Gene therapy, the correction of genetic defects by site-specific transgene expression in vivo, will revolutionize the treatment of human disease. The cystic fibrosis transmembrane conductance regulator (CFTR) gene has been clones and specific CFTR mutations which result in cystic fibrosis (CF) have been characterized in detail. However, the inability to achieve extended, high level expression of transgenes in the lung has precluded translation of this molecular cloning achievement into effective gene therapy for CF. Recently, we have demonstrated high levels of chloramphenicol acetyltransferase (CAT) reporter gene expression in the lungs of mice for up to 3 weeks following either aerosolization or intravenous (iv) injection of CAT expression vectors complexed to cationic liposomes. Immunostaining revealed that the majority of airway epithelial cells strongly express the CAT gene in vivo. We observed no histologic evidence of toxicity in any treated mice. We now propose to further develop our in vivo gene delivery technology, in order to create effective gene therapy for CF. Our 3 main goals are: 1) To maximize the level, duration and cellular-specificity of CAT reporter gene expression in the lungs of mice, using cationic liposome- mediated gene delivery by aerosol or iv administration. To accomplish these goals, we will first determine the parameters which maximize cationic liposome-mediated transfection of selected cultured cells. We will then use this data to maximize CA gene expression in mice by optimizing: A) the heterologous promoter element, B) cationic liposome formulation, C) DNA to liposome ratio, D) gene dosage and E) mean aerosol particle size and F) frequency/duration of administration. We will use immunostaining for intracellular CAT protein to identify the specific lung cell types and percentage of these cells transfected in vivo. 2) Maximize the level of duration of CFTR transgene expression in the lung using heterologous promoter elements. We will use the optimized parameters determined for the CAT reporter gene, including the most active viral promoter elements tested, to ten maximize CFTR transgene expression in the lung. 3) Use the CFTR 5' regulatory region to target CFTR transgene expression to appropriate lung cell types in vivo. We will use selected regulatory sequences derived from the 5' CFTR untranslated region fused to the CFTR coding region to mimic the endogenous pattern of CFTR gene expression in normal individuals. In summary, we will optimize the level, duration and cellular specificity of cationic liposome-mediated CFTR transgene expression we can achieve in vivo, while minimizing host toxicity. Our goal is to develop safe and effective, genetically-based in vivo therapy for CF. The ability to express the CFTR transgene in airway epithelial and sub- mucosal cells in vivo is an essential step towards achieving this goal.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
Project #
Application #
Study Section
Diabetes, Endocrinology and Metabolic Diseases B Subcommittee (DDK)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of California San Francisco
Internal Medicine/Medicine
Schools of Medicine
San Francisco
United States
Zip Code
Yu, W H; Kashani-Sabet, M; Liggitt, D et al. (1999) Topical gene delivery to murine skin. J Invest Dermatol 112:370-5
Gaensler, K M; Tu, G; Bruch, S et al. (1999) Fetal gene transfer by transuterine injection of cationic liposome-DNA complexes. Nat Biotechnol 17:1188-92
Mounkes, L C; Zhong, W; Cipres-Palacin, G et al. (1998) Proteoglycans mediate cationic liposome-DNA complex-based gene delivery in vitro and in vivo. J Biol Chem 273:26164-70
Liu, Y; Mounkes, L C; Liggitt, H D et al. (1997) Factors influencing the efficiency of cationic liposome-mediated intravenous gene delivery. Nat Biotechnol 15:167-73
Friend, D S; Papahadjopoulos, D; Debs, R J (1996) Endocytosis and intracellular processing accompanying transfection mediated by cationic liposomes. Biochim Biophys Acta 1278:41-50
Solodin, I; Brown, C S; Bruno, M S et al. (1995) A novel series of amphiphilic imidazolinium compounds for in vitro and in vivo gene delivery. Biochemistry 34:13537-44
Liu, Y; Liggitt, D; Zhong, W et al. (1995) Cationic liposome-mediated intravenous gene delivery. J Biol Chem 270:24864-70