Long-term efficacy of systemic multiexon skipping targeting dystrophin exons 45-55 with a cocktail of vivo-morpholinos in mdx52 mice.

Antisense-mediated exon skipping, which can restore the reading frame, is a most promising therapeutic approach for Duchenne muscular dystrophy. Remaining challenges include the limited applicability to patients and unclear function of truncated dystrophin proteins. Multiexon skipping targeting exons 45–55 at the mutation hotspot of the dystrophin gene could overcome both of these challenges. Previously, we described the feasibility of exons 45–55 skipping with a cocktail of Vivo-Morpholinos in vivo; however, the long-term efficacy and safety of Vivo-Morpholinos remains to be determined. In this study, we examined the efficacy and toxicity of exons 45–55 skipping by intravenous injections of 6 mg/kg 10-Vivo-Morpholino cocktail (0.6 mg/kg each vPMO) every 2 weeks for 18 weeks to dystrophic exon-52 knockout (mdx52) mice. Systemic skipping of the entire exons 45–55 region was induced, and the Western blot analysis exhibited the restoration of 5–27% of normal levels of dystrophin protein in skeletal muscles, accompanied by improvements in histopathology and muscle strength. No obvious immune response and renal and hepatic toxicity were detected at the end-point of the treatment. We demonstrate our new regimen with the 10-Vivo-Morpholino cocktail is effective and safe for long-term repeated systemic administration in the dystrophic mouse model

Long-Term Efficacy of Systemic Multiexon Skipping Targeting Dystrophin Exons 45–55 With a Cocktail of Vivo-Morpholinos in Mdx52 Mice

Antisense-mediated exon skipping, which can restore the reading frame, is a most promising therapeutic approach for Duchenne muscular dystrophy. Remaining challenges include the limited applicability to patients and unclear function of truncated dystrophin proteins. Multiexon skipping targeting exons 45–55 at the mutation hotspot of the dystrophin gene could overcome both of these challenges. Previously, we described the feasibility of exons 45–55 skipping with a cocktail of Vivo-Morpholinos in vivo; however, the long-term efficacy and safety of Vivo-Morpholinos remains to be determined. In this study, we examined the efficacy and toxicity of exons 45–55 skipping by intravenous injections of 6 mg/kg 10-Vivo-Morpholino cocktail (0.6 mg/kg each vPMO) every 2 weeks for 18 weeks to dystrophic exon-52 knockout (mdx52) mice. Systemic skipping of the entire exons 45–55 region was induced, and the Western blot analysis exhibited the restoration of 5–27% of normal levels of dystrophin protein in skeletal muscles, accompanied by improvements in histopathology and muscle strength. No obvious immune response and renal and hepatic toxicity were detected at the end-point of the treatment. We demonstrate our new regimen with the 10-Vivo-Morpholino cocktail is effective and safe for long-term repeated systemic administration in the dystrophic mouse model

Mutation- and age-related expression of dystrophin-positive revertant fibers in TA and GC muscles from <i>mdx</i> and <i>mdx52</i> mice.

<p>(A) The number of RFs in one TA or GC section. (B) The number of RF clusters containing 2 or more positive fibers. (C) The maximum number of RFs in a single cluster. <i>Mdx</i> mice have a significantly higher number of RFs in all criteria than <i>mdx52</i> mice except for 2<b> </b>months of age in maximum number of RFs per cluster. The number of RFs in all criteria increases with age. Values are mean ± S.D. (<i>n</i> = 3–6 mice per each group). *<i>P</i><0.05, **<i>P</i><0.01 between <i>mdx</i> and <i>mdx52</i> mice; †<i>P</i><0.05, ††<i>P</i><0.05 compared to 2<b> </b>months old. M: months.</p

Dystrophin-positive revertant fibers with central nuclei at ages of 2, 6, 12, and 18 months in <i>mdx</i> and <i>mdx52</i> mice.

<p>Representative immunohistochemical images of maximum clusters of RFs in TA muscles are shown in each group. <i>Mdx</i> shows a higher maximum number of RFs than <i>mdx52</i> in all age groups. Wild-type C57BL/6 muscle at 2<b> </b>months old is displayed as a control. An anti-dystrophin C-terminal antibody (green) and DAPI staining (blue) were used. M: months. 20x objective lens, scale bar  = 100<b> </b>μm.</p

No expression of eMHC in RFs and attenuation of ongoing muscle regeneration in aged <i>mdx</i> and <i>mdx52</i> mice.

<p>(A) Triple staining of <i>mdx</i> and <i>mdx52</i> mice for RF (green), eMHC (red), and nucleus (blue). Revertant dystrophin is not co-localized with newly regenerated eMHC-positive fibers in TA and GC muscles from <i>mdx</i> and <i>mdx52</i> mice at any age. The pictures are representative GC muscles from <i>mdx</i> and <i>mdx52</i> mice at each age. 20x objective lens, scale bar  = 100<b> </b>μm. (B) The number of eMHC-positive fibers. Values are mean ± S.D. (<i>n</i> = 3–6 mice per group). A significant decrease in the number of eMHC-positive fibers is found only at 18<b> </b>months old in <i>mdx</i> mice (**<i>P</i><0.01 compared to 2<b> </b>months old, †<i>P</i><0.05 compared to 6 and 12<b> </b>months old). Symbol colors are accordant with the color of mice (red; <i>mdx</i>, blue; <i>mdx52</i>).</p

Distinct changes in the percentage of centrally nucleated fibers by mutations and age in <i>mdx</i> and <i>mdx52</i> mice.

<p>(A) Representative images of TA muscles from <i>mdx</i> and <i>mdx52</i> mice at ages 2, 6, 12 and 18<b> </b>months with hematoxylin and eosin staining. Wild-type C57BL/6 muscle at 2<b> </b>months of age is displayed as a control. M: months. Scale bar  = 100<b> </b>μm. (B) The percentage of centrally nucleated fibers in TA and GC muscles from <i>mdx</i> and <i>mdx52</i> mice. Three hundred to one thousand myofibers were counted in left and right muscles and the percentage of CNFs was averaged between the two muscles per mouse. Values are mean ± S.D. (<i>n</i> = 3–6 mice per group). *<i>P</i><0.05, **<i>P</i><0.01 between <i>mdx</i> and <i>mdx52</i> mice; †<i>P</i><0.05, ††<i>P</i><0.01 compared to 2<b> </b>months old; ‡<i>P</i><0.05, ‡‡<i>P</i><0.01 compared to 6<b> </b>months old. Symbol colors are accordant with the color of mice (red; <i>mdx</i>, blue; <i>mdx52</i>).</p