(582ao) Enhanced and Sustained Transgene Expression Mediated By a Binary Baculoviral System

Baculovirus (BV) mediates transient expression (< 7 days) partly due to its large genome. To prolong the expression, we developed a binary system whereby the transgene (flanked by attP/B, loxP or Frt sites) in the substrate BV was excised by the recombinase (ΦC31o, Cre or FLPo) expressed by a second BV and recombined into smaller minicircles. The recombination efficiency conferred by ΦC31o was lower (≈40~75%), but approached ≈90~95% by Cre and FLPo in various cell lines (HEK293, HeLa, BHK, etc.) and stem cells (e.g. human adipose-derived stem cells, hASCs). Compared with FLPo, Cre exerted higher expression level and cell viability (≈95~100%), thus the Cre-based BV was engineered to incorporate additional cis-acting element to help the replication of the minicircles, inculuding: oriP/EBNA1, S/MAR or human origin of replication (ori). In HEK293 cells, only oriP/EBNA1 prolonged the transgene expression and maintained the episomal minicircles for 30 days without inadvertent integration, while the BV genome was degraded in 10 days. Upon delivering bmp2 or vegf genes, the efficient recombination/minicircle formation substantially prolonged and enhanced the growth factor expression in hASCs, and the prolonged BMP2 expression concomitantly ameliorated the osteogenesis of hASCs, a stem cell with poor osteogenesis potential. Altogether, this binary BV system exploiting Cre-mediated recombination and oriP/EBNA1 effectively enhanced and sustained the transgene expression in dividing and non-dividing cells, thereby broadening the applications of BV.