Enhanced and prolonged baculovirus-mediated expression by incorporating recombinase system and in cis elements: a comparative study

Abstract

Baculovirus (BV) is a promising gene vector but mediates transient expression. To prolong the expression, we developed a binary system whereby the transgene in the substrate BV was excised by the recombinase (ΦC31o, Cre or FLPo) expressed by a second BV and recombined into smaller minicircle. The recombination efficiency was lower by ΦC31o (≈40–75%), but approached ≈90–95% by Cre and FLPo in various cell lines and stem cells [e.g. human adipose-derived stem cells (hASCs)]. Compared with FLPo, Cre exerted higher expression level and lower negative effects; thus, we incorporated additional cis-acting element [oriP/Epstein–Barr virus nuclear antigen 1 (EBNA1), scaffold/matrix attached region or human origin of replication (ori)] into the Cre-based BV system. In proliferating cells, only oriP/EBNA1 prolonged the transgene expression and maintained the episomal minicircles for 30 days without inadvertent integration, whereas BV genome was degraded in 10 days. When delivering bmp2 or vegf genes, the efficient recombination/minicircle formation prolonged and enhanced the growth factor expression in hASCs. The prolonged bone morphogenetic protein 2 expression ameliorated the osteogenesis of hASCs, a stem cell with poor osteogenesis potential. Altogether, this BV vector exploiting Cre-mediated recombination and oriP/EBNA1 conferred remarkably high recombination efficiency, which prolonged and enhanced the transgene expression in dividing and non-dividing cells, thereby broadening the applications of BV.

Synthetic switch-based baculovirus for transgene expression control and selective killing of hepatocellular carcinoma cells

“…The resultant BVs were amplified by infecting Sf-9 cells and the virus titer (pfu/ml) was determined as described (36). …”

Healing of massive segmental femoral bone defects in minipigs by allogenic ASCs engineered with FLPo/Frt-based baculovirus vectors

“…Baculovirus vectors BacFLPo (expressing FLPo [14]) and the substrate vectors BacFCBW (expressing human BMP2 [22]) and BacFCVW (expressing human VEGF [22]) were constructed previously (Fig. 1A).…”

“…Importantly, baculovirus transduces rabbit chondrocytes [13], bone marrowderived mesenchymal stem cells (BMSCs) and adipose-derived stem cells (ASCs) at efficiencies exceeding 95% [14]. Both BMSCs and ASCs have been extensively exploited for tissue regeneration, but ASCs are inferior to BMSCs in osteogenesis potential [15,16], although ASCs can be easily be isolated in abundance.…”

“…However, baculovirus does not integrate the transgene into chromosomes [14] and baculovirus genome is degraded with time [17,18]. Therefore, baculovirus mediates short-term (<7 days) transgene expression in mammalian cells [8,19,20].…”

“…The key to the prolonged BMP2/VEGF expression was the FLP/Frt-mediated DNA minicircle formation which, however, occurred in only z40e50% of rabbit ASCs. To improve the efficiency, we recently developed a new version of baculoviral vector (BacFLPo) that exploited a codonoptimized version of FLP recombinase (FLPo), which tremendously enhanced the recombination efficiency in rabbit ASCs and BMSCs to >90% [14]. This FLPo/Frt-based hybrid baculovirus system has been exploited for prolonged and enhanced expression of bone morphogenetic protein 6 (BMP-6) and transforming growth factor b3 (TGF-b3) [23].…”