Duchenne Muscular Dystrophy Research Articles

DMD mutations in pediatric patients with phenotypes of Duchenne/Becker muscular dystrophy

Abstract Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are common X-inherited neuromuscular diseases. The genetic diagnosis has been used as the diagnostic choice for DMD/BMD. The study subjects consisted of 37 patients from Southwest China. Peripheral blood was collected for the extraction of genomic DNA. DMD mutation was sequenced using the next-generation sequencing approach. The detected mutation was validated using the multiplex ligation-dependent probe amplification or Sanger sequencing methods. Variation annotation and pathogenicity prediction were performed using the online databases. Pathogenic mutations were identified 3 splicing site, 7 single nucleotide, 1 indel, 23 deletion, and 3 duplication mutations. Novel DMD variants were discovered, including two novel splicing variations (c.1890 + 1G>T; c.1923 + 1G>A), one missense mutation (c.1946G>T), one nonsense mutation (c.7441G>T), one indel mutation (INDEL EX20), and one duplication mutation (DUP EX75-78). The current study provides mutation information of DMD for the genetic diagnosis of DMD/BMD.

RNA sequencing facilitates the identification of genetic causes of Duchenne muscular dystrophy and proposes a stepwise DMD diagnostic procedure.

A certain percentage of Duchenne muscular dystrophy (DMD) patients remain genetically undiagnosed after routine genetic testing. Accurate genetic diagnosis is crucial for determining eligibility for mutation-specific therapies and providing relatives with reliable genetic and reproductive counselling. In this study, we utilized RNA sequencing to achieve precise genetic diagnoses in three DMD patients. We identified a deep intronic variant, NC_000023.11:g. 32644691A>C (NM_004006.3:c.1149+273T>G), responsible for creating a novel exon in one patient. An abnormal splicing event was also observed in the second patient. Additionally, RNA sequencing of the pathological muscle samples revealed differentially expressed genes. Based on these findings, we proposed a comprehensive, stepwise diagnostic procedure for DMD. Our study suggests that RNA sequencing can be instrumental in diagnosing disease-causing intronic variants, and the proposed procedure aims to enhance the clarity and accuracy of genetic diagnoses in DMD.

Protective effect of maltol on pathological response of cardiomyocyte in dystrophic mice.

Heart diseases are a significant contributor to global morbidity and mortality, and despite their diverse and complex mechanisms, treatment options remain limited. Maltol, a natural compound with antioxidant and anti-inflammatory activities, exhibits potential for addressing this need. This study evaluates the cardioprotective effects of maltol in isoproterenol (ISO)-induced cardiac stress models and Duchenne muscular dystrophy (DMD). Maltol's cardiac cytotoxicity was assessed in rodent (H9c2) and human (AC16) cells and compared with that of dapagliflozin to illustrate its cardiac safety. In ISO-induced stress models, maltol significantly reduced hypertrophic markers and inflammation while enhancing autophagy and antioxidant pathways. In the mdx mice, a DMD model, maltol treatment improved cardiac contractility and reduced pathogenic remodeling. Enhanced phosphorylation of phospholamban and trends toward higher SERCA2a expression indicated enhanced Ca2+ handling, which is crucial in DMD cardiomyopathy. This study demonstrated that maltol has the potential to provide therapeutic benefits for DMD and other cardiac conditions characterized by hypertrophy and inflammation, as evidenced by its well-known antioxidant properties, low cytotoxicity, and capacity to enhance cardiac function and Ca2+ handling.

Evaluation of a six-minute walk test in the DE50-MD canine model of Duchenne muscular dystrophy and its effect on blood-borne biomarkers

Background Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease caused by mutations in the dystrophin gene resulting in cycles of muscle degeneration, inflammation and regeneration. The 6-minute walk test (6MWT) is a key functional outcome measure for DMD patient clinical trials and has been adapted for use in animal models of the disease. The DE50-MD dog model of DMD closely reflects the DMD patient phenotype prior to loss of ambulation. For pre-clinical trials using this model, functional outcome measures must be established. Methods This longitudinal study compared distance walked in a 6MWT by DE50-MD and WT control dogs and assessed the utility of the 6MWT as a functional biomarker. Dogs underwent two 6MWTs conducted approximately 48-hours apart, at 3, 6, 9, 12, 15 and 18 months of age. In addition, we evaluated the stability of selected blood-borne biomarkers in 12-month old DE50-MD and WT dogs 0, 3, 6, 24 and 48 hours following a 6MWT. Results DE50-MD dogs exhibited significantly shorter 6-minute walk distance (6MWD) than WT dogs at all timepoints (P<0.05), with no difference in 6MWD between the first and second 6MWT. C-C motif chemokine ligand 2 (CCL2), myomesin-3 (MYOM3) and myostatin (MSTN) were biomarkers of the DE50-MD phenotype that remained unchanged in DE50-MD dogs following the 6MWT, while creatine kinase (CK) activity significantly increased 3-hours following the test in DE50-MD dogs but remained unchanged in WT dogs. Conclusions The 6MWT effectively discriminates DE50-MD from WT dogs aged 3-18 months and a single 6MWT is sufficient for future studies. Serum MYOM3, CCL2 and MSTN are good biomarkers of the DE50-MD phenotype that are unaffected by this relatively low level exertion.

Hypertrophic cardiomyopathy in Duchenne muscular dystrophy: a case series.

Duchenne muscular dystrophy is characterised by fibrofatty replacement of muscle, resulting in dilated cardiomyopathy. Hypertrophic cardiomyopathy affects 1:200-1:500 people and is characterised by asymmetric ventricular septal hypertrophy. To date, there have been two separately reported cases describing the combined pathology of these disorders. Herein, we expand upon these reports with a case series describing longitudinal findings in three patients with Duchenne muscular dystrophy who developed hypertrophic cardiomyopathy.

Determination of qPCR reference genes suitable for normalizing gene expression in a novel model of Duchenne muscular dystrophy, the D2-mdx mouse.

Duchenne muscular dystrophy (DMD) is a X-linked neuromuscular disorder arising from mutations in the dystrophin gene, leading to a progressive muscle wasting and disability. Currently there is no universal therapy, and there is thus a strong interest in preclinical studies for finding novel treatments. The most widely used and characterized mouse model for DMD is the C57BL/10ScSn-Dmdmdx/J (BL10-mdx), but this model exhibits mild pathology and does not replicate key features of human disease. The D2.B10-Dmdmdx/J (D2-mdx) mouse is a more recent model which seems to better mimics the complex human DMD phenotype. However, the D2-mdx mouse remains less extensively characterised than its BL10-mdx counterpart. Quantitative PCR analysis of gene expression is an important tool to monitor disease progression and evaluate therapeutic efficacy, but measurements must be normalised to stably expressed reference genes, which should ideally be determined and validated empirically. We examined gene expression in the gastrocnemius (GC), diaphragm (DIA) and heart in the D2-mdx mouse, the BL10-mdx mouse, and appropriate strain-matched wild-type controls (D2-wt and BL10-wt), from 4 to 52 weeks of age, using a large panel of candidate references (ACTB, AP3D1, CSNK2A2, GAPDH, HPRT1, PAK1IP1, RPL13A, SDHA, and in the heart, also HTATSF1 and HMBS). Data was analyzed using GeNorm, Bestkeeper, deltaCt and Normfinder algorithms to identify stable references under multiple possible scenarios. We show that CSNK2A2, AP3D1 and ACTB represent strong universal reference genes in both GC and DIA, regardless of age, muscle type, strain and genotype, while HTATSF1 and SDHA are optimal for the heart. GAPDH, HPRT1 and RPL13A were conversely revealed to be poor references, showing tissue-, age- or disease-specific changes in expression. Our results illustrate the importance of determining appropriate reference genes for specific comparative scenarios, but also reconfirm that universal panels can nevertheless be identified for normalising gene expression studies in even complex pathological states.

Identification of novel transcription factors regulated by H3K27 acetylation in myogenic differentiation of porcine skeletal muscle satellite cells.

H3K27 acetylation (H3K27ac) is crucial in muscle development as it regulates gene expression. Dysregulation of H3K27ac level has been linked to muscle-related diseases such as Duchenne muscular dystrophy, yet the mechanisms through which H3K27ac influences myogenic differentiation are not fully understood. Here, we utilized the SGC-CBP30 drug, a CBP/p300 bromodomain inhibitor, to reduce H3K27ac level and investigated its effect on myogenic differentiation of porcine skeletal muscle satellite cells. The results demonstrated an increased H3K27ac level during normal muscle satellite cell differentiation. We found that the addition of SGC-CBP30 resulted in a reduced level of H3K27ac based on ATAC-seq and CUT&Tag data. Our analysis revealed that a cluster characterized by reduced levels of H3K27ac and increased levels of H3K27me3 was enriched with motifs corresponding to Bach2, MafK, and Fosl2 transcription factors. Furthermore, knockdown of Bach2, MafK, and Fosl2 produced a similar suppression effect on myogenic differentiation. Taken together, our study contributes to a better understanding of how H3K27ac influences myogenic differentiation.

Abstract 4125188: Cardiac Specific Biomarkers of Progression in Duchenne Muscular Dystrophy Detected Using Aptamer-Based Proteomics

Background: Duchenne muscular dystrophy (DMD) is an X-linked, progressive neuromuscular disorder characterized by cardiac and skeletal myopathy. The cardiac and skeletal muscle manifestations have different ages of onset and rates of progression, suggesting variability in the pathophysiology of dystrophin loss within the respective muscle groups. Hypothesis: We hypothesized that proteomics with comprehensive clinical phenotyping would identify novel biomarkers that correlate with cardiac but not skeletal disease progression in boys with DMD. Methods: This single-center cohort study of 56 DMD subjects performed cardiac magnetic resonance (CMR), including left ventricular ejection fraction (LVEF), late gadolinium enhancement (LGE), and myocardial circumferential strain (ECC). Fifty-one subjects underwent quantitative muscle testing, and 33 underwent actigraphy (MSK indices). Aptamer-based technology (SomaScan, SomaLogic, Boulder, CO) evaluated 1128 proteins in plasma sampled at the time of CMR. Correlations between serum protein levels and cardiac or MSK indices were tested using Benjamini-Hochberg corrected p-values (q-values). Results: The mean age of DMD subjects was 15.5 years old +/- 4.30. Twenty-six subjects had left ventricular dysfunction (LVEF <55%), 45 subjects had LGE, and 45 were non-ambulatory. Of 1128 proteins analyzed, 208 demonstrated a significant correlation for cardiac but not MSK indices (Figure 1). Figure 2 lists the 20 proteins with the strongest correlations, which included biomarkers of inflammation, calcium regulation in myocytes, and cell adhesion. Conclusion: We detected 208 novel proteins that correlate with cardiac-specific DMD disease progression. These proteins provide a better understanding of the underlying differences of cardiac disease, compared to MSK disease in DMD and provide clues to potential cardiac therapeutic options.

Abstract 4118084: Targeting Monoamine Oxidase B in Dystrophic mdx Hearts Dampens Inflammation and Fibrosis

Background: Cardiomyopathy is a major cause of death for Duchenne muscular dystrophy (DMD) patients. Current treatments cannot prevent cardiac tissue remodeling. Oxidative stress, inflammation and fibrosis are early events in DMD, preceding heart dysfunction. Monoamine Oxidase B (MAOB), which forms H 2 O 2 by oxidizing amines, is overactivated in inflammatory conditions and overcomes cell antioxidant defenses, thus altering redox homeostasis and eliciting harmful effects. We showed that targeting MAOB with inhibitors (iMAOB) improves skeletal muscle function in dystrophic mice by lowering oxidative stress, and reduces inflammation in murine models of sepsis and arthritis by dampening NF-kB, a redox-sensitive transcription factor. Aim: We explore the therapeutic potential of iMAOB to alleviate cardiomyopathy using dystrophin-deficient mdx mice. We hypothesize that iMAOB treatment could dampen oxidative stress, inflammation and fibrosis in dystrophic mdx hearts by modulating the phenotype of cells that are crucial in cardiac remodeling. Methods: Three-month-old mdx mice, that already show signs of fibrosis but no cardiac dysfunction, were orally treated with iMAOB or vehicle for one month (n≥ 6). Heart ventricular mononucleated cells were obtained by enzymatic digestion. Myeloid, endothelial and fibroblast cells were isolated by cell sorting by FACS and analysed by RT-PCR. Oxidative stress, inflammation and fibrosis were measured in whole tissue by immunohistochemistry. Results: The expression of proinflammatory and profibrotic genes was markedly increased in myeloid [Interleukin (Il)-1b, Il-6, Tgf-b and Spp1, the gene coding for osteopontin], endothelial [Il-1b, Il-6, mmp2 and nos3] and cardiac fibroblast cells [Tgf-b, Spp1, Timp1 and Col1] isolated from mdx hearts, as compared to wild type mice. All these genes were significantly dampened by iMAOB treatment. In parallel, once again iMAOB treatment blunted the increase in oxidative stress, inflammation and fibrosis observed in mdx cardiac sections. Of notice, the differences were not linked to changes in the percentage of the various cell types, as they were unmodified amongst wild type, mdx and iMAOB-treated mdx hearts. Conclusions: We show that iMAOB can positively affect the phenotype of cells that are important in cardiac tissue remodeling. Our data suggest that iMAOB can be a viable therapeutic tool in DMD cardiomyopathy. As iMAOB are already in clinical use, such approach could be easily translated to patients.

Lipin1 as a therapeutic target for respiratory insufficiency of duchenne muscular dystrophy

In Duchenne muscular dystrophy (DMD), diaphragm muscle dysfunction results in respiratory insufficiency which is a leading cause of death in patients. Mutations to the dystrophin gene result in myocyte membrane instability, contributing to the structural deterioration of the diaphragm muscle tissues. With previous works suggesting the importance of lipin1 for maintaining skeletal muscle membrane integrity, we explored the roles of lipin1 in the dystrophic diaphragm. We found that the protein expression levels of lipin1 were reduced by 60% in the dystrophic diaphragm. While further knockdown of lipin1 in the dystrophic diaphragm leads to increased necroptosis, restoration of lipin1 in the dystrophic diaphragm results in reduced inflammation and fibrosis, decreased myofiber death, and improved respiratory function. Our results demonstrated that lipin1 restoration improved respiratory function by enhancing membrane integrity and suggested that lipin1 could be a potential therapeutic target for preventing respiratory insufficiency and respiratory failure in DMD. Continued investigation is required to better understand the mechanisms behind these findings, and to determine the role of lipin1 in maintaining muscle membrane stability.

Abstract 4132820: S-nitrosylation of cardiac Cx43 hemichannels at Cys271 promotes arrhythmogenicity and myocardial injury upon cardiac stress in Duchenne Muscular Dystrophy

Connexin-43 (Cx43) plays a critical role in the propagation of action potentials among cardiomyocytes and proper cardiac contractility. In healthy cardiomyocytes, Cx43 is located at the intercalated disk; however, Cx43 remodeling is observed in cardiac pathologies and is linked with arrhythmogenesis and sudden cardiac death. Utilizing a mouse model of Duchenne muscular dystrophy (DMD mdx ), we have previously demonstrated that cardiac Cx43 is laterally localized, forming undocked hemichannels that activate via S-nitrosylation in response to isoproterenol-evoked cardiac stress. This activation leads to the disruption of cardiac membrane permeability, triggered activity, and deadly arrhythmogenic behaviors. To establish the direct role of S-nitrosylated Cx43 in DMD cardiomyopathy, we developed a specific knock-in mouse line in which the single Cx43 site for S-nitrosylation, cysteine 271 (Cys271), was substituted with a serine (C271S +/- ). Here, we developed a DMD mdx :C271S +/- line (4–6 months old), exhibiting reduced levels of S-nitrosylated Cx43 after crossing DMDmdx mice and C271S +/- mouse lines to assess the effect of β-adrenergic stimulation-induced cardiac stress and heart dysfunction. We show that cardiac Cx43 remodeling was not prevented in DMD mdx :C271S +/- , similar to what was shown in DMD mdx mice via immunofluorescence analysis. In addition, DMD mdx mice displayed an increased number of deadly arrhythmogenic events, increased Ca 2+ signaling, and prolonged action potentials in Langendorff-perfused whole hearts via optical mapping, compared to wild-type and DMD mdx :C271S +/- mice. Similarly, isoproterenol treatment evoked severe myocardial injury, increased levels of plasmatic cardiac troponin I (cTnI), and 40% mortality in DMD mdx mice. Notably, DMD mdx :C271S +/- mice, similar to DMD mdx mice treated with the Cx43 hemichannel blocker Gap19, exhibited cardioprotection compared to the cardiac dysfunction observed in DMD mdx mice. Therefore, these findings strongly suggest that S-nitrosylation of Cx43 proteins at site Cys271 represents a fundamental NO-mediated mechanism involved in the induction of arrhythmias and myocardial injury in DMD mdx after β-adrenergic stress.

Abstract 4147633: Structural and Electrophysiological Cardiac Changes in Children with Osteogenesis Imperfecta (OI)

Introduction: Osteogenesis imperfecta (OI) is an inherited type 1 collagen disorder leading to bone fragility and increased fractures. Cardiovascular disease is the leading cause of death of adults with OI, but studies on cardiac changes from this collagen defect in children are sparse. We aim to fill this gap by assessing changes in EKG and echocardiogram (ECHO) variables in children with OI. Hypothesis: Children with OI will exhibit arrhythmogenic EKG changes and/or ECHO abnormalities which will precede changes that are documented in adults. Methods: 53 children with OI receiving care at Children’s Mercy-Kansas City were identified via ICD-10 codes. 64 with Duchenne Muscular Dystrophy (DMD, expected cardiac abnormalities) and 55 with non-accidental trauma (NAT, no expected cardiac abnormalities) served as controls. We specifically compared the most recent EKG and ECHO metrics extracted from medical charts by Kruskal-Wallis testing. Results: EKG: Children with OI differed from both NAT and DMD. OI had shorter median PR intervals (p<0.001) plus longer median QRS (p<0.001) and QTc intervals (p=0.002) than NAT. DMD had the shortest PR and longest QRS and QTc (see Table). Echo: The three groups showed no difference in systolic/diastolic BP or LV thickness. However, OI differed from NAT and/or DMD in other metrics. OI had higher end diastolic volume (p<0.001), end systolic volume (p<0.001) and stroke volume (p<0.05) than NAT, but lower values than the DMD group. Similarly, OI children had higher LV mass and LV internal diastolic and systolic diameters than NAT, but lower than DMD (p<0.001). OI LVOT diameter and Aortic root diameter were lower than NAT, but not as low as DMD (p<0.05 and p<0.001, respectively). Conclusion: Children with OI exhibit EKG and ECHO abnormalities that are similar to but less severe than for DMD, a group in which cardiovascular abnormalities are well documented. These abnormalities may be predictive of arrythmia/sudden cardiac death and/or functional decline in heart function. Our data suggest that OI children could benefit from ongoing routine cardiology studies along with orthopedic/metabolic management. Future studies would assess effects of age and OI treatments on cardiac abnormalities.

Myocardial Strain Assessment for Early Duchenne Muscular Dystrophy Diagnosis in Pediatric Patients Using Cardiac MRI

Assessing myocardial strain remains challenging, particularly in the pediatric population, due to the smaller heart sizes, higher heart rates, and variability in strain parameters compared to adult populations. This study aimed to investigate the utility of myocardial strain measurements using cardiac magnetic resonance-feature tracking (CMR-FT) for early diagnosis of Duchenne muscular dystrophy (DMD) in pediatric patients. Twenty-eight DMD patients and 20 healthy controls were involved in this study. Global circumferential, longitudinal, and radial strain (GCS, GLS, and GRS) were measured for the left ventricle (LV) using CMR-FT. Segmental strain values only of the inferolateral and anterolateral LV segments in DMD patients without late gadolinium enhancement (LGE) and DMD patients with LGE were compared to the healthy controls. Strain measurements using CMR-FT in DMD patients were considerably lower than those of healthy controls, with all p-values lower than 0.001. DMD patients without LGE showed decreased inferolateral and anterolateral segmental values only relative to healthy controls. The same behavior was maintained for the LV geometry. Multivariable linear regression demonstrated that the end-systole (ES) wall thicknesses and thickening were associated with decreased GCS and GLS. CMR-FT is crucial in detecting cardiac abnormalities in patients with DMD. It represents an innovative imaging biomarker that can detect initial myocardial alterations in DMD cardiomyopathy without relying on gadolinium.

Macrophages producing chondroitin sulfate proteoglycan-4 induce neuro-cardiac junction impairment in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is caused by the absence of the full form of the dystrophin protein, which is essential for maintaining the structural integrity of muscle cells, including those in the heart and respiratory system. Despite progress in understanding the molecular mechanisms associated with DMD, myocardial insufficiency persists as the primary cause of mortality, and existing therapeutic strategies remain limited. This study investigates the hypothesis that a dysregulation of the biological communication between infiltrating macrophages (MPs) and neurocardiac junctions exists in dystrophic cardiac tissue. In a mouse model of DMD (mdx), this phenomenon is influenced by the over-release of chondroitin sulfate proteoglycan-4 (CSPG4), a key inhibitor of nerve sprouting and a modulator of the neural function, by MPs infiltrating the cardiac tissue and associated with dilated cardiomyopathy, a hallmark of DMD. Givinostat, the histone deacetylase inhibitor under current development as a clinical treatment for DMD, is effective at both restoring a physiological microenvironment at the neuro-cardiac junction and cardiac function in mdx mice in addition to a reduction in cardiac fibrosis, MP-mediated inflammation, and tissue CSPG4 content. This study provides novel insight into the pathophysiology of DMD in the heart, identifying potential new biological targets. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Assessment of Phosphorodiamidate Morpholino Oligomer Treatment Patterns for Patients with Duchenne Muscular Dystrophy: A MarketScan Claims Analysis.

Phosphorodiamidate morpholino oligomers (PMOs), weight-based treatments administered weekly by intravenous infusion, are approved in the US for patients with Duchenne muscular dystrophy (DMD) amenable to certain exon skipping. Evidence regarding PMO treatment patterns in real-world settings is limited. This study used longitudinal administrative claims data to characterize PMO treatment patterns among US patients with DMD. MarketScan® commercial and Medicaid data (January 1, 2018-December 31, 2021) were used to identify claims for PMO treatments (eteplirsen, golodirsen, viltolarsen, casimersen). The proportion of days covered (PDC), proportion with continuous PMO claims coverage (no gaps in claims ≥ 30days), and time to subsequent PMO claims after a ≥ 30-day gap in PMO claims were described. One hundred thirty-three patients with ≥ 1 PMO claim were identified. Multiple codes were needed to identify PMO treatment coverage. Mean age was 14.1years; all patients were male. Mean continuous follow-up duration was 669.3days. Median PDC was 83.4%. Seventy-four (55.6%) patients had continuous PMO claims coverage (no ≥ 30-day gaps in claims). Of the 59 patients with ≥ 1 gap in PMO claims of ≥ 30days, 39 had ≥ 1 subsequent PMO claim. Accounting for censoring via Kaplan-Meier analysis, 75.5% had a subsequent PMO claim within 1 year after a ≥ 30-day gap, with a median time of 64days (including the qualifying 30days). Understanding treatment patterns is important for characterizing real-world utilization of precision genetic medicines. This study observed a high PDC for PMO treatments for DMD. Most patients had continuous PMO claims coverage, and most patients with a gap in PMO claims had a subsequent PMO claim. Nonetheless, the observed persistence may have been underestimated given shortcomings of claims data and payer coverage considerations. Caution should be exercised when inferring treatment effectiveness or tolerability based on observed treatment patterns from claims data alone for weight-based, infused PMO treatments.

Characterization of Nonclinical Drug Metabolism and Pharmacokinetic Properties of Phosphorodiamidate Morpholino Oligonucleotides, a Novel Drug Class for Duchenne Muscular Dystrophy.

Eteplirsen, golodirsen, and casimersen are phosphorodiamidate morpholino oligomers (PMOs) that are approved in the United States for the treatment of patients with Duchenne muscular dystrophy (DMD) with mutations in the DMD gene that are amenable to exon 51, 53, and 45 skipping, respectively. Here we report a series of in vivo and in vitro studies characterizing the drug metabolism and pharmacokinetic (DMPK) properties of these three PMOs. Following a single intravenous dose, plasma exposure was consistent for all three PMOs in mouse, rat, and nonhuman primate (NHP), and plasma half-lives were similar for eteplirsen (2.0-4.1 h) and golodirsen (2.1-8.7 h) across species and more variable for casimersen (3.2-18.1 h). Plasma protein binding was low (<40%) for all three PMOs in mouse, rat, NHP, and human and was largely concentration independent. In the mdx mouse model of DMD, following a single intravenous injection, extensive biodistribution was observed in the target skeletal muscle tissues and the kidney for all three PMOs; consistent with the latter finding, the predominant route of elimination was renal. In vitro studies using liver microsomes showed no evidence of hepatic metabolism, and none of the PMOs were identified as inhibitors or inducers of the human cytochrome P450 enzymes or membrane drug transporters tested at clinically relevant concentrations. These findings suggest that key DMPK features are consistent for eteplirsen, golodirsen, and casimersen and provide evidence for the concept of a PMO drug class with potential application to novel exon-skipping drug candidates. SIGNIFICANCE STATEMENT: The PMOs eteplirsen, golodirsen, and casimersen share similar absorption, distribution, metabolism, and excretion and DMPK properties, which provides evidence for the concept of a PMO treatment class. A PMO drug class may support a platform approach to enhance understanding of the pharmacokinetic and pharmacodynamic behavior of these molecules. The grouping of novel agent series into platforms could be beneficial in the development of drug candidates for populations in which traditional clinical trials are not feasible.

Robust and Explainable Stage Prediction in Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is one of the life-threatening rare genetic disease affecting millions of male minors across the globe. Given its progressive nature, we can demarcate the various stages of DMD through the loss of muscular movements, ambulation, respiratory difficulties, and cardiac dysfunction. In this work, we employ machine learning models for understanding the progression of DMD through the prediction of its stages. Our attempts to predict the stages of DMD on the data collected by Molecular Diagnostics, Counseling, Care and Research Center (MDCRC) from 223 visits of 90 subjects demonstrate more than 80% accuracy with the state-of-the-art methods. We further study the biological/physiological importance of features in characterizing the stages of DMD.

First report of PURA syndrome in a Colombian patient with de novo missense variant c.692T>C (p.Phe231Ser)

We present the first documented case of PURA syndrome in Colombia. This rare neurological disease results from mutations in the PURA gene located on chromosome 5, leading to haploinsufficiency of the PUR-α protein. This protein is essential for early brain development and neuronal function. The patient, a seven-years-old boy, started showing dystonic hand movements at 14 days of age; at six, he had neurodevelopmental delay, generalized hypotonia, frequent episodes of apnea, and swallowing difficulties. Although other conditions were initially considered, such as Duchenne muscular dystrophy and neuronal ceroid lipofuscinosis, a whole exome sequencing revealed the pathogenic variant c.692T>C (p.Phe231Ser) in the exon 1 of the PURA gene, not previously reported in other patients. With this finding, we adopted a comprehensive management approach addressing the patient’s characteristics and alterations. Since the PURA syndrome is not on the list of orphan/rare diseases recognized by the Colombian Ministerio de Salud y Protección Social, we hope our report will contribute to its official recognition. The case shows the importance of considering rare diagnoses in patients with uncommon neurological symptoms, underlining the usefulness of genomic sequencing in diagnosis and the need for collaboration to optimize healthcare for patients with PURA syndrome and similar diseases.

In Vitro Gene Therapy Using Human iPS-Derived Mesoangioblast-Like Cells (HIDEMs) Combined with Microdystrophin (μDys) Expression as the New Strategy for Duchenne Muscular Dystrophy (DMD) Experimental Treatment

Duchenne Muscular Dystrophy (DMD) is a genetic disorder characterized by disruptions in the dystrophin gene. This study aims to investigate potential a therapeutic approach using genetically modified human iPS-derived mesoangioblast-like cells (HIDEMs) in mdx mouse model. This study utilizes patient-specific myoblasts reprogrammed to human induced pluripotent stem cells (iPSCs) and then differentiated into HIDEMs. Lentiviral vectors carrying microdystrophin sequences have been employed to deliver the genetic construct to express a shortened, functional dystrophin protein in HIDEMs. The study indicated significant changes within redox potential between healthy and pathological HIDEM cells derived from DMD patients studied by catalase and superoxide dismutase activities. Microdystrophin expressing HIDEMs also improved expression of genes involved in STARS (striated muscle activator of Rho signaling) pathway albeit in selective DMD patients (with mild phenotype). Although in vivo observations did not bring progress in the mobility of mdx mice with HIDEMs, microdystrophin interventions this may argue against “treadmill test” as suitable for assessment of mdx mice recovery. Low-level signaling of the Rho pathway and inflammation-related factors in DMD myogenic cells can also contribute to the lack of success in a functional study. Overall, this research contributes to the understanding of DMD pathogenesis and provides insights into potential novel therapeutic strategy, highlighting the importance of personalized gene therapy.

First, do no harm: the role of preclinical animal models in predicting adverse events in gene therapy clinical trials for Duchenne muscular dystrophy and X-Linked myotubular myopathy

The occurrence of severe adverse events (SAEs) in patients with Duchenne muscular dystrophy (DMD), X-linked myotubular myopathy (XLMTM), and other neuromuscular diseases treated with adeno-associated virus (AAV) constructs has prompted studies to improve the safety and efficacy of gene therapy. Physicians have weighed the medical tenet of “first, do no harm” against the perspective of patients with progressive life-threatening conditions who may accept greater risk. Regarding SAE pathogenesis, discussion has focused on total AAV exposure and patient mutations more likely to induce immunity, while stressing the limitations of animal models in predicting adverse events. Therapeutic strategies for reducing side effects have employed more myotropic AAV serotypes and efficient transgenes. Other recommendations include excluding certain DMD gene mutations associated with SAEs and substituting less immunogenic transgenes such as utrophin (DMD) and myotubularin-related protein (XLMTM). For the sake of preclinical studies, emphasis has been placed on outbred rodents and larger animals that better predict immunity. Here, the absence of side effects in canine DMD and XLMTM models might be explained partly by phenotypic differences between affected humans and dogs. Specifically, dystrophin- and myotubularin-deficient dogs exhibit milder lesions, including less muscle fat deposition and the absence of hepatopathy, respectively, which could lead to reduced immune responses to AAV constructs. To better predict future problems, thought should be given to tracking early subclinical markers of the innate immune response, especially complement activation. Regardless of steps taken to improve the predictive value of animal models for SAEs, some questions will only be answered through human clinical trials after carefully considering the risk-benefit ratio.