Journal
HUMAN MOLECULAR GENETICS
Volume 22, Issue 24, Pages 4914-4928Publisher
OXFORD UNIV PRESS
DOI: 10.1093/hmg/ddt341
Keywords
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Funding
- Intramural Research Grant for Neurological and Psychiatric Disorders of the National Center of Neurology and Psychiatry [22-5]
- Ministry of Health, Labour, and Welfare of Japan [H21-Translational Research-011, H21-Clinical Research-015, H23-Neuromuscular Disease-005]
- MRC Confidence in Concept Award [CiC17]
- Medical Research Council [G0900887] Funding Source: researchfish
- MRC [G0900887] Funding Source: UKRI
- Grants-in-Aid for Scientific Research [25460666] Funding Source: KAKEN
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Phosphorodiamidate morpholino oligomer (PMO)-mediated exon skipping is among the more promising approaches to the treatment of several neuromuscular disorders including Duchenne muscular dystrophy. The main weakness of this approach arises from the low efficiency and sporadic nature of the delivery of charge-neutral PMO into muscle fibers, the mechanism of which is unknown. In this study, to test our hypothesis that muscle fibers take up PMO more efficiently during myotube formation, we induced synchronous muscle regeneration by injection of cardiotoxin into the tibialis anterior muscle of Dmd exon 52-deficient mdx52 and wild-type mice. Interestingly, by in situ hybridization, we detected PMO mainly in embryonic myosin heavy chain-positive regenerating fibers. In addition, we showed that PMO or 2'-O-methyl phosphorothioate is taken up efficiently into C2C12 myotubes when transfected 24-72 h after the induction of differentiation but is poorly taken up into undifferentiated C2C12 myoblasts suggesting efficient uptake of PMO in the early stages of C2C12 myotube formation. Next, we tested the therapeutic potential of PMO for laminin-alpha 2 chain-null dy(3K)/dy(3K) mice: a model of merosin-deficient congenital muscular dystrophy (MDC1A) with active muscle regeneration. We confirmed the recovery of laminin-alpha 2 chain and slightly prolonged life span following skipping of the mutated exon 4 in dy(3K)/dy(3K) mice. These findings support the idea that PMO entry into fibers is dependent on a developmental stage in myogenesis rather than on dystrophinless muscle membranes and provide a platform for developing PMO-mediated therapies for a variety of muscular disorders, such as MDC1A, that involve active muscle regeneration.
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