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.
