ABSTRACT
BACKGROUND: Occluded arteries and ischemic tissues cannot always be treated by angioplasty, stenting or by-pass-surgery. Under such circumstances, viral gene therapy may be useful in inducing increased blood supply to ischemic area. There is evidence of improved blood flow in ischemic skeletal muscle and myocardium in both animal and human studies using adenoviral vascular endothelial growth factor (VEGF) gene therapy. However, the expression is transient and repeated gene transfers with the same vector are inefficient due to immune responses. METHODS: Different baculoviral vectors pseudotyped with or without vesicular stomatitis virus glycoprotein (VSV-G) and/or carrying woodchuck hepatitis virus post-transcriptional regulatory element (Wpre) were tested both in vitro and in vivo. VEGF-D(ΔNΔC) was used as therapeutic transgene and lacZ as a control. In vivo efficacy was evaluated as capillary enlargement and transgene expression in New Zealand White (NZW) rabbit skeletal muscle. RESULTS: A statistically significant capillary enlargement was detected 6 days after gene transfer in transduced areas compared to the control gene transfers with baculovirus and adenovirus encoding ß-galactosidase (lacZ). Substantially improved gene transfer efficiency was achieved with a modified baculovirus pseudotyped with VSV-G and carrying Wpre. Dose escalation experiments revealed that either too large volume or too many virus particles caused inflammation and necrosis in the target tissue, whereas 10(9) plaque forming units injected in multiple aliquots resulted in transgene expression with only mild immune reactions. CONCLUSIONS: We show the first evidence of biologically significant baculoviral gene transfer in skeletal muscle of NZW rabbits using VEGF-D(ΔNΔC) as a therapeutic transgene.
Subject(s)
Baculoviridae/genetics , Gene Transfer Techniques , Muscle, Skeletal/blood supply , Neovascularization, Physiologic/genetics , Vascular Endothelial Growth Factor D/genetics , Vascular Endothelial Growth Factor D/therapeutic use , Animals , Capillaries/growth & development , Capillary Permeability , Female , Gene Transfer Techniques/adverse effects , Hep G2 Cells , Humans , Muscle, Skeletal/pathology , Perfusion , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Protein Isoforms/genetics , Rabbits , Recombinant Proteins/biosynthesis , Transduction, Genetic , Treatment OutcomeABSTRACT
BACKGROUND: Baculovirus expression vector system (BEVS) has become a standard in recombinant protein production and virus-like particle preparation for numerous applications. FINDINGS: We describe here protocols which adapt baculovirus generation into 96-well format. CONCLUSION: The established methodology allows simple baculovirus generation, fast virus titering within 18 h and efficient recombinant protein production in a high-throughput format. Furthermore, the produced baculovirus vectors are compatible with gene expression in vertebrate cells in vitro and in vivo.
ABSTRACT
Baculoviruses can express transgenes in a wide range of vertebrate cells. However, in some cells transgene expression is weak. To enhance transgene expression, we studied the effect of the Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) on baculovirus (BV)-mediated gene expression of several transgenes. A significant increase in BV-mediated gene expression was detected in several cell lines. A 10-fold increase in transgene expression was observed with the WPRE as determined by the percentage of positive cells and mean fluorescence intensity (MFI). Furthermore, a combination of optimized cell culture medium and WPRE virus led to more than a 60-fold increase in gene expression. In accordance, elevated mRNA and protein levels were detected in WPRE-virus transduced cells. In HepG2 and RaaSMC, WPRE-mediated enhancement was comparable to the previously shown positive effect of sodium butyrate on BV-mediated gene expression. Thus, inclusion of the WPRE into a baculovirus vector provides a simple means to improve BV-mediated gene expression in vertebrate cells.
