Exon-Skipping Using Antisense Oligonucleotides for Laminin-Alpha2-Deficient Muscular Dystrophy

Phosphorodiamidate morpholino oligomer (PMO)-mediated exon-skipping is among the more promising approaches available for the treatment of several neuromuscular disorders, including Duchenne muscular dystrophy. The main weakness of this treatment arises from the low efficiency and sporadic nature of the delivery of neutrally charged PMOs into muscle fibres, the mechanism of which is unknown. Recently, using wild-type and dystrophic mdx52 mice, we showed that muscle fibres took up PMOs more efficiently during myotube formation. Interestingly, we detected PMO mainly in embryonic myosin heavy chain-positive regenerating fibres through in situ hybridisation. Next, we tested the therapeutic potential of PMOs in laminin-alpha2 (laminin-α2) chain-null dy^3K/dy^3K mice, a model of merosin-deficient congenital muscular dystrophy 1A (MDC1A or LAMA2-related muscular dystrophy: LAMA2-MD) with active muscle regeneration. We confirmed the recovery of the laminin-α2 chain following the skipping of the mutated exon 4 in dy^3K/dy^3K mice, which prolonged the lifespan of the animals slightly. These findings support the theory that PMO entry into fibres is dependent on the developmental stage in myogenesis rather than on dystrophin-deficient muscle membranes, and recommend a platform for the future development of PMO-mediated therapies for a variety of muscular disorders, such as LAMA2-MD, that involve active muscle regeneration. Herein, we describe the methods for PMO transfection/injection and the evaluation of the efficacy of exon-skipping in the laminin-α2-deficient dy^3K/dy^3K mouse model both in vitro and in vivo.