Baculovirus is a promising gene delivery vector but its widespread application is impeded as it only mediates transient transgene expression in mammalian cells. To prolong the expression, we developed a dual baculovirus system whereby one baculovirus expressed FLP recombinase while the other harbored an Frt-flanking cassette encompassing the transgene and oriP/EBNA1 derived from Epstein-Barr virus. After cotransduction of cells, the expressed FLP cleaved the Frt-flanking cassette off the baculovirus genome and catalyzed circular episome formation, then oriP/EBNA1 within the cassette enabled the self-replication of episomes. The excision/recombination efficiency was remarkably enhanced by sodium butyrate, reaching 75% in human embryonic kidney-293 (HEK293) cells, 85% in baby-hamster kidney (BHK) cells, 77% in primary chondrocytes, and 48% in mesenchymal stem cells (MSCs). The hybrid baculovirus substantially prolonged the transgene expression to approximately 48 days without selection and >63 days with selection, thanks to the maintenance of replicons and transgene transcription. In contrast to the replicating episomes, the baculovirus genome was rapidly degraded. Furthermore, an osteoinductive growth factor gene was efficiently delivered into MSCs using this system, which not only prolonged the growth factor expression but also potentiated the osteogenesis of MSCs. These data collectively implicate the potential of this hybrid baculovirus system in gene therapy applications necessitating sustained transgene expression.
The production of recombinant adeno-associated virus (rAAV) commonly requires plasmid cotransfection, which hinders its mass production. Herein we describe the development of a novel process for rAAV production by combining the advantages of baculovirus-mediated gene delivery and BelloCell bioreactor (a novel packed-bed reactor for animal cell culture; CESCO Bioengineering, Hsinchu, Taiwan). We constructed three baculoviral vectors: Bac-LacZ carries the lacZ gene flanked by AAV inverted terminal repeats, Bac-RC harbors AAV rep and cap genes, and Bac-Helper carries helper genes derived from adenovirus. Cotransduction of HEK-293 cells with these three baculoviruses resulted in successful production of rAAV, and the protein and rAAV yield did not decrease with Bac-RC passage for up to four passages. By adjusting the dose ratio of Bac-LacZ to Bac-RC, adding sodium butyrate, and transferring the production process to the BelloCell-500-AP (500 ml), which allowed for high-density culture and effective baculovirus-mediated transduction of HEK-293 cells, the maximal specific rAAV yield reached approximately 3.8 x 10(4) vector genome (VG) or 247 infectious viral particles (IVP) per cell, which corresponded to approximately 1 x 10(14) VG or 8.5 x 10(11) IVP per reactor run. The yield was comparable or superior to those obtained with other production systems. Baculoviral transduction is simple and cost-effective and the BelloCell-500-AP offers high-density culture of HEK-293 cells. Altogether, the combination of baculoviral transduction and BelloCell reactor culture provides a novel and economically viable approach for rAAV production.
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