Development of molecular medicine approaches for neuromuscular disease
Keywords: Neuromuscular disease
Gene editing
uORF
CRISPR/Cas9
Extracellular vesicles
AAV9
Duchenne muscular dystrophy
Translation upregulation

Neuromuscular diseases are a broad group of debilitating disorders that impair muscle functioning. Duchenne muscular dystrophy (DMD) is the most common hereditary form of neuromuscular disease, for which there is currently no cure. DMD is caused by null mutations in the dystrophin gene (DMD), which encodes an essential cytoskeletal protein required for myofibre integrity and function. The leading DMD therapeutic strategy is antisense oligonucleotide-mediated ‘exon skipping’, however, this requires lifelong repeat administration. Single intervention strategies for the permanent correction of disease-causing mutations in the genomic DNA (such as CRISPR/Cas9, clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) are important avenues for DMD therapeutic development. An AAV9-delivered CRISPR/Cas9 system was employed to excise a disease-causing Dmd exon in the severely affected double knockout (dKO) mouse (lacking dystrophin and its developmental paralogue, utrophin). Dystrophin protein expression was partially restored in the muscle of dKO mice, although no extension in lifespan was observed. Within-fibre patchiness of dystrophin expression was observed in longitudinal skeletal muscle sections, an as of yet unreported consequence of CRISPR-mediated Dmd gene editing in myofibres. This provides an insight into the myonuclear domain structure of dystrophic fibres, and raises concerns about the possible functionality of gene edited myofibres that contain interspersed dystrophin-positive and -negative regions.
The development of non-viral therapeutic strategies focused on improving muscle architecture are also of particular interest for neuromuscular diseases. Extracellular vesicles (EVs) are membrane-enclosed nanoparticles involved in natural cell-cell communication, believed to transfer pro-myogenic and anti-fibrotic factors in the muscle niche. Both myotube- and myoblast-derived EVs enhanced myogenic differentiation at lower, rather than higher, doses in recipient C2C12 muscle cell culture. Highly pro-myogenic effects observed in non-vesicular transfer experiments were attributed to the co-purification of insulin within the isolation medium, highlighting important methodological considerations for the EV field.
Strategies that enable the specific activation of gene expression by translational modulation are currently lacking. Upstream open reading frames (uORFs) present within the 5ʹ untranslated regions of mRNA transcripts function in cis to repress translation of the downstream primary ORF. The in situ disruption of uORFs would be therapeutically beneficial for upregulating expression of a disease-relevant protein or protective factor. Validation of a panel of bioinformatically predicted uORFs revealed that not all are functional, including two uORFs with relevance to neuromuscular disease (i.e. SMN2 and UTRN for SMA and DMD respectively). A complex interplay of uORF features was found to influence uORF activity, where initiation of uORF translation was essential for functionality. The biophysical properties of poly-amino acid residues in synthetic uORF micropeptides were found to be major determinants of uORF activity. A synthetic poly-acid uORF sequence was highly repressive, and truncations of this sequence resulted in predictable levels of transgene attenuation. Such uORFs could be useful in biotechnology for the development of tunable, inducible, and tissue-specific transgene expression systems. Finally, CRISPR/Cas13b-mediated RNA editing was applied for the upregulation of a reporter transgene by uORF start codon disruption.