Exon skipping peptide-conjugated morpholinos downregulate dynamin 2 to rescue centronuclear myopathy.

Abstract

Centronuclear myopathies (CNM) are rare congenital disorders characterized by muscle weakness and disorganization of myofibers. These conditions can result from dominant mutations in the DNM2 gene encoding the GTPase dynamin, making them potential targets for antisense therapy. Preclinical studies suggested decreasing DNM2 as a therapy but a recent clinical trial with antisense oligonucleotides did not effectively address the disease and showed some non-muscle toxicity. Here, to promote DNM2 downregulation in muscle versus other tissues, we used an exon skipping peptide-conjugated phosphorodiamidate morpholino (PPMO) targeting Dnm2 exon 6 splicing in the Dnm2R369W/+ mouse model for the moderate CNM form. PPMOs intravenous administration at early age (4 weeks) significantly downregulated intact (i.e. normally spliced) Dnm2 mRNA (∼50%) and DNM2 protein levels in muscle. This intervention led to a rescue of muscle force, thereby preventing disease progression. PPMO administration at a later age (8 weeks), when mice demonstrated established phenotypes, efficiently decreased intact Dnm2 mRNA and protein levels in muscle, resulting in reversal of the disease phenotype and significant improvement in muscle force (from 11 mN/mg to nearly 16 mN/mg). Overall, our results indicate that PPMOs targeting Dnm2 splicing effectively decrease intact Dnm2 mRNA and protein levels in muscle and rescue muscle force in Dnm2R369W/+ mice, suggesting a promising translational approach for patients with DNM2 mutations and potentially other forms of CNM. More generally, it provides the concept of using the exon skipping strategy to decrease the protein expression of a target gene, rather than producing a shorter functional protein as is generally done.