The Effect of 6-Thioguanine on Alternative Splicing and Antisense-Mediated Exon Skipping Treatment for Duchenne Muscular Dystrophy

Ingrid E C Verhaart,Annemieke Aartsma-Rus

doi:10.1371/currents.md.597d700f92eaa70de261ea0d91821377

Ingrid E C Verhaart, Annemieke Aartsma-Rus

Open Access

https://doi.org/10.1371/currents.md.597d700f92eaa70de261ea0d91821377

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Journal: PLOS Currents	Publication Date: Jan 1, 2012
Citations: 7	License type: CC BY 4.0

Affiliation: Leiden University Medical Center

Abstract

The severe muscle wasting disorder Duchenne muscular dystrophy (DMD) is caused by genetic defects in the DMD gene, leading to a complete absence of dystrophin protein. Of the therapeutic approaches addressing the underlying genetic defect, exon skipping through antisense oligonucleotides (AONs) is the closest to clinical application. Several strategies to improve the efficiency of this approach are currently being investigated, such as the use of small chemical compounds that improve AONmediated exon skipping levels. Recently, enhanced exon skipping in combination with a guanine analogue, 6-thioguanine (6TG) was reported for phosphorodiamidate morpholino oligomers (PMO). Here the effect of 6TG on the exon skipping efficacy of 2’-O-methyl phosphorothioate RNA (2OMePS) and PMO AONs in vitro and in vivo was further evaluated, as well as the effect of 6TG by itself. Results confirm an increase of exon skipping levels in vitro, however, in contrast to the previous report, no effect was observed in vivo. Importantly, 6TG treatment in vitro resulted in numerous additional DMD exon skipping events. This, in combination with the known cytotoxic effects of 6TG after incorporation in DNA, warrants reconsidering of the use of 6TG as enhancer of AON efficiency in DMD, were chronic treatment will be required.

Highlights

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are both muscle wasting disorders caused by mutations in the DMD gene
The effect of 6TG on exon skipping by 2’-O-methyl phosphorothioate RNA (2OMePS) antisense oligonucleotides (AONs) was first tested in vitro in cultured human muscle cells derived from a healthy control
The same effect was seen for the AONs h45AON5, h53AON1 and h55AON, where low levels of exon skipping were detected without or with doses ?25nM of AONs and an improvement in skipping levels was seen for 50-200 nM

Summary

Introduction

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are both muscle wasting disorders caused by mutations in the DMD gene. DMD is characterized by a much more severe and progressive phenotype than BMD. The BMD phenotype is much more variable with an average age of onset around 12, wheelchair-dependency in their twenties and death in their forties for the most severely affected patients[1]. Only around 50% of BMD patients develops dilated cardiomyopathy[1]. The differences between both phenotypes are due to the different nature of the underlying genetic defects. The DMD gene, located on Xp21, encodes dystrophin, an important protein for muscle fibres. In BMD mutations do not affect the reading frame, allowing protein translation to continue, resulting in internally deleted dystrophin proteins, which are partly functional. In BMD patients the disease progression is slower[4]

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