Baculovirus-mediated vascular endothelial growth factor-D(ΔNΔC) gene transfer induces angiogenesis in rabbit skeletal muscle.

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.

Baculovirus is an efficient vector for the transduction of the eye: comparison of baculovirus- and adenovirus-mediated intravitreal vascular endothelial growth factor D gene transfer in the rabbit eye.

BACKGROUND: The present study aimed to determine the efficiency and safety of baculovirus-mediated intravitreal gene transfer in rabbit eye and to compare its efficiency with adenovirus. We also studied how an intravitreal injection of vectors producing vascular endothelial growth factor D (VEGF-D) impacts the vasculature of rabbit eye. METHODS: Baculoviral (BacVEGF-D) or adenoviral VEGF-D (AdVEGF-D) were administered intravitreally into the right eye at different doses (10(8), 10(9) and 10(10) IU/ml) to 24 animals. Left eyes were injected with control viruses. To determine how long transgene expression lasted, we injected BacVEGF-D or BacLacZ to the vitreous humour of 11 animals and followed them for 4 weeks. Vitreous samples were taken after sacrifice for enzyme-linked immunosorbent assays and eyes were removed and fixed for histological analyses. RESULTS: Both baculoviruses and adenoviruses caused efficient expression of VEGF-D in the rabbit eyes. BacVEGF-D caused a dose-dependent vascular leakage and a moderate dilation of the capillaries. The highest effect was seen 6 days after gene transfer and was detectable for 2 weeks. Intravitreal injection of baculovirus caused expression of VEGF-D in the inner retina, photoreceptor cells and in retinal pigment epithelium cells, whereas adenovirus-mediated VEGF-D expression was detected in the nerve fiber layer and ganglion cell layer. Baculovirus caused a transient inflammation similar to adenoviruses. CONCLUSIONS: The study suggests that baculoviruses are efficient vectors for ocular gene transfer, especially if deeper retinal layers need to be transduced. In addition, intravitreal VEGF-D gene transfer caused blood-retina barrier breakdown but not neovessel formation in the rabbit eye.

Post-transcriptional regulatory element boosts baculovirus-mediated gene expression in vertebrate cells.

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.

A multipurpose vector system for the screening of libraries in bacteria, insect and mammalian cells and expression in vivo.

We have constructed a novel tetra-promoter vector (pBVboostFG) system that enables screening of gene/cDNA libraries for functional genomic studies. The vector enables an all-in-one strategy for gene expression in mammalian, bacterial and insect cells and is also suitable for direct use in vivo. Virus preparation is based on an improved mini Tn7 transpositional system allowing easy and fast production of recombinant baculoviruses with high diversity and negligible background. Cloning of the desired DNA fragments or libraries is based on the recombination system of bacteriophage lambda. As an example of the utility of the vector, genes or cDNAs of 18 different proteins were cloned into pBVboostFG and expressed in different hosts. As a proof-of-principle of using the vector for library screening, a chromophoric Thr65-Tyr-Gly67-stretch of enhanced green fluorescent protein was destroyed and subsequently restored by novel PCR strategy and library screening. The pBVboostFG enables screening of genome-wide libraries, thus making it an efficient new platform technology for functional genomics.

Adenovirus-mediated VEGF-A gene transfer induces bone formation in vivo.

Osteoporosis is a major problem in elderly population. We tested the hypothesis whether vascular endothelial growth factor (VEGF-A) gene transfer is an appropriate way to enhance bone formation and recruitment of osteoblasts in vivo. Adenovirus vectors containing VEGF-A or lacZ cDNAs (1.4x10(10) pfu) were injected locally into right distal femurs of New Zealand White rabbits. Saline was injected into all contralateral distal femurs. One and three weeks after the gene transfers femurs were collected for analyses. X-Gal staining showed that up to 20% of the bone marrow cells were transfected although gene transfer also resulted in biodistribution of the vector and expression of the transgene in liver and spleen. Trabecular bone hard tissue histomorphometry of the distal femurs was performed to analyze the effect of gene transfer on bone turnover. When compared with unilateral lacZ transfected trabecular bone at one-week and three-week time points, VEGF-A gene transfer significantly increased bone formation parameters, such as osteoblast number, osteoid volume, and bone volume. Also, bone resorption surface was greatly reduced. It is concluded that injection of adenovirus vector can transfect bone marrow cells in vivo with a relatively high efficiency. Our results suggest that adenovirus-mediated VEGF-A gene transfer induces bone formation via increasing osteoblast activity and may be useful for the treatment of osteoporosis and other diseases that require efficient osteogenic therapy.

Coxsackievirus B3 VLPs purified by ion exchange chromatography elicit strong immune responses in mice.

Coxsackievirus B3 (CVB3) is an important cause of acute and chronic viral myocarditis, and dilated cardiomyopathy (DCM). Although vaccination against CVB3 could significantly reduce the incidence of serious or fatal viral myocarditis and various other diseases associated with CVB3 infection, there is currently no vaccine or therapeutic reagent in clinical use. In this study, we contributed towards the development of a CVB3 vaccine by establishing an efficient and scalable ion exchange chromatography-based purification method for CVB3 virus and baculovirus-insect cell-expressed CVB3 virus-like particles (VLPs). This purification system is especially relevant for vaccine development and production on an industrial scale. The produced VLPs were characterized using a number of biophysical methods and exhibited excellent quality and high purity. Immunization of mice with VLPs elicited a strong immune response, demonstrating the excellent vaccine potential of these VLPs.

TiO2 coating promotes human mesenchymal stem cell proliferation without the loss of their capacity for chondrogenic differentiation.

Human mesenchymal stem cells (hMSCs) are used in applications, which may require a large amount of cells; therefore, efficient expansion of the cells is desired. We studied whether TiO2 coating on plastic cell culture dishes could promote proliferation of hMSCs without adverse effects in chondrogenic differentiation. TiO2-films were deposited on polystyrene dishes and glass coverslips using an ultrashort pulsed laser deposition technique. Human MSCs from three donors were expanded on them until 95% confluence, and the cells were evaluated by morphology, immunocytochemistry and quantitative RT-PCR (qRT-PCR). The chondrogenic differentiation in pellets was performed after cultivation on TiO2-coated dishes. Chondrogenesis was evaluated by histological staining of proteoglycans and type II collagen, and qRT-PCR. Human MSC-associated markers STRO-1, CD44, CD90 and CD146 did not change after expansion on TiO2-coated coverslips. However, the cell number after a 48h-culture period was significantly higher on TiO2-coated culture dishes. Importantly, TiO2 coating caused no significant differences in the proteoglycan and type II collagen staining of the pellets, or the expression of chondrocyte-specific genes in the chondrogenesis assay. Thus, the proliferation of hMSCs could be significantly increased when cultured on TiO2-coated dishes without weakening their chondrogenic differentiation capacity. The transparency of TiO2-films allows easy monitoring of the cell growth and morphology under a phase-contrast microscope.

Baculoviruses mediate efficient gene expression in a wide range of vertebrate cells.

Baculovirus expression vector system (BEVS) is well known as a feasible and safe technology to produce recombinant (re-)proteins in a eukaryotic milieu of insect cells. However, its proven power in gene delivery and gene therapy is still poorly recognized. The basis of BEVS lies in large enveloped DNA viruses derived from insects, the prototype virus being Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Infection of insect cell culture with a virus encoding a desired transgene under powerful baculovirus promoter leads to re-protein production in high quantities. Although the replication of AcMNPV is highly insect specific in nature, it can penetrate and transduce a wide range of cells of other origin. Efficient transduction requires only virus arming with an expression cassette active in the cells under investigation. The inherent safety, ease and speed of virus generation in high quantities, low cytotoxicity and extreme transgene capacity and tropism provides many advantages for gene delivery over the other viral vectors typically derived from human pathogens.

In vivo application and tracking of baculovirus.

Baculoviruses are safe and high-capacity vectors for gene delivery which have matured from the initial successful experiments performed in liver cells into convenient tools to transduce almost any cell from any origin in vitro and ex vivo. This is a result of 15 years of intensive vector development as well as studies performed in vertebrate cells to reveal important factors affecting the transduction efficacy. Now, at the stage when the first evidence of meaningful use of baculoviruses for therapeutic applications has been reported, there is no doubt that the technology will meet the expectations as highly useful platform for many applications of gene delivery. This review summarizes the pre-clinical in vivo work carried out with baculoviruses and discusses remaining challenges which still need to be solved.

Culture medium induced vimentin reorganization associates with enhanced baculovirus-mediated gene delivery.

Baculoviruses can express transgenes under mammalian promoters in a wide range of vertebrate cells. However, the success of transgene expression is dependent on both the appropriate cell type and culture conditions. We studied the mechanism behind the substantial effect of the cell culture medium on efficiency of the baculovirus transduction in different cell lines. We tested six cell culture mediums; the highest transduction efficiency was detected in the presence of RPMI 1640 medium. Vimentin, a major component of type III intermediate filaments, was reorganized in the optimized medium, which associated with enhanced nuclear entry of baculoviruses. Accordingly, the phosphorylation pattern of vimentin was changed in the studied cell lines. These results suggest that vimentin has an important role in baculovirus entry into vertebrate cells. Enhanced gene delivery in the optimized medium was observed also with adenoviruses and lentiviruses. The results highlight the general importance of the culture medium in the assembly of the cytoskeleton network and in viral gene delivery.

A 96-well format for a high-throughput baculovirus generation, fast titering and recombinant protein production in insect and mammalian cells.

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.

Short and long-term effects of hVEGF-A(165) in Cre-activated transgenic mice.

We have generated a transgenic mouse where hVEGF-A(165) expression has been silenced with loxP-STOP fragment, and we used this model to study the effects of hVEGF-A(165) over-expression in mice after systemic adenovirus mediated Cre-gene transfer. Unlike previous conventional transgenic models, this model leads to the expression of hVEGF-A(165) in only a low number of cells in the target tissues in adult mice. Levels of hVEGF-A(165) expression were moderate and morphological changes were found mainly in the liver, showing typical signs of active angiogenesis. Most mice were healthy without any major consequences up to 18 months after the activation of hVEGF-A(165) expression. However, one mouse with a high plasma hVEGF-A(165) level died spontaneously because of bleeding into abdominal cavity and having liver hemangioma, haemorrhagic paratubarian cystic lesions and spleen peliosis. Also, two mice developed malignant tumors (hepatocellular carcinoma and lung adenocarcinoma), which were not seen in control mice. We conclude that long-term uncontrolled hVEGF-A(165) expression in only a limited number of target cells in adult mice can be associated with pathological changes, including possible formation of malignant tumors and uncontrolled bleeding in target tissues. These findings have implications for the design of long-term clinical trials using hVEGF-A(165) gene and protein.

Optimized self-excising Cre-expression cassette for mammalian cells.

We observed that overexpression of Cre recombinase in 293 T cells has toxic effects and that the chicken beta-actin promoter is active in Escherichia coli, causing expression of Cre in bacteria. This led to significant problems in the cloning of Cre/loxP constructs. Leaky Cre-expression in E. coli, and toxicity of the Cre overexpression in mammalian cells, were solved by constructing a novel silent self-inactivating Cre (SSi-Cre) expression cassette. The SSi-Cre is based on modified loxP sites flanking the Cre/Int/DsRed fusion gene containing a Cre coding sequence interrupted by an intron, which prevents leaky expression of Cre in E. coli. Additionally, this system contains a reporter gene to visualize Cre activity by fluorescent microscopy. The SSi-Cre cassette provides a universal strategy for the generation of Cre/loxP constructs, as well as a solution to the toxicity caused by the overexpression of Cre in target cells. SSi-Cre should thus provide a useful tool for various applications based on the Cre/loxP system.

Adeno-associated virus-mediated gene transfer of a secreted decoy human macrophage scavenger receptor reduces atherosclerotic lesion formation in LDL receptor knockout mice.

Macrophage scavenger receptors (MSR) promote atherosclerotic lesion formation, and modulation of MSR activity has been shown to influence atherosclerosis. Soluble receptors are effective in inhibiting receptor-mediated functions in various diseases. We have generated a secreted macrophage scavenger receptor (sMSR) that consists of the bovine growth hormone signal sequence and the human MSR A I extracellular domains. sMSR reduces degradation of atherogenic modified low-density lipoproteins and monocyte/macrophage adhesion on endothelial cells in vitro. To test long-term effects of sMSR, atherosclerosis-susceptible LDLR knockout mice were transduced via the tail vein with an adeno-associated virus (AAV) expressing sMSR or control enhanced green fluorescent protein (EGFP), and a Western-type diet was started. Gene transfer caused a temporary elevation in alkaline phosphatase and aspartate amino transferase values without a change in C-reactive protein. sMSR protein was detected in the plasma of the transduced mice by a specific ELISA 6 months after the gene transfer. AAV-mediated sMSR gene transfer reduced atherosclerotic lesion area in the aorta by 21% (P < 0.05) compared to EGFP-transduced control mice. Even though eradication of established disease was not possible, atherosclerotic lesion formation could be modified using AAV-mediated gene transfer of the decoy sMSR.

Enhanced gene delivery by avidin-displaying baculovirus.

Flexible alteration of virus surface properties would be beneficial for enhanced and targeted gene delivery. A useful approach could be based on a high-affinity receptor-ligand pair, such as avidin and biotin. In this study, we have constructed an avidin-displaying baculovirus, Baavi. Avidin display was expected to enhance cell transduction due to the high positive charge of avidin in physiological pH and to provide a binding site for covering the virus with desired biotinylated ligands. Successful incorporation of avidin on the virus envelope was detected by immunoblotting and electron microscopy. Multiple biotin-binding sites per virus were detected with fluorescence-correlation spectroscopy and tight biotin binding was observed using an optical biosensor, IAsys. Baavi showed a 5-fold increase in transduction efficiency in rat malignant glioma cells (BT4C) and a 26-fold increase in rabbit aortic smooth muscle (RAASMC) cells compared to wild-type baculovirus. Enhanced transduction was also observed with biotinylated target cells. Biotinylated epidermal growth factor (EGF) enabled specific targeting of the virus with high efficiency to EGF receptor-expressing (SKOV-3) cells. An additional advantage of the avidin display was demonstrated with biotinylated paramagnetic particles, which enabled magnetic targeting. Altogether, we show that avidin display is a rapid and versatile method to improve viral properties for gene delivery.

Baculovirus capsid display: a novel tool for transduction imaging.

Baculoviruses are enveloped insect viruses that can carry large quantities of foreign DNA in their genome. Baculoviruses have proved to be very promising gene therapy vectors but little is known about their transduction mechanisms in mammalian cells. We show in this study that Autographa californica multiple nuclear polyhedrosis virus capsid is compatible with the incorporation of desired proteins in large quantities. Fusions can be made to the N-terminus or C-terminus of the major capsid protein vp39 without compromising the viral titer or functionality. As an example of the baculovirus capsid display we show a tracking of the baculovirus transduction in mammalian cells by an enhanced green fluorescent protein (EGFP)-displaying virus. Our confocal and electron microscopy results suggest that the transduction block in mammalian cells is not in the endosomal escape, as previously proposed, but rather in the cytoplasmic transport or nuclear entry of the virus capsid. Our results also suggest that the EGFP-tagged virus can be used for visualization of the virus biodistribution in vivo. Furthermore, capsid-modified baculoviruses hold great promise for the nuclear and subcellular targeting of transgenes and as a novel peptide display system for a variety of eukaryotic applications.