Treatment Of Duchenne Muscular Dystrophy Research Articles

Therapeutic phosphorodiamidate morpholino oligonucleotides: Physical properties, solution structures, and folding thermodynamics

Elucidating the structure-function relationships for therapeutic RNA mimicking phosphorodiamidate morpholino oligonucleotides (PMOs) is challenging due to the lack of information about their structures. While PMOs have been approved by the US Food and Drug Administration for treatment of Duchenne muscular dystrophy, no structural information on these unique, charge-neutral, and stable molecules is available. We performed circular dichroism and solution viscosity measurements combined with molecular dynamics simulations and machine learning to resolve solution structures of 22-mer, 25-mer, and 30-mer length PMOs. The PMO conformational dynamics are defined by the competition between non-polar nucleobases and uncharged phosphorodiamidate groups for shielding from solvent exposure. PMO molecules form non-canonical, partially helical, stable folded structures with a small 1.4- to 1.7-nm radius of gyration, low count of three to six base pairs and six to nine base stacks, characterized by -34 to -51kcal/mol free energy, -57 to -103kcal/mol enthalpy, and -23 to -53kcal/mol entropy for folding. The 4.5- to 6.2-cm3/g intrinsic viscosity and Huggins constant of 4.5-9.9 are indicative of extended and aggregating systems. The results obtained highlight the importance of the conformational ensemble view of PMO solution structures, thermodynamic stability of their non-canonical structures, and concentration-dependent viscosity properties. These principles form a paradigm to understand the structure-properties-function relationship for therapeutic PMOs to advance the design of new RNA-mimic-based drugs.

Urine titin as a novel biomarker for Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is the most severe form of muscular dystrophy that is caused by lack of dystrophin, a critical structural protein in skeletal muscle. DMD treatments, and quantitative biomarkers to assess the efficacy of potential treatments, are urgently needed. Previous evidence has shown that titin, a muscle cell protein, is increased in the urine of patients with DMD, suggesting its usefulness as a DMD biomarker. Here, we demonstrated that the elevated titin in urine is directly associated with the lack of dystrophin and urine titin responses to drug treatment. We performed a drug intervention study using mdx mice, a DMD mouse model. We showed that mdx mice, which lack dystrophin due to a mutation in exon 23 of the Dmd gene, have elevated urine titin. Treatment with an exon skipper that targets exon 23 rescued muscle dystrophin level and dramatically decreased urine titin in mdx mice and correlates with dystrophin expression. We also demonstrated that titin levels were significantly increased in the urine of patients with DMD. This suggests that elevated urine titin level might be a hallmark of DMD and a useful pharmacodynamic marker for therapies designed to restore dystrophin levels.

Promising Treatments for Duchenne Muscular Dystrophy: Restoring Dystrophin Protein Expression Using Nucleic Acid Therapeutics

Review Promising Treatments for Duchenne Muscular Dystrophy: Restoring Dystrophin Protein Expression Using Nucleic Acid Therapeutics Guo Hu and Chen Chen * Division of Cardiology and Hubei Key Laboratory of Genetics and Molecular Mechanisms of Cardiological Disorders, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. * Correspondence: chenchen@tjh.tjmu.edu.cn; Tel. & Fax: 86-27-6937-8422 Received: 10 October 2022 Accepted: 4 November 2022 Published: 11 January 2023 Abstract: Duchenne muscular dystrophy is caused by inadequate generation of functional dystrophin protein. Traditional clinical treatments can only slightly mitigate the progression of the disease, but not completely stem or reverse the decline in muscle function. Therapies aimed at dystrophin recovery are currently under development, among which are exon skipping and stop codon readthrough therapies. They are now used in clinics, while gene addition therapies are in phase III clinical trials. Gene editing therapies have also been approved for the first clinical trial recently. This review will discuss these emerging therapies, clinical trials, and directions for future developments.

Time to diagnosis of Duchenne muscular dystrophy in Austria and Germany

Duchenne muscular dystrophy (DMD) is an X-linked genetic disorder manifesting in early childhood with progressive muscular weakness and atrophy, and resulting in early loss of ambulation. The collection and evaluation of epidemiological data for this disease is crucial for an early diagnosis and disease management. In Germany, data are collected via the TREAT-NMD DMD patient registry (www.dmd-register.de). In contrast, data collection in Austria has not yet been performed systematically. For collecting data from Austrian DMD patients, an online survey of the patient’s caregivers was conducted. Data of 57 patients were collected entailing initial symptoms, diagnosis and therapeutic measures. Comparable data has been collected for Germany via the TREAT-NMD DMD patient registry. 57 DMD patients aged 4–34 years completed the Austrian survey. On average, first symptoms of the disease appeared at the age of 3.1 years. As the most frequent first symptom, 46% of the patients described problems in climbing stairs. In 40% of the patients, DMD was diagnosed early due to an accidentally detected hyperCKemia in infancy or early childhood. Corticosteroids represented the main therapeutic option in our cohort. At the time of the survey, only 52% of the patients were treated with corticosteroids. Patients from Germany reported that first symptoms appeared at the age of 3.06 years. Diagnosis was established by genetic testing or muscle biopsy. 47% of the patients were treated with corticosteroids. Time between first symptoms and diagnosis was 7 months in Austria, and 4.7 months in Germany, respectively. Compared to earlier international studies, the Austrian data show encouraging results regarding earlier start of corticosteroid therapy in a larger percentage of patients. Austrian and German data show a trend towards an earlier diagnosis of DMD, while the age at symptom onset was similar to previous studies. The collection and evaluation of epidemiological data of DMD patients is important and will hopefully contribute to accelerate DMD diagnosis and treatment access for the patients.

Newborn Screening for the Diagnosis and Treatment of Duchenne Muscular Dystrophy.

A pilot newborn screening (NBS) program for Duchenne muscular dystrophy (DMD) study proposes to assess the feasibility of the screening procedure, temporal course of the various steps of screening, and the public acceptability of the program. This is particularly vital to ascertain as DMD is considered a 'non-treatable' disease and thus does not fit the traditional criteria for newborn screening. However, modern perspectives of NBS for DMD are changing and point to possible net benefits for children and their families undertaking NBS for DMD. The aim of this workshop was to establish pathways for the successful implementation and evaluation of a pilot NBS for DMD program in Australia. Consensus was reached as to the rationale for, potential benefits, risks, barriers and facilitators of screening, alongside the establishment of screening protocols and clinical referral pathways.

Updates in the Use of Vamorolone and Steroids in the Treatment of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a devastating, life-limiting neuromuscular disorder that has a global prevelance of 21.4 cases per 100,000 live male births. DMD is caused by mutations in the dystrophin gene, which is integral to muscle membrane stability. Mutations in the dystrophin gene lead to inflammation and muscle fibrosis. Current standard-of-care treatments include the use of chronic (or classic) corticosteroids, which can cause significant side effects. Vamorolone is a first-in-class dissociative steroid that acts as a partial agonist to glucocorticoid receptors but as an antagonist to mineralocorticoid receptors, leading to a more favourable side effect profile. Clinical data has shown that vamorolone has similar anti-inflammatory benefits to classic corticosteroids but an improved side effect profile.

State of the Art Procedures for the Isolation and Characterization of Mesoangioblasts.

Adult skeletal muscle is a dynamic tissue able to regenerate quite efficiently, thanks to the presence of stem cell machinery. Besides the quiescent satellite cells that are activated upon injury or paracrine factors, other stem cells are described to be directly or indirectly involved in adult myogenesis. Mesoangioblasts (MABs) are vessel-associated stem cells originally isolated from embryonic dorsal aorta and, at later stages, from the adult muscle interstitium expressing pericyte markers. Adult MABs entered clinical trials for the treatment of Duchenne muscular dystrophy and the transcriptome of human fetal MABs has been described. In addition, single cell RNA-seq analyses provide novel information on adult murine MABs and more in general in interstitial muscle stem cells. This chapter provides state-of-the-art techniques to isolate and characterize murine MABs, fetal and adult human MABs.

A Comprehensive Review of Duchenne Muscular Dystrophy: Genetics, Clinical Presentation, Diagnosis, and Treatment

Duchenne Muscular Dystrophy (DMD) is a genetic disorder involving progressive muscle deterioration leading to loss of mobility, cardiomyopathy, and respiratory complications leading to an early death by the fourth decade of life. Males are affected more often as DMD results from a mutation in the dystrophin gene residing on the X chromosome. The DMD genetic mutation results in a complete functional lack of dystrophin, which culminates as an inadequate connection between the intracellular actin filaments and the extracellular skeleton of muscle. Boys affected by DMD clinically present with muscle weakness before age five, are often wheelchair-bound by age 12, and rarely survive beyond the third decade of life.
 Traditional treatment strategies have focused primarily on quality-of-life improvement and have included the use of glucocorticoids and physical therapy. No cure currently exists, however many novel treatments for DMD are currently being explored. Some of these involve gene therapy, exon skipping, stop codon skipping, CRISPR technology interventions, and the use of a retinal dystrophin isoform. In this comprehensive review, we recapitulate the literature findings to summarize the history, epidemiology, genetics, clinical presentation, diagnosis, and current and future strategies for the treatment of Duchenne Muscular Dystrophy.

Synthesis of 5'-Thiol Functionalized Morpholino Oligo-Nucleotide and Subsequent Conjugation with IGT to Improve Delivery and Antisense Efficacy In Vitro.

Thiol functionalized oligonucleotides are useful intermediates for a wide range of applications including DNA nanobiotechnology field through conjugation with various types of probes and cargos. Due to the limitation of synthetic process, phosphorodiamidate morpholino oligonucleotides (PMOs) have not been explored like other oligonucleotides through SH conjugation as mentioned above. In this paper, we report the synthesis of 5'-SH functionalized PMO using a solid support synthesis protocol with an optimized cysteine derived linker so that loading and coupling efficiency of morpholino monomers were effective enough to get a 25-mer 5'-SH functionalized PMO against human Nanog. The PMO with SH functionality was subsequently conjugated with our previously reported Internal Oligo-guanidinium Transporter (IGT) in solution phase to obtain the IGT-PMO conjugate. Interestingly, 5'-conjugated PMO (IGT-PMO) showed 2.5 times better antisense efficacy than 3'-conjugated PMO with IGT (PMO-IGT). 5'-Conjugation enables us to use IGT-PMO for further conjugation at the 3'-N terminal of PMO which was not possible earlier with 5'-OH-PMO-IGT. PMO has become an important class of antisense reagents because four PMO-based drugs have been approved for the treatment of Duchenne muscular dystrophy; hence such an improved result with 5'-modified PMO could be useful for enhancing the therapeutic efficacy of DMD drugs. Similarly, thiol-modified PMO could also be explored like other thiol-containing oligonucleotides for various other applications.

PCR143 Project Hercules - Burden of Steroid Use in the Treatment of Duchenne Muscular Dystrophy: Findings From Patient and Carer Dyads and a Focus Group

To gather qualitative evidence from patients and their parents on the impact of long-term, high-dose steroid use side effects in Duchenne Muscular Dystrophy (DMD), including patient and parent health-related quality of life (HRQoL) impact, personal carer burden, and health service utilisation. This study was conducted as part of Project HERCULES.

Histone deacetylase inhibitors improve antisense-mediated exon-skipping efficacy in mdx mice

Antisense-mediated exon skipping is one of the most promising therapeutic strategies for Duchenne muscular dystrophy (DMD), and some antisense oligonucleotide (ASO) drugs have already been approved by the US FDA despite their low efficacy. The potential of this therapy is still limited by several challenges, including the reduced expression of the dystrophin transcript and the strong 5'-3' imbalance in mutated transcripts. We therefore hypothesize that increasing histone acetylation using histone deacetylase inhibitors (HDACi) could correct the transcript imbalance, offering more available pre-mRNA target and ultimately increasing dystrophin rescue. Here, we evaluated the impact of such a combined therapy on the Dmd transcript imbalance phenomenon and on dystrophin restoration levels in mdx mice. Analysis of the Dmd transcript levels at different exon-exon junctions revealed a tendency to correct the 5'-3' imbalance phenomenon following treatment with HDACi. Significantly higher levels of dystrophin restoration (up to 74% increase) were obtained with givinostat and valproic acid compared with mice treated with ASO alone. Additionally, we demonstrate an increase in H3K9 acetylation in human myocytes after treatment with valproic acid. These findings indicate that HDACi can improve the therapeutic potential of exon-skipping approaches, offering promising perspectives for the treatment of DMD.

Viltolarsen: From Preclinical Studies to FDA Approval.

Viltolarsen is a phosphorodiamidate morpholino antisense oligonucleotide (PMO) designed to skip exon 53 of the DMD genefor the treatment of Duchenne muscular dystrophy (DMD), one of the most common lethal genetic disorders characterized by progressive degeneration of skeletal muscles and cardiomyopathy. It was developed by Nippon Shinyaku in collaboration withthe National Center of Neurology and Psychiatry (NCNP)in Japan based on the preclinical studies conducted in the DMDdog model at the NCNP. After showing hopeful results in pre-clinical trials and several clinical trials across North America and Japan, it received US Food and Drug Administration (FDA) approval for DMDin 2020. Viltolarsen restores the reading frame of the DMD gene by skipping exon 53and produces a truncated but functional form of dystrophin. It can treat approximately 8-10% of the DMD patient population.This paper aims to summarize the development of viltolarsen from preclinical trials to clinical trials to, finally, FDA approval, and discusses the challenges that come with fighting DMD using antisense therapy.

High-Throughput Screening to Identify Modulators of Sarcospan.

High-throughput screening enables the discovery of disease-modifying small molecules. Here, we describe the development of a scalable, cell-based assay to screen for small molecules that modulate sarcospan for the treatment of Duchenne muscular dystrophy. We detail the hit validation pipeline, which includes secondary screening, gene/protein quantification, and an in vitro membrane stability assay.

Restoring Dystrophin Expression by Skipping Exons 6 and 8 in Neonatal Dystrophic Dogs.

Duchenne muscular dystrophy (DMD) is caused by the mutations in the DMD gene resulting in no dystrophin production. Skipping DMD exons using phosphorodiamidate morpholino oligomers (PMOs) is an emerging treatment strategy that can restore the reading frame of the mutated gene and produce truncated but functional dystrophin protein. To date, four PMOs, including eteplirsen, casimersen, viltolarsen, and golodirsen, have been conditionally approved by the FDA for the treatment of DMD. Since degeneration of muscle fibers and irreversible fibrosis occur from childhood, the earlier treatment is preferred. The canine X-linked muscular dystrophy in Japan (CXMDj), a dog model of DMD, produces no dystrophin and exhibits a severe phenotype similar to human patients from early childhood. As such, CXMDj, which harborsa splice site mutation in intron 6, is a useful model for examining the long-term effects of early PMO treatment. In this chapter, we describe the systemic delivery of a cocktail of four PMOs that can successfully induce multiple exon skipping (exons 6-9) in neonataldystrophic dogs. We also describe the procedures to evaluate the efficacy and toxicity, including clinical grading of dystrophic dogs, ELISA-based quantification of PMOs, histology, RT-PCR, and western blotting.

A state-of-the-art review of tamoxifen as a potential therapeutic for duchenne muscular dystrophy.

Introduction: This systematic review analyzes the state-of-art repurposing of the drug tamoxifen (TAM) in the treatment of Duchenne Muscular Dystrophy (DMD), including its mechanism of action, toxicological findings, and past and ongoing clinical trials. A parallel aim of this work was to explore whether evidence exists to support further funding of investigation on TAM treatment for DMD patients with a pivotal trial in young patients. Bringing evidence and answering the scientific question of whether this treatment could improve the quality-of-life of DMD patients is needed to establish guidelines and accelerate access to promising therapies for DMD patients. Methods: The search was conducted in January 2022 utilizing PubMed. All MeSH terms for "Duchenne Muscular Dystrophy" and "tamoxifen" were used. The inclusion and exclusion criteria were defined according to the PICOS framework. Results: The included publications all explored the use of TAM with promising outcomes in muscular strength recovery and a decrease in pathology biomarkers. Two reviews recognize TAM as a potential treatment for DMD patients and state that drug repurposing plays a crucial role in the quest for a drug candidate to treat this rare disease. Conclusion: According to available data, TAM shows promise as a treatment for DMD, both pharmacologically and clinically. However, published data to date are insufficient to definitively conclude the beneficial effect of TAM on quality-of-life and ultimately survival, particularly in the youngest patients diagnosed with DMD.

Identification of 2,6-Diaminopurine As a Candidate for Hemophilia a Ribosomal Readthrough Therapy

Nonsense mutations introduce premature termination codons (PTCs) and are responsible for 11% hereditary genetic disorders including hemophilias. Drug-induced ribosomal readthrough over PTCs is an emerging strategy to treat the genetic disorders caused by nonsense mutations. Ribosomal readthrough therapy aims to allow translation beyond the PTC, creating a full-length protein and possible phenotypic rescue. Ataluren (PTC124), which induces readthrough by binding to the ribosomal 60S subunit, was recently granted conditional approval in the European Union for the treatment of Duchenne muscular dystrophy caused by nonsense mutations. Most nonsense mutations in FVIII and FIX lead to severe diseases. In theory, hemophilias are ideally suited for drug-induced readthrough therapy, because even a small increase in FVIII or FIX activity to over 1% could significantly improve bleeding symptoms in patients with nonsense mutations. Thus, ribosomal readthrough therapy has the potential to provide a small-molecule, personalized treatments based on patient's mutation profile. We previously investigated different readthrough drugs on different FVIII nonsense mutations and found that geneticin, but not gentamicin and Ataluren, is an effective readthrough agent for a number of such mutations. Recently, 2,6-diaminopurine (DAP), a natural compound found in extracts of the edible mushroom Lepista inversa, has shown high-efficiency correction of UGA nonsense mutations and low toxicity. DAP is thought to act as an inhibitor of a tRNA-specific 2'-O-methyltransferase and promotes the interaction of the near-cognate tRNATrp with the UGA stop codon. We introduced 22 UGA mutations, including the most common recurring mutations from hemophilia A (HA) patients into the B domain deleted FVIII-Gassia luciferase fusion (BDD-Gluc) protein. Cells stably expressing these mutant fusion proteins were treated with DAP and geneticin. DAP induced higher secreted luciferase activity, FVIII antigen and cofactor activity levels in cell culture media than geneticin treatments. A majority of DAP-treated mutants produced FVIII co-factor activities and antigens that are over 2% of wild-type level, with several mutants reaching 10-15% levels. Using an immunoprecipitation-immunoblotting assay, we detected full-length BDD-Gluc fusion proteins in culture media and cell lysates of both DAP- and geneticin-treated cells expressing nonsense mutants. Two major factors affect the outcome of readthrough therapies are the readthrough efficiency of drugs and the nature of replacement of amino acid (AA) residues. Studies had shown that a cytosine at +4 position is a favorable nucleotide sequence context. Readthrough efficiency of F8 mRNA is influenced, but not determined by nucleotide sequence contexts of the PTC. It was reported that only tryptophan was replaced with DAP-induced readthrough while three potential AAs can be replaced with geneticin treatment. We systematically substituted PTCs with potential replacement AAs and found that activities of readthrough products are determined by the compatibility of replacement AA residues with FVIII secretion and activity. Our results suggest that DAP, with its high readthrough efficiency and low toxicity, is a promising drug candidate for HA patients carrying susceptible UGA mutations. This cell-based study identifies candidate patient mutations and provides a rationale for a future clinical study of DAP or one of its derivatives as a therapeutic for these patients.

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

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

Transplantation of Differentiated Tonsil-Derived Mesenchymal Stem Cells Ameliorates Murine Duchenne Muscular Dystrophy via Autophagy Activation.

Skeletal muscles play many important roles in the human body and any malfunction or disorder of the skeletal muscles can lead to a reduced quality of life. Some skeletal dysfunctions are acquired, such as sarcopenia but others are congenital. Duchenne muscular dystrophy (DMD) is one of the most common forms of hereditary muscular dystrophy and is caused by a deficiency of the protein, Dystrophin. Currently, there is no clear treatment for DMD, there are only methods that can alleviate the symptoms of the disease. Mesenchymal stem cells, including tonsil-derived mesenchymal stem cells (TMSCs) have been shown to differentiate into skeletal muscle cells (TMSC-myocyte) and can be one of the resources for the treatment of DMD. Skeletal muscle cell characteristics of TMSC-myocytes have been confirmed through changes in morphology and expression of skeletal muscle markers such as Myogenin, Myf6, and MYH families after differentiation. Based on these characteristics, TMSC-myocytes have been transplanted into mdx mice, a mouse model of DMD, to investigate whether they can help improve the symptoms of DMD. The red fluorescent protein gene was transduced into TMSC (TMSC-R) for tracking transplanted cells. Prior to transplantation (TP), it was confirmed whether TMSC-R-myocytes had the same differentiation potential as TMSC-myocytes. Increased expression of dystrophin and autophagy markers in the TP group compared with the sham group was confirmed in the gastrocnemius muscle 12weeks after TP. These results demonstrate muscle regeneration and functional recovery of mdx via autophagy activation following TMSC-myocyte TP.

I.12 Modulating muscle stem cells to enhance regeneration to ameliorate DMD disease progression

Duchenne muscular dystrophy (DMD) is a lethal X-linked muscle-wasting disease. Dystrophin functions as an essential component of the large oligomeric dystrophin-glycoprotein complex (DGC). The DGC acts to connect the actin cytoskeleton of the myofiber to the surrounding extracellular matrix through the sarcolemma. In the absence of dystrophin, DGC assembly is impaired which weakens the muscle fibers rendering them highly susceptible to injury, causing accumulation of inflammation and fibrosis, and progressive loss of muscle mass and function. We discovered that dystrophin is highly expressed in activated muscle stem cells and associates with the serine-threonine kinase Mark2/Par1b, a regulator of cell polarity. In the absence of dystrophin, Mark2 is downregulated, resulting in the inability to localize the cell polarity regulator Pard3 to the opposite side of the cell. Consequently, the number of asymmetric divisions and hence the generation of myogenic progenitors is significantly reduced. Our findings indicate that muscle wasting in DMD not only is caused by myofiber fragility, but also is exacerbated by impaired regeneration owing to intrinsic satellite cell dysfunction. Through a small-molecule screen, we made the discovery of a novel signal transduction pathway involving epidermal growth factor receptor (EGFR) and Aurora kinase A (Aurka) as key regulators of asymmetric satellite cell divisions. Inhibiting EGFR causes a substantial shift from asymmetric to symmetric division modes, whereas EGF treatment increases asymmetric divisions. EGFR activation acts through Aurka to orient mitotic centrosomes, and inhibiting Aurka blocks EGF stimulation-induced asymmetric division. In vivo EGF treatment markedly activates asymmetric divisions of dystrophin-deficient satellite cells in mdx mice, increasing progenitor numbers, enhancing regeneration, and restoring muscle strength. Therefore, activating an EGFR-dependent polarity pathway promotes functional rescue of dystrophin-deficient satellite cells and enhances muscle force generation. In collaboration with Satellos Bioscience, we have been developing small molecule drugs that stimulate asymmetric division by modulating the EGFR/Aurka and other pathways. Our goal is to develop small molecule modulators of muscle stem cell asymmetric division as a treatment for DMD.

P.130 RGX-202: An investigational AAV8 gene therapy coding for a novel microdystrophin as a treatment for Duchenne muscular dystrophy

RGX-202 is an investigational one-time gene therapy for the treatment of Duchenne muscular dystrophy (Duchenne) that utilizes a NAV AAV8 vector, a muscle specific promoter (Spc5-12), and an optimized microdystrophin transgene. The microdystrophin coding sequence involves codon optimization, depletion of CpG dinucleotides, and an extended coding region for the C-Terminal (CT) domain found in naturally occurring dystrophin. Preclinical studies in mdx mice demonstrated a dose proportional increase of RGX-202 vector DNA and microdystrophin in skeletal and heart muscle and restoration of the dystrophin-associated complex (DAPC) at the sarcolemma, indicating the potential benefit of the CT domain. Moreover, after a single intravenous (IV) injection of RGX-202, mdx mice showed significant improvements in dystrophic muscle pathology (muscle lesions and fibrosis) and function (in vitro force, and automated gait analysis) at doses > 1e14 GC/kg. A Phase I/II trial, AFFINITY DUCHENNE, is a multicenter, open-label dose escalation study to evaluate the safety, tolerability and clinical efficacy of a one-time IV dose of RGX-202 in patients with Duchenne. Six ambulatory, pediatric patients (ages 4 to 11 years old) with Duchenne are expected to enroll in 2 cohorts at doses of 1x10¹⁴ genome copies (GC)/kg body weight (n=3) and 2x10¹⁴ GC/kg body weight (n=3). An expansion may allow for up to 6 additional participants to be enrolled at each dose for a total of up to 9 participants at each dose and 18 participants in the study. Additional inclusion criteria are based on dystrophin gene mutations in exons 18-58, a functional assessment, and AAV seronegative status. Endpoints include safety, immunogenicity assessments, RGX-202 microdystrophin levels in muscle, functional assessments (North Star Ambulatory Assessment and timed function tests), and muscle preservation (MRI).