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

Abstract

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 delivery of the neutrally charged PMO into muscle fibers, the mechanism of which is unknown. Recently, using wild-type and dystrophic mdx52 mice, we showed that muscle fibers took up PMO more efficiently during myotube formation. Interestingly, through in situ hybridization, we detected PMO mainly in embryonic myosin heavy chain-positive regenerating fibers. Next, we tested the therapeutic potential of PMO in laminin-alpha2 (laminin-α2) chain-null dy 3K /dy 3K mice, a model of merosin-deficient congenital muscular dystrophy 1A (MDC1A) with active muscle regeneration. We confirmed the recovery of the laminin-α2 chain following skipping of the mutated exon 4 in dy 3K /dy 3K mice, which prolonged the life span of the animals slightly. These findings support the theory that PMO entry into fibers is dependent on the developmental stage in myogenesis rather than on dystrophinless muscle membranes, and provide a platform for the future development of PMO-mediated therapies for a variety of muscular disorders, such as MDC1A, that involve active muscle regeneration. Herein, we describe the methods for PMO transfection/injection and evaluation of the efficacy of exon skipping in the laminin-α2-deficient dy 3K /dy 3K mouse model both in vitro and in vivo.