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PURPOSE OF WORK. The efficiency of wild-type AdV5 infection is mainly determined by CAR expression. However, tumor cells very often do not express CAR or express this receptor at low levels. By using capsid genetic engineering AdV5 can be retargeted toward new receptors/molecules specifically expressed on target cells/tissues. Previously, we have constructed several AdV5 retargeted towards aminopeptidase N (CD13) and/or αvβ3 integrin by incorporating NGR peptide either into AdV5 fiber (AdFNGR) or hexon (AdHNGR) protein. Both CD13 and αvβ3 are highly expressed on tumor vasculature. In this study we investigate the type of endocytosis pathway used by these NGR-modified AdVs. MATERIALS AND METHODS. Transduction efficiency was assessed by determining β-galactosidase activity. For inhibiting specific endocytic pathways cells were pre-treated with drugs (dyngo 4a, dynasore, pitstop 2 or filipin III) or transfected with siRNA targeting dynamin-2, clathrin or caveolin-1. Confocal microscopy was used to measure/monitor internalization of fluorescently labelled AdV. RESULTS. By using pharmacological or genetic means, we show that AdFNGR and AdHNGR transduce CAR-negative cells in both dynamin- and lipid rafts/caveolin-dependent manner. Moreover, by using confocal microscopy we studied viral internalization and observed retention of AdFNGR and AdHNGR at the cell membrane after inhibiting lipid rafts. Inhibition of clathrin mediated endocytosis, either by specific inhibitors or clathrin downregulation, had no influence on transduction efficiency of AdFNGR and AdHNGR. CONCLUSION. Our results point out the important role of lipid raft-mediated endocytosis in NGR- bearing AdV5 transduction of CAR-negative cells. Better understanding of endocytic pathway of NGR modified AdV vectors could help in tailoring efficient vectors aimed at targeting tumor vasculature. To the best of our knowledge, this is the first study describing endocytic pathways used by NGR-bearing AdV5 vectors. MAIN BIBLIOGRAPHY. Majhen, D., et al., Disulfide bond formation in NGR fiber-modified adenovirus is essential for retargeting to aminopeptidase N. Biochem Biophys Res Commun, 2006. 348(1): p. 278- 87. Jullienne, B., et al., Efficient delivery of angiostatin K1-5 into tumors following insertion of an NGR peptide into adenovirus capsid. Gene Ther, 2009. 16(12): p. 1405-15. |
