Randomised trial of Aureobasidium pullulans-produced beta 1,3-1,6-glucans in patients with Duchenne muscular dystrophy: favourable changes in gut microbiota and clinical outcomes indicating their potential in epigenetic manipulation

Gene therapy in the treatment of disease

CRISPR/Cas9-Mediated Genome Editing Corrects Dystrophin Mutation in Skeletal Muscle Stem Cells in a Mouse Model of Muscle Dystrophy.

Patients with Duchenne muscular dystrophy (DMD) tend to bleed more during surgery than do patients with other conditions. A retrospective analysis of blood loss after spinal surgery for scoliosis was therefore undertaken in 102 patients undergoing surgery in the senior author's unit. These included 48 patients with DMD, 26 patients with spinal muscular atrophy, and a miscellaneous group of 28 other patients most of whom had idiopathic scoliosis. For each patient the age at surgery, estimated blood volume, duration of operation, Cobb angle, and number of vertebrae fused were recorded and compared. Expression of dystrophin in skeletal muscle and the underlying gene mutation were also determined. The estimated blood loss in patients with DMD was significantly higher than that in patients with spinal muscular atrophy undergoing the same or similar procedure (P < 0.005) and was also significantly greater than that of the third group, which consisted mostly of patients with idiopathic scoliosis (P < 0.0005). Blood loss in the patient group with DMD showed a significant relationship with duration of surgery (P < 0.05). As most patients expressed no dystrophin, this did not correlate with the estimated blood loss. There was also no correlation between the estimated blood loss and the type of gene mutation found causing DMD. The authors' previous observations confirm the increased blood loss in patients with DMD who undergo scoliosis surgery. Because children with DMD lack dystrophin in all muscle types, including smooth muscle, the excessive blood loss may be because of a poor vascular smooth muscle vaso-constrictive response due to a lack of dystrophin.

Good news for the mdx mouse community: Improved dystrophin restoration after skipping mouse dystrophin exon 23

Gene-mediated Restoration of Normal Myofiber Elasticity in Dystrophic Muscles

Duchenne muscular dystrophy (DMD) is caused by mutations in the X-linked dystrophin (DMD) gene. The absence of dystrophin protein leads to progressive muscle weakness and wasting, disability and death. To establish a tailored large animal model of DMD, we deleted DMD exon 52 in male pig cells by gene targeting and generated offspring by nuclear transfer. DMD pigs exhibit absence of dystrophin in skeletal muscles, increased serum creatine kinase levels, progressive dystrophic changes of skeletal muscles, impaired mobility, muscle weakness and a maximum life span of 3 months due to respiratory impairment. Unlike human DMD patients, some DMD pigs die shortly after birth. To address the accelerated development of muscular dystrophy in DMD pigs when compared with human patients, we performed a genome-wide transcriptome study of biceps femoris muscle specimens from 2-day-old and 3-month-old DMD and age-matched wild-type pigs. The transcriptome changes in 3-month-old DMD pigs were in good concordance with gene expression profiles in human DMD, reflecting the processes of degeneration, regeneration, inflammation, fibrosis and impaired metabolic activity. In contrast, the transcriptome profile of 2-day-old DMD pigs showed similarities with transcriptome changes induced by acute exercise muscle injury. Our studies provide new insights into early changes associated with dystrophin deficiency in a clinically severe animal model of DMD.

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

Dystrophinopathy is caused by alterations in the dystrophin gene. The severe phenotype, Duchenne muscular dystrophy (DMD), is caused by a lack of dystrophin in skeletal muscles, resulting in necrosis and regenerating fibers, inflammatory cells, and muscle fibrosis. Progressive muscle weakness is a characteristic finding of this condition. Here, we encountered a rare case of a 10-year-old patient with asymptomatic dystrophinopathy with no dystrophin expression and investigated the reason for the absence of muscle weakness to obtain therapeutic insights for DMD. Using RNA-seq analysis, gene expression in skeletal muscles was compared among patients with asymptomatic dystrophinopathy, three patients with typical DMD, and two patients without dystrophinopathy who were leading normal daily lives. Cathepsin K (CTSK), myosin heavy chain 3 (MYH3), and nodal modulator 3-like genes exhibited a >8-fold change, whereas crystallin mu gene (CRYM) showed a <1/8-fold change in patients with typical DMD compared with their expression in the patient with asymptomatic dystrophinopathy. Additionally, CTSK and MYH3 expression exhibited a >16-fold change (P < 0.01), whereas CRYM expression showed a <1/16-fold change (P < 0.01) in patients with typical DMD compared with their expression in those without dystrophinopathy. CTSK plays an essential role in skeletal muscle loss, fibrosis, and inflammation in response to muscles injected with cardiotoxin, one of the most common reagents that induce muscle injury. Increased CTSK expression is associated with muscle injury or necrosis in patients with DMD. The lack of muscle weakness in the patient with asymptomatic dystrophinopathy might be attributed to the low CTSK expression in the muscles. To the best of our knowledge, this is the first report to demonstrate that CTSK expression was significantly higher in the skeletal muscles of patients with DMD with a typical phenotype than in those without dystrophinopathy.

Efficacy of Multi-exon Skipping Treatment in Duchenne Muscular Dystrophy Dog Model Neonates

G.P.82: Dystrophin-deficient pigs provide new insights into the hierarchy of physiological derangements of dystrophic muscle

Transduction of Full-length Dystrophin to Multiple Skeletal Muscles Improves Motor Performance and Life Span in Utrophin/Dystrophin Double Knockout Mice

ABSTRACTIntroductionIdentifying serum biomarkers that reflect the restoration of dystrophin in skeletal muscle is important for evaluating the effect of dystrophin‐restoring therapies in preclinical and clinical trials. Many potential blood biomarkers have been identified in Duchenne muscular dystrophy (DMD) patients, which change with disease progression or respond to pharmacological treatment. In this study, it was suggested that a panel of such blood biomarker candidates could be used to monitor dystrophin rescue in mdx mice treated with microdystrophin based therapies.MethodsPlasma samples from mdx mice treated with the microdystrophin therapy SGT‐001 were analysed with an antibody suspension bead array consisting of 87 antibodies. The array targets 83 unique proteins previously identified as biomarker candidates for DMD. Each sample was assayed at two different plasma dilutions to cover a broader concentration range. Protein concentrations estimated as Median fluorescent intensities (MFI) were correlated to dystrophin expression in muscle tissue, as measured by immunohistochemistry and Western blot. Thirteen of the targets were selected and analysed in a DMD and Becker muscular dystrophy (BMD) longitudinal natural history cohort using a suspension bead array.ResultsTen proteins were found to be significantly elevated in untreated mdx mice compared with C57 wild‐type mice and to correlate with dystrophin expression (Spearman's correlation, FDR &lt; 0.05) upon gene transfer in mdx mice. Translatability of these biomarkers from animal models to patients was evaluated by exploring abundance trajectories over time in both DMD and BMD patients and association with dystrophin expression in BMD patients. Consistent with the observations in mouse, six of these biomarker candidates were more abundant in DMD patients compared with controls, and six were also differentially abundant between BMD and DMD patients. Among them, serum titin was shown to be associated with dystrophin expression in BMD patients, having a steeper decline over time in patients with low dystrophin expression in tibialis anterior compared with patients with high expression. Myosine light chain 3 had a steeper decline with time in DMD patients compared with BMD patients.ConclusionsThe 10 biomarker candidates identified in mouse plasma are related to muscle contraction, glycolysis, microtubule formation and protein degradation. Human titin and myosine light chain 3 were the most interesting candidates as explorative biomarkers to monitor microdystrophin expression in gene therapies. If confirmed, these biomarkers could be used to detect restoration of dystrophin expression per se, monitor changes in dystrophin expression over time and potentially confirm disease phenotype changes from severe to mild disease forms.

Duchenne muscular dystrophy (DMD) is a progressive disease caused by the complete loss of the protein dystrophin. This loss leads to the development of cardiomyopathy which is fatal. It has been shown that without dystrophin, the muscle membrane becomes de-stabilized, leading to damage resulting in muscle weakening and cardiac failure. This makes understanding the mechanisms driving cardiomyopathy in DMD at the cellular level critical for the design of effective treatments. The bulk of the work to address DMD progression and treatment has focused on the loss of dystrophin in skeletal muscles. Presently, there is limited understanding of the development of cardiomyopathy, although it is the leading cause of death, and the pathology is unknown. This severely limits the ability to develop novel therapeutics to ameliorate cardiac failure. This project implements a novel biosensor to monitor, in live cardiac muscle, thin filament activation in real-time. The biosensor fuses fluorescent donor and acceptor proteins to the calcium-sensing cardiac troponin C (TnC). Using the biosensor, cardiac muscle activation will be monitored based on the detection of Förster Resonance Energy Transfer (FRET). In this system, all essential features of the muscle remain intact, including excitation-contraction coupling and muscle load. By utilizing the biosensor, this work will examine changes in myofilament states in DMD models in a loaded system, which closely represents in vivo cardiac muscle function. We have already seen a significant decrease in force in mdx papillary muscles as compared to controls. Additionally, the use of sarcomere specific small molecules targeting both the thin and thick filament will be used to examine the role of these sarcomeric components in the development of DMD-driven cardiomyopathy. The results of this project will provide new knowledge of the mechanisms of sarcomere activation in DMD-driven heart disease and provide a platform to test novel small molecules for the treatment of the lethal cardiomyopathy.

Murine models of Duchenne muscular dystrophy: is there a best model?

236. Delivery of VEGF Using AAV Vectors Stimulates Skeletal Muscle Regeneration In Vivo