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K. Nagel-Wolfrum, T. Goldmann, A. Rebbibo-Sabbah, T. Ben-Yosef, U. Wolfrum; Suppression of a Nonsense Mutation in Ush1c by Aminoglycosides - A Gene Based Therapy. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5353.
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The human Usher syndrome (USH) is the most frequent cause of inherited combined deaf-blindness. It is clinically and genetically heterogeneous and is assigned to three clinical USH types. The most severe type is USH1, characterized by profound inner ear defects and retinitis pigmentosa. The USH1C gene encodes harmonin. A USH1C point mutation leads to a premature translation stop (R31X) in a German family. The auditory deficit is successfully treated with cochlear implants. No effective treatment for the the ophthalmic component of USH exists. Gene based strategies are attractive for the treatment of hereditary retinal degenerations. Here, we evaluated a strategy mediated on aminoglycosides to cure the retinal phenotype. Aminoglycosides reduce the fidelity of translation of mRNA, leading to readthroughs of nonsense mutations and expression of full-length proteins. We estimated the suppression of aminoglycosides on the R31X mutation in vitro and in vivo. Furthermore, retinal toxicity of them was analyzed.
in vitro translation assays; transient transfection of HEK293T cells with expression wt-USH1C or mutated USH1C; application of aminoglycosides; immunocytochemistry; organotypic retina cultures; TUNEL assays.
In vitro translation assays revealed a dose-dependent suppression of the R31X mutation mediated by aminoglycosides with a maximum suppression rate of 65% for G418. Harmonin was not expressed in untransfected controls and untreated R31X transfected HEK293T cells. Aminoglycosides applications revealed dose-dependent harmonin expression in R31X transfected cells. In organotypic retina cultures, an increase of apoptosis was observed in all retinal layers after G418 application. In addition, the morphologic analyses of treated retinas indicated dysintegration of the overall organization of the retina layers. Furthermore, immunofluorescence analyses revealed decrease of GFAP expression in G418 treated retina cultures.
The present study revealed a dose-dependent suppression of the R31X nonsense mutation of USH1C by aminoglycosides in vitro and in vivo, indicating high therapeutic potential of aminoglycosides for USH. The fading of GFAP expression in G418 treated organotypic retina cultures indicates a selective toxic effect of aminoglycosides on Müller glia cells. The loss of the integrative function of Müller glia cells may also be the reason for the retinal disorganisation and the increase of apoptosis. Nevertheless, the design of less toxic aminoglycosides may increase the high potential for aminoglycoside gene based therapy.
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