(Taken directly from the application) Our major objective in this proposal is to design, construct, and test replication-incompetent adenovirus vectors for cystic fibrosis gene therapy. The replication-incompetent vectors should elicit less inflammation after transduction than the replication-defective vectors currently in use, and should therefore increase the efficiency of transduction and the length of time that the transduced gene is expressed. The replication-incompetent vectors will be constructed by deleting adenovirus genes whose products are essential for virus replication and complementing the defective viruses using cell lines that express the virus gene product(s). The effects of E1A, E1B, and E3 expression on the efficiency of transduction and the degree of inflammation will be examined by constructing and comparing viruses that either have these genes present or deleted. The Escherichia coli LacZ gene will be introduced into the viruses as a reporter. The replication-incompetent viruses will then be tested in an inflamed rat lung model of cystic fibrosis to determine the level of inflammation elicited (by staining sections of the transduced lung for inflammatory cells and comparing to untransduced control lung tissue), the efficiency of transduction, the length of expression of the LacZ gene, and the ability of the vector to permit repeated administration without losing transduction efficiency (the latter three tested by staining for beta-galactosidase activity). Using the data generated, the most efficient vector will be designed and constructed. These studies will serve as a prelude to testing of the virus as a gene therapy vector for cystic fibrosis.
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