[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Baculovirus is an insect virus that is non-pathogenic to humans and has emerged as a promising gene therapy vector. Since solid tumor growth/metastasis critically relies on angiogenesis and hEA, a fusion protein comprising human endostatin and angiostatin, exhibits potent antiangiogenic and antitumor efficacy in mouse models; this study aimed to evaluate the feasibility of baculovirus for hEA expression and antiangiogenesis-based cancer gene therapy. Toward this end, we constructed Bac-hEA that mediated transient hEA expression and Bac-ITR-hEA that exploited the adeno-associated virus inverted terminal repeats (ITRs) for prolonged hEA expression. Western blot and ELISA analyses showed that both Bac-hEA and Bac-ITR-hEA expressed hEA in transduced mammalian cells, yet Bac-ITR-hEA only marginally prolonged the hEA expression. In comparison with Bac-hEA, nonetheless, Bac-ITR-hEA significantly enhanced the hEA expression level that concurred with augmented antiangiogenic properties, as demonstrated by cell proliferation, migration and tubule network formation assays. Importantly, intratumoral injection of Bac-ITR-hEA into prostate cancer mouse models, when compared with Bac-hEA, exerted stronger antiangiogenic effects in vivo, more potently inhibited tumor growth and significantly prolonged mouse survival. This study collectively supported the notion that hEA is an effective antiangiogenic protein and proved the potential of baculovirus as a vector for antiangiogenesis-based cancer therapy, which may be combined with chemotherapy, radiotherapy or gene therapies using other vectors.
Cancer gene therapy 06/2011; 18(9):637-45. DOI:10.1038/cgt.2011.35 · 2.42 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.