Severe Duchenne Muscular Dystrophy Research Articles

Treadmill running and mechanical overloading improved the strength of the plantaris muscle in the dystrophin-desmin double knockout (DKO) mouse.

Limited knowledge exists regarding the chronic effect of muscular exercise on muscle function in a murine model of severe Duchenne muscular dystrophy (DMD). Here we determined the effects of 1month of voluntary wheel running (WR), 1month of enforced treadmill running (TR) and 1month of mechanical overloading resulting from the removal of the synergic muscles (OVL) in mice lacking both dystrophin and desmin (DKO). Additionally, we examined the effect of activin receptor administration (AR). DKO mice, displaying severe muscle weakness, atrophy and greater susceptibility to contraction-induced functional loss, were exercised or treated with AR at 1month of age and in situ force production of lower leg muscle was measured at the age of 2months. We found that TR and OVL increased absolute maximal force and the rate of force development of the plantaris muscle in DKO mice. In contrast, those of the tibialis anterior (TA) muscle remained unaffected by TR and WR. Furthermore, the effects of TR and OVL on plantaris muscle function in DKO mice closely resembled those in mdx mice, a less severe murine DMD model. AR also improved absolute maximal force and the rate of force development of the TA muscle in DKO mice. In conclusion, exercise training improved plantaris muscle weakness in severely affected dystrophic mice. Consequently, these preclinical results may contribute to fostering further investigations aimed at assessing the potential benefits of exercise for DMD patients, particularly resistance training involving a low number of intense muscle contractions. KEY POINTS: Very little is known about the effects of exercise training in a murine model of severe Duchenne muscular dystrophy (DMD). One reason is that it is feared that chronic muscular exercise, particularly that involving intense muscle contractions, could exacerbate the disease. In DKO mice lacking both dystrophin and desmin, characterized by severe lower leg muscle weakness, atrophy and fragility in comparison to the less severe DMD mdx model, we found that enforced treadmill running improved absolute maximal force of the plantaris muscle, while that of tibialis anterior muscle remained unaffected by both enforced treadmill and voluntary wheel running. Furthermore, mechanical overloading, a non-physiological model of chronic resistance exercise, reversed plantaris muscle weakness. Consequently, our findings may have the potential to alleviate concerns and pave the way for exploring the prescription of endurance and resistance training as a viable therapeutic approach for the treatment of dystrophic patients. Additionally, such interventions may serve in mitigating the pathophysiological mechanisms induced by physical inactivity.

DMD deletions underlining mild dystrophinopathies: literature review highlights phenotype-related mutation clusters and provides insights about genetic mechanisms and prognosis.

DMD gene pathogenic variations cause a spectrum of phenotypes, ranging from severe Duchenne muscular dystrophy, the Becker milder cases, the intermediate or very mild muscle phenotypes invariably characterized by high CK, and the ultrarare fully-asymptomatic cases. Besides these phenotypes, X-linked dilated cardiomyopathy is also caused by DMD mutations. Males carrying DMD deletions with absent or very mild phenotypes have been sparsely described. We performed a horizon scan on public datasets to enroll males with the above phenotypes and carrying DMD deletions to delineate myopathic genotype-phenotype relationships. We inventoried 81 males, who were divided into the following clinical categorization: fully-asymptomatic males aged >43 years (A, N = 22); isolated hyperCKemia (CK, N = 35); and mild weakness (any age) with or without high CK (WCK, N = 24). In all cases, deleted intervals were exons 2 to 55, and no downstream exons were ever involved, apart from an exon 78 deletion in a WCK patient. All deletions were in-frame apart from the known exception to the rule of exon 2 and exon 78. We correlated the mild phenotypes (A and CK) to deleted exons, intronic breakpoints, exon-exon junctions, 3' isoforms rule, and protein epitopes, and we found that some genetic profiles are exclusively/mainly occurring in A/CK phenotypes, suggesting they are compatible with a quasi-normal muscular performance. We discussed diverse pathogenic mechanisms that may contribute to mild dystrophinopathic phenotypes, and we tried to address some "critical" genetic configurations or exon content needed to preserve a semi-functional DMD gene.

Higher Prevalence of Nonsense Pathogenic DMD Variants in a Single-Center Cohort from Brazil: A Genetic Profile Study That May Guide the Choice of Disease-Modifying Treatments.

Dystrophinopathies are muscle diseases caused by pathogenic variants in DMD, the largest gene described in humans, representing a spectrum of diseases ranging from asymptomatic creatine phosphokinase elevation to severe Duchenne muscular dystrophy (DMD). Several therapeutic strategies are currently in use or under development, each targeting different pathogenic variants. However, little is known about the genetic profiles of northeast Brazilian patients with dystrophinopathies. We describe the spectrum of pathogenic DMD variants in a single center in northeast Brazil. This is an observational, cross-sectional study carried out through molecular-genetic analysis of male patients diagnosed with dystrophinopathies using Multiplex Ligation-dependent Probe Amplification (MLPA) followed by Next-Generation Sequencing (NGS)-based strategies. A total of 94 male patients were evaluated. Deletions (43.6%) and duplications (10.6%) were the most recurring patterns of pathogenic variants. However, small variants were present in 47.1% of patients, most of them nonsense variants (27.6%). This is the largest South American single-center case series of dystrophinopathies to date. We found a higher frequency of treatment-amenable nonsense single-nucleotide variants than most previous studies. These findings may have implications for diagnostic strategies in less-known populations, as a higher frequency of nonsense variants may mean a higher possibility of treating patients with disease-modifying drugs.

Systemic deletion of DMD exon 51 rescues clinically severe Duchenne muscular dystrophy in a pig model lacking DMD exon 52

Duchenne muscular dystrophy (DMD) is a fatal X-linked disease caused by mutations in the DMD gene, leading to complete absence of dystrophin and progressive degeneration of skeletal musculature and myocardium. In DMD patients and in a corresponding pig model with a deletion of DMD exon 52 (DMDΔ52), expression of an internally shortened dystrophin can be achieved by skipping of DMD exon 51 to reframe the transcript. To predict the best possible outcome of this strategy, we generated DMDΔ51-52 pigs, additionally representing a model for Becker muscular dystrophy (BMD). DMDΔ51-52 skeletal muscle and myocardium samples stained positive for dystrophin and did not show the characteristic dystrophic alterations observed in DMDΔ52 pigs. Western blot analysis confirmed the presence of dystrophin in the skeletal muscle and myocardium of DMDΔ51-52 pigs and its absence in DMDΔ52 pigs. The proteome profile of skeletal muscle, which showed a large number of abundance alterations in DMDΔ52 vs. wild-type (WT) samples, was normalized in DMDΔ51-52 samples. Cardiac function at age 3.5 mo was significantly reduced in DMDΔ52 pigs (mean left ventricular ejection fraction 58.8% vs. 70.3% in WT) but completely rescued in DMDΔ51-52 pigs (72.3%), in line with normalization of the myocardial proteome profile. Our findings indicate that ubiquitous deletion of DMD exon 51 in DMDΔ52 pigs largely rescues the rapidly progressing, severe muscular dystrophy and the reduced cardiac function of this model. Long-term follow-up studies of DMDΔ51-52 pigs will show if they develop symptoms of the milder BMD.

Altered muscle niche contributes to myogenic deficit in the D2-mdx model of severe DMD

Lack of dystrophin expression is the underlying genetic basis for Duchenne muscular dystrophy (DMD). However, disease severity varies between patients, based on specific genetic modifiers. D2-mdx is a model for severe DMD that exhibits exacerbated muscle degeneration and failure to regenerate even in the juvenile stage of the disease. We show that poor regeneration of juvenile D2-mdx muscles is associated with an enhanced inflammatory response to muscle damage that fails to resolve efficiently and supports the excessive accumulation of fibroadipogenic progenitors (FAPs), leading to increased fibrosis. Unexpectedly, the extent of damage and degeneration in juvenile D2-mdx muscle is significantly reduced in adults, and is associated with the restoration of the inflammatory and FAP responses to muscle injury. These improvements enhance regenerative myogenesis in the adult D2-mdx muscle, reaching levels comparable to the milder B10-mdx model of DMD. Ex vivo co-culture of healthy satellite cells (SCs) with juvenile D2-mdx FAPs reduces their fusion efficacy. Wild-type juvenile D2 mice also manifest regenerative myogenic deficit and glucocorticoid treatment improves their muscle regeneration. Our findings indicate that aberrant stromal cell responses contribute to poor regenerative myogenesis and greater muscle degeneration in juvenile D2-mdx muscles and reversal of this reduces pathology in adult D2-mdx muscle, identifying these responses as a potential therapeutic target for the treatment of DMD.

Valproic acid reduces muscle susceptibility to contraction-induced functional loss but increases weakness in two murine models of Duchenne muscular dystrophy.

Skeletal muscles in animal models of Duchenne muscular dystrophy (DMD) are more susceptible to contraction-induced functional loss, which is not related to fatigue. Valproic acid (VPA) reportedly improves serological and histological markers of damage in dystrophin-deficient murine muscle. Here, we tested whether VPA would reduce the susceptibility to contraction-induced functional loss in two murine DMD models. Adult female mdx (mild) and D2-mdx (severe) DMD murine models were administered VPA (240 mg/kg) or saline for 7 days. Some VPA-treated mdx mice also performed voluntary running in a wheel, which is known to reduce the susceptibility to contraction-induced functional loss; that is, isometric force drop following eccentric contractions. In situ muscle function was assessed before, during and after eccentric contractions. Muscle utrophin and desmin expression were also evaluated using immunoblotting. Interestingly, VPA reduced the isometric force drop following eccentric contractions in both murine models, without change in the relative eccentric maximal force and in the expression of utrophin and desmin. VPA for 7 days combined with voluntary running had no additive effect compared to VPA alone. Furthermore, VPA reduced the absolute isometric maximal force before eccentric contractions in both murine models. The results of our study indicated that VPA in both murine DMD models reduced the susceptibility to contraction-induced functional loss but increased muscle weakness.

Thyroid-stimulating hormone receptor signaling restores skeletal muscle stem cell regeneration in rats with muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe and progressive myopathy leading to motor and cardiorespiratory impairment. We analyzed samples from patients with DMD and a preclinical rat model of severe DMD and determined that compromised repair capacity of muscle stem cells in DMD is associated with early and progressive muscle stem cell senescence. We also found that extraocular muscles (EOMs), which are spared by the disease in patients, contain muscle stem cells with long-lasting regenerative potential. Using single-cell transcriptomics analysis of muscles from a rat model of DMD, we identified the gene encoding thyroid-stimulating hormone receptor (Tshr) as highly expressed in EOM stem cells. Further, TSHR activity was involved in preventing senescence. Forskolin, which activates signaling downstream of TSHR, was found to reduce senescence of skeletal muscle stem cells, increase stem cell regenerative potential, and promote myogenesis, thereby improving muscle function in DMD rats. These findings indicate that stimulation of adenylyl cyclase leads to muscle repair in DMD, potentially providing a therapeutic approach for patients with the disease.

Dystrophin Restoration after Adeno-Associated Virus U7–Mediated Dmd Exon Skipping Is Modulated by Muscular Exercise in the Severe D2-Mdx Duchenne Muscular Dystrophy Murine Model

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disease caused by Dmd mutations, resulting in the absence of dystrophin in skeletal muscle, with a greater susceptibility to damage during contraction (exercise). In this study, we evaluated whether voluntary exercise impacts a Dmd exon skipping approach and muscle physiology in a severe DMD murine model. We intramuscularly injected an adeno-associated virus (AAV) U7 snRNA that aimed to correct Dmd reading frame in D2-mdx mice, and they voluntary ran in a wheel for 1 month. Our results suggested that voluntary running did not induce muscle fiber regeneration, considering the percentage of centronucleated fibers, Myh3 and Myh4 expression, or maximal force production, and thus possibly not compromise a gene therapy approach. With the AAV-U7 injected just before exercise initiation, we reported that voluntary running did not impact the number of viral genomes and the expression of U7 and Dmd 1 month after injection of AAV-U7, but reduced the amount of dystrophin, from 80% to 65% of the muscle cross-sectional area with dystrophin-expressing fibers. In conclusion, voluntary running did not induce muscle damage and had no drastic detrimental effect on the AAV gene therapy exon skipping approach in a severe murine DMD model. Moreover, our results suggested that exercise should be an additional element to consider in the design and conception of future therapeutic approaches for DMD.

Differentiation of Pediatric-Onset Duchenne and Becker Muscular Dystrophy Subphenotypes Using Data from the Muscular Dystrophy Surveillance Tracking and Research Network (MD STARnet).

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) phenotypes are used to describe disease progression in affected individuals. However, considerable heterogeneity has been observed across and within these two phenotypes, suggesting a spectrum of severity rather than distinct conditions. Characterizing the phenotypes and subphenotypes aids researchers in the design of clinical studies and clinicians in providing anticipatory guidance to affected individuals and their families. Using data from the Muscular Dystrophy Surveillance, Tracking, and Research Network (MD STARnet), we used K-means cluster analysis to group phenotypically similar males with pediatric-onset dystrophinopathy. We identified four dystrophinopathy clusters: Classical BMD, Classical DMD, late ambulatory DMD, and severe DMD. The clusters that we identified align with both 'classical' and 'non-classical' dystrophinopathy described in the literature. Individuals with dystrophinopathies have heterogenous clinical presentations that cluster into phenotypically similar groups. Use of clinically-derived phenotyping may provide a clearer understanding of disease trajectories, reduce variability in study results, and prevent exclusion of certain cohorts from analysis. Findings from studying subphenotypes may ultimately improve our ability to predict disease progression.

A clinical case of severe Duchenne muscular dystrophy caused by a nonsense mutation in the DMD gene in a girl

Duchenne muscular dystrophy (DMD) is a hereditary progressive muscular dystrophy, mainly manifested in boys, is characterized by the onset at an early age, gradual symmetrical atrophy of the striated musculature of the limbs, trunk, as well as damage to the heart muscle. As a rule, girls and women inheriting a pathological mutation are classified only as its carriers and do not have clinical manifestations of the disease. Rare cases when women or girls show clinical manifestations of DMD may be due to chromosomal rearrangements involving the region of the short arm of the X chromosome (Xp21.2), deletions of this region, complete loss of the X chromosome (Shereshevsky-Turner syndrome), homogenous X chromosome dysomnia, compound heterozygous state for two pathogenic mutations in the DMD gene, nonequilibrium inactivation of the X chromosome. When female mutation carriers have DMD clinical symptoms, they usually manifest much milder than boys and young males. Descriptions of patients with the severe course and rapid progression of the disease, comparable in the rate of progression with boys, are rare. In this article, the authors share their experience of observing a girl patient who suffered from a severe form of DMD.

Cholesterol absorption blocker ezetimibe prevents muscle wasting in severe dysferlin-deficient and mdx mice.

BackgroundMuscular dystrophy (MD) causes muscle wasting and is often lethal in patients due to a lack of proven therapies. In contrast, mouse models of MD are notoriously mild. We have previously shown severe human‐like muscle pathology in mdx [Duchenne MD (DMD)] and dysferlin‐deficient limb‐girdle MD type 2B (LGMD2B) mice by inactivating the gene encoding for apolipoprotein E (ApoE), a lipid transporter synthesized by the liver, brain and adipocytes to regulate lipid and fat metabolism. Having recently established that human DMD is a novel type of primary genetic dyslipidaemia with elevated cholesterol, we sought to determine whether cholesterol could exacerbate the muscle wasting process observed in severe rodent MD.MethodsSevere mdx and dysferlin knock‐out mice lacking ApoE were treated with ezetimibe (15 mg/kg/day), a clinically approved drug exhibiting few pleiotropic effects. In separate studies, dietary cholesterol was raised (from 0.2% to 2% cholesterol) in combination with experimental micro‐injury and direct cholesterol injection assays. Muscles were assessed histologically for changes in collagen and adipocyte infiltration and both transcriptomic and cellular changes by RNA‐seq and fluorescence‐activated cell sorting analysis.ResultsTreatment of severe DMD and LGMD2B mice with ezetimibe completely prevented clinical signs of ambulatory dysfunction (0% incidence vs. 33% for vehicle treatment; P < 0.05). Histological analyses revealed that ezetimibe‐reduced fibro‐fatty infiltration up to 84% and 63% in severely affected triceps (P ≤ 0.0001) and gastrocnemius (P ≤ 0.003) muscles, resulting in a respective 1.9‐fold and 2.2‐fold retention of healthy myofibre area (P ≤ 0.0001). Additionally, raising dietary cholesterol and thus concentrations of plasma low‐density lipoprotein‐associated cholesterol (by 250%; P < 0.0001) reduced overall survivability (by 100%; P < 0.001) and worsened muscle damage in the LGMD2B triceps by 767% (P < 0.03). Micro‐pin‐induced mechanical injury in LGMD2B mice fed a high cholesterol diet exacerbated muscle damage by 425% (P < 0.03) and increased macrophage recruitment (by 98%; P = 0.03) compared with those injured on a chow diet. Parallel RNA‐seq analyses revealed that injury in cholesterol‐fed mice also modulated the expression of 3671 transcripts (1953 up‐regulated), with fibrogenic, inflammatory and programmed cell death‐associated pathways among the most enriched. Mice lacking dysferlin also displayed heightened muscle necrosis (by 123%; P < 0.0001) following a direct intramuscular injection of cholesterol compared with control mice.ConclusionsCholesterol exacerbates rodent MD. Specific inhibition of cholesterol absorption with ezetimibe may safely attenuate human MD severity and delay death.

Abstract 11387: Four-Dimensional Spatiotemporal Characterization of Cardiomyopathy in Duchenne Muscular Dystrophy Using Cardiac Magnetic Resonance Imaging

Introduction: Cardiomyopathy (CM) is the most common cause of mortality in Duchenne muscular dystrophy (DMD). Because CM progression is variable, there is a critical need for biomarkers to detect early onset or rapid progression. Our objective was to evaluate localized kinematic parameters that correlate with function utilizing spatiotemporal analysis of 4D (3D plus time) cardiac magnetic resonance (CMR). We hypothesized that novel regional strain metrics would correlate with left ventricular ejection fraction (LVEF), providing insight for future DMD CM characterization studies . Methods: Sequential short axis cine CMR images of DMD patients (n=10) with a range of CM severity were compiled into 4D sequences. Epi- and endocardial borders were then segmented across one cardiac cycle for kinematic analysis. We evaluated Green-Lagrange circumferential strain (ε cc ) at 60 equally spaced short axis slices and across 60 time points to generate a spatiotemporal map. We also evaluated a novel “hybrid strain index” combining basal ε cc with posterior free-wall longitudinal strain. Spearman’s rho was used to determine statistical correlation. Results: DMD subjects had a median age of 11 years (8-24); 3 had LVEF&lt;55% and 7 had late gadolinium enhancement. Regional peak averages of ε cc decreased with LVEF for basal ( rho =-0.94, p &lt;.001) and mid-LV ( rho =-0.84, p =.004) but not at the apex ( rho =-0.55, p =.106). The hybrid strain index also decreased with LVEF ( rho =0.89, p &lt;.001). Qualitative spatiotemporal ε cc maps of mild, moderate, and severe DMD cases demonstrate stark differences in regional wall motion (Figure 1). Conclusion: 4D spatiotemporal analysis of CMR images in DMD patients allows for robust comparison of regional kinematic parameters that correlate with LVEF. Figure 1: 4D CMR spatiotemporal analysis of DMD cardiomyopathy. A,B) Schematic of circumferential strain (ε cc ). C,D,E) Example mild, moderate, and severe patient spatiotemporal ε cc maps.

The Donnan-dominated resting state of skeletal muscle fibers contributes to resilience and longevity in dystrophic fibers.

Duchenne muscular dystrophy (DMD) is an X-linked dystrophin-minus muscle-wasting disease. Ion homeostasis in skeletal muscle fibers underperforms as DMD progresses. But though DMD renders these excitable cells intolerant of exertion, sodium overloaded, depolarized, and spontaneously contractile, they can survive for several decades. We show computationally that underpinning this longevity is a strikingly frugal, robust Pump-Leak/Donnan (P-L/D) ion homeostatic process. Unlike neurons, which operate with a costly "Pump-Leak-dominated" ion homeostatic steady state, skeletal muscle fibers operate with a low-cost "Donnan-dominated" ion homeostatic steady state that combines a large chloride permeability with an exceptionally small sodium permeability. Simultaneously, this combination keeps fiber excitability low and minimizes pump expenditures. As mechanically active, long-lived multinucleate cells, skeletal muscle fibers have evolved to handle overexertion, sarcolemmal tears, ischemic bouts, etc.; the frugality of their Donnan dominated steady state lets them maintain the outsized pump reserves that make them resilient during these inevitable transient emergencies. Here, P-L/D model variants challenged with DMD-type insult/injury (low pump-strength, overstimulation, leaky Nav and cation channels) show how chronic "nonosmotic" sodium overload (observed in DMD patients) develops. Profoundly severe DMD ion homeostatic insult/injury causes spontaneous firing (and, consequently, unwanted excitation-contraction coupling) that elicits cytotoxic swelling. Therefore, boosting operational pump-strength and/or diminishing sodium and cation channel leaks should help extend DMD fiber longevity.

A Genotype-Phenotype Correlation Study of Exon Skip-Equivalent In-Frame Deletions and Exon Skip-Amenable Out-of-Frame Deletions across the DMD Gene to Simulate the Effects of Exon-Skipping Therapies: A Meta-Analysis.

Dystrophinopathies are caused by mutations in the DMD gene. Out-of-frame deletions represent most mutational events in severe Duchenne muscular dystrophy (DMD), while in-frame deletions typically lead to milder Becker muscular dystrophy (BMD). Antisense oligonucleotide-mediated exon skipping converts an out-of-frame transcript to an in-frame one, inducing a truncated but partially functional dystrophin protein. The reading frame rule, however, has many exceptions. We thus sought to simulate clinical outcomes of exon-skipping therapies for DMD exons from clinical data of exon skip-equivalent in-frame deletions, in which the expressed quasi-dystrophins are comparable to those resulting from exon-skipping therapies. We identified a total of 1298 unique patients with exon skip-equivalent mutations in patient registries and the existing literature. We classified them into skip-equivalent deletions of each exon and statistically compared the ratio of DMD/BMD and asymptomatic individuals across the DMD gene. Our analysis identified that five exons are associated with significantly milder phenotypes than all other exons when corresponding exon skip-equivalent in-frame deletion mutations occur. Most exon skip-equivalent in-frame deletions were associated with a significantly milder phenotype compared to corresponding exon skip-amenable out-of-frame mutations. This study indicates the importance of genotype-phenotype correlation studies in the rational design of exon-skipping therapies.

The Expanding Spectrum of Dystrophinopathies: HyperCKemia to Manifest Female Carriers.

ABSTRACTBackground:X-linked dystrophinopathies have a wide spectrum of manifestation. The most common forms are severe Duchenne muscular dystrophy (DMD) and Becker’s muscular dystrophy (BMD). However, less common manifestations are isolated cardiomyopathy, myalgia, cramps, rhabdomyolysis, hyperCKemia, and manifest female carriers. Materials and Methods:This case series is a part of an ongoing long-term prospective cohort of children with DMD and BMD from the year 2013. The clinical details are maintained in the clinic files and standard management protocols are followed. For this case series, clinical details were collected from the clinic files and recorded on a case record proforma. Details of cardiology, radiology, and genetic investigations were collected. Results:We report cases of classical DMD, BMD, manifest female carrier with proximal pelvic girdle weakness, a female carrier with isolated dilated cardiomyopathy, and infantile-onset asymptomatic hyperCKemia. We also report less common but notable clinical presentations of DMD, autism, intellectual disability, epilepsy, and asymptomatic transaminitis. Conclusions:It is important for clinicians to be aware of these less common clinical presentations for prompt diagnosis, and to avoid unnecessary investigations. Here, we report the clinical spectrum of dystrophinopathies seen in pediatric neuromuscular clinic and emphasize the variability and expanding knowledge about different manifestations of dystrophinopathies.

Gene modification strategies using AO-mediated exon skipping and CRISPR/Cas9 as potential therapies for Duchenne muscular dystrophy patients.

Duchenne muscular dystrophy (DMD) is an X-linked genetic disease affecting 1 in 5000 young males worldwide annually. Patients experience muscle weakness and loss of ambulation at an early age, with ∼75% reduced life expectancy. Recently developed genetic editing strategies aim to convert severe DMD phenotypes to a milder disease course. Among these, the antisense oligonucleotide (AO)-mediated exon skipping and the adeno-associated viral-delivered clustered regularly interspaced short palindromic repeat (CRISPR) associated protein 9 (adeno-associated viral (AAV)-delivered CRISPR/Cas9) gene editing have shown promising results in restoring dystrophin protein expression and functionality in skeletal and heart muscle in both animals and human cells in vivo and in vitro. However, therapeutic benefits currently remain unclear. The aim of this review is to compare the potential therapeutic benefits, efficacy, safety, and clinical progress of AO-mediated exon skipping and CRISPR/Cas9 gene-editing strategies. Both techniques have demonstrated therapeutic benefit and long-term efficacy in clinical trials. AAV-delivery of CRISPR/Cas9 may potentially correct disease-causing mutations following a single treatment compared to the required continuous AO/PMO-delivery of exon skipping drugs. The latter has the potential to increase the dystrophin expression in skeletal/heart muscle with sustained effects. However, therapeutic challenges including the need for optimised delivery must be overcome in to advance current clinical data.

Therapeutic potential of miRNAs in Duchenne muscular dystrophy and other neuromuscular diseases.

Therapeutic potential of miRNAs in Duchenne muscular dystrophy and other neuromuscular diseases.

Very Low Residual Dystrophin Quantity Is Associated with Milder Dystrophinopathy.

ObjectiveThis study was undertaken to determine whether a low residual quantity of dystrophin protein is associated with delayed clinical milestones in patients with DMD mutations.MethodsWe performed a retrospective multicentric cohort study by using molecular and clinical data from patients with DMD mutations registered in the Universal Mutation Database–DMD France database. Patients with intronic, splice site, or nonsense DMD mutations, with available muscle biopsy Western blot data, were included irrespective of whether they presented with severe Duchenne muscular dystrophy (DMD) or milder Becker muscular dystrophy (BMD). Patients were separated into 3 groups based on dystrophin protein levels. Clinical outcomes were ages at appearance of first symptoms; loss of ambulation; fall in vital capacity and left ventricular ejection fraction; interventions such as spinal fusion, tracheostomy, and noninvasive ventilation; and death.ResultsOf 3,880 patients with DMD mutations, 90 with mutations of interest were included. Forty‐two patients expressed no dystrophin (group A), and 31 of 42 (74%) developed DMD. Thirty‐four patients had dystrophin quantities < 5% (group B), and 21 of 34 (61%) developed BMD. Fourteen patients had dystrophin quantities ≥ 5% (group C), and all but 4 who lost ambulation beyond 24 years of age were ambulant. Dystrophin quantities of <5%, as low as <0.5%, were associated with milder phenotype for most of the evaluated clinical outcomes, including age at loss of ambulation (p < 0.001).InterpretationVery low residual dystrophin protein quantity can cause a shift in disease phenotype from DMD toward BMD. ANN NEUROL 2021;89:280–292

DMD – BIOMARKERS & OUTCOME MEASURES: P.127 Identification of biallelic DMD variants during maternal carrier testing for Becker muscular dystrophy

Dystrophinopathies are allelic X-linked recessive muscular dystrophies caused by pathogenic variants in the dystrophin (DMD) gene. In males, out-of-frame DMD variants typically cause more severe Duchenne muscular dystrophy (DMD), while in-frame variants typically result in Becker muscular dystrophy (BMD). Females who carry a DMD variant are at increased risk for cardiomyopathy and may have skeletal muscle symptoms. We report a mother (proband) and three sons, the oldest (10-year-old) and youngest (8-year-old) of whom have a pathogenic in-frame deletion of exons 45-47 and diagnoses of Becker muscular dystrophy. Both have progressive muscle weakness, recurrent rhabdomyolysis, and elevated creatine phosphokinase (CPK) levels ranging from 2,500 – 18,000 unit/L (ref 22-250 unit/L). During confirmatory carrier testing, the mother was found to carry the familial exon 45-47 deletion in addition to an unexpected second pathogenic in-frame DMD deletion of exons 49-51, which was not present in her two previously tested sons. She reports muscle cramps and pain but no muscle weakness. Cardiac MRI shows no current evidence of cardiomyopathy. DMD testing for the family's 9-year-old son detected the exon 49-51 deletion, which supported a diagnosis of BMD for him and confirmed the two DMD changes are situated on opposite alleles in his mother. He had an existing diagnosis of Perthes disease, but had otherwise not come to clinical attention for BMD-related symptoms. The family had reported that CPK levels measured locally had been within normal limits. CPK levels measured subsequent to genetic testing were mildly elevated (up to 321 unit/L), and muscle pain and fatigue were noted during clinical visit, consistent with BMD. This case highlights unexpected outcomes of maternal dystrophinopathy carrier testing, namely the detection of a second variant not previously known in the family, and subsequent diagnosis of BMD in a son who had been presumed to be unaffected. These results underscore the importance of genetic counselling in this setting. This case also describes an additional example of mild BMD in the setting of exon 49-51 deletion, as well as a further example of biallelic DMD variants in a female, of which only a few cases have been described.

DMD – ANIMAL MODELS & PRECLINICAL TREATMENT: P.207 AAV.U7 technology for two mutational hotspots of the DMD gene (∼6%) results in efficient exon skipping, protein restoration and force improvement

Mutations in the DMD gene cause two disorders, the severe Duchenne muscular dystrophy (DMD), associated with no protein expression and the milder Becker muscular dystrophy (BMD) associated with the production of an internally truncated but semi-functional protein. Genotype-phenotype correlation of DMD and BMD prompted a promising therapeutic strategy named exon skipping that intends to convert a DMD to a BMD phenotype. This strategy makes use of antisense oligonucleotides (AONs) to hide exon definition. This should transform an out-of-frame DMD reading frame into an in-frame one, leading into expression of an internally truncated but functional protein. AONs are promising but have two major drawbacks: 1. stability which required repeated injection; 2. limited penetration in heart. Alternatively, an antisense sequence can be carried by a small nuclear RNA (U7snRNA) and delivered using a viral vector such as adeno-associated virus (AAV). This approach (AAV.U7) has been demonstrated by several group including ours, to overcome AON's drawbacks. In this study, we focused on skipping of exon 2 and exon 44, two mutational hotspots of the DMD gene as their skipping could be beneficial to ∼12% of patients. Our objective was to evaluate the efficiency of an AAV.U7 exon skipping strategy in these hotspot mutations both in vitro and in vivo. For in vitro studies, we used skin punches from several DMD patients and transdifferentiated them into myotubes. Using those cells, we showed that our constructs efficiently mediated exons 2 or 44 skipping (at least ≥50%) and induced expression of a slightly truncated dystrophin. For in vivo studies, we used either a new mouse model carrying an exon 2 duplication (referred to as Dup 2 mice) or more recently a new humanized DMD mouse model that is missing human exon 45 disrupting reading frame of hDMD. This new model (referred to as hDMD/mdx del45 mice) does not express mouse and human dystrophins and presents a dystrophic muscle pathology in multiple muscles. In vivo, following intramuscular or intravenous injection, we observed an efficient exon 2 or 44 skipping (at least ≥50%) and expression of a truncated dystrophin (at least ≥40%). Muscle strength using different tests was improved (by ∼50% compared to an untreated mouse). These AAV.U7- could help ∼12% of DMD patients. One of those vectors is being evaluated in a clinical trial.