Abstract
ABSTRACTExon skipping is a promising strategy for Duchenne muscular dystrophy (DMD) disease-modifying therapy. To make this approach safe, ensuring that excluding one or more exons will restore the reading frame and that the resulting protein will retain critical functions of the full-length dystrophin protein is necessary. However, in vivo testing of the consequences of skipping exons that encode the N-terminal actin-binding domain (ABD) has been confounded by the absence of a relevant animal model. We created a mouse model of the disease recapitulating a novel human mutation, a large de novo deletion of exons 8-34 of the DMD gene, found in a Russian DMD patient. This mutation was achieved by deleting exons 8-34 of the X-linked mouse Dmd gene using CRISPR/Cas9 genome editing, which led to a reading frame shift and the absence of functional dystrophin production. Male mice carrying this deletion display several important signs of muscular dystrophy, including a gradual age-dependent decrease in muscle strength, increased creatine kinase, muscle fibrosis and central nucleation. The degrees of these changes are comparable to those observed in mdx mice, a standard laboratory model of DMD. This new model of DMD will be useful for validating therapies based on skipping exons that encode the N-terminal ABD and for improving our understanding of the role of the N-terminal domain and central rod domain in the biological function of dystrophin. Simultaneous skipping of exons 6 and 7 should restore the gene reading frame and lead to the production of a protein that might retain functionality despite the partial deletion of the ABD.
Highlights
Duchenne muscular dystrophy (DMD) is an X-linked recessive disease affecting approximately 1 in 5000 newborn males (Mendell and Lloyd-Puryear, 2013)
Identification of a new mutation in the DMD gene leading to the development of Duchenne muscular dystrophy We identified a new mutation in the DMD gene of a male patient of Russian origin
A novel, de novo mutation in the DMD gene leading to a frameshift deletion of exons 8-34 was found in a Russian patient with muscular dystrophy
Summary
Duchenne muscular dystrophy (DMD) is an X-linked recessive disease affecting approximately 1 in 5000 newborn males (Mendell and Lloyd-Puryear, 2013). The disease is characterized by progressive muscle wasting, leading to patients becoming fully dependent on mobility aids early in life. The disease is caused by the absence of the functional dystrophin protein in muscle cells. Dystrophin is a large (3685 amino acids in humans), evolutionarily conserved protein. It is a crucial component of a molecular complex, the main function of which is to ensure muscle membrane integrity and stability by playing the role of a ‘molecular spring’, connecting a cytoskeletal network to the sarcolemma and extracellular matrix. When dystrophin is absent or aberrant, the sarcolemma anchoring of actomyosin complexes becomes weaker, which effectively disrupts membrane integrity during muscle contraction and leads to myofibre necrosis and inflammation. Regeneration compensates for the loss of myofibres until the myoblast pool becomes depleted, which is associated with muscle degeneration and disease progression (Davies and Nowak, 2006)
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