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

Recombinant adeno-associated virus (rAAV) vectors are under consideration for a wide variety of gene therapy applications. One of the limitations of the rAAV vector system has been the difficulty in producing the vector in sufficient quantity for adequate preclinical and clinical evaluation. A common method for vector production involves large-scale transient transfection of multiple plasmids into cultured cells. Because this approach might not be feasible for clinical scale manufacturing, we have sought approaches for rAAV vector production that avoid transient transfection procedures. In previously reported work, we generated an AAV packaging cell line that produces infec- tious rAAV when the vector genome is transfected into the cell line as plasmid DNA. We have now extended this approach by constructing a hybrid recombinant adenovirus (rAd) that contains a complete rAAV vector genome in the E1 region. This hybrid virus is used to deliver the rAAV genome to the packaging cell line (in the place of plasmid transfection). rAAV is produced 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.