Platelet-derived growth factor receptors and ligands are up-regulated in paediatric fibromatoses

AIMS: Platelet-derived growth factors (PDGF) and their receptors (PDGFR) play an essential role in pathways involved in the regulation of cell proliferation, growth and function. Overexpression of PDGF/R is reported in a wide range of solid tumours. The aim was to determine levels of PDGF/R expression in paediatric fibromatoses and myofibromatosis. METHODS AND RESULTS: Quantitative real-time polymerase chain reaction was used to examine the expression level of alpha and beta isoforms of PDGF/R in 17 fibromatoses, four myofibromatoses and three dermatofibrosarcoma protuberans (DFSP) in children. Fifteen of 17 (88%) fibromatoses and all myofibromatoses and DFSPs demonstrated increased expression of PDGFalpha and beta compared with a panel of normal tissues. In terms of the cognate receptors, 13/17 (76%) fibromatoses demonstrated increased expression for PDGFRalpha and Rbeta, whereas 3/4 myofibromatoses demonstrated increased expression of PDGFRalpha and all four had increased PDGFRbeta expression. All DFSPs were associated with increased expression of both PDGFRalpha and PDGFRbeta compared with normal control tissues. CONCLUSIONS: Increased expression of PDGF/Ralphabeta may play an important role in the mechanism of growth of these paediatric fibromatous lesions and warrants further investigation, since novel therapeutic interventions could potentially be developed in the light of the expression patterns.     

Merosin (laminin-2) localization in basal lamina of normal skeletal muscle fibers and changes in plasma membrane of merosin-deficient skeletal muscle fibers

Primary deficiency of merosin causes a severe congenital muscular dystrophy (CMD) and a mouse dystrophy (dy/dy mouse). Also, its secondary deficiency is seen in some CMD with abnormal glycosylation of -dystroglycan, an extracellular membrane protein, which is the receptor of merosin and binds to dystrophin underlying the sarcolenma via -dystroglycan, a transmembrane protein. In immunogold and freeze-etch electron microscopic studies, merosin in basal lamina of normal skeletal muscles has a zonation in the distribution and is localized at the lamina lucida of muscle basal lamina, and dystrophin molecules are often closed to merosin molecules at the inside and outside surface of muscle plasma membrane. Moreover, merosin molecules exist as the short fine cross-bridge fibrils connecting the basal lamina to the neighboring outer leaflet of the muscle plasma membrane. In freeze-fracture studies, the changes in muscle plasma membranes of dy/dy mice reveal a markedly decreased density of orthogonal arrays (OAs) but normal density of intramembranous particles (IMPs), whereas depletions of IMPs with decreased OAs have been found in Fukyama-type congenital muscular dystrophy, Duchenne muscular dystrophy, and mdx mice. Thus, further studies including the functional role of OAs would be required to understand the pathomechanism of merosin-deficient CMD.     

PDGF-A/PDGF alpha-receptor signaling is required for lung growth and the formation of alveoli but not for early lung branching morphogenesis.

Platelet-derived growth factors (PDGF) constitute a family of four gene products (PDGF-A-D) acting by means of two receptor tyrosine kinases, PDGFR alpha and beta. Three of the ligands (PDGF-A, -B, and -C) bind to PDGFR alpha with high affinity. Knockout of pdgf-a in mice has demonstrated a role for PDGF-A in the recruitment of smooth muscle cells to the alveolar sacs and their further compartmentalization into alveoli. Although this is a late, postnatal step in lung development, pdgf-a antisense oligonucleotides were previously shown to inhibit epithelial branching in rat lung explants in vitro, which reflects an early embryonic process. These conflicting results may be explained by substitution of genetic loss of pdgf-a by maternal transfer of PDGF-A to the knockout embryo or the presence of other PDGFR alpha agonists (PDGF-B and -C) in vivo, potentially masking an effect of PDGF-A on branching morphogenesis. Alternatively, the administration of pdgf-a antisense oligonucleotides affected other processes than the intended. To discriminate between these opposing possibilities, we have analyzed lung development in pdgfr alpha -/- embryos and lung primordia grown in vitro. Our analysis shows that, while the pdgfr alpha -/- lungs and explanted lung rudiments were smaller than normal, branching morphogenesis appears qualitatively intact and proceeds until at least embryonic day 15.5, generating both prospective conducting and respiratory airways. We conclude that, although PDGF-AA signaling over PDGFR alpha may have direct or indirect roles in overall lung growth, it does not specifically control early branching of the lung epithelium.     

Transgenic expression of {alpha}7{beta}1 integrin maintains muscle integrity, increases regenerative capacity, promotes hypertrophy, and reduces cardiomyopathy in dystrophic mice

We previously reported that enhanced expression of the alpha7beta1 integrin ameliorates the development of muscular dystrophy and extends longevity in alpha7BX2-mdx/utr(-/-) transgenic mice (Burkin DJ, Wallace GQ, Nicol KJ, Kaufman DJ, Kaufman SJ: Enhanced expression of the alpha7beta1 integrin reduces muscular dystrophy and restores viability in dystrophic mice. We now report on the mechanism by which these mice were rescued by the integrin. As a result of increased integrin in alpha7BX2-mdx/utr(-/-) mice the structural integrity of the myotendinous and neuromuscular junctions are maintained. A twofold increase in satellite cells in alpha7BX2-mdx/utr(-/-) skeletal muscle was detected by immunofluorescence using the satellite cell marker c-met. These cells enhanced the regenerative capacity of muscle in the transgenic animals as determined by fusion of BrdUrd-labeled cells into muscle fibers. Increased integrin also leads to hypertrophy. Finally, transgenic expression of alpha7BX2 integrin chain in skeletal muscle secondarily reduces the development of cardiomyopathy, the ultimate cause of death in these animals. We believe this multiplicity of responses to increased alpha7beta1 integrin collectively inhibits the development of muscle disease and increases longevity in these mice.     

The physiological effects of IGF-1 (class 1:Ea transgene) over-expression on exercise-induced damage and adaptation in dystrophic muscles of mdx mice

Duchenne muscular dystrophy (DMD) is a genetic disorder in which muscle weakness and fragility contribute to ongoing muscle degeneration. Although exercise-induced muscle damage is associated with adaptation that protects normal muscle from further damage, exploiting this process to protect dystrophic muscle has been avoided for fear of inducing excessive muscle degeneration. However, muscle-specific over-expression of the class 1:Ea isoform of insulin-like growth factor-1 (IGF-1) reduces myofibre necrosis in dystrophic mdx mice (a model for DMD) and, therefore, may enhance the adaptation process in response to eccentric exercise. To test this hypothesis, we evaluated the effect of transgenic class 1:Ea IGF-1 over-expression on the susceptibility to muscle damage and subsequent adaptation in 12-week-old dystrophic mdx and non-dystrophic control mice. Experiments were conducted in vivo using a custom-built isokinetic mouse dynamometer to measure the deficit in joint torque (indicating muscle damage) after 20 maximal lengthening (eccentric) contractions. Adaptation to this damaging exercise was evaluated by repeating the protocol 7 days after the initial exercise. The over-expression of IGF-1 significantly increased the normalised joint torque in non-dystrophic mice and appeared to ameliorate the muscle weakness in dystrophic mice. All mice displayed a marked reduction in the susceptibility to muscle damage on day 7; however, this adaptation was unaffected by IGF-1, showing that IGF-1 does not protect the dystrophic muscles of adult mdx mice against damage resulting from maximal lengthening contractions.     

Laminin alpha1 chain reduces muscular dystrophy in laminin alpha2 chain deficient mice

Laminin (LN) alpha2 chain deficiency in humans and mice leads to severe forms of congenital muscular dystrophy (CMD). Here, we investigated whether LNalpha1 chain in mice can compensate for the absence of LNalpha2 chain and prevent the development of muscular dystrophy. We generated mice expressing a LNalpha1 chain transgene in skeletal muscle of LNalpha2 chain deficient mice. LNalpha1 is not normally expressed in muscle, but the transgenically produced LNalpha1 chain was incorporated into muscle basement membranes, and normalized the compensatory changes of expression of certain other laminin chains (alpha4, beta2). In 4-month-old mice, LNalpha1 chain could fully prevent the development of muscular dystrophy in several muscles, and partially in others. The LNalpha1 chain transgene not only reversed the appearance of histopathological features of the disease to a remarkable degree, but also greatly improved health and longevity of the mice. Correction of LNalpha2 chain deficiency by LNalpha1 chain may serve as a paradigm for gene therapy of CMD in patients.     

Fibrinogen drives dystrophic muscle fibrosis via a TGFbeta/alternative macrophage activation pathway

In the fatal degenerative Duchenne muscular dystrophy (DMD), skeletal muscle is progressively replaced by fibrotic tissue. Here, we show that fibrinogen accumulates in dystrophic muscles of DMD patients and mdx mice. Genetic loss or pharmacological depletion of fibrinogen in these mice reduced fibrosis and dystrophy progression. Our results demonstrate that fibrinogen-Mac-1 receptor binding, through induction of IL-1beta, drives the synthesis of transforming growth factor-beta (TGFbeta) by mdx macrophages, which in turn induces collagen production in mdx fibroblasts. Fibrinogen-produced TGFbeta further amplifies collagen accumulation through activation of profibrotic alternatively activated macrophages. Fibrinogen, by engaging its alphavbeta3 receptor on fibroblasts, also directly promotes collagen synthesis. These data unveil a profibrotic role of fibrinogen deposition in muscle dystrophy.     

Platelet-derived growth factor isoform expression in carbon tetrachloride-induced chronic liver injury

Platelet-derived growth factor (PDGF) has an essential role in liver fibrogenesis, as PDGF-B and -D both act as potent mitogens on culture-activated hepatic stellate cells (HSCs). Induction of PDGF receptor type-beta (PDGFR beta) in HSC is well documented in single-dose carbon tetrachloride (CCl(4))-induced acute liver injury. Of the newly discovered isoforms PDGF-C and -D, only PDGF-D shows significant upregulation in bile duct ligation (BDL) models. We have now investigated the expression of PDGF isoforms and receptors in chronic liver injury in vivo after long-term CCl(4) treatment and demonstrated that isolated hepatocytes have the requisite PDGF signaling pathways, both in the naive state and when isolated from CCl(4)-treated rats. In vivo, PDGF gene expression showed upregulation of all PDGF isoforms and receptors, with values peaking at 4 weeks and decreasing to near basal levels by 8 and 12 weeks. Interestingly, PDGF-C increased significantly when compared to BDL-models. PDGF-A, PDGF-C and PDGF receptor type-alpha (PDGFR alpha) correlated closely with inflammation and steatosis. Immunohistochemistry revealed expression of PDGF-B, -C and -D in areas corresponding to centrilobular necrosis, inflammation and fibrosis, whereas PDGF-A localized in regenerative hepatocytes. PDGFR beta was identified along the fibrotic septa, whereas PDGFR alpha showed positive staining in fibrotic septa and regenerative hepatocytes. Despite a significant decline of PDGF isoforms, hepatocyte regeneration peaked at 8 weeks. A marked difference in the degree of fibrosis was observed amongst the individual animals. In summary, PDGF expression in liver damage primarily parallels mesenchymal cell proliferation and extracellular matrix production, rather than hepatocyte regeneration. We conclude that PDGF levels in chronic liver injury peak at 4 weeks after onset of injury, and that the outcome of chronic toxic liver injury strongly depends on the individual capacity for tissue regeneration in the weeks following the peak of PDGF expression.     

Cilostazol attenuates oxidative stress and apoptosis in the quadriceps muscle of the dystrophic mouse experimental model

Duchenne muscular dystrophy (DMD) is the most severe and frequent form of muscular dystrophy. The mdx mouse is one of the most widely used experimental models to understand aspects of the biology of dystrophic skeletal muscles and the mechanisms of DMD. Oxidative stress and apoptosis are present in early stages of the disease in mdx mice. The high production of reactive oxygen species (ROS) causes activation of apoptotic death regulatory proteins due to DNA damage and breakdown of nuclear and mitochondrial membranes. The quadriceps (QUA) muscle of the mdx mouse is a good tool to study oxidative events. Previous studies have demonstrated that cilostazol exerts an anti-oxidant effect by decreasing the production of reactive oxygen species (ROS). The present study aimed to evaluate the ability of cilostazol to modulate oxidative stress and apoptosis in the QUA muscle of mdx mice. Fourteen-day-old mdx mice received cilostazol or saline for 14 days. C57BL/10 mice were used as a control. In the QUA muscle of mdx mice, cilostazol treatment decreased ROS production (−74%), the number of lipofuscin granules (−47%), lipid peroxidation (−11%), and the number of apoptotic cells (−66%). Thus cilostazol showed anti-oxidant and anti-apoptotic action in the QUA muscle of mdx mice.     

Immunoproteasome in animal models of Duchenne muscular dystrophy

Increased proteasome activity has been implicated in the atrophy and deterioration associated with dystrophic muscles of Duchenne muscular dystrophy (DMD). While proteasome inhibitors show promise in the attenuation of muscle degeneration, proteasome inhibition-induced toxicity was a major drawback of this therapeutic strategy. Inhibitors that selectively target the proteasome subtype that is responsible for the loss in muscle mass and quality would reduce side effects and be less toxic. This study examined proteasome activity and subtype populations, along with muscle function, morphology and damage in wild-type (WT) mice and two murine models of DMD, dystrophin-deficient (MDX) and dystrophin- and utrophin-double-knockout (DKO) mice. We found that immunoproteasome content was increased in dystrophic muscles while the total proteasome content was unchanged among the three genotypes of mice. Proteasome proteolytic activity was elevated in dystrophic muscles, especially in DKO mice. These mice also exhibited more severe muscle atrophy than either WT or MDX mice. Muscle damage and regeneration, characterized by the activity of muscle creatine kinase in the blood and the percentage of central nuclei were equally increased in dystrophic mice. Accordingly, the overall muscle function was similarly reduced in both dystrophic mice compared with WT. These data demonstrated that there was transformation of standard proteasomes to immunoproteasomes in dystrophic muscles. In addition, DKO that showed greatest increase in proteasome activities also demonstrated more severe atrophy compared with MDX and WT. These results suggest a putative role for the immunoproteasome in muscle deterioration associated with DMD and provide a potential target for therapeutic intervention.     

Alpha7beta1 integrin does not alleviate disease in a mouse model of limb girdle muscular dystrophy type 2F

Transgenic expression of the alpha7beta1 integrin in the dystrophic mdx/utr-/- mouse decreases development of muscular dystrophy and enhances longevity. To explore the possibility that elevating alpha7beta1 integrin expression could also ameliorate different forms of muscular dystrophy, we used transgenic technology to enhance integrin expression in mice lacking delta-sarcoglycan (delta sgc), a mouse model for human limb girdle muscular dystrophy type 2F. Unlike alpha7 transgenic mdx/utr-/- mice, enhanced alpha7beta1 integrin expression in the delta sgc-null mouse did not alleviate muscular dystrophy in these animals. Expression of the transgene in the delta sgc-null did not alleviate dystrophic histopathology, nor did it decrease cardiomyopathy or restore exercise tolerance. One hallmark of integrin-mediated alleviation of muscular dystrophy in the mdx/utr-/- background is the restoration of myotendinous junction integrity. As assessed by atomic force microscopy, myotendinous junctions from normal and delta sgc-null mice were indistinguishable, thus suggesting the important influence of myotendinous junction integrity on the severity of muscular dystrophy and providing a possible explanation for the inability of enhanced integrin expression to alleviate dystrophy in the delta sgc-null mouse. These results suggest that distinct mechanisms underlie the development of the diseases that arise from deficiencies in dystrophin and sarcoglycan.     

PDGFRα signalling promotes fibrogenic responses in collagen‐producing cells in Duchenne muscular dystrophy

Fibrosis is a characteristic of Duchenne muscular dystrophy (DMD), yet the cellular and molecular mechanisms responsible for DMD fibrosis are poorly understood. Utilizing the Collagen1a1‐GFP transgene to identify cells producing Collagen‐I matrix in wild‐type mice exposed to toxic injury or those mutated at the dystrophin gene locus (mdx) as a model of DMD, we studied mechanisms of skeletal muscle injury/repair and fibrosis. PDGFRα is restricted to Sca1+, CD45? mesenchymal progenitors. Fate‐mapping experiments using inducible CreER/LoxP somatic recombination indicate that these progenitors expand in injury or DMD to become PDGFRα+, Col1a1‐GFP+ matrix‐forming fibroblasts, whereas muscle fibres do not become fibroblasts but are an important source of the PDGFRα ligand, PDGF‐AA. While in toxin injury/repair of muscle PDGFRα, signalling is transiently up‐regulated during the regenerative phase in the DMD model and in human DMD it is chronically overactivated. Conditional expression of the constitutively active PDGFRα D842V mutation in Collagen‐I+ fibroblasts, during injury/repair, hindered the repair phase and instead promoted fibrosis. In DMD, treatment of mdx mice with crenolanib, a highly selective PDGFRα/β tyrosine kinase inhibitor, reduced fibrosis, improved muscle strength, and was associated with decreased activity of Src, a downstream effector of PDGFRα signalling. These observations are consistent with a model in which PDGFRα activation of mesenchymal progenitors normally regulates repair of the injured muscle, but in DMD persistent and excessive activation of this pathway directly drives fibrosis and hinders repair. The PDGFRα pathway is a potential new target for treatment of progressive DMD. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Absence of alpha 7 integrin in dystrophin-deficient mice causes a myopathy similar to Duchenne muscular dystrophy

Both the dystrophin-glycoprotein complex and alpha7beta1 integrin have critical roles in the maintenance of muscle integrity via the provision of mechanical links between muscle fibres and the basement membrane. Absence of either dystrophin or alpha7 integrin results in a muscular dystrophy. To clarify the role of alpha7 integrin and dystrophin in muscle development and function, we generated integrin alpha7/dystrophin double-mutant knockout (DKO) mice. Surprisingly, DKO mice survived post-natally and were indistinguishable from wild-type, integrin alpha7-deficient and mdx mice at birth, but died within 24-28 days. Histological analysis revealed a severe muscular dystrophy in DKO mice with endomysial fibrosis and ectopic calcification. Weight loss was correlated with the loss of muscle fibres, indicating that progressive muscle wasting in the double mutant was most likely due to inadequate muscle regeneration. The data further support that premature death of DKO mice is due to cardiac and/or respiratory failure. The integrin alpha7/dystrophin-deficient mouse model, therefore, resembles the pathological changes seen in Duchenne muscular dystrophy and suggests that the different clinical severity of dystrophin deficiency in human and mouse may be due to a fine-tuned difference in expression of dystrophin and integrin alpha7 in both species. Together, these findings indicate an essential role for integrin alpha7 in the maintenance of dystrophin-deficient muscles.     

Elimination of myostatin does not combat muscular dystrophy in dy mice but increases postnatal lethality

Myostatin is a TGF-beta family member and a negative regulator of skeletal muscle growth. It has been proposed that reduction or elimination of myostatin could be a treatment for degenerative muscle diseases such as muscular dystrophy. Laminin-deficient congenital muscular dystrophy is one of the most severe forms of muscular dystrophy. To test the possibility of ameliorating the dystrophic phenotype in laminin deficiency by eliminating myostatin, we crossed dy(W) laminin alpha2-deficient and myostatin null mice. The resulting double-deficient dy(W)/dy(W);Mstn(-/-) mice had a severe clinical phenotype similar to that of dy(W)/dy(W) mice, even though muscle regeneration was increased. Degeneration and inflammation of muscle were not alleviated. The pre-weaning mortality of dy(W)/dy(W);Mstn(-/-) mice was increased compared to dy(W)/dy(W), most likely due to significantly less brown and white fat in the absence of myostatin, and postweaning mortality was not significantly improved. These results show that eliminating myostatin in laminin-deficiency promotes muscle formation, but at the expense of fat formation, and does not reduce muscle pathology. Any future therapy based on myostatin may have undesirable side effects.     

EDL and soleus muscles of the C57BL6J/dy2j laminin-alpha 2-deficient dystrophic mouse are not vulnerable to eccentric contractions

Many muscular dystrophies arise as a consequence of mutations in a series of interconnected proteins associated with the sarcolemma. This group of proteins is collectively referred to as the 'dystrophin-associated complex'. We used the C57BL6J/dy(2j), dystrophia muscularis, dystrophic mouse, in which the laminin-alpha(2) component of the dystrophin-associated complex is mutated, to test the hypothesis that the disruption of this complex will destabilize the lipid bilayer, rendering it more susceptible to damage during eccentric contractions. We demonstrated that neither slow- nor fast-twitch dystrophic muscles were more susceptible to eccentric contractions when compared with controls. Only fast-twitch extensor digitorum longus (EDL) muscles (from both dystrophic and control mice) showed an irreversible loss of force with our eccentric contraction protocol, suggesting that it is the fast 11b fibres (not present in slow-twitch soleus) which are most susceptible to eccentric damage. We used the general anaesthetic halothane to increase the fluidity of the lipid bilayer to see if this would uncover any greater susceptibility of the dystrophic muscle to eccentric damage. This also did not reveal any greater fragility of fast- and slow-twitch dystrophic muscles. We did, however, demonstrate that halothane made both control and dystrophic fast- and slow-twitch muscles more susceptible to eccentric contraction damage. The C57BL6J/dy(2j) dystrophic laminopathy produced the pathophysiological and pathohistological characteristics associated with muscular dystrophy: the fast- and slow-twitch dystophic muscles produced only 55 and 53%, respectively, of the force of control muscles and 34 and 40%, respectively, of the dystrophic muscle fibres were branched. The presence of the branched fibres in the dystrophic muscles did not make them more susceptible to eccentric damage but may have contributed to the reduction in maximal force in the dystrophic muscles. We conclude that our data do not support the structural hypothesis that the dystrophin-associated complex acts as a scaffolding to support the lipid bilayer, but are consistent with channel-based hypotheses put forward to explain the dystrophic process.     

Interleukin-15 administration improves diaphragm muscle pathology and function in dystrophic mdx mice

Interleukin (IL)-15, a cytokine expressed in skeletal muscle, has been shown to have muscle anabolic effects in vitro and to slow muscle wasting in rats with cancer cachexia. Whether IL-15 has therapeutic potential for diseases such as Duchenne muscular dystrophy (DMD) is unknown. We examined whether IL-15 administration could ameliorate the dystrophic pathology in the diaphragm muscle of the mdx mouse, an animal model for DMD. Four weeks of IL-15 treatment improved diaphragm strength, a highly significant finding because respiratory function is a mortality predictor in DMD. Enhanced diaphragm function was associated with increased muscle fiber cross-sectional area and decreased collagen infiltration. IL-15 administration was not associated with changes in T-cell populations or alterations in specific components of the ubiquitin proteasome pathway. To determine the effects of IL-15 on myofiber regeneration, muscles of IL-15-treated and untreated wild-type mice were injured myotoxically, and their functional recovery was assessed. IL-15 had a mild anabolic effect, increasing fiber cross-sectional area after 2 and 6 days but not after 10 days. Our findings demonstrate that IL-15 administration improves the pathophysiology of dystrophic muscle and highlight a possible therapeutic role for IL-15 in the treatment of neuromuscular disorders especially in which muscle wasting is indicated.     

Muscle-specific BCL2 expression ameliorates muscle disease in laminin {alpha}2-deficient, but not in dystrophin-deficient, mice

To examine the role of apoptosis in neuromuscular disease progression, we have determined whether pathogenesis in dystrophin-deficient (mdx) and laminin alpha2-deficient (Lama2-null) mice is ameliorated by overexpression of the anti-apoptosis protein BCL2 in diseased muscles. The mdx mice are a model for the human disease, Duchenne muscular dystrophy (DMD), and the Lama2-null mice are a model for human congenital muscular dystrophy type 1A (MDC1A). For these studies, we generated transgenic mice that overexpressed human BCL2 under control of muscle-specific MyoD or MRF4 promoter fragments. We then used cross-breeding to introduce the transgenes into diseased mdx or Lama2-null mice. In mdx mice, we found that overexpression of BCL2 failed to produce any significant differences in muscle pathology. In contrast, in the Lama2-null mice, we found that muscle-specific expression of BCL2 led to a several-fold increase in lifespan and an increased growth rate. Thus, BCL2-mediated apoptosis appears to play a significant role in pathogenesis of laminin alpha2 deficiency, but not of dystrophin deficiency, suggesting that therapies designed to ameliorate disease by inhibition of apoptosis are more likely to succeed in MDC1A than in DMD.     

Rescue of severely affected dystrophin/utrophin-deficient mice through scAAV-U7snRNA-mediated exon skipping

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder caused by mutations in the dystrophin gene that result in the absence of functional protein. Antisense-mediated exon skipping is one of the most promising approaches for the treatment of DMD and recent clinical trials have demonstrated encouraging results. However, antisense oligonucleotide-mediated exon skipping for DMD still faces major hurdles such as extremely low efficacy in the cardiac muscle, poor cellular uptake and relatively rapid clearance from circulation, which means that repeated administrations are required to achieve some therapeutic efficacy. To overcome these limitations, we previously proposed the use of small nuclear RNAs (snRNAs), especially U7snRNA to shuttle the antisense sequences after vectorization into adeno-associated virus (AAV) vectors. In this study, we report for the first time the efficiency of the AAV-mediated exon skipping approach in the utrophin/dystrophin double-knockout (dKO) mouse which is a very severe and progressive mouse model of DMD. Following a single intravenous injection of scAAV9-U7ex23 in dKO mice, near-normal levels of dystrophin expression were restored in all muscles examined, including the heart. This resulted in a considerable improvement of their muscle function and dystrophic pathology as well as a remarkable extension of the dKO mice lifespan. These findings suggest great potential for AAV-U7 in systemic treatment of the DMD phenotype.