Entwicklung eines standardisierbaren Systems zum quantitativen Nachweis von DNAzym-Aktivität in vitro für den Einsatz in der Therapie des allergischen Asthma bronchiale

Allergisches Asthma bronchiale gehört zu den häufigsten chronisch-entzündlichen Erkrankungen in den industrialisierten Ländern. Die Betroffenen leiden unter einer Hyperreagibilität des Bronchialssytems, rezidivierender bronchialer Obstruktion und Entzündung und schließlich einem sogenannten “Airway...

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Bibliographic Details
Main Author: Casola, Lisa Maria
Contributors: Garn, Holger (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2018
Online Access:PDF Full Text
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Allergic asthma is one of the most common chronic-inflammatory diseases in industralized countries. Patients suffer from a hyperreactivity, recurrent bronchial obstruction and inflammation; and finally so-called “airway remodelling”, which represents irreversible structural changes. Genesis of the disease is multifactorial. In its pathophysiology, contact with allergens leads the initiation a cascade of immunological events. Downstream this causes the degranulation of mast cells with massive liberation of preformed substances that arouse the typical allergic symptoms. During this process, a misbalance in the equilibrium of different T-helper-cell subtypes with a shift towards the subpopulation of Th2-cells plays a crucial role. The differentiation into this direction is essentially mediated by the transcription factor GATA-3. To date, allergic asthma may only be treated symptomatically, for example using ß2-mimetics or glucocorticosteroids. Thus, there is a significant medical need for causative therapeutic approaches, which is further underlined by unwanted side effects of current therapy options. In this matter, antisense strategies might be an approach to fill the still existing gap. They can inhibit translation and subsequent protein overexpression via interference with mRNA. DNAzymes, for example, bind mRNA and later degrade it by their inheritant catalytic activity. Unfortunately, so far no simple in vitro test system is available to make a reliable statement about the efficiency of individual DNAzymes. This dissertation aimed to develop two test systems and compare them concerning their suitability for daily use in research. In both cases, the transcription factor GATA-3 was chosen as a target for the DNAzyme hgd40 as model system. In one experimental setting, its genetic sequence was cloned into a vector, which additionally contains the gene locus for a fluorescent protein (mCherry). In the second approach, a reporter gene system was used, which included sequences for luciferases (vector psiCheck with the luciferases Firefly and Renilla). Degradation of the GATA-3 mRNA by hgd40 should cause a reduction of the expression of Cherry or the luciferases respectively, so that the reduced signal detection should demonstrate the efficacy of the DNAzyme. Finally, it was possible to create the appropriate expression vectors and transfer them into cells (HEK-293) via transfection. At the same time, a successful transfection of the cells with the DNAzyme could be shown. However, after treatment with hgd40 there was no measurable suppression of the signal. The reasons for this failure might be multiplex. First of all, the choice of the cell line and transfection medium is very important, because problems may occur at the point of uptake or liberation of the DNAzyme in the adequate compartment of the cell. Furthermore, the structure of the target mRNA plays a crucial role for a successful interaction. In addition, the expression of the target structure can be prohibited by intracellular or environmental factors as well as obstacles can arise during signal detection. To avoide aforementioned problems different assay protocols were tested. It becomes apparent that every step to create such a test system for DNAzymes is afflicted with complications, and that there is a clear need for further experiments. Both test systems did not show any target down-regulation after treatment with the DNAzyme. However, sufficient expression of the luciferases and GATA-3 as well as an intracellular uptake of the DNAzyme was detectable in the system working with the psiCheck-GATA-3 vector. Therefore, this test system seems to be more suitable for further studies.