Untersuchungen zum Einsatz nanopartikulärer Trägersysteme zur Transfektion von Immun- und Alveolarzellen mit therapeutischen DNAzymen

Asthma bronchiale gehört weltweit zu den häufigsten chronischen Lungenerkrankungen. Eine wichtige Rolle in der Pathogenese des allergischen Asthmas spielen T-Helferzellen (TH2) für deren Differenzierung und Aktivierung der Transkriptionsfaktor GATA-3 essentiell ist. Durch l...

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Bibliographic Details
Main Author: Paul, Christoph
Contributors: Garn, Holger (Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2012
Online Access:PDF Full Text
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Bronchial Asthma is one of the most common chronic pulmonary diseases worldwide. T-helper cells (TH2) have been shown to play a central role in the pathogenesis of allergic asthma. Differentiation and activation of TH2 cells necessarily depends on the transcription factor GATA-3. It has been shown that local administration of a GATA-3 mRNA cleaving DNAzyme – a DNA-oligonucloetide with inherent catalytic activity – may significantly improve an asthmatic phenotype in mice. However, effective administration of “naked” DNAzymes represent a challenge since their cellular uptake is hindered by size and anionic character of the molecules. Hence, it was the goal of the present work to investigate whether natural surfactant or artificial vectors could improve DNAzyme uptake and thereby the therapeutic efficacy of this type of novel drugs. In our in vitro experimental setup using different cell lines we analysed the uptake of a fluorochrome-labelled DNAzymes by flow cytometry under the influence of surfactant, Polyethylenimine (PEI) and Polyethylenimine-Polyethylene glycol (PEI-PEG) polymers. The two investigated surfactant preparations Alveofact® and Curosurf® did not influence DNAzyme uptake in our experiments. Consequently, our data do not support the hypothesis that surfactant could improve the uptake of DNA-based molecules in vitro. We did however observe an effective transfection of cells when PEI or PEI-PEG polymers were applied in the same experimental setup. All PEI-PEG polymers showed significantly less cytotoxicity compared to the unmodified PEI molecules. Among the PEI-PEG polymers PEI(25kDa)-g-PEG(5kDa)4 with four 5 kDa PEG side chains proved to be the most efficient vector, and was therefore selected for an initial in vivo experiment. Mice with an experimentally induced allergic asthma phenotype were treated intratracheally with a complex of GATA-3 specific DNAzyme and PEI-PEG polymer once daily over a period of 4 days during the time of local allergen challenge. Unfortunately, this treatment regime led to severe, in several animals even fatal, lung inflammation. This effect depended on the administered polymer dose, and was most pronounced in animals treated with uncomplexed PEI-PEG without DNAzyme. Taken together, the investigated DNAzyme/PEI-PEG complexes – similar to effects already known for siRNA/PEI-PEG complexes - proved to be efficient transfection vectors in vitro. The observed adverse reactions in vivo however, suggest a significant toxic potential of these complexes. As the chronic disease condition bronchial asthma is expected to require long-term treatments, toxicologically safe systems are an absolute prerequisite for novel therapeutic options. Thus, future development of vector systems for an effective local application of nucleic acid-based drugs must aim at a significant reduction of toxic properties of such complexes paralleled by comparable or even improved transfection efficacies.