Antisense oligonucleotide-mediated exon skipping is able to correct out-of-frame mutations in Duchenne muscular dystrophy and restore truncated yet functional dystrophins. However, its application is limited by low potency and inefficiency in systemic delivery, especially failure to restore dystrophin in heart. Here, we conjugate a phosphorodiamidate morpholino oligomer with a designed cellpenetrating peptide (PPMO) targeting a mutated dystrophin exon. Systemic delivery of the novel PPMO restores dystrophin to almost normal levels in the cardiac and skeletal muscles in dystrophic mdx mouse. This leads to increase in muscle strength and prevents cardiac pump failure induced by dobutamine stress in vivo. Muscle pathology and function continue to improve during the 12-week course of biweekly treatment, with significant reduction in levels of serum creatine kinase. The high degree of potency of the oligomer in targeting all muscles and the lack of detectable toxicity and immune response support the feasibility of testing the novel oligomer in treating Duchenne muscular dystrophy patients.M utations in the dystrophin gene underlie two forms of muscular dystrophy: Duchenne and Becker muscular dystrophy (DMD and BMD). DMD is caused mainly by nonsense and frame-shift mutations with little or no production of functional dystrophin protein, leading to disease onset in early childhood with lethal consequences. BMD is caused by mutations that typically create shortened but in-frame transcripts with production of partially functional dystrophin, leading to variable and often overt symptoms (1-3). Most DMD mutations occur within the rod domain, which spans more than half the length of the protein, but seems to have limited functional importance (4, 5). Antisense therapy uses specific oligomers to remove the mutated or additional exon(s) that disrupt the reading frame, thus restoring the expression of shortened forms of dystrophin protein retaining critical functions (6-11).We previously demonstrated that i.m. delivery of a specific 2Ј-O-methyl phosphorothioate antisense oligonucleotide (2ЈOMeAON) was able to skip targeted dystrophin exon 23 in mdx mouse, a model of DMD (9). This mouse carries a nonsense point mutation within exon 23 and lacks dystrophin expression (except in a few rare revertant fibers) in all muscles, including the heart (12, 13). Skipping the mutated exon 23 restored both the reading frame and dystrophin expression, with functional improvement of the treated muscles (14) [supporting information (SI) Fig. S1a]. Recently we showed that a phosphorodiamidate morpholino oligomer (PMO), E23ϩ7-18 targeting the junction of exon 23 and intron 23 of mouse dystrophin (referred to as PMOE23 hereafter), was able to induce up to functional levels of dystrophin expression in some skeletal muscles by regular i.v. injections in mdx mice (15). However, dystrophin expression induced by both 2ЈOMeAON and PMO required high doses and was highly variable between muscles and myofibers in terms of observed efficacy. Of greater conc...

Absence of dystrophin makes skeletal muscle more susceptible to injury, resulting in breaches of the plasma membrane and chronic inflammation in Duchenne muscular dystrophy (DMD). Current management by glucocorticoids has unclear molecular benefits and harsh side effects. It is uncertain whether therapies that avoid hormonal stunting of growth and development, and/or immunosuppression, would be more or less beneficial. Here, we discover an oral drug with mechanisms that provide efficacy through anti-inflammatory signaling and membrane-stabilizing pathways, independent of hormonal or immunosuppressive effects. We find VBP15 protects and promotes efficient repair of skeletal muscle cells upon laser injury, in opposition to prednisolone. Potent inhibition of NF-κB is mediated through protein interactions of the glucocorticoid receptor, however VBP15 shows significantly reduced hormonal receptor transcriptional activity. The translation of these drug mechanisms into DMD model mice improves muscle strength, live-imaging and pathology through both preventive and post-onset intervention regimens. These data demonstrate successful improvement of dystrophy independent of hormonal, growth, or immunosuppressive effects, indicating VBP15 merits clinical investigation for DMD and would benefit other chronic inflammatory diseases.

The availability of animal models for Duchenne muscular dystrophy has led to extensive preclinical research on potential therapeutics. Few studies have focused on reliability and sensitivity of endpoints for mdx mouse drug trials. Therefore, we sought to compare a wide variety of reported and novel endpoint measures in exercised mdx and normal control mice at 10, 20, and 40 weeks of age. Statistical analysis as well as power calculations for expected effect sizes in mdx preclinical drug trials across different ages showed that body weight, normalized grip strength, horizontal activity, rest time, cardiac function measurements, blood pressure, total central/peripheral nuclei per fiber, and serum creatine kinase are the most effective measurements for detecting drug-induced changes. These data provide an experimental basis upon which standardization of preclinical drug testing can be developed.

Recent studies showed that chronic administration of losartan, an angiotensin II type I receptor antagonist, improved skeletal muscle function in dystrophin-deficient mdx mice. In this study, C57BL/10ScSn-Dmdmdx/J female mice were either untreated or treated with losartan (n = 15) in the drinking water at a dose of 600 mg/L over a 6-month period. Cardiac function was assessed via in vivo high frequency echocardiography and skeletal muscle function was assessed using grip strength testing, Digiscan monitoring, Rotarod timing, and in vitro force testing. Fibrosis was assessed using picrosirius red staining and Image J analysis. Gene expression was evaluated using real-time polymerized chain reaction (RT-PCR). Percentage shortening fraction was significantly decreased in untreated (26.9% ± 3.5%) mice compared to losartan-treated (32.2% ± 4.2%; P < .01) mice. Systolic blood pressure was significantly reduced in losartan-treated mice (56 ± 6 vs 69 ± 7 mm Hg; P < .0005). Percentage cardiac fibrosis was significantly reduced in losartan-treated hearts (P < .05) along with diaphragm (P < .01), extensor digitorum longus (P < .05), and gastrocnemius (P < .05) muscles compared to untreated mdx mice. There were no significant differences in skeletal muscle function between treated and untreated groups. Chronic treatment with losartan decreases cardiac and skeletal muscle fibrosis and improves cardiac systolic function in dystrophin-deficient mdx mice.