Research Article: Identification of Small Molecule and Genetic Modulators of AON-Induced Dystrophin Exon Skipping by High-Throughput Screening

Date Published: December 17, 2009

Publisher: Public Library of Science

Author(s): Debra A. O’Leary, Orzala Sharif, Paul Anderson, Buu Tu, Genevieve Welch, Yingyao Zhou, Jeremy S. Caldwell, Ingo H. Engels, Achim Brinker, Cathal Seoighe.

Abstract: One therapeutic approach to Duchenne Muscular Dystrophy (DMD) recently entering clinical trials aims to convert DMD phenotypes to that of a milder disease variant, Becker Muscular Dystrophy (BMD), by employing antisense oligonucleotides (AONs) targeting splice sites, to induce exon skipping and restore partial dystrophin function. In order to search for small molecule and genetic modulators of AON-dependent and independent exon skipping, we screened ∼10,000 known small molecule drugs, >17,000 cDNA clones, and >2,000 kinase- targeted siRNAs against a 5.6 kb luciferase minigene construct, encompassing exon 71 to exon 73 of human dystrophin. As a result, we identified several enhancers of exon skipping, acting on both the reporter construct as well as endogenous dystrophin in mdx cells. Multiple mechanisms of action were identified, including histone deacetylase inhibition, tubulin modulation and pre-mRNA processing. Among others, the nucleolar protein NOL8 and staufen RNA binding protein homolog 2 (Stau2) were found to induce endogenous exon skipping in mdx cells in an AON-dependent fashion. An unexpected but recurrent theme observed in our screening efforts was the apparent link between the inhibition of cell cycle progression and the induction of exon skipping.

Partial Text: Duchenne Muscular Dystrophy (DMD) is the most common of nine categories of muscular dystrophy, occurring at an incidence of 1/3500 live born males [1]. All cases of DMD are caused by a loss of dystrophin protein expression, however the underlying genetic mutations for the disease vary greatly between individuals and can include deletions, insertions or point mutations throughout the dystrophin gene (DMD), which is the largest gene in the human genome (spanning 2.4 Mb of the X chromosome) [2], [3]. The severity of muscle wasting in DMD means that most patients die in the second decade of their lives due to respiratory and cardiac failure, as a consequence of loss of dystrophin expression in both cardiac and skeletal muscle [4].

We have used an unbiased screening approach to identify small molecule and genetic regulators of AON-induced exon skipping of Dystrophin. Specific enhancement of the ratio of exon skip/full-length transcripts was determined by qPCR analysis, eliminating hits that simply influence transcriptional activation or luciferase stability, a concern for any HTS using luciferase reporters [57]. Ideally a screen for modulators of DMD exon skipping would be performed in myotubes, since the tissue most affected in DMD patients is skeletal muscle. However, given the variable nature of myoblast differentiation, this was not technically feasible for a robust HTS assay. The fact that an equivalent number of compounds identified via HTS in HEK 293 cells showed specific enhancement of AON-induced exon skipping of endogenous Dmd in the mdx myoblast cellular context as were observed using the hE72-Luc reporter in HEK 293 cells indicates that the mechanisms of action involve ubiquitous molecules. In agreement with our results, Stoilov et al. also found that compounds identified via HTS with a minigene reporter construct in HEK 293 cells were capable of specifically enhancing inclusion of exon 10 in endogenous transcripts of MAPT[36].



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