Bifunctional SuperCD suicide gene expression is highly effective in a rat hepatoma model, thereby significantly improving both the therapeutic index and the efficacy of hepatocellular carcinoma killing by fluorocytosine.
Synthetic transcriptional control unit constructs not only exhibit a superb degree of structural compactness, but also provide new means for liver-directed expression of therapeutic genes.
The intercellular trafficking property of the herpes simplex virus type 1 tegument protein VP22 makes it a promising tool for overcoming low transduction efficiencies in gene therapy. However, recent reports suggest not only that VP22 cannot facilitate intercellular spreading and that trafficking of VP22 fusion proteins results from artifacts of cell fixation only. To provide direct evidence for the presence or absence of VP22-mediated intercellular trafficking, we generated an adenoviral vector with a dual expression cassette for VP22 fused to green fluorescent protein (VP22 GFP) and DsRed under the control of distinct human cytomegalovirus immediate-early enhancer/promoter regions. Using this vector, we were able to distinguish clearly between primary transduced cells and cells taking up VP22GFP by intercellular trafficking. To our knowledge, for the first time, we could demonstrate by live-cell confocal fluorescence microscopy that VP22GFP can be found intracellularly in unfixed recipient cells. The extent of VP22 spread was similar in paraformaldehyde-fixed cells and unfixed cells as demonstrated by fluorescence-activated cell sorting analysis. We thus confirmed the ability of VP22-mediated intercellular trafficking in live unfixed cells.
Poor treatment results in advanced hepatoblastoma (HB) made alternative treatment approaches desirable. Gene-directed tumor therapy is increasingly investigated in different malignancies. The aim of this study was to analyze possible alternatives of gene transfer into HB cells and to study therapeutic applications based on different strategies. Liposomal transfection of HB cells was assessed using liver-specific promoters, and adenovirus and Sendai virus transductions were performed in vitro. Transfer efficiencies were measured via flow cytometry determining expression of vector-encoded marker gene green fluorescent protein. Gene silencing of the anti-apoptotic bcl-2 gene in HUH6 cells was performed using lipofection of small interfering RNA (siRNA). Additionally, suicide gene therapy was carried out through a yeast-derived cytosine deaminase (YCD)-combined yeast uracil phosphoribosyltransferase (YUPRT)-based adenovirus-mediated gene transfer, leading to a potent intracellular prodrug transformation of 5-fluorocytosine into 5-fluorouracil. Treatment efficiencies were monitored via MTT viability assay. Highest gene transfer rates (86%) were observed using adenovirus transduction. We furthermore observed a significant therapeutic effect of adenovirus-mediated YCD::YUPRT suicide gene transfer. Liposomal-mediated anti-bcl-2 siRNA transfer led to a significant improvement of cisplatin treatment in HUH6 cells. Liver-specific promoters were found to be strongly active in HUH6 cells (mixed HB-derived), but less active in HepT1 cells (embryonal HB-derived). Liposomal transfection and viral transduction are effective approaches to genetically manipulate HB cells in vitro. For the first time, we demonstrate a positive effect of siRNA gene silencing in this malignancy. Additionally, we successfully investigated a model of adenovirus-based suicide gene therapy in HB cell cultures. Our data strongly encourage further studies assessing these alternative treatment approaches.
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