Production of recombinant adeno-associated virus vectors using a packagingcell line and a hybrid recombinant adenovirus

Recombinant adeno-associated virus (rAAV) vectors are under consideration f or a wide variety of gene therapy applications. One of the limitations of t he rAAV vector system has been the difficulty in producing the vector in su fficient quantity for adequate preclinical and clinical evaluation. A commo n method for vector production involves large-scale transient transfection of multiple plasmids into cultured cells. Because this approach might not b e feasible for clinical scale manufacturing we have sought approaches for r AAV vector production that avoid transient transfection procedures. In prev iously reported work, we generated an AAV packaging cell line that produces infectious rAAV when the vector genome is transfected into the cell line a s plasmid DNA. We have now extended this approach by constructing a hybrid recombinant adenovirus (rAd) that contains a complete rAAV vector genome in the El region. This hybrid virus is used to deliver the rAAV genome to the packaging cell line (in the place of plasmid transfection). rAAV is produc ed when the packaging cell line is infected with the hybrid adenovirus and wild-type adenovirus. This method avoids the need for plasmid transfection and is adaptable to large-scale manufacturing processes.