884. Integration of a Helper-Dependent Adenovirus Mediated by the Phage |[Phi]|C31 Integrase

Adenoviral vectors (Ad) deleted of all viral genes, known as helper-dependent Ad (HD) or gutted Ad, possess a linear genomic DNA that remains as a non-integrated episome in the nucleus of the infected cells. Because HD can be grown to very high titers, are weakly immunogenic and can carry up to 35-kb of foreign DNA, they are very attractive vectors for gene therapy applications. Under certain circumstances (for example to stably express large cDNAs such as dystrophin into stem cells or myoblasts) it could be valuable to promote integration of HD into the cell chromosomes. The phage C31 integrase normally mediates integration of a phage DNA containing an attP site into the attB site of the bacterial chromosome. Interestingly, when the C31 integrase is expressed in mammalian cells, it can mediate integration of a circular DNA containing an attB site into pseudo attP sites of the cell chromosomes. In the present study, we tested whether the C31 integrase could promote integration of an HD into mammalian cells. We have thus constructed an HD containing an attB site, which carries as a gene reporter, a green fluorescent protein (GFP) expression cassette. In addition, the GFP expression cassette was flanked by two loxP sites to permit its circularization upon expression of the Cre recombinase. HeLa cells were transfected with plasmids expressing Cre and the C31 integrase. The next day, the cells were transduced with HD-GFPattB and at selected time points, without any selective pressure, the expression of GFP was quantified by flow cytometry. At twelve days post-transduction, the fluorescence index ([% of GFP positive cells] X [intensity of fluorescence]) was significantly higher in cells transfected with Cre and the C31 integrase. This observation suggests these two enzymes are able to promote integration of HD-GFPattB. Experiments are in progress to optimize the integration efficiency and to test if Cre expression is essential for this process to occur.