. The method of claim 1, wherein the cationic liposome is between about 100 nm and 500 nm in diameter.

32. The method of claim 1, wherein the selected nucleic acid comprises DNA.

33. The method of claim 1, wherein the selected nucleic acid encodes a detectable or selectable marker.

34. The method of claim 1, wherein the selected nucleic acid encodes the human CFTR protein.

35. The method of claim 1, wherein the selected nucleic acid encodes the human CFTR protein, which protein is expressed under the control of the HCMV immediate early enhancer and promoter.

36. The method of claim 1, wherein the selected nucleic acid encodes one or more of:

a toxic peptide, a tumor suppressor, an anti-sense molecule, an anti-bacterial agent, and a cytokine.

37. The method of claim 1, wherein the cationic lipid-nucleic acid complex comprises a targeting agent.

38. The method of claim 1, wherein the nucleic acid comprises a plasmid.