Therapy For Duchenne Muscular Dystrophy Research Articles

AAV gene therapy for Duchenne Muscular Dystrophy: lessons learned from a phase 3 trial.

AAV gene therapy for Duchenne Muscular Dystrophy: lessons learned from a phase 3 trial.

430P The damaged lysosome is a therapeutic target for combined therapy in Duchenne muscular dystrophy

430P The damaged lysosome is a therapeutic target for combined therapy in Duchenne muscular dystrophy

CRISPR-mediated megabase-scale transgene de-duplication to generate a functional single-copy full-length humanized DMD mouse model

BackgroundThe development of sequence-specific precision treatments like CRISPR gene editing therapies for Duchenne muscular dystrophy (DMD) requires sequence humanized animal models to enable the direct clinical translation of tested strategies. The current available integrated transgenic mouse model containing the full-length human DMD gene, Tg(DMD)72Thoen/J (hDMDTg), has been found to have two copies of the transgene per locus in a tail-to-tail orientation, which does not accurately simulate the true (single) copy number of the DMD gene. This duplication also complicates analysis when testing CRISPR therapy editing outcomes, as large genetic alterations and rearrangements can occur between the cut sites on the two transgenes.ResultsTo address this, we performed long read nanopore sequencing on hDMDTg mice to better understand the structure of the duplicated transgenes. Following that, we performed a megabase-scale deletion of one of the transgenes by CRISPR zygotic microinjection to generate a single-copy, full-length, humanized DMD transgenic mouse model (hDMDTgSc). Functional, molecular, and histological characterisation shows that the single remaining human transgene retains its function and rescues the dystrophic phenotype caused by endogenous murine Dmd knockout.ConclusionsOur unique hDMDTgSc mouse model simulates the true copy number of the DMD gene, and can potentially be used for the further generation of DMD disease models that would be better suited for the pre-clinical assessment and development of sequence specific CRISPR therapies.

Heteroduplex oligonucleotide technology boosts oligonucleotide splice switching activity of morpholino oligomers in a Duchenne muscular dystrophy mouse model

The approval of splice-switching oligonucleotides with phosphorodiamidate morpholino oligomers (PMOs) for treating Duchenne muscular dystrophy (DMD) has advanced the field of oligonucleotide therapy. Despite this progress, PMOs encounter challenges such as poor tissue uptake, particularly in the heart, diaphragm, and central nervous system (CNS), thereby affecting patient’s prognosis and quality of life. To address these limitations, we have developed a PMOs-based heteroduplex oligonucleotide (HDO) technology. This innovation involves a lipid-ligand-conjugated complementary strand hybridized with PMOs, significantly enhancing delivery to key tissues in mdx mice, normalizing motor functions, muscle pathology, and serum creatine kinase by restoring internal deleted dystrophin expression. Additionally, PMOs-based HDOs normalized cardiac and CNS abnormalities without adverse effects. Our technology increases serum albumin binding to PMOs and improves blood retention and cellular uptake. Here we show that PMOs-based HDOs address the limitations in oligonucleotide therapy for DMD and offer a promising approach for diseases amenable to exon-skipping therapy.

Positive experience in treating patients with Duchenne muscular dystrophy caused by a nonsense mutation: family clinical case

Duchenne muscular dystrophy is a severe genetic disease caused by mutations in the dystrophin gene (DMD), which lead to a significant decrease or complete absence of the protein of the same name in muscle fibers. The disease manifests itself in boys from the age of 2–5 years in the form of progressive muscular weakness, cardiomyopathy and respiratory failure. As a result of progression, patients lose the ability to move by adolescence and die in the third decade from cardiopulmonary complications. However, timely pathogenetic therapy for Duchenne muscular dystrophy can prolong the life of patients and improve its quality. In this regard, early diagnosis of the disease and early initiation of pathogenetic therapy are very important.The article describes two clinical cases of the disease in siblings with onset at 2 years of age and different ages of diagnosis. Pathogenetic therapy in the first case made it possible to prolong the patient’s outpatient stay and improve the quality of life, and early diagnosis and initiation of pathogenetic therapy in the second case avoided disease progression and contributed to the development of new motor skills. The presented clinical case demonstrates the need for early diagnosis of the disease even before the onset of clinical manifestations.

Possibility of exon skipping therapy for Duchenne muscular dystrophy in Russian patients: present and future

Background. Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy in children, that occurs between one and three years of age. DMD is caused by pathogenic and likely pathogenic variants in the DMD gene, which lead to a deficit of various isoforms of the dystrophin protein, the main protein of the muscle cytoskeleton. Drugs aimed at slowing the progression of the disease are being actively developed around the world. One of the perspective approaches to pathogenetic therapy is therapy using exon skipping. As a result of this treatment, the reading frame is restored due to the exon skipping enabling the production of truncated dystrophin.Aim. To evaluate the applicability of exon skipping therapy in Russian patients with DMD.Materials and methods. The applicability of therapy aimed at exon skipping was analyzed for a sample of 1519 patients admitted to the laboratory of DNA diagnostics of the Research Centre for Medical Genetics with a referral diagnosis of Duchenne/Becker muscular dystrophy from October 1, 2018 to September 1, 2023.Results. As a result of the study and analysis of the spectrum of mutations in the DMD gene among patients with DMD in the Russian Federation, the theoretical applicability of exon skipping therapy was assessed: for 29.3 % of patients this approach to treatment is applicable. The proportions of patients for whom existing exon skipping therapies are available were also estimated. In total, skipping of frequent exons 51, 53, 45 is applicable for 14.6 % of patients. Conclusion. One of the effective and accessible types of therapy for DMD is exon skipping. This type of therapy is mutation-specific. In this regard, the assessment of applicability will allow us to estimate the proportion of patients for whom a particular exon skipping will be available.

Clinician Perspectives of Gene Therapy as a Treatment Option for Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a progressive, life-limiting, neuromuscular disorder. Clinicians play an important role in informing families about therapy options, including approved gene therapies and clinical trials of unapproved therapies. This study aimed to understand the perspectives of clinicians about gene therapy for DMD, which has not previously been studied. We conducted interviews with specialist clinicians treating patients with DMD in the United States (n = 8) and United Kingdom (n = 8). Interviews were completed in 2022, before any approved gene therapies, to gain insight into barriers and facilitators to implementing gene therapy and educational needs of clinicians. Most respondents expressed cautious optimism about gene therapy. Responses varied regarding potential benefits with most expecting delayed progression and duration of benefit (1 year to lifelong). Concern about anticipated risks also varied; types of anticipated risks included immunological reactions, liver toxicity, and cardiac or renal dysfunction. Clinicians generally, but not uniformly, understood that gene therapy for DMD would not be curative. Most reported needing demonstrable clinical benefit to justify treatment-related risks. Our data demonstrate variability in knowledge and attitudes about gene therapy among clinicians who follow patients with DMD. As our knowledge base about DMD gene therapy grows, clinician education is vital to ensuring that accurate information is communicated to patients and families.

Gene therapy for Duchenne Muscular Dystrophy: assessing the readiness of Italian centres of expertise.

Duchenne muscular dystrophy (DMD) is a heritable disorder that causes a rapid and progressive loss of ambulatory skills. There is no curative therapy for this pathology, that is currently managed with a combination of physiotherapy and pharmacological interventions limiting the progression of the disease (e.g. corticosteroids, cardiac medications). However, a new opportunity is represented by gene therapy, a promising treatment that, however, requires significant expertise during the whole delivery of care and a solid organisational infrastructure. An organisational strategy that could effectively support its delivery to DMD patients in Italy is the hub-and-spoke model. However, an accurate portrait of the present network of DMD centres of expertise in Italy and of their readiness in the delivery of gene therapy is paramount, to facilitate access to this experimental medicine in the future. In this context, the present study aimed to map the DMD centres of expertise in Italy and later evaluate their preparedness in terms of gene therapy delivery. For this purpose, a series of items was proposed to 30 centres in Italy, of which 20 responded. After assessing the readiness of the involved centres in terms of patient preparation, therapy infusion, close surveillance, and long-term follow-up, we proposed a suitable organizational model, namely a flexible hub-and-spoke model, for the delivery of gene therapy in the Italian DMD network and solutions to tackle the challenges emerged from the survey. Overall, the present study detected an adequate readiness of the Italian DMD centres of expertise, despite observing a significant room for improvement in digital infrastructures, culture, and training.

Duchenne muscular dystrophy skeletal muscle cells derived from human induced pluripotent stem cells recapitulate various calcium dysregulation pathways

Duchenne muscular dystrophy (DMD) is an X-linked progressive muscle degenerative disease, caused by mutations in the dystrophin gene and resulting in premature death. As a major secondary event, an abnormal elevation of the intracellular calcium concentration in the dystrophin-deficient muscle contributes to disease progression in DMD. In this study, we investigated the specific functional features of induced pluripotent stem cell-derived muscle cells (hiPSC-skMCs) generated from DMD patients to regulate intracellular calcium concentration. As compared to healthy hiPSC-skMCs, DMD hiPSC-skMCs displayed specific spontaneous calcium signatures with high levels of intracellular calcium concentration. Furthermore, stimulations with electrical field or with acetylcholine perfusion induced higher calcium response in DMD hiPSC-skMCs as compared to healthy cells. Finally, Mn2+ quenching experiments demonstrated high levels of constitutive calcium entries in DMD hiPSC-skMCs as compared to healthy cells. Our findings converge on the fact that DMD hiPSC-skMCs display intracellular calcium dysregulation as demonstrated in several other models. Observed calcium disorders associated with RNAseq analysis on these DMD cells highlighted some mechanisms, such as spontaneous and activated sarcoplasmic reticulum (SR) releases or constitutive calcium entries, known to be disturbed in other dystrophin-deficient models. However, store operated calcium entries (SOCEs) were not found to be dysregulated in our DMD hiPSC-skMCs model. These results suggest that all the mechanisms of calcium impairment observed in other animal models may not be as pronounced in humans and could point to a preference for certain mechanisms that could correspond to major molecular targets for DMD therapies.

Systemic delivery of full-length dystrophin in Duchenne muscular dystrophy mice

Current gene therapy for Duchenne muscular dystrophy (DMD) utilizes adeno-associated virus (AAV) to deliver micro-dystrophin (µDys), which does not provide full protection for striated muscles as it lacks many important functional domains of full-length (FL) dystrophin. Here we develop a triple vector system to deliver FL-dystrophin into skeletal and cardiac muscles. We split FL-dystrophin into three fragments linked to two orthogonal pairs of split intein, allowing efficient assembly of FL-dystrophin. The three fragments packaged in myotropic AAV (MyoAAV4A) restore FL-dystrophin expression in both skeletal and cardiac muscles in male mdx4cv mice. Dystrophin-glycoprotein complex components are also restored at the sarcolemma of dystrophic muscles. MyoAAV4A-delivered FL-dystrophin significantly improves muscle histopathology, contractility, and overall strength comparable to µDys, but unlike µDys, it also restores defective cavin 4 localization and associated signaling in mdx4cv heart. Therefore, our data support the feasibility of a mutation-independent FL-dystrophin gene therapy for DMD, warranting further clinical development.

Chimeric Cell Therapy Transfers Healthy Donor Mitochondria in Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a severe X-linked disorder characterized by dystrophin gene mutations and mitochondrial dysfunction, leading to progressive muscle weakness and premature death of DMD patients. We developed human Dystrophin Expressing Chimeric (DEC) cells, created by the fusion of myoblasts from normal donors and DMD patients, as a foundation for DT-DEC01 therapy for DMD. Our preclinical studies on mdx mouse models of DMD revealed enhanced dystrophin expression and functional improvements in cardiac, respiratory, and skeletal muscles after systemic intraosseous DEC administration. The current study explored the feasibility of mitochondrial transfer and fusion within the created DEC cells, which is crucial for developing new therapeutic strategies for DMD. Following mitochondrial staining with MitoTracker Deep Red and MitoTracker Green dyes, mitochondrial fusion and transfer was assessed by Flow cytometry (FACS) and confocal microscopy. The PEG-mediated fusion of myoblasts from normal healthy donors (MBN/MBN) and normal and DMD-affected donors (MBN/MBDMD), confirmed the feasibility of myoblast and mitochondrial fusion and transfer. The colocalization of the mitochondrial dyes MitoTracker Deep Red and MitoTracker Green confirmed the mitochondrial chimeric state and the creation of chimeric mitochondria, as well as the transfer of healthy donor mitochondria within the created DEC cells. These findings are unique and significant, introducing the potential of DT-DEC01 therapy to restore mitochondrial function in DMD patients and in other diseases where mitochondrial dysfunction plays a critical role.Graphical

Molecular and Biochemical Therapeutic Strategies for Duchenne Muscular Dystrophy.

Significant progress has been achieved in understanding Duchenne muscular dystrophy (DMD) mechanisms and developing treatments to slow disease progression. This review article thoroughly assesses primary and secondary DMD therapies, focusing on innovative modalities. The primary therapy addresses the genetic abnormality causing DMD, specifically the absence or reduced expression of dystrophin. Gene replacement therapies, such as exon skipping, readthrough, and gene editing technologies, show promise in restoring dystrophin expression. Adeno-associated viruses (AAVs), a recent advancement in viral vector-based gene therapies, have shown encouraging results in preclinical and clinical studies. Secondary therapies aim to maintain muscle function and improve quality of life by mitigating DMD symptoms and complications. Glucocorticoid drugs like prednisone and deflazacort have proven effective in slowing disease progression and delaying loss of ambulation. Supportive treatments targeting calcium dysregulation, histone deacetylase, and redox imbalance are also crucial for preserving overall health and function. Additionally, the review includes a detailed table of ongoing and approved clinical trials for DMD, exploring various therapeutic approaches such as gene therapies, exon skipping drugs, utrophin modulators, anti-inflammatory agents, and novel compounds. This highlights the dynamic research field and ongoing efforts to develop effective DMD treatments.

Recent Advances in Pre-Clinical Development of Adiponectin Receptor Agonist Therapies for Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is caused by genetic mutations in the cytoskeletal-sarcolemmal anchor protein dystrophin. Repeated cycles of sarcolemmal tearing and repair lead to a variety of secondary cellular and physiological stressors that are thought to contribute to weakness, atrophy, and fibrosis. Collectively, these stressors can contribute to a pro-inflammatory milieu in locomotor, cardiac, and respiratory muscles. Given the many unwanted side effects that accompany current anti-inflammatory steroid-based approaches for treating DMD (e.g., glucocorticoids), there is a need to develop new therapies that address inflammation and other cellular dysfunctions. Adiponectin receptor (AdipoR) agonists, which stimulate AdipoR1 and R2 isoforms on various cell types, have emerged as therapeutic candidates for DMD due to their anti-inflammatory, anti-fibrotic, and pro-myogenic properties in pre-clinical human and rodent DMD models. Although these molecules represent a new direction for therapeutic intervention, the mechanisms through which they elicit their beneficial effects are not yet fully understood, and DMD-specific data is limited. The overarching goal of this review is to investigate how adiponectin signaling may ameliorate pathology associated with dystrophin deficiency through inflammatory-dependent and -independent mechanisms and to determine if current data supports their future progression to clinical trials.

РАННЯЯ ДИАГНОСТИКА МИОДИСТРОФИИ ДЮШЕННА: АКТУАЛЬНОСТЬ, ПРОБЛЕМЫ, ВОЗМОЖНОСТИ И ПЕРСПЕКТИВЫ

Duchenne muscular dystrophy (DMD) is an orphan neuromuscular progressive genetic disease that causes muscle breakdown and subsequent loss. Provided early diagnosis, complex standard and pathogenetic therapy significantly changes the trajectory of the disease, making it more benign. Diagnosis of DMD is a two-pillar system with creatine kinase (CK) level measured at the first stage followed by molecular genetic study in case the enzyme threshold values. The quality of genetic diagnostics in Russia has reached the best worldwide standards recently though identifying children with elevated CK levels is yet difficult. The number of diagnosed DMD patients in Russia is significantly lower than the statistically estimated global amount. The average age for diagnosis is not decreasing either. Thus, the median age for DMD and Becker muscular dystrophy (BMD) molecular diagnosis is 7 years and 8 months old. The number of identified children below the age of 2 y/o in Russia has remained consistently low over the past three years. According to the results of a familial cases survey, the correct primary diagnosis of DMD was established in less than half of all cases, 47.1%. The most common incorrect diagnoses were as follows: asymptotic hepatitis (25.1%), perinatal damage to the central nervous system (6.4%), cerebral palsy (2.7%), autism spectrum disorder (ASD) (2.7%) and flat feet, hallux valgus etc. (16% in total). The median duration between the initial DMD manifestation and molecular diagnosis is 3 years and 8 months as yet whilst the age-specific guidelines suggest prescribing standard and pathogenetic therapy during the 2 to 5 y/o period of life. Therefore, the Russia’s average age of DMD diagnosis above 7 y/o lies far beyond the optimal age for initiating of the DMD therapy with the purpose of gaining its maximum possible clinical benefit. The accumulated international and Russian experience demonstrates the possibility for improving of the primary DMD and BMD diagnosis in early preschool age and therefore requires its nationwide introduction.

Access to novel therapies for Duchenne muscular dystrophy—Insights from expert treating physicians

Access to novel therapies for Duchenne muscular dystrophy—Insights from expert treating physicians

BREATHE DMD: boosting respiratory efficacy after therapeutic hypoxic episodes in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a fatal genetic neuromuscular disorder, characterised by progressive decline in skeletal muscle function due to the secondary consequences of dystrophin deficiency. Weakness extends to the respiratory musculature, and cardiorespiratory failure is the leading cause of death in men with DMD. Intermittent hypoxia has emerged as a potential therapy to counteract ventilatory insufficiency by eliciting long-term facilitation of breathing. Mechanisms of sensory and motor facilitation of breathing have been well delineated in animal models. Various paradigms of intermittent hypoxia have been designed and implemented in human trials culminating in clinical trials in people with spinal cord injury and amyotrophic lateral sclerosis. Application of therapeutic intermittent hypoxia to DMD is considered together with discussion of the potential barriers to progression owing to the complexity of this devastating disease. Notwithstanding the considerable challenges and potential pitfalls of intermittent hypoxia-based therapies for DMD, we suggest it is incumbent on the research community to explore the potential benefits in pre-clinical models. Intermittent hypoxia paradigms should be implemented to explore the proclivity to express respiratory plasticity with the longer-term aim of preserving and potentiating ventilation in pre-clinical models and people with DMD.

Two operational modes of atomic force microscopy reveal similar mechanical properties for homologous regions of dystrophin and utrophin.

Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by the absence of the protein dystrophin. Dystrophin is hypothesized to work as a molecular shock absorber that limits myofiber membrane damage when undergoing reversible unfolding upon muscle stretching and contraction. Utrophin is a dystrophin homologue that is under investigation as a protein replacement therapy for DMD. However, it remains uncertain whether utrophin can mechanically substitute for dystrophin. Here, we compared the mechanical properties of homologous utrophin and dystrophin fragments encoding the N terminus through spectrin repeat 3 (UtrN-R3, DysN-R3) using two operational modes of atomic force microscopy (AFM), constant speed and constant force. Our comprehensive data, including the statistics of force magnitude at which the folded domains unfold in constant speed mode and the time of unfolding statistics in constant force mode, show consistent results. We recover parameters of the energy landscape of the domains and conducted Monte Carlo simulations which corroborate the conclusions drawn from experimental data. Our results confirm that UtrN-R3 expressed in bacteria exhibits significantly lower mechanical stiffness compared to insect UtrN-R3, while the mechanical stiffness of the homologous region of dystrophin (DysN-R3) is intermediate between bacterial and insect UtrN-R3, showing greater similarity to bacterial UtrN-R3.

Identification of hub genes and therapeutic siRNAs to develop novel adjunctive therapy for Duchenne muscular dystrophy

ObjectiveDuchenne muscular dystrophy (DMD) is a devastating X-linked neuromuscular disorder caused by various defects in the dystrophin gene and still no universal therapy. This study aims to identify the hub genes unrelated to excessive immune response but responsible for DMD progression and explore therapeutic siRNAs, thereby providing a novel treatment.MethodsTop ten hub genes for DMD were identified from GSE38417 dataset by using GEO2R and PPI networks based on Cytoscape analysis. The hub genes unrelated to excessive immune response were identified by GeneCards, and their expression was further verified in mdx and C57 mice at 2 and 4 months (M) by (RT-q) PCR and western blotting. Therapeutic siRNAs were deemed as those that could normalize the expression of the validated hub genes in transfected C2C12 cells.Results855 up-regulated and 324 down-regulated DEGs were screened from GSE38417 dataset. Five of the top 10 hub genes were considered as the candidate genes unrelated to excessive immune response, and three of these candidates were consistently and significantly up-regulated in mdx mice at 2 M and 4 M when compared with age-matched C57 mice, including Col1a2, Fbn1 and Fn1. Furthermore, the three validated up-regulated candidate genes can be significantly down-regulated by three rational designed siRNA (p < 0.0001), respectively.ConclusionCOL1A2, FBN1 and FN1 may be novel biomarkers for DMD, and the siRNAs designed in our study were help to develop adjunctive therapy for Duchenne muscular dystrophy.

Chimeric Cell Therapies as a Novel Approach for Duchenne Muscular Dystrophy (DMD) and Muscle Regeneration.

Chimerism-based strategies represent a pioneering concept which has led to groundbreaking advancements in regenerative medicine and transplantation. This new approach offers therapeutic potential for the treatment of various diseases, including inherited disorders. The ongoing studies on chimeric cells prompted the development of Dystrophin-Expressing Chimeric (DEC) cells which were introduced as a potential therapy for Duchenne Muscular Dystrophy (DMD). DMD is a genetic condition that leads to premature death in adolescent boys and remains incurable with current methods. DEC therapy, created via the fusion of human myoblasts derived from normal and DMD-affected donors, has proven to be safe and efficacious when tested in experimental models of DMD after systemic-intraosseous administration. These studies confirmed increased dystrophin expression, which correlated with functional and morphological improvements in DMD-affected muscles, including cardiac, respiratory, and skeletal muscles. Furthermore, the application of DEC therapy in a clinical study confirmed its long-term safety and efficacy in DMD patients. This review summarizes the development of chimeric cell technology tested in preclinical models and clinical studies, highlighting the potential of DEC therapy in muscle regeneration and repair, and introduces chimeric cell-based therapies as a promising, novel approach for muscle regeneration and the treatment of DMD and other neuromuscular disorders.

Spending on Targeted Therapies for Duchenne Muscular Dystrophy

This study estimates public and private spending on genetically targeted treatments for Duchenne muscular dystrophy during years in which the drugs were marketed without completed confirmatory studies.