Recombinant adenoviruses are promising vectors for gene therapy for a number of reasons: they can be grown to high titers, they infect both resting and dividing cells efficiently, and they can accommodate reasonable-sized inserts. First generation adenovirus vectors contain deletions in the El region (and sometimes in the E3 region) and are grown in a helper cell line, 293 cells, which expresses the viral E1A and E1B genes. Work with these viruses has pointed towards directions for vector improvement. First, the construction of recombinant viruses requires recombination in human cells in culture, which is inefficient and results in the need to screen numerous plaques for the desired recombinant. Second, deletion of El does not render the virus completely replication-defective in most cell types, resulting in low levels of viral replication leading to an immune response. A number of studies in a variety of experimental systems, however, have shown that removal of additional viral genes in not always beneficial with respect to gene delivery. Moreover, there is no simple way to predict which vector backbones will be optimal for any given system. In this project, experiments are proposed to optimize vector production so as to be able to generate sets of vectors, using different backbones but carrying the same transgene, to test for cardiovascular gene transfer. The basis for these experiments is the development of an in vitro system for generating recombinant viruses, based on the Cre-lox recombination reaction. Using this reaction, various transgene constructs will be built into El-, E1/E2- and E1/E4-defective backbones, the latter two of which will be generated in this project using novel strategies. The transgenes carried by these vectors will incorporate modifications that improve expression. Finally, experiments are proposed to manipulate the packaging of the adenovirus genome. These studies are based on the recent isolation of a mutant virus, pm8001, which is unable to package viral DNA due to the lack of expression of the L1 52/55 kDa protein. Experiments are described in which attempts will be made to modulate specific packaging of recombinant vector genomes at the expense of wild type genomes, with the ultimate goal of using this system to selectively package so-called gutted adenovirus vectors. Together, these vector improvements will facilitate gene therapy of a range of cardiovascular diseases.
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