Self-amplification system for recombinant adeno-associated virus production.
A recently reported system for recombinant adeno-associated virus (rAAV) production does not require infection of a helper virus and depends on the transfection 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 plasmid as a source of rAAV DNA and determined the optimal timing of infection and dose of the input rAAV. When 293 cells were infected just after transfection with 100 particles/cell of rAAV, irrespective of the purity, CsCl-purified or crude, up to 2000 particles/cell of rAAV were produced (9- to 20-fold self-amplification), a yield comparable to that obtained by an adenovirus-free transfection. These results indicate that infection of rAAV can greatly reduce the amount of plasmid DNA for a large-scale transfection. This strategy will also be useful when applied to packaging cell lines inducibly expressing Rep and Cap proteins.
Urabe
M
M
Division of Genetic Therapeutics, Jichi Medical School, Tochigi, Japan.
Shimazaki
K
K
Saga
Y
Y
Okada
T
T
Kume
A
A
Tobita
K
K
Ozawa
K
K
D002478:Cells, Cultured; D004274:DNA, Recombinant / Q000502:physiology; D004279:DNA, Viral / Q000502:physiology; D000229:Dependovirus / Q000235:genetics / Q000502:physiology*; D005822:Genetic Vectors; D006801:Humans; D014161:Transduction, Genetic; D014162:Transfection; D014779:Virus Replication
D004274:DNA, Recombinant; D004279:DNA, Viral