Atelocollagen-mediated Applications of Myostatin-targeting siRNA Increase Skeletal Muscle Mass

Objectives: In craniofacial region, skeletal deformities and malocclusion are often caused by hyper- and hypotrophy of masticatory muscles or tongue. RNA interference (RNAi) offers a novel therapeutic strategy based on the highly specific and efficient silencing of a target gene. In this study, we report the effectiveness of in vivo small interfering RNAs (siRNAs) delivery into skeletal muscles of normal or diseased mice through siRNAs targeting myostatin (Mst), a negative regulator of skeletal muscle formation, with atelocollagen (ATCOL). Methods: We prepared the synthetic 19-nt RNAs for Mst (Mst-siRNA)/ATCOL complex (10mM) and injected into the masseter and biceps femoris muscles of 20-week old C57BL/6, or tibial muscles of 20-week old dystrophin deficient mdx mice, an animal model for DMD. Two weeks after injection, we observed gross morphology of the muscles and dissected the muscle tissues. We further examined the effect of systemic administration of the Mst-siRNA/ATCOL complex applied intravenously into normal mice four times in three weeks. Results: Local application of the Mst-siRNA/ATCOL complex caused a marked increase in the muscle mass within a few weeks after application, showing Mst down-regulation by a western blot analysis (P<0.01, Student's t-test). In the case of mdx mice, a dramatically increased muscle mass was also observed and the protein levels of Mst were significantly decreased in the Mst-siRNA/ATCOL-treated muscle (P<0.05). Immunohistochemical analysis on the masseter using an anti-laminin a2 antibody showed increase in the myofibril size of the Mst-siRNA/ATCOL-treated muscle, as is the case for the wild-type. Furthermore, by systemic application of the Mst-siRNA/ATCOL complex, we observed an obvious enlargement of skeletal muscles of lower limbs, masseters, etc. and confirmed reduction of Mst proteins in the Mst-siRNA/ATCOL-treated muscle (P<0.01). Conclusion: These results imply that ATCOL-mediated application of siRNAs is a powerful tool for future therapeutic use for diseases including muscular atrophy.