Development of an insect cell-based adeno-associated virus packaging cell line employing advanced gene expression control system.

The insect cell-based baculovirus expression vector (BEV) system is a leading platform for scalable production of adeno-associated viruses (AAVs). The previously described One-Bac system consists of an insect packaging cell line harboring the AAV and genes and a BEV carrying the transgene and AAV inverted terminal repeats. Here we describe a new system where we successfully translated the molecular design of a double AAV expression cassette to inducible plasmid vectors. These optimized plasmid vectors employ non-canonical late promoters and alternative start codons that alleviate promoter-promoter competition. Because too much expression can be toxic to the host cells, tighter regulation of AAV expression is warranted. This has been achieved by adopting alternate baculovirus homologous region enhancers. Inoculation of the resultant stable insect packaging cell line by a recombinant BEV produced high-titer recombinant AAV (rAAV) preparations (1 × 10 genome copies/mL). Sequential batch reactor experiments indicate that this system is amenable to large-scale AAV production. We generated an insect packaging cell line that employs an optimized gene control system, ensuring stable and appropriate Rep expression. This platform produces potent and high-yield AAV particles and demonstrates potential for scale up.