Gentherapeutische Vektoren für die Tumortherapie

Die vorliegende Arbeit beschäftigt sich generell mit der Entwicklung von Gentherapeutischen Transfektionssystemen mit dem vorrangigen Ziel die Behandlungsmöglichkeiten bei Tumorerkrankungen zu optimieren und auszubauen. Durch die Weiterentwicklung und Verknüpfung etablierter Systeme sollen neue Mögl...

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Bibliographic Details
Main Author: Hubing, Sascha
Contributors: Bakowsky, Udo (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2023
Online Access:PDF Full Text
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The present work is generally concerned with the development of gene therapy transfection systems with the primary goal of optimizing and expanding the treatment options for tumor diseases. By further developing and linking established systems, new possibilities are to be created to make the therapy of oncological diseases more targeted and efficient. The three projects described below address this goal through different approaches. In the 1st project in chapter 4 we describe polyethylenimine silica nanoparticles as efficient gene therapy vectors. By combining two established representatives from the group of non-viral vectors, we were able to achieve a significant improvement with regard to important properties. With different toxicity studies in cell culture we could prove that the cytotoxic side effects are much less pronounced compared to the individual components. For later use in practice, this optimization is of particular relevance because toxicity in particular is a major limiting factor for the use of non-viral gene therapeutics. Consequently, reduced side effects and improved compliance can be expected in future applications. Furthermore, we have demonstrated an increased transfection efficiency of about 40% compared to polyplexes. Especially this outstanding performance makes our vector an attractive candidate for gene therapy applications. We have demonstrated the maintenance of structural integrity of our vector over several days by a multi-day series of measurements to test the resilience to agglomeration events. The tendency to form agglomerates, which we observed to a particular extent with silica-NP, could thus be prevented. In terms of subsequent manageability and storage, this conversion is an enormous gain. The 2nd project in chapter 5 deals with the suitability of bioluminescence for self-induced photodynamic therapy. For this purpose, we transfected cells with pCMV-luc loaded lipopolyplexes to enable them for bioluminescence. The light beams emitted by the BL are expected to activate the photosensitizer hypericin embedded in the envelope of the transport system and consequently lead to apoptosis of the adjacent tissue through its toxic response. By studying the reciprocal effects of different amounts of hypericin and D-luciferin on the light emission from a luciferase-expressing target line, we were able to draw conclusions about the activation of hypericin by cell line bioluminescence. Comparing the emitted light beams in the absence and presence of different hypericin enrichments, we observed the absorption of a subset of the emitted light. However, the light absorption was not affected by the increase in hypericin concentration. As a result of light absorption by hypericin, we did not observe any toxic effects on the cell line used. Significant differences that we observed in MTT experiments were only due to the amount of D-luciferin used. Despite demonstrated absorption by the photosensitizer, no measurable toxic effects occurred. Consequently, the light released during the bioluminescence reaction is not sufficient to achieve efficient activation of the photosensitizer hypericin. In the 3rd project in Chapter 6, we describe lipopolyplexes as effective vectors for MDR1 knockdown by RNA interference, as a result of which cytostatic resistance mediated by overexpression of P-glycoprotein is reversed. Using RT-qPCR, we detected a reduction of MDR1 mRNA by approximately 60%. Polyplexes achieved only half the reduction with the same siMDR1 concentration. Especially for low DOX concentrations, we were able to show a pronounced recovery of sensitivity as a consequence of MDR1 knockdown. From no toxic effect at a concentration of 125 µg DOX / ml to a cell survival rate of almost 50% with preceding MDR1 knockdown with lipopolyplexes. Especially for clinical practice, the efficacy at low concentrations is important, since dose increases are often not possible due to pronounced side effects. Since a number of other factors are also discussed in close connection with the concentration of MDR1, which significantly influence the course and lethality of tumor disease, we investigated the effect of MDR1 knockdown on these characteristics, which include cell migration, invasion, proliferation and colonization. For all factors, we found a significant reduction in the respective trend correlating with the extent of MDR1 reduction through various in vitro experiments. These findings are generally supportive of the MDR1 knockdown therapy approach, as tumor cell spread is impaired in addition to improved efficacy of cytostatic therapy.