The Absence of Accessible Vitronectin Receptors in Differentiated Tissue Hinders Adenoviral-Mediated Gene Transfer to the Intestinal Epithelium In Vitro
- 46 Downloads
Purpose. Adenoviral (Ad) vectors have been used as efficient tools for gene therapy in various tissues, whereas in some differentiated epithelium transduction efficiency is almost abolished.
Methods. Caco-2 cell monolayers were chosen as an in vitro model for the differentiated intestinal epithelium. Fluorescence-labeled adenoviral particles were used for binding studies to cell surfaces. Internalization receptors for adenoviral uptake were decteted by a fluorescence-labeled vitronectin antibody. Gene expression was studied by using the β-galactosidase reporter gene. All experiments were done on undifferentiated and differentiated Caco-2 cells. Furthermore, adenoviral particles were allowed to bind to differentiated Caco-2 monolayers followed by a trypsinization step that disintegrates the monolayers and result in a cell suspension. Gene expression was tested after reseeding the cells into dishes.
Results. The results from adenoviral binding studies, vitronectin immunofluorescence detection and gene expression are in good agreement and indicate that virion binding as well as the expression of internalization receptors almost disappear in fully differentiated cells. Nonetheless, adenoviral binding to differentiated monolayers seems to be sufficient to cause up to 53% gene expression, but only if internalization of the vector can be induced by disintegrating the monolayers and releasing free vitronectin receptors.
Conclusions. These findings indicate that gene transfer to the intestinal epithelium utilizing adenoviral vectors is poor and ineffective, because of the lack of sufficient internalization receptors. If these receptors can be exposed in differentiated epithelium, transduction can be made more efficicient. Alternatively, a viral vector must be developed whose uptake mechanism is independent of integrin receptor expression like the enteral virus Ad40, or Ad5 could be conjugated to ligands that trigger viral internalization by receptor-mediated endocytosis.
Unable to display preview. Download preview PDF.
- 1.R. J. Guzman, P. Lemarchand, R. G. Crystal, S. E. Epstein, and T. Finkel. Circulation 88:2838–2848 (1993).Google Scholar
- 2.A. Kass-Eisler, E. Falck-Pedersen, M. Alvira, J. Rivera, P. M. Buttrick, B. A. Wittenberg, L. Cipriani, and L. A. Leinwand. Proc. Natl. Acad. Sci. USA 90:11498–11502 (1993).Google Scholar
- 3.J. A. Wolff. Gene Therapeutics. Methods an applications of direct gene transfer, Birkhaeuser, Boston, 1994.Google Scholar
- 4.B. J. Roessler, J. W. Hartman, D. V. Vallance, J. M. Latta, S. L. Janich, and B. L. Davidson. Hum. Gene Ther. 6:307–316 (1995).Google Scholar
- 5.B. N. Fields, and D. M. Knipe. Fundamental Virology, Raven Press, New York, 1994.Google Scholar
- 6.U. F. Greber, M. Willetts, P. Webster, and A. Helenius. Cell 75:477–486 (1993).Google Scholar
- 7.U. Swensson, R. Petersson, and E. Everitt. J. Virol. 38:70–91 (1981).Google Scholar
- 8.T. J. Wickham, R. Mathias, D. A. Cheresh, and G. R. Nemerow. Cell 73:309–319 (1993).Google Scholar
- 9.S. Huang, R. I. Endo, and G. R. Nemerow. J. Virol. 69:2257–2263 (1995).Google Scholar
- 10.G. Acsadi, B. Massie, M. Simoneau, P. Holland, K. Blaschuk, and G. Karpati. Hum. Mol. Gen. 3:579–584 (1994).Google Scholar
- 11.M. J. Goldman and M. J. Wilson. J. Virol. 69:5951–5958 (1995).Google Scholar
- 12.E. Walter, M. A. Croyle, B. L. Davidson, B. J. Roessler, J. M. Hilfinger, and G. L. Amidon. J. Control. Rel. 46:75–87.Google Scholar
- 13.I. J. Hidalgo, T. J. Raub, and R. T. Borchardt. Gastroenterology 96:736–749 (1989).Google Scholar
- 14.P. Artursson. Crit. Rev. Ther. Drug Carrier Syst. 8:305–330 (1991).Google Scholar
- 15.B. J. Roessler, J. W. Hartman, D. V. Vallance, J. M. Latta, S. L. Janich, and B. L. Davidson. Hum. Gene Ther. 6:307–316 (1995).Google Scholar
- 16.F. L. Graham and A. J. van Eb. Virology 52:456–467 (1973).Google Scholar
- 17.B. Precious and W. C. Russell. Growth, purification and titration of adenoviruses. In B. W. J. Mahy (ed.), Virology: a practical approach, IRL Press, Washington 1986, pp. 193.Google Scholar
- 18.J. H. Miller. A short course in bacterial genetics, CSHL Press, New York, 1972.Google Scholar
- 19.P. H. Vachon, and J. F. Beaulieu. Gastroenterology 103:414–423 (1992).Google Scholar
- 20.M. D. Peterson, W. M. Bement, and M. S. Mooseker. J. Cell. Sci. 105:461–472 (1993).Google Scholar
- 21.G. Wilson, I. F. Hassan, C. J. Dix, I. Williamson, and M. Mackay. J. Control. Rel. 11:25–40 (1990).Google Scholar
- 22.A. R. Hilgers, R. A. Conradi, and P. S. Burton. Pharm. Res. 9:902–910 (1990).Google Scholar
- 23.M. Pinto, S. Robine-Leon, M. D. Appay, M. Kedinger, N. Triadou, E. Dussaulx, B. Lacroix, P. Simon-Assmann, K. Haffen, J. Fogh, and A. Zweibaum. Biol. Cell 47:323–330 (1983).Google Scholar
- 24.R. Persson, U. Svensson, and E. Everitt. J. Virol. 46:956–963 (1993).Google Scholar
- 25.M. H. Coconnier, M. F. Bernet-Camard, and A. L. Servin. Differentiation 58:87–94 (1994).Google Scholar
- 26.J. F. Beaulieu. J. Cell Sci. 102:427–436 (1992).Google Scholar
- 27.M. Y. Choy, P. I. Richman, M. A. Horton, and T. T. Macdonald. J. Pathology 160:35–40 (1990).Google Scholar
- 28.P. H. Vachon, J. Durand, and J. F. Beaulieu. Anat. Rec. 235:567–576 (1993).Google Scholar
- 29.P. Mathias, T. Wickham, M. Moore, and G. Nemerow. J. Virol. 68:6811–6814 (1994).Google Scholar