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Dystrophin is a large sub-sarcolemmal protein. Its absence leads to Duchenne muscular dystrophy (DMD). Binding to the sarcolemma is essential for dystrophin to protect muscle from contraction-induced injury. It has long been thought that membrane binding of dystrophin depends on its cysteine-rich (CR) domain. Here, we provide in vivo evidence suggesting that dystrophin contains three additional membrane-binding domains including spectrin-like repeats (R)1-3, R10-12 and C-terminus (CT). To systematically study dystrophin membrane binding, we split full-length dystrophin into ten fragments and examined subcellular localizations of each fragment by adeno-associated virus-mediated gene transfer. In skeletal muscle, R1-3, CR domain and CT were exclusively localized at the sarcolemma. R10-12 showed both cytosolic and sarcolemmal localization. Importantly, the CR-independent membrane binding was conserved in murine and canine muscles. A critical function of the CR-mediated membrane interaction is the assembly of the dystrophin-associated glycoprotein complex (DGC). While R1-3 and R10-12 did not restore the DGC, surprisingly, CT alone was sufficient to establish the DGC at the sarcolemma. Additional studies suggest that R1-3 and CT also bind to the sarcolemma in the heart, though relatively weak. Taken together, our study provides the first conclusive in vivo evidence that dystrophin contains multiple independent membrane-binding domains. These structurally and functionally distinctive membrane-binding domains provide a molecular framework for dystrophin to function as a shock absorber and signaling hub. Our results not only shed critical light on dystrophin biology and DMD pathogenesis, but also provide a foundation for rationally engineering minimized dystrophins for DMD gene therapy.

The ultimate goal of muscular dystrophy gene therapy is to treat all muscles in the body. Global gene delivery was demonstrated in dystrophic mice more than a decade ago using adeno-associated virus (AAV). However, translation to affected large mammals has been challenging. The only reported attempt was performed in newborn Duchenne muscular dystrophy (DMD) dogs. Unfortunately, AAV injection resulted in growth delay, muscle atrophy and contracture. Here we report safe and bodywide AAV delivery in juvenile DMD dogs. Three ∼2-m-old affected dogs received intravenous injection of a tyrosine-engineered AAV-9 reporter or micro-dystrophin (μDys) vector at the doses of 1.92-6.24 × 10(14) viral genome particles/kg under transient or sustained immune suppression. DMD dogs tolerated injection well and their growth was not altered. Hematology and blood biochemistry were unremarkable. No adverse reactions were observed. Widespread muscle transduction was seen in skeletal muscle, the diaphragm and heart for at least 4 months (the end of the study). Nominal expression was detected in internal organs. Improvement in muscle histology was observed in μDys-treated dogs. In summary, systemic AAV gene transfer is safe and efficient in young adult dystrophic large mammals. This may translate to bodywide gene therapy in pediatric patients in the future.