Paediatrics neuromuscular disorders, genetics. Title: Effect of myonuclear domain structure on therapies for Duchenne muscular dystrophy

Effect of myonuclear domain structure on therapies for Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is the most prevalent genetic myopathy affecting children caused by mutations which disrupt expression of the dystrophin gene. Dystrophin performs structural and signalling roles and is crucial for protecting the muscle cell membrane (sarcolemma) from contractile damage. The re-expression of dystrophin protein in muscles of DMD patients remains a significant technical challenge. Accelerated approval of exon skipping drug eteplirsen by US FDA generated controversy in the field1, since the drug was shown to restore less than 1% of normal dystrophin protein expression. Therefore, the minimal levels of dystrophin required for therapeutic benefit is a question of key importance. An allelic condition, Becker muscular dystrophy (BMD) is characterized by later onset and slower progression. In fact, some BMD patients are near-asymptomatic as a consequence of ~30% of normal dystrophin expression from birth2. Further studies from the Wood group suggest that therapeutic re-introduction of as little as ~15% of normal dystrophin in adult dystrophic (mdx) mice is sufficient to provide protection against contractile damage3. Others, suggest that homogenous expression of dystrophin at 20-30% can significantly reduce muscle pathology4. The amount, quality, and correct localization of dystrophin are fundamental issues that underlie the success of dystrophin rescue therapies. There are a plethora of approaches which aim to restore dystrophin (e.g. exon skipping, gene therapy, cell therapy, stop codon read-through, gene editing)5. Each strategy has distinct characteristics in terms of the efficacy of protein restoration, the quality (i.e. the size, or amount of truncation), and the localisation of dystrophin within the myofibre. The latter point in particular is a relatively neglected area of study. Myofibres are long syncytial structures consisting of a multitude of myoblast-derived nuclei, each serving its own proximal region of cytoplasm (i.e. myonuclear domain). To investigate differences in dystrophin localisation researchers from the Wood group have previously generated a genetic mouse model that expresses varying levels of dystrophin from birth in a mosaic pattern as a consequence of skewed X-chromosome inactivation (the mdx-Xist mouse)6. These mice exhibit a within-fibre patchy dystrophin distribution. In contrast, exon skipping therapy in mdx mice using peptide-PMO (PPMO) conjugates resulted in a uniform pattern of dystrophin expression (despite total dystrophin protein levels being similar to the mdx-Xist mice). Importantly, stabilisation of muscle turnover was observed in the PPMO-treated animals, but was still apparent in the mdx-Xist mice. These findings suggest that the pattern of dystrophin localisation is a critical factor for the correction of dystrophic pathology. Furthermore, strategies such as cell therapy and CRISPR/Cas9 are likely to also generate chimeric myofibres whereby some nuclei express dystrophin and some do not. This situation is analogous to that observed in our mdx-Xist mice, and highlights an important limitation of these therapeutic approaches. Lastly, our data strongly suggest that dystrophin mRNA and protein are not free to diffuse throughout a myofibre, but may instead be limited to the cytoplasm/sarcolemma immediately surrounding the myonuclei from which they originate, consistent with the myonuclear domain hypothesis. This project aims to characterise the heterogeneity in gene expression between dystrophin-positive and dystrophinnegative myonuclear domains contained within myofibres, and to determine the relevance of myonuclear domain structures in the context of DMD therapies. We propose to explore this area using a number of parallel approaches