This project of the gene therapy center grant is focused on developing and testing practical techniques for administering genes to the lungs. The project will evaluate the efficiency of delivery of gene vectors to the lungs of adult rabbits by installation and aerosolization techniques. Aerosolized adenovirus will be studied to evaluate the infectivity as a function of particle size and type of aerosol generator. The quantity of vector delivered to the lungs will be measured, and the efficiency of gene expression then will be measured using a reporter gene. The distribution of gene expression along the airways will be evaluated together with the cells types expressing the reporter gene. The initial vector to be studied will be an adenovirus-LacZ construct with beta-gal measured to monitor gene expression. The relative efficiencies of instilled versus aerosolized virus will be quantified, and the possible beneficial effects of surfactant (synthetic lipid and natural surfactant lipid extract) on viral spreading and gene expression will be evaluated. The cell types expressing the gene product will be evaluated by histologic and in situ techniques to achieve adequate gene expression are in hand, the time course of gene expression and possible lung toxicity will be determined. These measurements then will be repeated using an adenovirus construct containing the human CFTR gene and using plasmid and protein-DNA conjugate-vectors. The overall goal is to identify the best administration techniques to deliver vectors to the lungs that will result in efficient and persistent expression of CFTR.
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