Self-amplification system for recombinant adeno-associated virus production

A recently reported system for recombinant adenoassociated virus (rAAV) pro duction does not require infection of a helper virus and depends on the tra nsfection with a huge amount of three plasmids: AAV-vector, AAV-helper, and adenovirus-helper plasmids. Toward simplifying rAAV production, as a first step, we tested the use of the rAAV itself instead of the AAV-vector plasm id as a source of rAAV DNA and determined the optimal timing of infection a nd dose of the input rAAV. When 293 cells were infected just after transfec tion with 100 particles/cell of rAAV, irrespective of the purity, CsCl-puri fied or crude, up to 2000 particles/cell of rAAV were produced (9- to 20-fo ld self-amplification), a yield comparable to that obtained by an adenoviru s-free transfection. These results indicate that infection of rAAV can grea tly reduce the amount of plasmid DNA for a large-scale transfection. This s trategy will also be useful when applied to packaging cell lines inducibly expressing Rep and Cap proteins. (C) 2000 Academic Press.