Neue Gentargeting-Strategien auf Basis der RNA interference RNAi

RNA Interferenz ist ein natürlicher Prozess in eukaryoten Zellen, der zur spezifischen Herunterregulation der Genexpression (Gen-Silencing) führt. Der Mechanismus der RNAi wird durch 21 - 23 nt lange doppelsträngige RNA-Moleküle (siRNA) vermittelt, welche sequenzspezifisch den Abbau und die Degradat...

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Bibliographic Details
Main Author: Werth, Stephanie
Contributors: Aigner, Achim (Prof. Dr.) (Thesis advisor)
Format: Dissertation
Published: Philipps-Universität Marburg 2013
Pharmakologisches Institut
Online Access:PDF Full Text
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Table of Contents: RNA interference is a natural process in eukaryotic cells leading to the specific down-regulation of gene expression (gene silencing). The mechanism of RNAi is mediated through 21-23 nt long double-stranded RNA molecules (siRNA) which sequence-specifically induce the degradation of their target-mRNA. One critical factor that determines the success of RNAi is the rapid degradation of nucleic acids by ribonucleases and reduced transfection efficiency. Polyethylenimines are synthetic polymers which are present in different molecular weights and which have a high cationic charge density. Due to their protonation they are able to form complexes with DNA or siRNA. As positively charged polyplexes they interact with the cell membrane and are absorbed by endocytosis into the cytosol. By fractionation of a commercially available 25kDa PEI using gel permeation chromatography, a low molecular weight polyethylenimine (F25-LMW-PEI) with superior transfection efficacy and low toxicity in the cell lines SKOV3, U87 and PC3 was obtained. Upon lyophilization and reconstitution of F25-LMW-PEI/siRNA/DNA complexes, siRNAs are still able to efficiently induce RNAi. To further demonstrate their applicability, lyophilized PEI/siRNA complexes are employed for targeting the growth factors VEGF and PTN. Treatment of the PC3 prostate carcinoma cells or U87 glioblastoma cells with fresh or with lyophilized complexes results in decreased cell proliferation in different assays due to the siRNA-mediated downregulation of VEGF and PTN. In conclusion, siRNAs can be applied in lyophilizes formulations, and lyophilized F25-LMW-PEI-based siRNA complexes represent a powerful, inexpensive, non-toxic and simple ready-to-use platform for the specific and efficient targeting of genes in vitro.