Wen-Hsin Lo

Baculovirus as a new gene delivery vector for stem cell engineering and bone tissue engineering

Baculovirus has emerged as a novel vector for in vitro and in vivo gene delivery due to its low cytotoxicity and non-replication nature in mammalian cells, but the applications of baculovirus in the genetic modification of human mesenchymal stem cells (hMSCs) and tissue engineering are yet to be reported. In this study, we genetically engineered hMSCs with a baculovirus (Bac-CB) expressing bone morphogenetic protein-2 (BMP-2). Bac-CB transduction of hMSCs at a multiplicity of infection of 40 triggered effective differentiation of hMSCs into osteoblasts. Supertransduction at day 6 after initial transduction enhanced the BMP-2 expression and further accelerated the in vitro osteogenesis, as confirmed by alkaline phosphatase assay, Alizarin red staining and reverse transcription-polymerase chain reaction analysis of osteoblastic genes. Implantation of the supertransduced cells at ectopic sites in the nude mice resulted in efficient cell differentiation into osteoblasts at week 2 and induced progressive mineralization and partial bone formation at week 6, as confirmed by hematoxylin and eosin, immunohistochemical and Alizarin red staining. These data collectively demonstrated, for the first time, the potential of baculovirus in hMSCs engineering and implicated its use in bone tissue engineering.

The healing of critical-sized femoral segmental bone defects in rabbits using baculovirus-engineered mesenchymal stem cells

Management of massive segmental bone defects remains a challenging clinical problem and bone marrow-derived mesenchymal stem cells (BMSCs) hold promise for bone regeneration. To explore whether BMSCs engineered by baculovirus (an emerging gene delivery vector) can heal large bone defects, New Zealand White (NZW) rabbit BMSCs were transduced with the BMP2-expressing baculovirus or VEGF-expressing baculovirus, and co-implanted into critical-sized (10mm) femoral segmental defects in NZW rabbits. X-ray analysis revealed that the baculovirus-engineered BMSCs not only bridged the defects at as early as week 2, but also healed the defects in 100% of rabbits (13/13) at week 4. The osteogenic metabolism, as monitored by positron emission tomography (PET) also suggested the completion of bone healing at week 8. When compared with other control groups, the BMP2/VEGF-expressing BMSCs remarkably enhanced the segmental bone repair and mechanical properties, as evidenced by micro-computed tomography (microCT), histochemical staining and biomechanical testing. The ameliorated bone healing concurred with the augmented angiogenesis. These data demonstrated, that BMSCs engineered to express BMP2 and VEGF accelerate the repair of large femoral bone defects and improve the quality of the regenerated bone, which paves an avenue to utilizing baculovirus as a vector for BMSCs modification and regenerative medicine.

Baculovirus transduction of mesenchymal stem cells triggers the toll-like receptor 3 pathway.

ABSTRACT Human mesenchymal stem cells (hMSCs) can be genetically modified with viral vectors and hold promise as a cell source for regenerative medicine, yet how hMSCs respond to viral vector transduction remains poorly understood, leaving the safety concerns unaddressed. Here, we explored the responses of hMSCs against an emerging DNA viral vector, baculovirus (BV), and discovered that BV transduction perturbed the transcription of 816 genes associated with five signaling pathways. Surprisingly, Toll-like receptor-3 (TLR3), a receptor that generally recognizes double-stranded RNA, was apparently upregulated by BV transduction, as confirmed by microarray, PCR array, flow cytometry, and confocal microscopy. Cytokine array data showed that BV transduction triggered robust secretion of interleukin-6 (IL-6) and IL-8 but not of other inflammatory cytokines and beta interferon (IFN-β). BV transduction activated the signaling molecules (e.g., Toll/interleukin-1 receptor domain-containing adaptor-inducing IFN-β, NF-κB, and IFN regulatory factor 3) downstream of TLR3, while silencing the TLR3 gene with small interfering RNA considerably abolished cytokine expression and promoted cell migration. These data demonstrate, for the first time, that a DNA viral vector can activate the TLR3 pathway in hMSCs and lead to a cytokine expression profile distinct from that in immune cells. These findings underscore the importance of evaluating whether the TLR3 signaling cascade plays roles in the immune response provoked by other DNA vectors (e.g., adenovirus). Nonetheless, BV transduction barely disturbed surface marker expression and induced only transient and mild cytokine responses, thereby easing the safety concerns of using BV for hMSCs engineering.

Development of a Hybrid Baculoviral Vector for Sustained Transgene Expression

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 role of adipose-derived stem cells engineered with the persistently expressing hybrid baculovirus in the healing of massive bone defects

Massive segmental defects arising from trauma or tumor resection remain a challenging clinical problem. To repair large, segmental bone defects using adipose-derived stem cells (ASCs) which alone cannot heal massive defects, we hypothesized that sustained expression of factors promoting osteogenesis (BMP2) and angiogenesis (VEGF) provides continuous stimuli to augment the healing. Baculovirus is a vector for gene delivery into stem cells, but it only mediates transient expression. Therefore we developed a dual system whereby one baculovirus expressed FLP recombinase (BacFLP) while the other hybrid baculovirus harbored an Frt-flanking transgene cassette. Within the ASCs transduced with BacFLP and the hybrid baculovirus, the transduction efficiency reached 98% and the FLP/Frt-mediated recombination efficiency approached 46%, leading to cassette excision off the baculovirus genome, enabling transgene persistence in episomal form and prolonging the expression to >28 days. ASCs engineered by the conventional baculovirus transiently expressing BMP2/VEGF (S group) only healed the critical-size (10mm) segmental femoral bone defects in 40% of New Zealand White rabbits at 12 weeks post-implantation, whereas ASCs engineered by the hybrid vectors persistently expressing BMP2/VEGF (L group) healed the critical-size defects in 12 out of 12 animals in 8 weeks. Compared with the S group, the L group not only accelerated the healing, but also ameliorated the bone quality (metabolism, volume, density, mechanical properties) and angiogenesis, thereby attesting our hypothesis that persistent BMP2/VEGF expression is essential. Use of ASCs engineered by the hybrid BV vector thus holds promise to treat massive segmental defects necessitating sustained stimuli.

Baculovirus transduction of human mesenchymal stem cell-derived progenitor cells: variation of transgene expression with cellular differentiation states.

We have previously demonstrated that baculovirus can efficiently transduce human mesenchymal stem cells (MSCs). In this study, we further demonstrated, for the first time, that baculovirus can transduce adipogenic, chondrogenic and osteogenic progenitors originating from MSCs. The transduction efficiency (21–90%), transgene expression level and duration (7–41 days) varied widely with the differentiation lineages and stages of the progenitors, as determined by flow cytometry. The variation stemmed from differential transgene transcription (as revealed by real-time reverse transcription-polymerase chain reaction), rather than from variability in virus entry or cell cycle (as determined by quantitative real-time PCR and flow cytometry). Nonetheless, the baculovirus-transduced cells remained capable of differentiating into adipogenic, osteogenic and chondrogenic pathways. The susceptibility to baculovirus transduction was higher for adipogenic and osteogenic progenitors, but was lower for chondrogenic progenitors. In particular, the duration of transgene expression was prolonged in the transduced adipogenic and osteogenic progenitors (as opposed to the MSCs), implicating the possibility of extending transgene expression via a proper transduction strategy design. Taken together, baculovirus may be an attractive alternative to genetically modify adipogenic and osteogenic progenitors in the ex vivo setting for cell therapy or tissue engineering.

Combination of baculovirus-expressed BMP-2 and rotating-shaft bioreactor culture synergistically enhances cartilage formation

Baculovirus is an emerging gene delivery vector, thanks to a number of unique advantages. Herein, we genetically modified the rabbit articular chondrocytes with a recombinant baculovirus (Bac-CB) encoding bone morphogenetic protein-2 (BMP-2), which conferred high level BMP-2 expression and triggered the re-differentiation of dedifferentiated third passage (P3) chondrocytes in the monolayer culture. The transduced and mock-transduced P3 cells were seeded into porous scaffolds and cultured in either the dishes or the rotating-shaft bioreactor (RSB), a novel bioreactor imparting a dynamic, two-phase culture environment. Neither mock-transduced constructs in the RSB culture nor the Bac-CB-transduced constructs in the static culture grew into uniform cartilaginous tissues. Only the Bac-CB-transduced constructs cultured in the RSB for 3 weeks resulted in cartilaginous tissues with hyaline appearance, uniform cell distribution, cartilage-specific gene expression and considerably enhanced cartilage-specific extracellular matrix deposition, as determined by histological staining, reverse transcription-PCR analyses and biochemical assays. This is the first study demonstrating that combination of baculovirus-mediated growth factor expression and RSB culture synergistically enhanced in vitro creation of cartilaginous tissues from dedifferentiated chondrocytes. Since baculovirus transduction is generally considered safe, this approach represents a viable alternative to stimulate the formation of engineered cartilage in a more cost-effective way than the growth factor supplementation.

Biosafety Assessment of Human Mesenchymal Stem Cells Engineered by Hybrid Baculovirus Vectors

Mesenchymal stem cells (MSCs) hold promise for cell therapy, and implantation of MSCs engineered with a baculovirus transiently expressing the growth factor can augment the bone repair. To prolong the baculovirus-mediated transgene expression, we developed hybrid baculovirus vectors exploiting the FLP/Frt-mediated recombination for circular episome formation. Transduction of human MSCs with the hybrid baculovirus vectors harboring the osteoinductive bmp2 gene substantially extended the BMP2 expression and improved the cellular osteogenic differentiation. To confirm the potential in the clinical setting, the present study evaluated the biosafety profile of human MSCs engineered by the hybrid vectors. We unraveled that transduction of MSCs with the hybrid baculovirus vectors slightly impeded the cell proliferation after transduction, probably due to the perturbation of cellular gene expression and induction of innate responses. Nonetheless, the hybrid baculovirus vectors did not compromise the cell viability and cellular differentiation. No transgene integration into the host chromosome and disruption of the karyotype of the MSCs were observed. Additionally, no upregulation of proto-oncogenes or downregulation of tumor suppressor genes occurred in the MSCs transduced with the hybrid baculovirus vectors. Neither did the transduced MSCs induce tumor formation in nude mice. This study not only supported the safety of MSCs for cell therapy but also implicated the potential of the human MSCs engineered by the hybrid baculovirus vectors for their applications in clinical scenarios necessitating sustained transgene expression.

Development of the hybrid Sleeping Beauty-baculovirus vector for sustained gene expression and cancer therapy

Antiangiogenesis is an appealing anticancer approach but requires continued presence of the antiangiogenic agents, which can be remedied by gene therapy. Baculovirus is an emerging gene delivery vector but only mediates transient expression (<7 days); thus, this study primarily aimed to develop a hybrid baculovirus for sustained antiangiogenic gene expression and cancer therapy. We first constructed plasmids featuring adeno-associated virus inverted terminal repeats (AAV ITRs), oriP/Epstein-Barr virus-expressed nuclear antigen 1 (EBNA1) or Sleeping Beauty (SB) transposon and compared their efficacies in terms of persistent expression. In human embryonic kidney (HEK293) cells, AAV ITR failed to prolong the expression while oriP/EBNA1 moderately extended the expression to 35 days. In contrast, the SB system led to stable expression beyond 77 days even without antibiotic selection. Given this finding, we constructed a hybrid SB baculovirus expressing the SB transposase and harboring the transgene cassette flanked by inverted repeat/direct-repeat (IR/DR) elements recognizable by SB. The hybrid SB baculovirus efficiently transduced mammalian cells and mediated an expression duration longer than that by conventional baculoviruses, thanks to the transgene persistence and integration. The SB baculovirus (Bac-SB-T2hEA/w) expressing the antiangiogenic fusion protein comprising endostatin and angiostatin (hEA) also enabled prolonged hEA expression. With sustained hEA expression, Bac-SB-T2hEA/w repressed the angiogenesis in vivo, hindered the growth of two different tumors (prostate tumor allografts and human ovarian tumor xenografts) in mice and extended the life span of animals. These data altogether implicated the potential of the hybrid SB-baculovirus vector for prolonged hEA expression and for the treatment of multiple types of angiogenesis-dependent tumors.

Baculovirus transduction of rat articular chondrocytes: roles of cell cycle

We have previously demonstrated highly efficient baculovirus transduction of primary rat articular chondrocytes, thus implicating the possible applications of baculovirus in gene‐based cartilage tissue engineering. However, baculovirus‐mediated gene expression in the chondrocytes is transient.