The identification and cloning of the CF gene and the prediction of the structure of the gene product, CFTR, present the opportunity to direct therapy of CF at the basic defect. We are focusing on the feasibility of delivering wild-type CFTR mRNA and/or cDNA to the airway epithelium of non-human primates. The monkey was chosen as the experimental animal model because of the similarity of its respiratory tract system with the human. The advantages of mRNA gene transfer include the lack of potential mutagenesis by recombination with chromosomal sequences, the higher level of transient protein production, and less obvious risk. The disadvantages are the instability of the message relative to DNA, and the synthetic expense. This study will test the hypothesis that transient expression of wild-type CFTR protein in airway epithelial cells can be achieved by CFTR mRNA transfer, using in vitro cell and organ culture systems, as well as in the monkey. To explore this hypothesis, this study will: 1) optimize cationic liposomal transfection conditions; 2) optimize CFTR mRNA production and increase the stability of the CFTR message in transfected cells; 3) apply these in vitro approaches to the animal. In collaboration with Dr. Joel Jessee (Director of Eukaryotic Gene Delivery Programs, Bethesda Research Laboratories), various cationic liposomes will be tested for their efficiency and toxicity in mediating mRNA transfer. Full-length cDNA clones of CFTR have been obtained and these clones will be used to generate full-length CFTR mRNA in a cell-free transcriptional system using T7 RNA polymerase. These cDNA clones will be further modified to generate a functional mRNA with greater stability in transfected cells. Once conditions for liposomal transfection are established and a stable CFTR message is obtained, we will apply the CFTR mRNA directly to the conducting airway epithelium. In the cell culture system, we will address whether the CFTR mRNA produced in this study is able to correct the CF phenotype in established CF cell lines through the functional analysis component of the cell biology core. The results of this study will then be applied to the airway organ culture to determine the targeted cell type. With this information, this mRNA gene transfer technique will be applied to a monkey model. The efficacy of the gene transfer and the stability of the transfection will be determined in monkeys that are acutely exposed to mRNA/liposome complexes. In the event that the necessary mRNA stabilization can not be achieved, the cationic liposome formulations which have been optimized will be used for plasmid DNA delivery and testing. This information will serve as the basis for applying gene therapy to CF patients.

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