Gentransfer und –regulation mit adeno-assoziierten Virusvektoren

Zusammenfassung Das Vektorsystem, basierend auf den adeno-assoziierten Viren, ist ein hoch attraktives Vektorsystem mit geringer biologischer Sicherheitsstufe (S1), da es keine bekannten Pathogenitätsfaktoren aufweist. Das Vektorsystem hat ein sehr breites Wirtsspektrum und ist aus diesem Grund un...

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Bibliographic Details
Main Author: Pinkenburg, Olaf
Contributors: Vogelmeier, Claus (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2007
Online Access:PDF Full Text
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Summary The vector system based on the adeno-associated virus is a highly attractive system with a low biological security level because of its lack of pathogenic factors. The vector system has a broad tropism and for this reason is a universal tool. A vector system of this kind should first be analysed for its basic properties. After this the TETON- OFF system and RNAi technology should be combined with the viral system. Finally gene transfer should be demonstrated in an in vivo animal model. Basic properties such as the time course of gene transfer were estimated first. Here,it was possible to determine the exact temporal profile of virus uptake and the resulting gene expression in detail. The stability of gene expression after viral transduction and also the amount of integration were also determined. With these recombinant viruses it was also possible to create stabile cell lines.It was possible to combine the tetracycline inducible system with the AAV system. For the TET-OFF system, a seven-fold stronger induction of luciferase could be shown compared to the TET-ON system. The TET-ON system showed a ten-fold higher basal activity than the TET-OFF system. Using the TET-OFF system, it was possible to produce the antimicrobial peptide hCAP18 in HEK-293 cells and to detect it immunochemically. RNAi technology was also combined with the AAV system; there it was possible to show a dose as well as a time-dependent regulation of luciferase expression in HEK-293 cells. In this overexpression model, an siRNA against luciferase and a control siRNA were used. It was possible to downregulate luciferase activity by 95 %. The RNAi technology was also used to reduce IL8 expression after TNFα stimulation of epithelial cells by decreasing the p65 protein. Strong expression of hCAP18, as well as its release into the medium, was shown with two different AAV constructs in HEK-293 cells and also in primary HBE cells. For gene transfer in the animal model, a self-complementing AAV was used. The angiogenic properties of the hCAP18/LL37 were be shown in the chronic ischemic rabbit hindlimb model. After the gene transfer, a higher blood flow and a higher capillary density could be verified in the hindlimb tissue.