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Adeno-associated virus (AAV)-mediated systemic micro-dystrophin (μDys) therapy is currently in clinical trials. The hope is to permanently improve the life quality of Duchenne muscular dystrophy (DMD) patients. Numerous preclinical studies have been conducted to support these trials. However, none examined whether a single therapy at a young age can lead to lifelong disease amelioration. To address this critical question, we injected 1 × 1013 vg particles/mouse of an AAV serotype-9 μDys vector to 3-month-old mdx mice through the tail vein. Therapeutic outcomes were evaluated at the age of 11 months (adulthood, 8 months postinjection) and 21 months (terminal age, 18 months postinjection). Immunostaining and Western blot showed saturated supraphysiological levels of μDys expression in skeletal muscle and heart till the end of the study. Treatment significantly improved grip force and treadmill running, and significantly reduced the serum creatine kinase level at both time points. Since cardiac death is a major threat in late-stage patients, we evaluated cardiac electrophysiology and hemodynamics by ECG and the closed-chest cardiac catheter assay, respectively. Significant improvements were observed in these assays. Importantly, many ECG and hemodynamic parameters (heart rate, PR interval, QRS duration, QTc interval, end-diastolic/systolic volume, dP/dt max and min, max pressure, and ejection fraction) were completely normalized at 21 months of age. Our results have provided direct evidence that a single systemic AAV μDys therapy has the potential to provide lifelong benefits in the murine DMD model. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy (DMD) is a fatal muscle disease caused by dystrophin deficiency. Dystrophin consists of the amino terminus, central rod domain with 24 spectrin-like repeats and four hinges (H), cysteine-rich domain, and carboxyl terminus. Several highly abbreviated micro-dystrophins (μDys) are currently in clinical trials. They all carry H1 and H4. In this study, we investigated whether these two hinges are essential for μDy function in murine DMD models. Three otherwise identical μDys were engineered to contain H1 and/or H4 and were named H1/H4 (with both H1 and H4), ΔH1 (without H1), and ΔH4 (without H4). These constructs were packaged in adeno-associated virus serotype-9 and delivered to the tibialis anterior muscle of 3-month-old male mdx4cv mice (1E12 vector genome particles/muscle). Three months later, we detected equivalent μDys expression in total muscle lysate. However, only H1/H4 and ΔH1 showed correct sarcolemmal localization. ΔH4 mainly existed as sarcoplasmic aggregates. H1/H4 and ΔH1, but not ΔH4, fully restored the dystrophin-associated protein complex and significantly improved the specific muscle force. Eccentric contraction-induced force decline was best protected by H1/H4, followed by ΔH1, but not by ΔH4. Next, we compared H1/H4 and ΔH1 in 6-week-old male mdx mice by intravenous injection (1E13 vector genome particles/mouse). Four months postinjection, H1/H4 significantly outperformed ΔH1 in extensor digitorum longus muscle force measurements but two constructs yielded comparable electrocardiography improvements. We conclude that H4 is essential for μDys function and H1 facilitates force production. Our findings will help develop next-generation μDys gene therapy. [ABSTRACT FROM AUTHOR]

The highly ordered cortical microtubule lattice of skeletal muscle is disorganized in dystrophin-deficient mdx mice. Implicated mechanisms include loss of dystrophin binding, altered α-tubulin posttranslational modification, expression of a β-tubulin involved in regeneration, and reactive oxygen species (ROS). Here we show that the transverse microtubules in mdx muscle expressing miniaturized dystrophins are rapidly lost after eccentric contraction. Analysis of mdx lines expressing different dystrophin constructs demonstrate that spectrin-like repeats R4-15 and R20-23 were required for mechanically stable microtubules. Microtubule loss was prevented by the non-specific antioxidant N-acetylcysteine while inhibition of NADPH oxidase 2 had only a partial effect, suggesting that ROS from multiple sources mediate the rapid loss of transverse microtubules after eccentric contraction. Finally, ablation of α-dystrobrevin, β- or γ-cytoplasmic actin phenocopied the transverse microtubule instability of miniaturized dystrophins. Our data demonstrate that multiple dystrophin domains, α-dystrobrevin and cytoplasmic actins are necessary for mechanically stable microtubules.

Micro-dystrophins are highly promising candidates for treating Duchenne muscular dystrophy, a lethal muscle disease caused by dystrophin deficiency. Here, we report robust disease rescue in the severe DBA/2J-mdx model with a neuronal nitric oxide synthase (nNOS)-binding micro-dystrophin vector. 2 × 1013 vector genome particles/mouse of the vector were delivered intravenously to 10-week-old mice and were evaluated at 6 months of age. Saturated micro-dystrophin expression was detected in all skeletal muscles and the heart and restored the dystrophin-associated glycoprotein complex and nNOS. In skeletal muscle, therapy substantially reduced fibrosis and calcification and significantly attenuated inflammation. Centronucleation was significantly decreased in the tibialis anterior (TA) and extensor digitorum longus (EDL) muscles but not in the quadriceps. Muscle function was normalized in the TA and significantly improved in the EDL muscle. Heart histology and function were also evaluated. Consistent with the literature, DBA/2J-mdx mice showed myocardial calcification and fibrosis and cardiac hemodynamics was compromised. Surprisingly, similar myocardial pathology and hemodynamic defects were detected in control DBA/2J mice. As a result, interpretation of the cardiac data proved difficult due to the confounding phenotype in control DBA/2J mice. Our results support further development of this microgene vector for clinical translation. Further, DBA/2J-mdx mice are not good models for Duchenne cardiomyopathy.