Adeno-associated virus (AAV) Rep protein enhances the generation of a recombinant mini-adenovirus (Ad) utilizing an Ad/AAV hybrid virus

Mini-adenoviruses (mAd) deleted of all viral coding regions represent an em erging approach for transgene expression. We have exploited the unique feat ures of the adeno-associated virus (AAV) terminal repeats within the contex t of an adenovirus-adeno-associated hybrid virus (Ad/AAV) as a strategy for rapid and efficient generation of mAd. Excision and generation of mAd from the parental Ad/AAV hybrid vector was achieved in 293 cells through recomb ination but without selection for mAd production. Analysis of mAd isolated from 293 cells indicated that mid DNA exists as monomer and dimer forms wit hin the recombinant viral capsid. Formation of recombinant mAd was signific antly increased using an AAV Rep78- or Rep68-expressing cell line through R ep-mediated excision utilizing the AAV terminal repeat sequences present in the Ad/AAV hybrid virus genome. The mAd viruses were infectious and able t o transfer functional gene to A549 and HeLa cells. This approach is rapid a nd efficient, thereby providing a simplified methodology for generating mAd ,vith functional transducing capabilities.