Mammalian transformation complex comprising a lipid carrier and DNA encoding CFTR

• US 6,620,795 B1
• Filed: 12/14/1999
• Issued: 09/16/2003
• 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., Zhu, Ning
• Primary Examiner(s): Wehbe' , Anne M.
• Assistant Examiner(s): LI, QIAN JANICE
• Application Number:
  US09/461,686
• Time in Patent Office: 1,372 Days
• Field of Search: 514/44, 435/320.1, 435/92.1, 435/458, 424/450, 536/23.1
• 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 47/54 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 47/544 Phospholipids
• 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 14/00 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• CPC Class Codes: C07K 14/4712 Cystic fibrosis
• CPC Class Codes: C07K 14/55 IL-2
• 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 or nucleic acid abre are prepared then delivered via aerosol or systemically to the lung abre or lung plus extrapulmonary tissues. The invention provides a direct method for transforming pulmonary cells as a means for treating the manifestations of CF in the lung and involved extrapulmonary tissues.

Claims

1. A pharmaceutical composition, said composition comprising a mammalian transcription complex in a pharmaceutically acceptable carrier or diluent;

said complex comprising a cationic lipid carrier and a plasmid having a transcription or expression cassette comprising a nucleotide sequence encoding a wild-type cystic fibrosis transmembrane conductance regulator protein, wherein said cassette is capable of being expressed to produce said protein in mammalian cells transfected with said cassette; and

wherein said lipid carrier comprises cationic and noncationic lipids, and wherein said complex has a ratio of between about 4;

1 to 1;

10 micrograms DNA to nanomoles cationic lipid; and

said complex does not aggregate in vitro.

2. The pharmaceutical composition according to claim 1, wherein said cassette comprises an inducible promoter.

3. The pharmaceutical 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 pharmaceutical composition according to claim 2, wherein said promoter is a promoter from a cystic fibrosis transmembrane conductance regulator gene.

5. The pharmaceutical composition according to claim 2, wherein said cassette comprises an SV40 enhancer element whereby transcription from said promoter is enhanced.

6. The pharmaceutical 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-dioleoyloxy-3-(trimethylammonio) propane (DOTAP);

lysinylphosphatidyl-ethanolamine (L-PE);

dioleoyl phosphatidylethanolamine (DOPE); and

cholesterol (Chol).

7. A method for increasing the amount of pulmonary cystic fibrosis transmembrane conductance regulator (CFTR) activity in a mammal in need thereof, said method comprising administering by inhalation to the lung of said mammal a therapeutically effective amount of a composition according to claim 6.

8. A method according to claim 7, wherein the mammal is a human.

9. A method according to claim 8, wherein said cystic fibrosis transmembrane conductance regulator (CFTR) protein is encoded by a human wild-type nucleotide sequence.

10. A method according to claim 9, wherein said human has cystic fibrosis.

11. The pharmaceutical 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).

12. The pharmaceutical composition according to claim 1, wherein said cationic lipid carrier comprises dioleoyl phosphatidylethanolamine (DOPE) 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).

13. The pharmaceutical composition according to claim 1, wherein said cationic lipid carrier is a small unilamellar vesicle and said complex is between about 100 nanometers and 10 microns in diameter.

14. The pharmaceutical composition according to claim 13, wherein said small unilamellar vesicle comprises (a) dioleoyl phosphatidylethanolamine (DOPE) and N[1-(2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) or (b) dimethyldioctadecyl-ammoniumbromide (DDAB) and cholesterol.

15. The pharmaceutical composition according to claim 1, wherein said cassette comprises an open reading frame nucleic acid sequence encoding a wild-type cystic fibrosis transmembrane conductance regulator protein that is intron-free.

16. The pharmaceutical composition according to claim 1, wherein said cassette comprises an open reading frame nucleic acid sequence encoding a wild-type cystic fibrosis transmembrane conductance regulator protein that has an intron 5′

to said open reading frame nucleic acid sequence.

17. The pharmaceutical composition according to claim 1, wherein said cassette comprises an open reading frame nucleic acid sequence encoding a wild-type cystic fibrosis transmembrane conductance regulator protein and has an expanded intron 3′

to said open reading frame nucleic acid sequence.

18. The pharmaceutical composition according to claim 1, wherein said pharmaceutically acceptable carrier or diluent is acceptable by intraoral or intranasal administration.

19. The pharmaceutical composition according to claim 1, wherein the pharmaceutically acceptable carrier or diluent is acceptable for intravenous administration.

20. The pharmaceutical composition according to claim 1, wherein the cystic fibrosis transmembrane conductance regulator (CFTR) protein is encoded by a human wild-type nucleotide sequence.

21. A method for increasing the amount of pulmonary cystic fibrosis transmembrane conductance regulator (CFTR) activity in a mammal in need thereof, said method comprising administering by inhalation to the lung of said mammal a therapeutically effective amount of a composition according to claim 1.

22. A method according to claim 21, wherein the mammal is a human.

23. A method according to claim 22, wherein said cystic fibrosis transmembrane conductance regulator (CFTR) protein is encoded by a human wild-type nucleotide sequence.

24. A method according to claim 22, wherein said human has cystic fibrosis.

25. A lit, said kit comprising:

a first container containing a plasmid having a transcription or expression cassette comprising a nucleotide sequence encoding a wild-type cystic fibrosis transmembrane conductance regulator protein and wherein said cassette is capable of being expressed to produce said protein in mammalian cells transfected with said cassette; and

a second container containing a specific amount of a cationic lipid carrier comprising cationic and noncationic lipids.

26. A kit according to claim 25, said kit further comprising a nebulizer.