Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder that affects dystrophin production, characterized by progressive neuromuscular dysfunction, often accompanied by osteoporosis. We prospectively evaluate the effects of bisphosphonates on bone micro-architecture reflected by trabecular bone score (TBS) of patients with DMD. A total of 72 male children or adolescents with DMD were included, with a mean age of 9.5 ± 1.8 years. They were divided into bisphosphonate treatment groups and control group based on areal bone mineral density (aBMD) and history of fragility fractures. Patients in bisphosphonate treatment groups randomly received intravenous infusion of 5 mg zoledronic acid (ZOL) annually or oral 70 mg alendronate weekly for three years. All patients took calcium 600 mg plus 125 IU vitamin D daily and calcitriol 0.25 μg every other day. TBS at the lumbar spine (LS) and aBMD at the LS, femoral neck (FN) and total hip (TH) were measured annually by dual-energy X-ray absorptiometry. Serum levels of β-isomerized carboxy-telopeptide of type I collagen and alkaline phosphatase were measured annually during the follow-up. A total of 25 (86.2%), 26 (92.9%) and 13 (86.7%) patients in the ZOL, alendronate and control groups completed the study. After 3 years, TBS Z-score increased from baseline by 1.13 (p < 0.01), 0.68 (p < 0.01) and 0.26 (p > 0.05) in the ZOL, alendronate and control groups, respectively. The mean increase in TBS Z-score from baseline was significantly greater in both bisphosphonate treatment groups compared to the control group (p < 0.05). No significant difference was found between the ZOL and alendronate groups. LS, FN and TH aBMD increased by 35.8%, 23.7% and 34.5% in the ZOL group (all p < 0.01 vs. baseline and control group) and by 21.5%, 29.3% and 25.0% in the alendronate group (all p < 0.05 vs. baseline and control group). LS and FN aBMD Z-scores increased by 1.56 and 1.63 in the ZOL group (all p < 0.01 vs. baseline), by 1.32 and 1.48 in the alendronate group (all p < 0.05 vs. baseline). Bisphosphonates demonstrated a favourable safety profile during the study period. This relatively long-term study confirms that zoledronic acid and alendronate are beneficial to improve micro-architecture reflected by TBS and aBMD of children or adolescents with DMD.

Delandistrogene moxeparvovec is a recombinant adeno-associated virus rhesus isolate serotype 74 vector-based gene therapy that addresses the absence of functional dystrophin in Duchenne muscular dystrophy (DMD). EMBARK is a phase 3, two-part, crossover, randomized, placebo-controlled trial assessing the safety and efficacy of delandistrogene moxeparvovec (single intravenous dose 1.33 × 1014vector genomes/kg) in ambulatory male patients with DMD aged 4 to < 8years; N = 125. One-year results demonstrated the manageable safety of delandistrogene moxeparvovec, consistent with previous clinical trials. The primary endpoint (change from baseline in North Star Ambulatory Assessment [NSAA] total score at 52weeks compared with placebo) did not meet statistical significance. However, key secondary endpoints, comprising timed function tests, suggested slowing or stabilization of disease progression with delandistrogene moxeparvovec, which could become increasingly evident over longer periods of time. We report 2-year follow-up of safety and functional outcomes in patients receiving delandistrogene moxeparvovec in EMBARK part1. As a result of the crossover study design, 2-year functional outcomes of patients receiving delandistrogene moxeparvovec in part1 of EMBARK were compared, by pre-specified analysis, with a matched propensity score-weighted external control (EC). At 2years, EMBARK patients showed statistically significant benefit versus the EC cohort in functional outcomes prognostic for delaying loss of ambulation (NSAA, Time to Rise, 10-m Walk/Run), demonstrating sustained stabilization or slowing of disease progression. Delandistrogene moxeparvovec micro-dystrophin expression and sarcolemmal localization were maintained over 64weeks. No new safety signals were observed between week52 and week104. Between baseline and week104, there were no treatment-related deaths, study discontinuations due to adverse events, or clinically significant complement-mediated adverse events. At 2years, stabilization or slowing of DMD disease progression was observed in ambulatory male patients with DMD aged 4 to < 8years receiving delandistrogene moxeparvovec versus a matched EC cohort. Safety was consistent with EMBARK 1-year data and manageable with appropriate monitoring. GOV: NCT05096221.

Does Motor Function Differ According to the Site of Mutation in Duchenne Muscular Dystrophy?

cPLA2 in musculoskeletal and autoimmune diseases: Molecular mechanisms and therapeutic insights.

Duchenne muscular dystrophy, caused by mutations in the DMD gene encoding dystrophin, is a severe progressive muscle-wasting disorder characterized by impaired muscle regeneration. We reveal the alternative splicing of transcription factor E2-alpha (encoding transcription factors E12 and E47) plays a pivotal role in myogenic progression. E47 is highly expressed in proliferating myoblasts and promotes proliferation, whereas E12 is upregulated during differentiation and drives myogenic commitment. Mechanistically, we identify the nuclear splicing factor polypyrimidine tract binding protein 1 as a key regulator of transcription factor E2-alpha mutually exclusive alternative splicing. Polypyrimidine tract binding protein 1 levels decline during normal myoblast differentiation, facilitating the switch from E47 to E12. However, in Duchenne muscular dystrophy patients and mdx mice, polypyrimidine tract binding protein 1 remains aberrantly elevated, resulting in dysregulated E47/E12 ratios (increased E47 and decreased E12), which disrupts myogenic differentiation and impairs muscle regeneration. Therapeutically, polypyrimidine tract binding protein 1 knockdown restores myoblast differentiation, enhances muscle repair, and improves muscle function in mdx mice. Furthermore, we demonstrate that dergrasyn, a deubiquitinase inhibitor, induces polypyrimidine tract binding protein 1 degradation, restores myogenic differentiation, and ameliorates dystrophic pathology. Our findings identify polypyrimidine tract binding protein 1 as a potential therapeutic target for Duchenne muscular dystrophy and highlight modulation of transcription factor E2-alpha splicing as a promising strategy to restore muscle regeneration.

Exon skipping antisense oligonucleotides (AONs) have been extensively studied as a promising method of treating Duchenne muscular dystrophy (DMD), yet the clinical efficacy of the conditionally approved AONs still remains low. Using phosphorothioated locked nucleic acid/2'-fluoro-RNA AONs, we aimed to increase AON efficiency by employing skeletal muscle-targeting conjugate molecules, cholesterol, and docosanoic acid to improve the biodistribution of the therapeutic. While conjugate molecules were able to induce high levels of skipping in an in vitro model, invivo studies in the hDMDdel52/mdx mouse model caused adverse symptomatic and systemic immune reactions, up to and including death, with little to no appreciable increase in exon skipping. Our study cautions against using these AON conjugates in an animal model due to severe toxicity.

Population-Based Investigation of DMD Genotype and Neurodevelopmental Concerns in Duchenne Muscular Dystrophy.

Muscle biopsy has long been regarded as the cornerstone for diagnosing pediatric muscular disorders; however, it is invasive and may be limited by sampling error and inconclusive histopathological findings. This study aimed to evaluate whether whole-exome sequencing (WES) can effectively replace muscle biopsy as a first-line diagnostic approach in children with suspected neuromuscular disorders. Between January 2018 and December 2025, we prospectively enrolled 47 pediatric patients presenting with clinical features suggestive of muscular disorders at a tertiary medical center in Taiwan. The cohort included patients with suspected muscular dystrophies (n = 21), congenital myopathies (n = 23), and multiplex ligation-dependent probe amplification (MLPA)-negative Duchenne muscular dystrophy (DMD; n = 3). All patients underwent WES as the initial diagnostic test without prior muscle biopsy. Trio-based analysis using parental samples was performed in 29.8% of cases. Variant interpretation followed the American College of Medical Genetics and Genomics (ACMG) guidelines. WES identified a definitive molecular diagnosis in 72.3% of patients (34/47). Diagnostic yields varied by subgroup: 100% (3/3) in MLPA-negative DMD, 71.4% (15/21) in muscular dystrophies, and 69.6% (16/23) in congenital myopathies. Pathogenic or likely pathogenic variants were detected in 31 distinct genes, including COL6A1 and COL6A3, which are associated with Ullrich congenital muscular dystrophy. Notably, 58.8% of diagnosed patients (20/34) received molecular diagnoses that differed from their initial clinical impression, encompassing conditions such as ZSWIM6-associated neurodevelopmental disorders, GJB2-related hearing loss, OCRL-associated Lowe syndrome, and various metabolic or syndromic disorders. In all three MLPA-negative DMD cases, WES identified point mutations amenable to mutation-specific therapies. No patient required a muscle biopsy for diagnostic confirmation during the study period. First-tier WES demonstrates high diagnostic utility in pediatric muscular disorders while avoiding invasive muscle biopsy. The high rate of diagnostic reclassification underscores the substantial phenotypic overlap between primary neuromuscular diseases and other neurological or systemic conditions. These findings support the early implementation of genetic testing to enable accurate diagnosis and timely initiation of targeted therapies.

A multidisciplinary care approach is fundamental in neuromuscular diseases due to their complex characteristics, requiring a timely diagnosis and optimization of the management plan. Diagnostic yield has greatly improved in the last years, due either to powerful genetic-molecular techniques or increased attention of physicians to red flags that can indicate a neuromuscular disorder for prompting a specialist evaluation. The improvement of standards of care and the availability of effective disease-modifying treatments such as enzymatic replacement therapies, RNA-based and also gene therapy, but also steroid therapy for Duchenne muscular dystrophy, have expanded life expectancy of patients, also improving their quality of life, although at the same time leading to new and more complex phenotypes. It also means that, for diseases with infantile onset, greater number of patients reach adulthood, this making it necessary to establish a “transition” process from pediatric to adult care centers for structured management plans and to mitigate the risk to be lost. We have now developed in Tuscany a program for transition involving two pediatric reference centers for rare and neuromuscular disorders (IRCCS Meyer, Florence and IRCCS Stella Maris, Calambrone, Pisa) and the adult reference center for neuromuscular disorder of Pisa, University Hospital, Ospedale Santa Chiara, ERN-NMD affiliated). This program, started in 2023, includes combined and integrated evaluation of adolescent-juvenile patients from childhood neuropsychiatrists and pediatricians and adult neurologists who, in exchanging their expertise, accompany affected people in this comprehensive journey for ensuring suitable standards of care. In total, 70 neuromuscular patients transitioned from the pediatric centers to the adult center, among them 11 affected by Duchenne muscular dystrophy and 30 by Becker muscular dystrophy, for which clinical and investigation data have been extensively collected for better understanding of disease natural history and responses to interventions and treatment.

Animal models and human 2D culture models have been instrumental for investigating skeletal muscle diseases and the development of therapeutics. However in vivo models and 2D cultures are limited in their translation to clinical application. These limitations are most evident through the success of myostatin inhibitors for improving mass and function in mice studies followed by unsuccessful clinical trials in patients with sarcopenia and Duchenne Muscular Dystrophy (DMD) (3-4). Although clinical trials of myostatin inhibitors have often reported increases in lean muscle mass, efficacy endpoints of improved muscle function are typically not achieved (3-4). Additionally, studies of age-related muscle atrophy, sarcopenia, have unique barriers to translation such as age-related gene expression changes and sex related muscle aging that is not conserved between species (1-2). Due to these challenges, our team developed a donor-derived 3D skeletal muscle platform housed in standard 24-well imaging plates. This platform was then utilized to investigate retention of sex- specific characteristics in 3D muscle cultures and the effectiveness of astaxanthin (ASTX) to improve contractile signaling and function in healthy and clinically sarcopenic 3D cultures. Initial characterization and validation were carried out in samples from healthy male and female donors. Contractile function recorded through digital image correlation (DIC) analysis during electrical stimulation was similar between male and female 3D muscles, but females displayed elevated type 1 fiber proportions compared to males. Female muscle also displayed elevated levels of OPA1 and TFAM protein levels along with decreased Akt signaling. Overall, female muscle exhibited a greater reliance on mitochondrial energy utilization and reduced protein synthesis indicating retention of sex-specific characteristics in 3D culture. Samples from young female and clinically sarcopenic female donors were studied in this platform for functional differences in force production, fatigue susceptibility, and contractile protein signaling following fatigue with or without astaxanthin antioxidant therapy. Astaxanthin was assessed at 1μM, 10μM, and 25μM in young cultures and only 10μM in sarcopenic cultures due to limitations in cell quantity for 7 days. Although there were no significant effects on force outputs or fatiguability for either group, 10μM rescued P38 and Akt signaling in the sarcopenic samples restoring levels to those exhibited by young DMSO- treated controls. Additionally, 10μM and 25μM in the young cohort suppressed Akt signaling indicating astaxanthin may negatively impact this critical pathway for exercise adaptation in healthy muscle. Although astaxanthin did not improve functional performance in the clinically sarcopenic or young female 3D muscle samples, clenbuterol was evaluated as a positive control for improving performance in young muscle and showed a nearly two-fold increase in both twitch and tetanic force response in 1μM treated samples compared to DMSO controls. These findings support the utilization of this muscle platform for assessing functional differences among drug treatments and untreated donor groups.

Safety and efficacy of fordadistrogene movaparvovec in ambulatory participants with Duchenne muscular dystrophy (CIFFREO): a phase 3, double-blind, randomised, placebo-controlled study.

Lumbopelvic stabilization-based physiotherapy and rehabilitation and urotherapy for lower urinary tract dysfunction in Duchenne Muscular Dystrophy: a randomized controlled trial.

Duchenne muscular dystrophy (DMD) is a severe X-linked recessive disorder caused by a deficiency of dystrophin, leading to progressive muscle degeneration and eventually cardiorespiratory failure. Exon-skipping therapies using cell-penetrating peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) restore production of a shortened but functional dystrophin protein. Since respiratory insufficiency is the leading cause of morbidity and mortality in DMD, we sought to examine the impact of PPMO on respiratory pathology. We evaluated the effects of RC-1001, a PPMO targeting a dystrophin mutation, in mdx mice, a preclinical model of DMD. These mice received monthly intravenous doses of RC-1001 (30, 50, or 100 mg/kg), starting at 2 months of age, and were monitored until the study endpoint at 12 months of age. Respiratory function was evaluated using whole-body plethysmography and the forced oscillometry technique, followed by histological and molecular analysis of respiratory muscles. PPMO-treated mice showed dose-independent improvements in respiratory function, with postmortem studies revealing significant dystrophin restoration, reduced inflammation, and decreased fibrosis in respiratory muscles. Additionally, dystrophin restoration and strength improvements were observed in limb muscles. Overall, PPMO-mediated exon skipping effectively targets respiratory pathology and is a promising therapy for respiratory insufficiency in patients with DMD.

With implementation of newborn screening (NBS) for Duchenne muscular dystrophy (DMD), creatine kinase-muscle (CK) values will be reported on newborns. Maternal, labor, delivery, and newborn factors may elevate CK levels, raising concern for DMD. Predictive modeling could aid hyperCKemia interpretation while awaiting diagnostic confirmation. In this single-center, prospective cohort study, parents of 8365 newborns were offered DMD-NBS. Electronic health records provided data on candidate predictors of hyperCKemia defined by values > 97th or 99th percentiles, or 2000 ng/mL in babies with normal DMD sequences. Exposures included maternal, newborn and perinatal factors. Associations between predictors and hyperCKemia were evaluated using univariate logistic regression. A multivariable prediction model for the 97th percentile was derived using backward stepwise logistic regression and externally validated in a cohort of 2672 newborns. HyperCKemia > 97th percentile was the main outcome. Univariate analyses revealed associations between hyperCKemia and maternal ethnicity, primiparity, labor and delivery complications, oxytocin induction or augmentation, duration of ruptured membranes, forceps or vacuum-assisted delivery, neonatal resuscitation, sex, gestational age, birth weight, and Apgar scores. Lower odds of hyperCKemia were associated with later hour-of-life at sample collection and birth by cesarean section. The final model included parity, shoulder dystocia, forceps or vacuum-assisted delivery, gestational age, neonatal resuscitation, Apgar score (1 min), and time of sample collection. Elevated CK levels may be used for DMD-NBS. Multiple perinatal factors are associated with transient non-DMD hyperCKemia. Our model considers the potential combined impact of such factors and generates a non-disease likelihood for preliminary hyperCKemia interpretation.

Meeting report: Expanding access to advanced cardiac therapies, including ventricular assist devices (VADs) and heart transplantation in muscular dystrophy.

Duchenne muscular dystrophy (DMD) is characterized by progressive muscle degeneration, with respiratory muscle weakness significantly impacting patient outcomes. Early detection of disease progression is critical for evaluating treatment strategies. This study investigates the feasibility of ultrasound shear wave elasticity imaging (US-SWEI) for assessing early-stage respiratory muscle changes in mdx mice, a model for DMD, aged 3 to 6 months. Longitudinal in vivo imaging evaluated the diaphragm's viscoelastic properties, with group and phase velocities measured to capture biomechanical changes. In mdx mice, group velocity increased from 3.47±0.15to4.20±0.20m/s, whereas wild-type values changed only modestly (2.76±0.11to3.01±0.16m/s). Histological analysis confirmed a significant positive correlation between group velocity and collagen deposition (R2=0.54,P=0.025), supporting fibrosis as a primary factor driving changes in diaphragm viscoelastic properties. These findings establish US-SWEI as a noninvasive, feasible approach for monitoring respiratory muscle health and advancing preclinical DMD research. By identifying early-stage changes in diaphragm properties, this approach enables the development of therapeutic interventions targeting respiratory complications. Furthermore, US-SWEI presents a potential avenue for assessing and monitoring neuromuscular diseases.

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder without an effective cure. Adeno-associated virus (AAV) based gene therapy has improved dystrophin function, with sub-optimal clinical outcomes. We reasoned that a combination of rational engineering of AAV9 capsids modified at the post-translational modification sites, optimal promoter selection, and codon-optimisation of the microdystrophin (μDys) can enhance the AAV9 vector functionality. Our initial promoter screening demonstrated improved dystrophin expression in muscle fibres with a ubiquitous CAG promoter (1.61-fold in CAG vs. MHCK7, p < 0.0001) in mdx mice. We then evaluated two engineered AAV9 capsids (N57Q, K51Q) containing CAG-μDys intramuscularly invivo, which demonstrated a significant improvement in grip strength 18 weeks after gene therapy. Subsequent evaluation of a codon-optimised microdystrophin transgene under the control of the optimal CAG promoter and capsid (AAV9K51Q) by intramuscular administration revealed enhanced muscle grip strength and dystrophin-glycoprotein complex restoration up to 4 months after gene therapy. Based on the improved performance of AAV9K51Q vectors during intramuscular gene transfer, we performed a systemic administration of these vectors alone, and a comparison with the control group revealed a significantly increased muscle contraction force by 1.6-1.7 fold and a 30%-60% dystrophin restoration in skeletal and cardiac muscles, up to 14 months after gene therapy. Collectively, our study underscores the therapeutic potential of engineered AAV9 vectors for potential clinical application in patients with DMD.

This commentary is on the original article by Schiava et al. on pages 429–440 of this issue.

Porcine models carrying DMD mutations recapitulate Duchenne and Becker muscular dystrophy (DMD and BMD), inherited disorders leading to progressive muscle weakness, with DMD presenting a far more severe and rapidly progressive phenotype. Accurate in vivo muscle function assessment is essential for monitoring disease progression and as an efficacy readout for therapeutic intervention. We present a protocol to evaluate muscle strength, dynamics, and fatigue in dystrophic pigs, including design setup, anesthesia, measurement, and data analysis. For complete details on the use and execution of this protocol, please refer to Blasi et al.1.

Acute liver injury (ALI) is a recognized adverse event with adeno-associated virus (AAV)-based gene therapies, including delandistrogene moxeparvovec, an AAVrh74-based gene therapy for Duchenne muscular dystrophy. Progression of ALI to acute liver failure (ALF) is rare. In clinical trials, ALF was defined as an international normalized ratio ≥1.5 with encephalopathy and/or ascites, occurring <26 weeks since product/treatment exposure, and in the absence of identified preexisting liver disease as assessed by the treating investigator. The two cases presented here represent the only known instances of ALF following delandistrogene moxeparvovec to date, both resulting in fatal outcomes. Both cases occurred in nonambulatory patients (ages 15 and 16 years). Both patients exhibited abrupt elevations in aminotransferases from baseline approximately 4 weeks after delandistrogene moxeparvovec administration. Over the subsequent weeks and despite interventions, aminotransferases, including gamma-glutamyl transferase (GGT), declined concurrently with a continued rise in total bilirubin, consistent with severe hepatocellular injury and a harbinger of ALF. An interdisciplinary expert panel was convened and concluded that no single biomarker or clinical feature consistently predicted the rapid progression of severe ALI to ALF. Advisors recommended basing treatment intervention for severe ALI on the rate and magnitude of changes from baseline in liver biomarkers (e.g., 2-3× increase in alanine aminotransferase within ≤1 week). For these severe cases, most advisors recommended intravenous methylprednisolone as the initial treatment whereas typical ALI may be managed with oral corticosteroid adjustment or may resolve with time, as seen in the clinical trial experience. Advisors hypothesized that delandistrogene moxeparvovec-related ALI is T-cell-mediated and discussed the potential of additional immunosuppression. Advisors also recommended enhancing baseline evaluation for hepatic comorbidities with lipid profiling, transient elastography, and abdominal ultrasound to further study potential risk factors for ALI progressing to ALF. Finally, there was strong support for generating additional real-world evidence and conducting prospective clinical trials to inform clinical management of ALI in practice.