Abstract
Baculovirus has been regarded safe and has captured growing attention as an promising gene delivery vector. However, the transient expression nature due to its inability to replicate in mammalian cells limits its widespread application. To prolong the expression, we developed a binary baculovirus system whereby one baculovirus expressed FLP recombinase while the other harbored an Frt-flanking cassette encompassing Epstein-Barr virus oriP/EBN-1, which are essential for the episomal maintenance of the EBV genome in latently infected cells, and the transgene expression cassette. 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 HEK293 cells, 85% in BHK cells, 77% in primary chondrocytes and 48% in mesenchymal stem cells (MSCs), and the episomally-maintained plasmids were rapidly generated at 2 days post-trasnduction. The hybrid baculovirus substantially prolonged the transgene expression to 48 days without selection and >63 days with selection (50 □g/ml G418). This results indicated that the system is capable of sustained transgene expression without integration into the host genome, 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 (bone morphogenetic protein-2) was efficiently delivered into MSCs using this system, which not only prolonged the growth factor expression but also potentiated the osteogenesis of MSCs. On the other hand, we constructed a baculovirus reprogramming system in which multiple reprogramming factor genes, which induce the reprogramming of somatic cells into induced pluripotent stem cells (iPSCs), are fused in-frame into a single open reading frame (ORF) via self-cleaving 2A sequences. This system was capable of efficiently expressed multiple reprogramming factors in somatic cells, suggested this system may provide a valuable tool for generation of iPSCs cells. These data collectively implicate the potential of this hybrid baculovirus system in gene therapy applications necessitating sustained transgene expression.