Transfection of lung via aerosolized transgene delivery

• US 5,641,662 A
• Filed: 03/10/1993
• Issued: 06/24/1997
• 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): RORIES, CHARLES
• Application Number:
  US08/029,022
• Time in Patent Office: 1,567 Days
• Field of Search: 514/44, 424/450, 435/172.1, 435/172.3, 435/320.1, 935/62, 935/54, 935/55, 128/200.14, 128/200.24, 536/24.1, 436/71
• 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. Lipid carrier-nucleic acid complexes are prepared then delivered via aerosol to the lung airway. The invention provides a direct method for transforming pulmonary cells as a means for treating disorders of the lung as for providing a means for delivering substances systematically following expression in the lung.

Claims

1. A composition comprising:

a nebulized transfection agent, prepared by nebulizing a mixture comprising complexes between DNA molecules comprising expression cassettes and cationic lipid carriers in a pharmaceutically acceptable carrier, wherein the cationic lipid carriers are in the form of vesicles having diameter of about 100-500 nm, wherein said expression cassettes and said cationic lipid carriers are present in said mixture in a ratio ranging from about 4;

1 to about 1;

2 mg DNA to micromole of cationic lipid, wherein said mixture is substantially free of macroaggregates of said complexes, and wherein said expression cassettes comprise a DNA sequence that is transcribed to produce a transcription product in vivo in a mammalian lung cell transfected by said nebulized transfection agent.

2. The composition according to claim 1, wherein said DNA sequence comprises an inducible promoter.

3. The composition according to claim 2, wherein said inducible promoter is a cell specific promoter, a tissue specific promoter or a hormone responsive promoter.

4. The composition according to claim 2, wherein said DNA sequence comprises an SV40 enhancer element whereby transcription from said promoter is enhanced.

5. A method of transfecting mammalian lung cells in vivo and obtaining synthesis of a polypeptide in said cells, said method comprising contacting said cells with a sufficient amount of a nebulized composition according to claim 1 to produce transfected cells that synthesize said polypeptide.

6. The method according to claim 5, wherein said lung cells are distal airway cells or proximal airway cells.

7. The method according to claim 5, wherein said lung cells are tracheal cells.

8. The method according claim 5, wherein said lung cells are normal lung cells.

9. The composition according to claim 1, wherein said cationic lipid carriers are small unilamellar vesicles.

10. The composition according to claim 9, wherein said small unilamellar vesicles comprise (a) dioleoyl-phosphatidylethanolamine (DOPE) and N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) or (b) dimethyldioctadecylammoniumbromide (DDAB) and cholesterol.

11. The composition according to claim 1, wherein said expression cassettes and said cationic lipid carriers are present in said mixture in a ratio in the range of from about 1:

1 to 1;

2 mg DNA to μ

mol of cationic lipid.

12. The composition according to claim 1, wherein said cationic lipid carrier comprises a lipid selected from the group consisting of N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA);

dimethyl dioctadecyl ammonium bromide (DDAB);

1,2-dioleyloxy-3-(trimethylammonio) propane (DOTAP);

lysinylphosphatidyl-ethanolamine (L-PE);

dioleoylphosphatidylethanolamine (DOPE); and

cholesterol.

13. The composition according to claim 1, wherein said cationic lipid carrier comprises cholesterol and a lipid selected from the group consisting of N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA);

dimethyldioctadecylammoniumbromide (DDAB);

1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP); and

lysinylphosphatidyl-ethanolamine (L-PE).

14. The composition according to claim 1, wherein said cationic lipid carrier comprises dioleoylphosphatidylethanolamine (DOPE) and a lipid selected from the group consisting of N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA);

dimethyldioctadecylammoniumbromide (DOTAP);

1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP); and

lysinylphosphatidyl-ethanolamine (L-PE).

15. A method for transfecting cells lining conducting airways and alveoli of a mammalian lung, said method comprising:

introducing a sufficient amount of a composition according to claim 1 into said lung via intraoral or intranasal delivery to transfect said airways and said alveoli.