Charakterisierung GATA-3-spezifischer DNAzyme und Analyse der therapeutischen Wirksamkeit in experimentellen Modellen des allergischen Asthma bronchiale

Das allergische Asthma bronchiale ist eine chronisch entzündliche Erkrankung der Atemwege begleitet von einer erhöhten Mukussekretion und Atemwegs-hyperreagibilität. Die häufigste Therapie basiert auf einer Kombination von inhalativen Kortikosteroiden (ICS) und langwirksamen β2-Sympathomimetika. Die...

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Bibliographic Details
Main Author: Dicke, Tanja Maria
Contributors: Garn, Holger (Dr.) (Thesis advisor)
Format: Doctoral Thesis
Language:German
Published: Philipps-Universität Marburg 2009
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Table of Contents: Allergic asthma is a chronic inflammatory disease, accompanied by increased mucus secretion and airway hyper responsiveness. The most common therapy comprises a combination of inhaled corticosteroids (ICS) and long-acting β2-agonists. However these drugs are only able to reduce the symptoms associated with the disease. Furthermore, they cause unwanted side-effects and the therapy fails to work in some patients. Therefore, new antisense-acting therapeutics need to be developed with no or little side-effects. Of the potential candidates we selected GATA-3 as the target molecule for intervention. GATA 3 is a key regulator of Th2 cell differentiation and activation, and is also expressed in eosinophils, mast cells and natural killer cells. Thus, GATA-3 plays a central role in allergic inflammation. Transcription factors like GATA 3 are intracellular molecules and consequently they are difficult to address therapeutically. However, antisense strategies provide new opportunities since they interact directly with the mRNA of the target molecule. As therapeutic strategy GATA-3-specific DNAzymes were developed in our group. These molecules are single-stranded desoxyoligonucleotide molecules with a high specificity to GATA 3 mRNA with an inherent RNA-cleaving enzymatic activity. We used the GATA 3-specific DNAzymes gd21 and hgd40 and additionally the control DNAzyme ODNg. The control DNAzyme possesses the catalytic sequence but it cannot bind the GATA-3 mRNA. The aim of the present work was to characterize GATA-3-specific DNAzymes with regard to their specificity, potential side-effects, stability, and bioavailability. Additionally, these DNAzymes were also tested for their effectiveness in an experimental models of allergic asthma. With regard to species specificity, the DNAzyme gd21 was able to cleave only the murine GATA-3 mRNA and the hgd40 could cleave the human as well as the murine and rat GATA-3 mRNA in a dose dependent fashion. The catalytic activity was found to be specific for GATA-3 mRNA, there was no influence on other mRNAs or GATA-3 DNA. With regard to potential off-target effects, we could exclude an activation of the innate immune system via Toll-like receptor-9 (TLR-9) in vitro and in vivo. To investigate the stability and bioavailability of GATA-3-specific DNAzymes, a sensitive hybridization ELISA and an anion exchange chromatography method was developed. Based on these methods, we could show that the investigated DNAzymes are stable over a long time at different storage temperatures. The detection of DNAzymes in serum after local application confirmed their absorption into the lung and that the DNAzymes became bioavailable. The effectiveness of GATA-3-specific DNAzymes was validated in vivo in experimental models of acute allergic airway inflammation. The preventive as well the therapeutic application of GATA-3-specific DNAzymes led to reduced inflammation in the lung and a clear improvement of the airway reactivity. A direct comparison of the hgd40 to the ICS budesonide revealed that the effectiveness of DNAzyme in a mouse model of acute allergic airway inflammation was at least comparable to budesonid. We further tested the effectiveness of GATA-3-specific DNAzymes in a model of chronic allergic bronchial asthma, with better represents the human situation. A positive therapeutic effect could be observed including a reduction in remodelling processes in the airways. In summary, we could establish that intranasal application of GATA-3-specific DNAzymes represents a novel promising strategy for therapy of bronchial allergic asthma, whose effectiveness needs now validation in human asthmatic patients.