Pharmacogenetic Abatement of a Nonsense Mutation in Usher Syndrome 1c in the Retina by Aminoglycosides

Abstract

Purpose: : The human Usher syndrome (USH) is the most frequent cause of inherited combined deaf-blindness. It is clinically and genetically heterogeneous, assigned to three clinical USH types. The most severe type is USH1. So far no effective treatment for the ophthalmic component of USH exists. A nonsense mutation in the USH1C gene (p.R31X) leads to a stop causing the USH1 disease. We investigated the efficacy of commercial aminoglycosides (e.g. G418, paromomycin) and modified aminoglycosides NB30, NB54 and the novel compound PTC124 to induce a read-through of this mutation as a treatment of USH1C in the retina.

Methods: : Aminoglycoside induced read-through of the p.R31X mutation was validated in HEK293T cells and mouse retinal explants. GST-pull downs were applied to test harmonin's scaffold function. Retinal toxicities of drugs were assessed by TUNEL-assays and immunoflourescene of molecular markers in retinal explants.

Results: : Dose-dependent read-throughs of the p.R31X mutation by aminoglycosides and PTC124 were observed not only in cultured cells but also in the retina. This read-through of p.R31X restored binding capacity of USH1C protein harmonin and therefore its scaffold function. TUNEL-assays of treated retinas revealed differential toxicity of the read-through compounds with a major effect on the inner retina. Müller glia cells were identified as primary target of the commercial aminoglycosides by immunofluorescence.

Conclusions: : The read-through of the p.R31X nonsense mutation of USH1C mediated by commercial aminoglycosides as well as the novel compounds effectively restored protein expression and function. In the retina, the primary cellular targets of the obvious toxicity of commercial aminoglycosides are Müller glia cells. Drastically reduced toxicity combined with their read-through efficacy reveals the high potential of NB30, NB54 and PTC124 as new therapeutic agents for USH1C and other genetic conditions.

Keywords: gene transfer/gene therapy • Muller cells • drug toxicity/drug effects