Mdx Mouse Model Research Articles

Duchenne muscular dystrophy trajectory in R-DMDdel52 preclinical rat model identifies COMP as biomarker of fibrosis

Duchenne muscular dystrophy (DMD) is a fatal muscle-wasting disorder caused by mutations in the Dystrophin gene and for which there is currently no cure. To bridge the gap between preclinical and therapeutic evaluation studies, we have generated a rat model for DMD that carries an exon 52 deletion (R-DMDdel52) causing a complete lack of dystrophin protein. Here we show that R-DMDdel52 animals recapitulated human DMD pathophysiological trajectory more faithfully than the mdx mouse model. We report that R-DMDdel52 rats displayed progressive and severe skeletal muscle loss associated with fibrotic deposition, fat infiltration and fibre type switch. Early fibrosis was also apparent in the cardiac muscle. These histological modifications led to severe muscle, respiratory and cardiac functional impairments leading to premature death around 1 year. Moreover, DMD muscle exhibited systemic inflammation with a mixed M1/M2 phenotype. A comparative single cell RNAseq analysis of the diaphragm muscle was performed, revealing cellular populations alteration and molecular modifications in all muscle cell types. We show that DMD fibroadipogenic progenitors produced elevated levels of cartilage oligomeric matrix protein, a glycoprotein responsible for modulating homeostasis of extracellular matrix, and whose increased concentration correlated with muscle fibrosis both in R-DMDdel52 rats and human patients. Fibrosis is a component of tissue remodelling impacting the whole musculature of DMD patients, at the tissue level but most importantly at the functional level. We therefore propose that this specific biomarker can optimize the prognostic monitoring of functional improvement of patients included in clinical trials.

Reduced Sarcolemmal Membrane Repair Exacerbates Striated Muscle Pathology in a Mouse Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a common X-linked degenerative muscle disorder that involves mutations in the DMD gene that frequently reduce the expression of the dystrophin protein, compromising the structural integrity of the sarcolemmal membrane and leaving it vulnerable to injury during cycles of muscle contraction and relaxation. This results in an increased frequency of sarcolemma disruptions that can compromise the barrier function of the membrane and lead to death of the myocyte. Sarcolemmal membrane repair processes can potentially compensate for increased membrane disruptions in DMD myocytes. Previous studies demonstrated that TRIM72, a muscle-enriched tripartite motif (TRIM) family protein also known as mitsugumin 53 (MG53), is a component of the cell membrane repair machinery in striated muscle. To test the importance of membrane repair in striated muscle in compensating for the membrane fragility in DMD, we crossed TRIM72/MG53 knockout mice into the mdx mouse model of DMD. These double knockout (DKO) mice showed compromised sarcolemmal membrane integrity compared to mdx mice, as measured by immunoglobulin G staining and ex vivo muscle laser microscopy wounding assays. We also found a significant decrease in muscle ex vivo contractile function as compared to mdx mice at both 6 weeks and 1.5 years of age. As the DKO mice aged, they developed more extensive fibrosis in skeletal muscles compared to mdx. Our findings indicate that TRIM72/MG53-mediated membrane repair can partially compensate for the sarcolemmal fragility associated with DMD and that the loss of membrane repair results in increased pathology in the DKO mice.

Effects of moderate aerobic exercise, low-level laser therapy, or their combination on muscles pathology, oxidative stress and irisin levels in the mdx mouse model of Duchenne muscular dystrophy.

This study aimed to investigate how the combined use of low-level laser therapy (LLLT) and exercise, to reduce the possible side effects and/or increase the benefits of exercise, would affect oxidative stress, utrophin, irisin peptide, and skeletal, diaphragmatic, and cardiac muscle pathologies. In our study, 20 mdx mice were divided into four groups. Groups; sedentary and placebo LLLT (SC), sedentary and LLLT (SL), 30-min swimming exercise (Ex), and 30-min swimming exercise and LLLT (ExL). After 8weeks of swimming exercise, muscle tests, biochemically; oxidative stress index (OSI), utrophin and irisin levels were measured. Skeletal, diaphragmatic and cardiac muscle histopathological scores, skeletal and cardiac muscle myocyte diameters were determined under the light and electron microscope. While only irisin levels were increased in group SL compared to SC, it was determined that OSI, heart muscle histopathological scores decreased and irisin levels increased in both exercise groups (p < 0.05). In addition, in the ExL group, an increase in rotarod and utrophin levels, and a decrease in muscle and diaphragm muscle histopathological scores were observed (p < 0.05). It was determined that the application of swimming exercise in the mdx mouse model increased the irisin level in the skeletal muscle, while reducing the OSI, degeneration in the heart muscle, inflammation and cardiopathy. When LLLT was applied in addition to exercise, muscle strength, skeletal muscle utrophin levels increased, and skeletal and diaphragmatic muscle degeneration and inflammation decreased. In addition, it was determined that only LLLT application increased the level of skeletal muscle irisin.

Wharton's Jelly-Derived Mesenchymal Stem Cells with High Aurora Kinase A Expression Show Improved Proliferation, Migration, and Therapeutic Potential.

Mesenchymal stem cells (MSCs) are effective therapeutic agents that contribute to tissue repair and regeneration by secreting various factors. However, donor-dependent variations in MSC proliferation and therapeutic potentials result in variable production yields and clinical outcomes, thereby impeding MSC-based therapies. Hence, selection of MSCs with high proliferation and therapeutic potentials would be important for effective clinical application of MSCs. This study is aimed at identifying the upregulated genes in human Wharton's jelly-derived MSCs (WJ-MSCs) with high proliferation potential using mRNA sequencing. Aurora kinase A (AURKA) and dedicator of cytokinesis 2 (DOCK2) were selected as the upregulated genes, and their effects on proliferation, migration, and colony formation of the WJ-MSCs were verified using small interfering RNA (siRNA) techniques. mRNA expression levels of both the genes were positively correlated with the proliferation capacity of WJ-MSCs. Moreover, AURKA from human WJ-MSCs regulated the antiapoptotic effect of skeletal muscle cells by upregulating the chemokine (C motif) ligand (XCL1); this was further confirmed in the mdx mouse model. Taken together, the results indicated that AURKA and DOCK2 can be used as potential biomarkers for proliferation and migration of human WJ-MSCs. In particular, human WJ-MSCs with high expression of AURKA might have therapeutic efficacy against muscle diseases, such as Duchenne muscular dystrophy (DMD).

Muscle Damage in Dystrophic mdx Mice Is Influenced by the Activity of Ca2+-Activated KCa3.1 Channels.

Duchenne muscular dystrophy (DMD) is an X-linked disease, caused by a mutant dystrophin gene, leading to muscle membrane instability, followed by muscle inflammation, infiltration of pro-inflammatory macrophages and fibrosis. The calcium-activated potassium channel type 3.1 (KCa3.1) plays key roles in controlling both macrophage phenotype and fibroblast proliferation, two critical contributors to muscle damage. In this work, we demonstrate that pharmacological blockade of the channel in the mdx mouse model during the early degenerative phase favors the acquisition of an anti-inflammatory phenotype by tissue macrophages and reduces collagen deposition in muscles, with a concomitant reduction of muscle damage. As already observed with other treatments, no improvement in muscle performance was observed in vivo. In conclusion, this work supports the idea that KCa3.1 channels play a contributing role in controlling damage-causing cells in DMD. A more complete understanding of their function could lead to the identification of novel therapeutic approaches.

Human pluripotent stem cell-derived myogenic progenitors undergo maturation to quiescent satellite cells upon engraftment.

Human pluripotent stem cell (hPSC)-derived myogenic progenitor cell (MPC) transplantation is a promising therapeutic approach for a variety of degenerative muscle disorders. Here, using an MPC-specific fluorescent reporter system (PAX7::GFP), we demonstrate that hPSC-derived MPCs can contribute to the regeneration of myofibers in mice following local injury and in mice deficient of dystrophin (mdx). We also demonstrate that a subset of PAX7::GFP MPCs engraft within the basal lamina of regenerated myofibers, adopt a quiescent state, and contribute to regeneration upon reinjury and in mdx mouse models. This subset of PAX7::GFP MPCs undergo a maturation process and remodel their molecular characteristics to resemble those of late-stage fetal MPCs/adult satellite cells following invivo engraftment. These in-vivo-matured PAX7::GFP MPCs retain a cell-autonomous ability to regenerate and can repopulate in the niche of secondary recipient mice, providing a proof of principle for future hPSC-based cell therapy for muscle disorders.

Description of Osmolyte Pathways in Maturing Mdx Mice Reveals Altered Levels of Taurine and Sodium/Myo-Inositol Co-Transporters.

Duchenne muscular dystrophy (DMD) is a genetic disorder characterized by progressive muscle degeneration. Osmotic stress participates to DMD pathology and altered levels of osmolyte pathway members have been reported. The goal of this study was to gain insight in osmoregulatory changes in the mdx mouse model by examining the expression of osmolyte pathway members, including taurine transporter (TauT), sodium myo-inositol co-transporter (SMIT), betaine GABA transporter (BGT), and aldose reductase (AR) in the skeletal muscles and diaphragm of mdx mice aged 4, 8, 12, and 26 weeks. Necrosis was most prominent in 12 week-old mdx mice, whereas the amount of regenerated fibers increased until week 26 in the tibialis anterior. TauT protein levels were downregulated in the tibialis anterior and gastrocnemius of 4 to 12 week-old mdx mice, but not in 26 week-old mice, whereas TauT levels in the diaphragm remained significantly lower in 26 week-old mdx mice. In contrast, SMIT protein levels were significantly higher in the muscles of mdx mice when compared to controls. Our study revealed differential regulation of osmolyte pathway members in mdx muscle, which points to their complex involvement in DMD pathogenesis going beyond general osmotic stress responses. These results highlight the potential of osmolyte pathway members as a research interest and future therapeutic target in dystrophinopathy.

Inhibition of PKCθ Improves Dystrophic Heart Phenotype and Function in a Novel Model of DMD Cardiomyopathy.

Chronic cardiac muscle inflammation and subsequent fibrotic tissue deposition are key features in Duchenne Muscular Dystrophy (DMD). The treatment of choice for delaying DMD progression both in skeletal and cardiac muscle are corticosteroids, supporting the notion that chronic inflammation in the heart plays a pivotal role in fibrosis deposition and subsequent cardiac dysfunction. Nevertheless, considering the adverse effects associated with long-term corticosteroid treatments, there is a need for novel anti-inflammatory therapies. In this study, we used our recently described exercised mdx (ex mdx) mouse model characterised by accelerated heart pathology, and the specific PKCθ inhibitor Compound 20 (C20), to show that inhibition of this kinase leads to a significant reduction in the number of immune cells infiltrating the heart, as well as necrosis and fibrosis. Functionally, C20 treatment also prevented the reduction in left ventricle fractional shortening, which was typically observed in the vehicle-treated ex mdx mice. Based on these findings, we propose that PKCθ pharmacological inhibition could be an attractive therapeutic approach to treating dystrophic cardiomyopathy

Co-Administration of Simvastatin Does Not Potentiate the Benefit of Gene Therapy in the mdx Mouse Model for Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is the most common and cureless muscle pediatric genetic disease, which is caused by the lack or the drastically reduced expression of dystrophin. Experimental therapeutic approaches for DMD have been mainly focused in recent years on attempts to restore the expression of dystrophin. While significant progress was achieved, the therapeutic benefit of treated patients is still unsatisfactory. Efficiency in gene therapy for DMD is hampered not only by incompletely resolved technical issues, but likely also due to the progressive nature of DMD. It is indeed suspected that some of the secondary pathologies, which are evolving over time in DMD patients, are not fully corrected by the restoration of dystrophin expression. We recently identified perturbations of the mevalonate pathway and of cholesterol metabolism in DMD patients. Taking advantage of the mdx model for DMD, we then demonstrated that some of these perturbations are improved by treatment with the cholesterol-lowering drug, simvastatin. In the present investigation, we tested whether the combination of the restoration of dystrophin expression with simvastatin treatment could have an additive beneficial effect in the mdx model. We confirmed the positive effects of microdystrophin, and of simvastatin, when administrated separately, but detected no additive effect by their combination. Thus, the present study does not support an additive beneficial effect by combining dystrophin restoration with a metabolic normalization by simvastatin.

Ursolic Acid Increases Strength in mdx Mice Model and may Decrease Fibrosis Deposition by TGF-ß Downregulation

Dystrophin disfunction results in sarcolemma destabilization, leading muscle cell damage by continuous degeneration cycles and limited regeneration. In muscle dystrophy, caused by dystrophin dysfunction, inflammation, necrosis and fibrosis are pathophysiological muscle function loss characteristics. As a genetic disease, this muscle dystrophy has no cure, however, advances in drug therapy using glucocorticoids can decrease the disease progression. Subsequently, alternative therapies were studied, such as ursolic acid (UA), that inhibits muscle atrophy and increases muscle mass and strength. Herein, we used 10 mg/kg daily supplementation in mdx mice for 4 weeks to evaluate serum creatine phosphokinase (CPK), muscle strength (Kondziela test), muscular organization (histology) and expression of fibrosis related genes (TGF-ß, TNF-α, mstn and ostn). UA supplementation increased muscle morphological organization, motor strength and decreased muscular TGF-ß expression. Altogether, the gene expression profile, histological organization and strength could suggest that UA treatment did not stop the fibrogenesis but decreased its progress.

Inhibition of sphingolipid de novo synthesis counteracts muscular dystrophy.

Duchenne muscular dystrophy (DMD), the most common muscular dystrophy, is a severe muscle disorder, causing muscle weakness, loss of independence, and premature death. Here, we establish the link between sphingolipids and muscular dystrophy. Transcripts of sphingolipid de novo biosynthesis pathway are up-regulated in skeletal muscle of patients with DMD and other muscular dystrophies, which is accompanied by accumulation of metabolites of the sphingolipid pathway in muscle and plasma. Pharmacological inhibition of sphingolipid synthesis by myriocin in the mdx mouse model of DMD ameliorated the loss in muscle function while reducing inflammation, improving Ca2+ homeostasis, preventing fibrosis of the skeletal muscle, heart, and diaphragm, and restoring the balance between M1 and M2 macrophages. Myriocin alleviated the DMD phenotype more than glucocorticoids. Our study identifies inhibition of sphingolipid synthesis, targeting multiple pathogenetic pathways simultaneously, as a strong candidate for treatment of muscular dystrophies.

Lipocalin 2 Influences Bone and Muscle Phenotype in the MDX Mouse Model of Duchenne Muscular Dystrophy.

Lipocalin 2 (Lcn2) is an adipokine involved in bone and energy metabolism. Its serum levels correlate with bone mechanical unloading and inflammation, two conditions representing hallmarks of Duchenne Muscular Dystrophy (DMD). Therefore, we investigated the role of Lcn2 in bone loss induced by muscle failure in the MDX mouse model of DMD. We found increased Lcn2 serum levels in MDX mice at 1, 3, 6, and 12 months of age. Consistently, Lcn2 mRNA was higher in MDX versus WT muscles. Immunohistochemistry showed Lcn2 expression in mononuclear cells between muscle fibres and in muscle fibres, thus confirming the gene expression results. We then ablated Lcn2 in MDX mice, breeding them with Lcn2−/− mice (MDXxLcn2−/−), resulting in a higher percentage of trabecular volume/total tissue volume compared to MDX mice, likely due to reduced bone resorption. Moreover, MDXxLcn2−/− mice presented with higher grip strength, increased intact muscle fibres, and reduced serum creatine kinase levels compared to MDX. Consistently, blocking Lcn2 by treating 2-month-old MDX mice with an anti-Lcn2 monoclonal antibody (Lcn2Ab) increased trabecular volume, while reducing osteoclast surface/bone surface compared to MDX mice treated with irrelevant IgG. Grip force was also increased, and diaphragm fibrosis was reduced by the Lcn2Ab. These results suggest that Lcn2 could be a possible therapeutic target to treat DMD-induced bone loss.

Reducing sarcolipin expression improves muscle metabolism in mdx mice.

Duchenne muscular dystrophy (DMD) is an inherited muscle wasting disease. Metabolic impairments and oxidative stress are major secondary mechanisms that severely worsen muscle function in DMD. Here, we sought to determine whether germline reduction or ablation of sarcolipin (SLN), an inhibitor of sarco/endoplasmic reticulum (SR) Ca2+ ATPase (SERCA), improves muscle metabolism and ameliorates muscle pathology in the mdx mouse model of DMD. Glucose and insulin tolerance tests show that glucose clearance rate and insulin sensitivity were improved in the SLN haploinsufficient mdx (mdx:sln+/-) and SLN-deficient mdx (mdx:sln-/-) mice. The histopathological analysis shows that fibrosis and necrosis were significantly reduced in muscles of mdx:sln+/- and mdx:sln-/- mice. SR Ca2+ uptake, mitochondrial complex protein levels, complex activities, mitochondrial Ca2+ uptake and release, and mitochondrial metabolism were significantly improved, and lipid peroxidation and protein carbonylation were reduced in the muscles of mdx:sln+/- and mdx:sln-/- mice. These data demonstrate that reduction or ablation of SLN expression can improve muscle metabolism, reduce oxidative stress, decrease muscle pathology, and protects the mdx mice from glucose intolerance.

Use of Small Animal Models for Duchenne and Parameters to Assess Efficiency upon Antisense Treatment.

Duchenne muscular dystrophy (DMD) is a rare genetic disease affecting 1 in 5000 newborn boys. It is caused by mutations in the DMD gene with a consequent lack of dystrophin protein that leads to deterioration of myofibers and their replacement with fibro-adipogenic tissue. Using antisense oligonucleotides (AONs) to modify out-of-frame mutations in the DMD gene, named exon skipping, is currently considered among the most promising treatments for DMD patients. The development of this strategy is rapidly moving forward, and AONs designed to skip exons 51 and 53 have received accelerated approval in the USA. In preclinical setting, the mdx mouse model, carrying a point mutation in exon 23 of the murine Dmd gene that prevents production of dystrophin protein, has emerged as a valuable tool, and it is widely used to study in vivo therapeutic approaches for DMD. Here we describe the methodology for intravenous delivery of AONs targeting dystrophin through tail vein of mdx mice. Furthermore, the most relevant functional analyses to be performed in living mice, and the most informative histopathological and molecular assays to evaluate the effect of this treatment are detailed.

Lifespan Analysis of Dystrophic mdx Fast-Twitch Muscle Morphology and Its Impact on Contractile Function.

Duchenne muscular dystrophy is caused by the absence of the protein dystrophin from skeletal muscle and is characterized by progressive cycles of necrosis/regeneration. Using the dystrophin deficient mdx mouse model, we studied the morphological and contractile chronology of dystrophic skeletal muscle pathology in fast-twitch Extensor Digitorum Longus muscles from animals 4–22 months of age containing 100% regenerated muscle fibers. Catastrophically, the older age groups lost ∼80% of their maximum force after one eccentric contraction (EC) of 20% strain with the greatest loss of ∼92% recorded in senescent 22-month-old mdx mice. In old age groups, there was minimal force recovery ∼24% after 120 min, correlated with a dramatic increase in the number and complexity of branched fibers. This data supports our two-phase model where a “tipping point” is reached when branched fibers rupture irrevocably on EC. These findings have important implications for pre-clinical drug studies and genetic rescue strategies.

Sensitivity to behavioral stress impacts disease pathogenesis in dystrophin-deficient mice.

Mutation to the gene encoding dystrophin can cause Duchenne muscular dystrophy (DMD) and increase the sensitivity to stress in vertebrate species, including the mdx mouse model of DMD. Behavioral stressors can exacerbate some dystrophinopathy phenotypes of mdx skeletal muscle and cause hypotension-induced death. However, we have discovered that a subpopulation of mdx mice present with a wildtype-like response to mild (forced downhill treadmill exercise) and moderate (scruff restraint) behavioral stressors. These "stress-resistant" mdx mice are more physically active, capable of super-activating the hypothalamic-pituitary-adrenal and renin-angiotensin-aldosterone pathways following behavioral stress and they express greater levels of mineralocorticoid and glucocorticoid receptors in striated muscle relative to "stress-sensitive" mdx mice. Stress-resistant mdx mice also presented with a less severe striated muscle histopathology and greater exercise and skeletal muscle oxidative capacity at rest. Most interestingly, female mdx mice were more physically active following behavioral stressors compared to male mdx mice; a response abolished after ovariectomy and rescued with estradiol. We demonstrate that the response to behavioral stress greatly impacts disease severity in mdx mice suggesting the management of stress in patients with DMD be considered as a therapeutic approach to ameliorate disease progression.

Hematopoietic Prostaglandin D Synthase Inhibitor PK007 Decreases Muscle Necrosis in DMD mdx Model Mice.

Duchenne muscular dystrophy (DMD) is characterized by progressive muscle weakness and wasting due to the lack of dystrophin protein. The acute phase of DMD is characterized by muscle necrosis and increased levels of the pro-inflammatory mediator, prostaglandin D2 (PGD2). Inhibiting the production of PGD2 by inhibiting hematopoietic prostaglandin D synthase (HPGDS) may alleviate inflammation and decrease muscle necrosis. We tested our novel HPGDS inhibitor, PK007, in the mdx mouse model of DMD. Our results show that hindlimb grip strength was two-fold greater in the PK007-treated mdx group, compared to untreated mdx mice, and displayed similar muscle strength to strain control mice (C57BL/10ScSn). Histological analyses showed a decreased percentage of regenerating muscle fibers (~20% less) in tibialis anterior (TA) and gastrocnemius muscles and reduced fibrosis in the TA muscle in PK007-treated mice. Lastly, we confirmed that the DMD blood biomarker, muscle creatine kinase activity, was also reduced by ~50% in PK007-treated mdx mice. We conclude that our HPGDS inhibitor, PK007, has effectively reduced muscle inflammation and fibrosis in a DMD mdx mouse model.

DMD – ANIMAL MODELS: EP.78 BCAA supplementation does not improve muscle function in mdx and D2-mdx mouse models for Duchenne muscular dystrophy

DMD – ANIMAL MODELS: EP.78 BCAA supplementation does not improve muscle function in mdx and D2-mdx mouse models for Duchenne muscular dystrophy

DMD – ANIMAL MODELS: EP.85 Oestrogen and HPA/renin-angiotensin-aldosterone pathways are associated with the behavioural sensitivity to stress in dystrophin-deficient mice

Mutation to the dystrophin gene can increase the sensitivity to behavioural stress in vertebrates, including the mdx mouse model of Duchenne muscular dystrophy. Hyper-sensitivity to behavioural stress in mdx mice can exacerbate some dystrophinopathy phenotypes and cause hypotension-induced death. However, using a large-scale screen of male and female mdx mice, we have discovered a sub-population that show wildtype-like responses to mild (forced downhill treadmill exercise) and moderate (scruff-restraint) behavioural stress. These “stress resistant” mdx mice are more physically active, capable of super-activating the hypothalamic-pituitary-adrenal (HPA) and renin-angiotensin-aldosterone (RAAS) pathways following behavioural stress and they express greater levels of mineralocorticoid and glucocorticoid receptors in striated muscle relative to “stress-sensitive” mdx mice. At rest, “stress-resistant” mdx mice had a less severe striated muscle histopathology, including fewer central nucleated fibres, less fibrosis, greater vascular density, elevated expression levels of utrophin and greater exercise and skeletal muscle oxidative capacity. Most interestingly, female mdx mice were more “stress resistant”, as indicated by a greater level of physically activity following forced downhill treadmill exercise and scruff-restraint, relative to male mdx mice. Stress-resistance was completely abolished after ovariectomy. Replacement of oestradiol in ovariectomized female mdx mice completely rescued their “stress resistance”, again indicated by a greater physical activity response following forced downhill treadmill exercise, but not to scruff-restraint. Our data demonstrate that the differential sensitivity to behavioural stress in mdx mice is linked to oestrogen, activation of the HPA axis and the RAAS pathway and associated with striated muscle pathophysiology.

DMD – ANIMAL MODELS: EP.81 Enhanced exon skipping and Dystrophin production in a mouse model of Duchenne muscular dystrophy with EEV-PMO treatment

Antisense oligonucleotide-induced (ASOs) exon skipping is a promising treatment strategy for Duchenne muscular dystrophy (DMD), an X-linked recessive and fatal neuromuscular disease. DMD is caused by mutations in the dystrophin gene and affects approximately 1 in 3,500-5,000 male births. By modulating the splicing of dystrophin pre-mRNA, ASOs can restore the mRNA reading frame and generate a truncated, functional dystrophin protein. This exon skipping approach can address ∼80% of the mutations that cause DMD. However, the poor exposure and endosomal escape in target tissue after systemic administration of ASOs remains the biggest hurdle for the treatment of patients with DMD and results in insufficient dystrophin protein restoration, especially in non-skeletal muscle tissues such as the heart. We have developed a novel ASO delivery system by conjugation of Entrada's proprietary Endosomal Escape Vehicle (EEVTM) to a phosphorodiamidate morpholino oligomer (PMO), an effective ASO for DMD treatment. The EEV is composed of a cyclic cell-penetrating peptide coupled with a nuclear localization sequence, which is rationally designed to facilitate intracellular delivery, endosomal escape and finally localization in the nucleus. The EEV-PMO significantly enhances the systematic delivery of PMOs and results in robust exon skipping and restoration of dystrophin protein in the mdx mouse model skeletal as well as cardiac muscle. A single dose (20 mg/kg) by intravenous administration of EEV-PMO led to >50% exon skipping and >15% correction of dystrophin protein in the heart seven days post injection, and dystrophin protein levels were sustained for up to four weeks. Repeat dosing (10 mg/kg, 4 QW) resulted in >30% of wild-type dystrophin protein in the heart and normalized serum creatine kinase level seven days post last injection. These results have confirmed the significant therapeutic potential of the EEV-PMO delivery system for DMD. Furthermore, these results demonstrate that EEVs can serve as a general and highly efficient delivery platform that possesses great potential for the delivery of other therapeutic ASOs beyond DMD.