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Generation of a shut-off system for Adeno-associated viral gene transfer vectors

Rohwedder, Carolin

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Abstract

Systems to regulate gene expression from an Adeno-associated viral (AAV) vector are widely used. In most cases, the transgene expression has to be switched on by applying a drug. In terms of safety of gene therapy, a shut-off system for AAV vectors would be beneficial to silence gene expression in case of side-effects, ideally by destruction of the vector genome. Therefore, the aim of the present study was to develop a system for elimination of gene expression from an AAV vector after systemic injection using an inducible Cre recombinase. In presence of tamoxifen, the inducible Cre recombinase is activated which should result in excision of DNA fragments flanked by loxP sites within the vector. Before evaluating the final shut-off system, several experiments were performed to analyze the background activity of the induced CreERT2 recombinase, the best suited positions of the loxP sites within the AAV genome and the influence of the loxP sites on the transgene expression. Moreover, a co-transduction of an AAV vector encoding the CreERT2 and a second AAV vector expressing the reporter gene flanked by loxP sites was tested. AAV vectors of serotype 9 were used for packaging the final shut-off system consisting of the inducible CreERT2 recombinase, a luciferase reporter gene, and different positions of loxP sites to investigate the effect of loxP localization within the vector genome. To drive reporter gene and CreERT2 expression, the CMV promoter was used. All vectors were first tested in vitro. Afterwards, the vectors which showed a significant down-regulation of transgene expression after tamoxifen administration were also analyzed in vivo. Here, a significant reduction in reporter gene activity could be detected in animals receiving AAV vectors containing loxP sites one week upon tamoxifen administration. Another finding was that the insertion of loxP sites has a negative influence on the expression levels of the transgene. Thereby, the vector expressing the reporter gene flanked by loxP sites showed the lowest expression but also the highest extent of down-regulation after tamoxifen treatment. Finally, the shut-off system used was improved in terms of coding capacity of the AAV genome used. Therefore, the “self-cleaving” peptide P2A of the Porcine Teschovirus-1 was used to replace the promoter driving CreERT2 expression to increase the limiting coding capacity for the gene of interest. Again, these AAV9 vectors were tested in vitro and in vivo, also showing a significant reduction in reporter gene expression after tamoxifen administration. Taken together, expression of an inducible Cre recombinase allows efficient inactivation of AAV-mediated gene expression on the expense of reduced overall expression efficiency due to insertion of loxP sites. These results contribute to the generation of a novel shut-off system for AAV-mediated gene transfer applicable for the use in combination with various promoters and AAV serotypes to target cell types or tissues of choice.

Item Type: Dissertation
Supervisor: Müller, Prof. Dr. Martin
Date of thesis defense: 27 March 2017
Date Deposited: 03 Apr 2017 07:21
Date: 2017
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 500 Natural sciences and mathematics
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