A Cell-Based High-Throughput Screening Assay for Posttranscriptional Utrophin Upregulation

Abstract

Duchenne muscular dystrophy (DMD) is a devastating muscle-wasting disease caused by mutations in the dystrophin gene. Utrophin is a homologue of dystrophin that can compensate for its absence when overexpressed in DMD animal models. Utrophin upregulation is therefore a promising therapeutic approach for DMD. Utrophin is regulated at both transcriptional and posttranscriptional levels. Transcriptional regulation has been studied extensively, and assays have been described for the identification of utrophin promoter-targeting molecules. However, despite the profound impact that posttranscriptional regulation has on utrophin expression, screening assays have not yet been described that could be used to discover pharmaceuticals targeting this key phase of regulation. We describe the development and validation of a muscle cell linebased assay in which a stably expressed luciferase coding sequence is flanked by the utrophin 5′-and 3′-untranslated regions (UTRs). The assay was validated using the posttranscriptional regulation of utrophin by miR-206. The assay has a Z′ of 0.7, indicating robust performance in high-throughput format. This assay can be used to study utrophin regulatory mechanisms or to screen chemical libraries for compounds that upregulate utrophin posttranscriptionally via its UTRs. Compounds identified via this assay, used alone or in a synergistic combination with utrophin promoter-targeting molecules, would be predicted to have therapeutic potential for DMD.

Muscle Structure Influences Utrophin Expression in mdx Mice

“…RhoA, a small GTPase also increases utrophin expression without apparently influencing transcription [97]. Stabilizing RNA, a known function for the type III intermediate filament protein vimentin [98], is another potential mechanism that can increase utrophin expression without changing transcription [99], [100]. Desmin may also influence protein degradation pathways by trafficking lysosomes through the muscle via its interaction with myospryn [101], [102].…”

Duchenne muscular dystrophy drug discovery – the application of utrophin promoter activation screening

“…recently described a utrophin 3¢5¢ UTR reporter cell line (Moorwood et al; in press [137]) that could be utilised for HTS and demonstrated proof of concept for upregulation using a SBO to mask let-7 miRNA-mediated repression.…”

A Cell-Based High-Throughput Screen Addressing 3′UTR-Dependent Regulation of the MYCN Gene

“…However, until recently this kind of assay has been mainly focused on the identification of promoter-targeting compounds [7]. A similar approach in which a reporter gene is flanked by the 5′UTR and/or 3′UTR of a gene of interest could become a valuable tool for identification of molecules targeting post-transcriptional control mechanisms [8–10]. …”