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Skeletal muscle is composed of long multinucleated cells, termed myofibers, that are formed through the activation and differentiation of resident muscle stem cells, called satellite cells. In healthy individuals, skeletal muscle enables voluntary locomotion, while also playing a role in energy metabolism and thermoregulation. As skeletal muscle is integral to everyday processes, perturbations to skeletal muscle function can have devastating consequences. Here we describe an integral tool in biomedical research of skeletal muscle regeneration and disease, the immunofluorescence staining of myogenic cells. We highlight useful techniques for immunostaining myogenic cells, and we list validated antibodies for the staining of muscle proteins across different species and multiple developmental time points. This includes methods for unmasking antigens following formaldehyde fixation (using Myosin Heavy Chain staining as an example), and practices for preserving endogenous fluorescent proteins by cardiac perfusion fixation.

Vector production scale-up is a major barrier in systemic adeno-associated virus (AAV) gene therapy. Many scalable manufacturing methods have been developed. However, the potency of the vectors generated by these methods has rarely been compared with vectors made by transient transfection (TT), the most commonly used method in preclinical studies. In this study, we blindly compared therapeutic efficacy of an AAV9 micro-dystrophin vector generated by the TT method and scalable herpes simplex virus (HSV) system in a Duchenne muscular dystrophy mouse model. AAV was injected intravenously at 5 × 1014 (high), 5 × 1013 (medium), or 5 × 1012 (low) viral genomes (vg)/kg. Comparable levels of micro-dystrophin expression were observed at each dose in a dose-dependent manner irrespective of the manufacturing method. Vector biodistribution was similar in mice injected with either the TT or the HSV method AAV. Evaluation of muscle degeneration/regeneration showed equivalent protection by vectors made by either method in a dose-dependent manner. Muscle function was similarly improved in a dose-dependent manner irrespective of the vector production method. No apparent toxicity was observed in any mouse. Collectively, our results suggest that the biological potency of the AAV micro-dystrophin vector made by the scalable HSV method is comparable to that made by the TT method., Graphical Abstract, Hakim and colleagues compared AAV9 micro-dystrophin vectors produced by transient transfection and HSV-based methods in the murine DMD model by systemic delivery. They found that these vectors had comparable biological potency. Their findings paved the way to translate an HSV-based AAV production system for clinical application.

Reduction in the expression of sarcolipin (SLN), an inhibitor of sarco(endo)plasmic reticulum (SR) Ca2+-ATPase (SERCA), ameliorates severe muscular dystrophy in mice. However, the mechanism by which SLN inhibition improves muscle structure remains unclear. Here, we describe the previously unknown function of SLN in muscle differentiation in Duchenne muscular dystrophy (DMD). Overexpression of SLN in C2C12 resulted in decreased SERCA pump activity, reduced SR Ca2+ load, and increased intracellular Ca2+ ([Formula: see text]) concentration. In addition, SLN overexpression resulted in altered expression of myogenic markers and poor myogenic differentiation. In dystrophin-deficient dog myoblasts and myotubes, SLN expression was significantly high and associated with defective [Formula: see text] cycling. The dystrophic dog myotubes were less branched and associated with decreased autophagy and increased expression of mitochondrial fusion and fission proteins. Reduction in SLN expression restored these changes and enhanced dystrophic dog myoblast fusion during differentiation. In summary, our data suggest that SLN upregulation is an intrinsic secondary change in dystrophin-deficient myoblasts and could account for the [Formula: see text] mishandling, which subsequently contributes to poor myogenic differentiation. Accordingly, reducing SLN expression can improve the [Formula: see text] cycling and differentiation of dystrophic myoblasts. These findings provide cellular-level supports for targeting SLN expression as a therapeutic strategy for DMD.

Duchenne muscular dystrophy (DMD) is a muscle-wasting disease caused by dystrophin deficiency. Vascular dysfunction has been suggested as an underlying pathogenic mechanism in DMD. However, this has not been thoroughly studied in a large animal model. Here we investigated structural and functional changes in the vascular smooth muscle and endothelium of the canine DMD model. The expression of dystrophin and endothelial nitric oxide synthase (eNOS), neuronal NOS (nNOS), and the structure and function of the femoral artery from 15 normal and 16 affected adult dogs were evaluated. Full-length dystrophin was detected in the endothelium and smooth muscle in normal but not affected dog arteries. Normal arteries lacked nNOS but expressed eNOS in the endothelium. NOS activity and eNOS expression were reduced in the endothelium of dystrophic dogs. Dystrophin deficiency resulted in structural remodeling of the artery. In affected dogs, the maximum tension induced by vasoconstrictor phenylephrine and endothelin-1 was significantly reduced. In addition, acetylcholine-mediated vasorelaxation was significantly impaired, whereas exogenous nitric oxide-induced vasorelaxation was significantly enhanced. Our results suggest that dystrophin plays a crucial role in maintaining the structure and function of vascular endothelium and smooth muscle in large mammals. Vascular defects may contribute to DMD pathogenesis. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Key points We developed a novel method to study sympatholysis in dogs. We showed abolishment of sarcolemmal nNOS, and reduction of total nNOS and total eNOS in the canine Duchenne muscular dystrophy (DMD) model. We showed sympatholysis in dogs involving both nNOS-derived NO-dependent and NO-independent mechanisms. We showed that the loss of sarcolemmal nNOS compromised sympatholysis in the canine DMD model. We showed that NO-independent sympatholysis was not affected in the canine DMD model. Abstract The absence of dystrophin in Duchenne muscular dystrophy (DMD) leads to the delocalization of neuronal nitric oxide synthase (nNOS) from the sarcolemma. Sarcolemmal nNOS plays an important role in sympatholysis, a process of attenuating reflex sympathetic vasoconstriction during exercise to ensure blood perfusion in working muscle. Delocalization of nNOS compromises sympatholysis resulting in functional ischaemia and muscle damage in DMD patients and mouse models. Little is known about the contribution of membrane-associated nNOS to blood flow regulation in dystrophin-deficient DMD dogs. We tested the hypothesis that the loss of sarcolemmal nNOS abolishes protective sympatholysis in contracting muscle of affected dogs. Haemodynamic responses to noradrenaline in the brachial artery were evaluated at rest and during contraction in the absence and presence of NOS inhibitors. We found sympatholysis was significantly compromised in DMD dogs, as well as in normal dogs treated with a selective nNOS inhibitor, suggesting that the absence of sarcolemmal nNOS underlies defective sympatholysis in the canine DMD model. Surprisingly, inhibition of all NOS isoforms did not completely abolish sympatholysis in normal dogs, suggesting sympatholysis in canine muscle also involves NO-independent mechanism(s). Our study established a foundation for using the dog model to test therapies aimed at restoring nNOS homeostasis in DMD.

Dual adeno-associated virus (AAV) technology was developed in 2000 to double the packaging capacity of the AAV vector. The proof of principle has been demonstrated in various mouse models. Yet, pivotal evidence is lacking in large animal models of human diseases. Here we report expression of a 7-kb canine ΔH2–R15 mini-dystrophin gene using a pair of dual AAV vectors in the canine model of Duchenne muscular dystrophy (DMD). The ΔH2–R15 minigene is by far the most potent synthetic dystrophin gene engineered for DMD gene therapy. We packaged minigene dual vectors in Y731F tyrosine-modified AAV-9 and delivered to the extensor carpi ulnaris muscle of a 12-month-old affected dog at the dose of 2 × 10(13) viral genome particles/vector/muscle. Widespread mini-dystrophin expression was observed 2 months after gene transfer. The missing dystrophin-associated glycoprotein complex was restored. Treatment also reduced muscle degeneration and fibrosis and improved myofiber size distribution. Importantly, dual AAV therapy greatly protected the muscle from eccentric contraction-induced force loss. Our data provide the first clear evidence that dual AAV therapy can be translated to a diseased large mammal. Further development of dual AAV technology may lead to effective therapies for DMD and many other diseases in human patients.

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.

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.

Adeno-associated virus (AAV) mediated gene replacement therapy holds great promise in treating inherited muscle diseases such as Duchenne muscular dystrophy (DMD). DMD is the most common lethal muscle disorder in children and is caused by mutations in the dystrophin gene. The full-length dystrophin coding sequence is approximately 11.2 kb. However, the maximal carrying capacity of the AAV vector is only 5 kb. To overcome this gene delivery conundrum, we developed a creative tri-AAV vector strategy. Specifically, we fragmented the full-length dystrophin cDNA expression cassette into three sections and engineered unique recombination signals for each section. Modified dystrophin cDNA fragments were independently packaged into three AAV vectors. Co-delivery of these vectors into dystrophin-deficient mice resulted in full-length dystrophin expression. Similar strategies can be used to deliver other large therapeutic genes.

Duchenne muscular dystrophy (DMD) is the most common lethal muscle disorder in children. It is caused by mutations of the dystrophin gene. Adeno-associated virus (AAV)-mediated gene replacement therapy has been actively pursued to treat DMD. However, this promising therapeutic modality has been challenged by the small packaging capacity of the AAV vector. The size of the full-length dystrophin cDNA is >11 kb, while an AAV virus can carry only a 5 kb genome. Innovative high-capacity AAV vectors may offer an opportunity to express the full-length dystrophin coding sequence. Here we describe several sets of tri-AAV vectors for full-length human dystrophin delivery. In each set, the full-length human dystrophin cDNA was split into three fragments and independently packaged into separate recombinant AAV vectors. Each vector was engineered with unique recombination signals for directional recombination. Tri-AAV vectors were coinjected into the tibialis anterior muscle of dystrophin-deficient mdx4cv mice. Thirty-five days after injection, dystrophin expression was examined by immunofluorescence staining. Despite low reconstitution efficiency, full-length human dystrophin was successfully expressed from the tri-AAV vectors. Our results suggest that AAV can be engineered to express an extra-large (up to 15 kb) gene that is approximately three times the size of the wild-type AAV genome. Further optimization of the trivector strategy may expand the utility of AAV for human gene therapy.

Duchenne muscular dystrophy (DMD) is a devastating muscle wasting disease caused by mutations in the dystrophin gene. Restoring dystrophin expression by adeno-associated virus mediated gene therapy holds great promise as a mutation-independent therapy for all DMD patients. The full-length dystrophin cDNA (≈11 kb) is far beyond the 5 kb packaging capacity of a single AAV vector. Consequently, truncated micro- and mini-dystrophin genes have been used. The micro- and mini-dystrophin genes encode ≈30% and ≈50% of the full-length dystrophin coding sequence, respectively. Mini-dystrophin is expected to provide better protection than micro-dystrophin. We have previously shown that dual AAV vectors can efficiently express a 6 to 8-kb mini-dystrophin in mdx mice. Dual AAV-mediated mini-dystrophin gene therapy significantly ameliorated histopathology and improved muscle function in mdx mice, a mouse model of DMD. To translate this promising therapy to large mammals, here we evaluated the reconstitution of a 7-kb minigene in the canine model of DMD by local injection. We engineered a pair of dual-AAV vectors to express a 7-kb canine codon-optimized ΔH2-R15 mini-dystrophin gene. For easy detection, a flag-tag and a GFP gene were fused to the N-terminal and C-terminal ends, respectively. To determine whether dual AAV vectors can lead to efficient mini-dystrophin expression, we co-delivered both vectors to the extensor carpi ulnaris muscle in the forelimb of DMD dogs at a dose of 2×013 vg particles/vector/muscle. Two months after gene transfer we evaluated transduction efficiency and observed successful expression of mini-dystrophin from the dual-AAV vectors. All flag-tag positive myofibers were also positive for minidystrophin, GFP and dystrophin-associated glycoprotein complex proteins. Importantly, mini-dystrophin gene therapy also reduced muscle force loss under the stress of repeated cycles of eccentric contraction. These results establish the proof-of-concept for mini-dystrophin gene therapy in dystrophic muscles of large mammals.

Adeno-associated virus serotype-8 and 9 (AAV-8 and 9) are the leading candidate vectors to test bodywide neonatal muscle gene therapy in large mammals. We have previously shown that systemic injection of 2-2.5×10(14) viral genome (vg) particles/kg of AAV-9 resulted in widespread skeletal muscle gene transfer in newborn dogs. However, nominal transduction was observed in the heart. In contrast, robust expression was achieved in both skeletal muscle and heart in neonatal dogs with 7.14-9.06×10(14) vg particles/kg of AAV-8. To determine whether superior cardiac transduction of AAV-8 is because of the higher vector dose, we delivered 6.14×10(14) and 9.65×10(14) vg particles/kg of AAV-9 to newborn puppies via the jugular vein. Transduction was examined 2.5 months later. Consistent with our previous reports, we observed robust bodywide transduction in skeletal muscle. However, increased AAV dose only moderately improved heart transduction. It never reached the level achieved by AAV-8. Our results suggest that differential cardiac transduction by AAV-8 and AAV-9 is likely because of the intrinsic property of the viral capsid rather than the vector dose.

Duchenne muscular dystrophy (DMD) is a progressive, muscle wasting disorder that affects all muscles in the body. An effective gene therapy for DMD will require efficient whole body muscle transduction. It was recently demonstrated that a single intravenous injection of adeno-associated virus (AAV) can lead to safe, bodywide muscle gene transfer in adolescent dogs affected by the canine model of DMD (cDMD) (Yue et al. 2015 Hum Mol Genet). Here we evaluated systemic gene therapy in three 3.5-m-old cDMD dogs using a novel canine codon-optimized micro-dystrophin vector. Transcriptional regulation is controlled by the muscle-specific CK8 promoter and a synthetic polyadenylation signal. All experimental subjects received transient immune suppression. One dog was administrated with 5×1013 viral genome (vg) particles/kg of the vector. Two dogs received 1×1014 vg particles/kg of the vector. All dogs tolerated injection well. Blood biochemistry (weekly in the first four weeks and biweekly thereafter) was unremarkable. Growth curve was nominally disturbed during the immunosuppression regimen, but recovered thereafter. Biopsy at 1,3 and 6 months after injection revealed widespread micro-dystrophin expression in 50-80% myofibers. The dystrophin-associated glycoprotein complex, including neuronal nitric oxide synthase (nNOS), was restored. While limited in sample size, muscle damage usually seen in young adult untreated dogs (inflammation, fibrosis, calcification) were rarely observed. CD4+, CD8+, and regulatory T cells were minimally detected. Night activity monitoring showed a trend of improvement. Limb muscle force (both forelimb and hind limb) was significantly enhanced compared to that of pre-injection. Our data suggest that systemic AAV micro-dystrophin therapy may translate to large mammals afflicted by DMD (Supported by Solid GT, NIH, DOD, Jesse's Journey).