An Adenoviral Expression System for AAV Rep78 Using Homologous Recombination

The construction and amplification of adenoviral (Ad) vectors expressing biologically active transgenes that are cytotoxic or inhibit Ad replication can be extremely difficult, if not impossible. In this study, we harnessed the ability of Ad genomes to undergo efficient homologous recombination to reconstitute the adeno-associated virus (AAV) rep78 gene, a cytotoxic gene that strongly inhibits Ad replication, which was divided between two parental, first-generation Ad vectors. A functional open reading frame was generated by recombination only upon co-infection of both parental vectors and after the onset of viral DNA replication. We were able to amplify both parental rep78 vectors to normal titers without any signs of inhibition or toxicity and could use them to generate progeny vectors containing a functional rep78 gene without any Ad genes. Using this vector recombination system in AAV rescue assays demonstrated that no Ad protein was essential for Rep78 mediated rescue of AAV ITR flanked DNA from plasmid or Ad backbones; the amount of rescue product generated was substantially greater in the presence of Ad infection; neither cellular nor viral DNA replication was necessary for rescue to occur; and progeny vector genomes were efficiently co-replicated along with conventional, first-generation Ad vectors.