Abstract
We recently developed hybrid baculovirus (BV) vectors that exploited FLPo/Frt-mediated DNA minicircle formation. Engineering of adipose-derived stem cells (ASCs) with the FLPo/Frt-based BV vectors enabled prolonged transgene expression and, after cell implantation into rabbits, ameliorated cartilage regeneration and bone repair. To translate the hybrid BV one step further towards clinical applications, here we assessed the biosafety profiles of the hybrid BV-engineered human ASCs (hASCs) in vitro and evaluated the immune responses elicited by the engineered porcine ASCs (pASCs) in large animals. We confirmed that the hybrid BV did not compromise the hASCs viability, immunosuppressive capacity and surface characteristics. Neither did the hybrid BV cause chromosomal abnormality/transgene integration in vitro nor induce tumorigenicity in vivo. In the large animal study, pASCs were engineered with the hybrid BV expressing BMP2/VEGF and implanted into femoral bone defects in mini pigs. The hybrid BV-engineered pASCs enabled prolonged BMP2/VEGF expression and triggered the healing of massive segmental bone defects, while only eliciting transient antibody, cytokine and local cellular immune responses stemming from the implantation procedure itself. These data altogether demonstrated the safety of the hybrid BV vectors for ASCs engineering and bone healing in large animals, hence implicating the potential in clinical applications. In the second part of study, we developed new BV vectors that exploit Cre/loxP-mediated microRNAs (miRNAs) sponge expression to treat osteoporotic bone defects. MicroRNAs (miRNAs) are a class of small non-coding, single-stranded RNAs, which are important regulators of various biological processes, including osteoclastogenesis, osteoblast differentiation and bone formation. Recently, many studies revealed aberrant miRNAs expression in elderly osteoporotic patients. Although many pharmacological agents prevent osteoporotic fractures, the repair of bone defects following fracture draws much less attention. Therefore, we have attempted to utilize miRNAs for repairing osteoporotic bone defects. Here, we found that bone marrow mesenchymal stem cells (BMSCs) harvested from rats with long-term estrogen deficiencies exhibited over-expression of miRNAs level (miR-30b, miR-138, miR-140 and miR-214). We unveiled that the osteogenic differentiation of osteoporotic BMSCs was enhanced by BV-mediated miR-140 or miR-214 sponges transduction, and also mitigated osteoclast maturation via a paracrine fashion after down-regulation of miR-140 or miR-214 in osteoporotic BMSCs by the BV transduction. Allotransplantation of the BMP2/miR-214 sponges-expressing osteoporotic BMSCs (LEBW/214S group) into the critical-size defect (3 mm in diameter) at the femur metaphysis of ovariectomised rat potentiated the bone healing and remodeling, filling 28% of bone volume/total volume (BV/TV) at 4 weeks in comparison to non-operated OVX group (22% of BV/TV). The LEBW/214S group not only accelerated the healing, but also ameliorated the bone quality (increasing density, trabecular number, trabecular thickness and decreasing trabecular space), as evaluated by micro computed tomography, histology and immunohistochemical staining. This study provided a new avenue to treatment of osteoporotic bone defects using miRNA-modulated BMSCs. Taken together, we demonstrated the safety of the FLPo/Frt hybrid BV vectors for ASCs engineering and bone healing in large animals. Furthermore, we also confirmed the feasibility of the Cre/loxP hybrid BV-mediated BMP2/miR-214 sponges-expressing osteoporotic BMSCs for osteoporotic bone healing.