Figure 4

Overview of the initial recombinant AAV production system. The generation of the first infectious clones of AAV permitted functional dissection of the virus genome. This allowed the construction of plasmids encoding rAAV genomes in which the minimal complement of wild-type sequences necessary for genome replication and packaging (i.e., the AAV ITRs) frame a gene of interest (transgene) instead of the AAV rep and cap genes. When these constructs are transfected into packaging cells together with a rep and cap expression plasmid they lead to the production of rAAV particles. Helper activities required for the activation and support of the productive phase of the AAV life cycle were originally introduced by infection of the packaging cells with wild-type Ad as depicted. Current transfection-based production methods make use of recombinant DNA encoding the helper activities instead of Ad infection. Cellular DNA polymerase activities together with the Rep78 and Rep68 proteins lead to the accumulation of replicative intermediates both in the duplex monomer (DM) and duplex dimer (DD) forms. A fraction of this de novo synthesized DNA is incorporated in the single-stranded format into preformed empty capsids most likely through the catalytic activities of the Rep52 and Rep40 proteins. The resulting infectious rAAV virions are released from the producer cells together with helper Ad particles. Sequential heat treatment and buoyant density centrifugation allows the selective elimination of the helper virus from the final rAAV preparation.