The goal of the present study was to explore the tropism of Virus-Like Particles (VLP) originating from the human polyoma JC- virus (JCV) in vitro and in vivo. VLP have been known for several years to be a promising alternative to the existing viral and non-viral DNA delivery systems. VLP are self-assembled structures composed only of proteins necessary for capsid formation, DNA packaging and gene delivery. They are replication deficient, exhibit a similar morphology and presumably share the same cell tropism, cellular uptake and intracellular trafficking mechanisms compared to their viral origin. VLP from JCV are being evaluated as a delivery vector for the central nervous system (CNS) because JCV preferentially infects both oligodendrocytes and astrocytes. VLP were produced by expressing VP1 from JCV in insect cells and purified by DEAE Sepharose chromatography. Before DNA packaging the VLP were dissociated into VP1 pentamers by a DTT/ EGTA treatment and contaminating DNA was removed. The purified VP1 pentamers were used for packaging, either EGFP or luciferase expressing DNA. Using VP1-VLP containing plasmid DNA expressing EGFP, we performed transduction assays in a panel of human and rodent brain-derived and non-brain derived cell lines in order to characterize the in vitro tropism and species specificity of VP1-VLP. Both human and rodent cell lines could be transduced. The highest transduction efficiency was observed in TC620 cells, a human oligodendroglioma line. In contrast, with the exception of one human prostate cell line (PC-3), VP1-VLP transduction was not observed in any non-brain-derived human or rodent cell line studied including those from kidney, liver, lung, skeletal muscle, and vascular endothelium. VP1-VLP transduction was also tested in primary brain cells from rats and humans. Transduction activity was detected in both human and rat primary brain cells. Among the brain cells from rats VP1-VLP preferentially transduced oligodendrocytes. To test transduction activity in vivo, VP1-VLP containing a luciferase expression plasmid were injected into the brain of mice. After stereotaxic injection, luciferase expression was measured non-invasively with a bioluminescence imaging system over a time period of 12 weeks. Luciferase expression reached peak levels at week 6 following injection. The level of expression then became relatively stable for another 6 weeks, when the experiment was terminated. In the present study we showed that JCV-derived VP1-VLP have a tropism for brain cells in vitro and in vivo. Tests with primary rat cells in vitro indicated that the VP1-VLP can be used to specifically transduce oligodendrocytes. Based on the in vivo data obtained in mice VP1-VLP may be used as an effective delivery vector to achieve long term expression in the brain.
Previous studies have shown that fibroblast growth factor (FGF)-1, FGF-2, and FGF-5 induce therapeutic angiogenesis. Here, we investigated the potential of FGF-4 for therapeutic neovascularization in comparison to vascular endothelial growth factor (VEGF), using adenoviral gene transfer in a novel rabbit hind limb ischemia model, with ischemia restricted to the calf. Magnetic resonance imaging and a modified Miles assay showed that both AdFGF-4 and AdVEGF given intramuscularly (i.m.) resulted in increases in vascular permeability and edema in transduced muscles 6 days after the gene transfer. In contrast, recombinant FGF-4 protein injected in the rabbit skin did not induce acute vascular permeability. Injections (i.m.) of AdFGF-4 and AdVEGF, but not intra-arterially administered AdVEGF, increased collateral growth, popliteal blood flow, and muscle perfusion compared with controls. The angiogenesis response consisted mainly of the enlargement of pre-existing vessels rather than an increase in capillary density. Adenoviral FGF-4 overexpression up-regulated endogenous VEGF, which may explain many of the effects thought to be specific for VEGF such as the increase in vascular permeability. This study demonstrates for the first time that FGF-4 induces vascular permeability, therapeutic angiogenesis, and arteriogenesis comparable to that of VEGF and could be useful for the treatment of peripheral vascular disease.
Recombinant retroviral vectors are useful tools for gene transfer in both gene therapy and research applications. An enhanced form of green fluorescent protein has been incorporated into recombinant retroviruses as a marker to follow infected cells. In this paper, we extended the use of the fluorescent reporter to quantify protein expression using such analytical tools as fluorescent microscopy, flow cytometry and fluorescent plate reader analysis. These tools enabled us to rapidly assess the titer of recombinant retrovirus harvested from packaging cells and to optimize parameters for infection of different cell lines.
