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

• US 5,756,353 A
• Filed: 06/07/1995
• Issued: 05/26/1998
• 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: Debs, Robert J.
• Primary Examiner(s): RORIES, CHARLES
• Application Number:
  US08/487,793
• Time in Patent Office: 1,084 Days
• Field of Search: 514/44, 424/450, 435/172.3, 435/6, 435/375, 435/377, 435/69.1, 435/91.1, 435/172.1, 435/320.1, 935/62, 935/54, 935/55, 935/70, 935/71, 128/200.14, 128/200.18, 128/200.23, 128/200.24, 536/24.1
• US Class Current: 514/44.00R
• 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 47/543 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: C07K 14/4712 Cystic fibrosis
• CPC Class Codes: C12N 15/88 using microencapsulation, e.g. using amphiphile liposome vesicle

Abstract

Methods for the delivery of genes directly to the lungs are disclosed. Liposome-nucleic acid complexes are delivered via aerosol with the subsequent in viva expression of a protein encoded by the delivered gene. The invention provides a convenient method for treating pulmonary disorders, as well as for delivering substances systemically via the lung.

Claims

1. A method of introducing a DNA molecule into a cell of the proximal or distal airways of a mammal, which DNA molecule comprises expression regulatory elements operatively linked to a nucleotide sequence to be expressed in said cells, the steps of the method comprising administering an aerosolized composition comprising the lipid-DNA complex to the nasal or oral passages of the mammal, wherein the composition comprising the lipid-DNA complex is substantially free of macroaggregates, and whereby the lipid-DNA complex contacts the airway of the mammal and the DNA molecule is introduced into a cell of the airway, and the nucleotide sequence is expressed in the cell.

2. The method of claim 1, wherein the DNA molecule is introduced into a lung cell in the lung of the mammal.

3. The method of claim 1, wherein the cationic lipid comprises a cationic lipid selected from the group consisting of DOTMA, DDAB, and DOTAP.

4. The method of claim 1, wherein the cationic lipid-DNA complex further comprises cholesterol.

5. The method of claim 4, wherein the cationic lipid and cholesterol are in a mass ratio of about 2:

1.

6. The method of claim 1, wherein the cationic lipid-DNA complex further comprises DOPE.

7. The method of claim 1, wherein the cationic lipid-DNA complex further comprises a neutral or anionic lipid.

8. The method of claim 1, wherein the cationic lipid-DNA complex further comprises a non-cationic lipid selected from the group consisting of phosphatidyl choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline, dioleoylphosphatidyl glycerol, and, dioleoylphosphatidyl ethanolamine.

9. The method of claim 1, the complex prepared from an MLV, an LUV, or an SUV.

10. The method of claim 1, wherein the complex comprises DNA and cationic lipid components in a ratio of about 1 mg DNA:

1 micromole cationic lipid.

11. The method of claim 1, wherein the complex is administered in a pharmaceutically acceptable excipient.

12. The method of claim 1, wherein the complex is substantially free of chelating agents.

13. The method of claim 1, wherein the complex is aerosolized in a nebulizer.

14. The method of claim 1, wherein the cationic lipid-DNA complex in aerosol form has a mean particle diameter of less than about 15 μ

m.

15. The method of claim 1, wherein the cationic lipid-DNA complex in aerosol form has a mean particle diameter of less than about 5 μ

m.

16. The method of claim 1, wherein the cationic lipid-DNA complex in aerosol form has a mean particle diameter of less than 2 μ

m.

17. The method of claim 1, wherein the cationic lipid-DNA complex comprises a nucleic acid base analog.

18. The method of claim 1, wherein the mammal is a mouse.

19. The method of claim 1 wherein expression of the nucleotide sequence results in production of a polypeptide encoded by the nucleotide sequence.

20. The method of claim 1, wherein the mammal has a disease selected from the group consisting of:

lung cancer, emphysema, asthma, bronchitis, pneumonia, cystic fibrosis and α

-1 antitrypsin deficiency.

21. The method of claim 1 wherein the expression regulatory elements include a promoter selected from the group consisting of:

an SV40 promoter, a CMV promoter, an HSV promoter, an RSV promoter, an MMTV promoter, a T7 promoter, and a T3 promoter.

22. The method of claim 1, wherein the complex comprises a DNA encoding a protein, which DNA comprises a promoter sequence which regulates transcription of an mRNA encoding the protein in a host cell in the mammal, wherein the protein comprises a secretory signal.

23. The method of claim 1, further comprising repeated administration of the complex in aerosol form to the mammal.

24. The method of claim 1, further comprising repeated administration of the complex in aerosol form to the mammal for between about 4 and about 20 days.

25. The method of claim 1, wherein the delivery of the complex to the lung provides for the transduction of cells in the lung, thereby making transformed cells, the method further comprising repeated administration of the complex in aerosol form to the mammal to replace transformed cells lost to natural turn over of lung cells.