A review on mechanistic insights into structure and function of dystrophin protein in pathophysiology and therapeutic targeting of Duchenne muscular dystrophy.

Duchenne Muscular Dystrophy (DMD) is an X-linked recessive genetic disorder characterized by progressive and severe muscle weakening and degeneration. Among the various forms of muscular dystrophy, it stands out as one of the most common and impactful, predominantly affecting boys. The condition arises due to mutations in the dystrophin gene, a key player in maintaining the structure and function of muscle fibers. The manuscript explores the structural features of dystrophin protein and their pivotal roles in DMD. We present an in-depth analysis of promising therapeutic approaches targeting dystrophin and their implications for the therapeutic management of DMD. Several therapies aiming to restore dystrophin protein or address secondary pathology have obtained regulatory approval, and many others are ongoing clinical development. Notably, recent advancements in genetic approaches have demonstrated the potential to restore partially functional dystrophin forms. The review also provides a comprehensive overview of the status of clinical trials for major therapeutic genetic approaches for DMD. In addition, we have summarized the ongoing therapeutic approaches and advanced mechanisms of action for dystrophin restoration and the challenges associated with DMD therapeutics.

Practical Considerations for Delandistrogene Moxeparvovec Gene Therapy in Patients With Duchenne Muscular Dystrophy.

BACKGROUND: Delandistrogene moxeparvovec is a gene transfer therapy approved in the United States, United Arab Emirates, and Qatar for the treatment of ambulatory patients aged four through five years with a confirmed Duchenne muscular dystrophy (DMD)-causing mutation in the DMD gene. This therapy was developed to address the underlying cause of DMD through targeted skeletal, respiratory, and cardiac muscle expression of delandistrogene moxeparvovec micro-dystrophin, an engineered, functional dystrophin protein. METHODS: Drawing on clinical trial experience from Study 101 (NCT03375164), Study 102 (NCT03769116), and ENDEAVOR (Study 103; NCT04626674), we outline practical considerations for delandistrogene moxeparvovec treatment. RESULTS: Before infusion, the following are recommended: (1) screen for anti-adeno-associated virus rhesus isolate serotype 74 total binding antibody titers <1:400; (2) assess liver function, platelet count, and troponin-I; (3) ensure patients are up to date with vaccinations and avoid vaccine coadministration with infusion; (4) administer additional corticosteroids starting one day preinfusion (for patients already on corticosteroids); and (5) postpone dosing patients with any infection or acute liver disease until event resolution. Postinfusion, the following are recommended: (1) monitor liver function weekly (three months postinfusion) and, if indicated, continue until results are unremarkable; (2) monitor troponin-I levels weekly (first month postinfusion, continuing if indicated); (3) obtain platelet counts weekly (two weeks postinfusion), continuing if indicated; and (4) maintain the corticosteroid regimen for at least 60 days postinfusion, unless earlier tapering is indicated. CONCLUSIONS: Although the clinical safety profile of delandistrogene moxeparvovec has been consistent, monitorable, and manageable, these practical considerations may mitigate potential risks in a real-world clinical practice setting.

Viltolarsen: a treatment option for Duchenne muscular dystrophy patients who are amenable to exon 53 skipping therapy.

INTRODUCTION: Duchenne muscular dystrophy (DMD) is a progressive genetic disease leading to muscular weakness. DMD is caused by mutations of the dystrophin gene on the X chromosome that is responsible for production of dystrophin protein. Dystrophin contributes to structural support in muscle cells and mutations result in dystrophin protein deficiency which causes muscle damage and the associated clinical presentation. Exon skipping medications, including the exon 53 targeting viltolarsen, are the first agents with the ability to partially restore dystrophin protein. AREAS COVERED: Herein, the authors profile viltolarsen for the DMD patients who are amenable to exon 53 skipping therapy and provide their expert perspectives on this subject. EXPERT OPINION: Current findings suggest that viltolarsen could play a role in the current and possible future treatment of DMD. Viltolarsen seems to be safe and restores dystrophin protein to around 6% of the normal level. Due to orphan drug status, after the completion of the phase 2 clinical trial, viltolarsen was granted accelerated approval in Japan and in the US. A phase 3 trial is currently in progress and needs to earn full approval. Although a multidisciplinary approach continues to be critical, the addition of exon skipping agents like viltolarsen may improve the quality of patients' lives. However, data on the long-term safety and efficacy of this medication are not yet available due to its recent accelerated approval.

Pharmacological management of dilated cardiomyopathy in Duchenne muscular dystrophy: A systematic review.

Duchenne muscular dystrophy is a fatal X-linked recessive disease affecting approximately 1 in 3500 births. It is characterized by a genetic lack of dystrophin, which is an essential protein for maintaining muscle integrity. The lack of dystrophin plays a pathophysiological role in the development of dilated cardiomyopathy in Duchenne muscular dystrophy. Currently, no consensus exists on specific pharmacological therapy guidelines for these patients; however, it centers around the guidelines for heart failure management. This systematic review investigated 12 randomized control trials dating back to 2005 in the pharmacotherapy of patients with dilated cardiomyopathy Duchenne muscular dystrophy. This review specifically included angiotensin-converting enzyme inhibitors, aldosterone receptor blockers, angiotensin receptor/neprilysin inhibitors, beta-blockers, and mineralocorticoid receptor antagonists. Despite their limitations, these studies have shown promising effects in improving the overall heart function and prognosis in patients with this condition. However, to attain higher statistical significance, future studies should investigate larger populations and for longer periods.

Emerging therapies for Duchenne muscular dystrophy.

Duchenne muscular dystrophy is an X-linked disease caused by the absence of functional dystrophin in the muscle cells. Major advances have led to the development of gene therapies, tools that induce exon skipping, and other therapeutic approaches, including treatments targeting molecular pathways downstream of the absence of functional dystrophin. However, glucocorticoids remain the only treatment unequivocally shown to slow disease progression, despite the adverse effects associated with their long-term use. Besides glucocorticoids, which are standard care, five compounds have received regulatory approval in some but not all jurisdictions, with further efficacy results being awaited. Several compounds with promising results in early-phase clinical trials have not met their efficacy endpoints in late-phase trials, but the clinical development of many other compounds is ongoing. The current landscape is complicated by the number of compounds in various stages of development, their various mechanisms of action, and their genotype-specific applicability. The difficulties of clinical development that arise from both the rarity and variability of Duchenne muscular dystrophy might be overcome in the future by use of sensitive biomarkers, natural history data, and ameliorated trial designs.

Clinical development on the frontier: gene therapy for duchenne muscular dystrophy.

Introduction: The development of adeno-associated virus (AAV) vectors as safe vehicles for in vivo delivery of therapeutic genes has been a major milestone in the advancement of gene therapy, enabling a promising strategy for ameliorating a wide range of diseases, including Duchenne muscular dystrophy (DMD).Areas covered: Based on experience with the development of a gene transfer therapy agent for DMD, we discuss ways in which gene therapy for rare disease challenges traditional clinical development paradigms, and recommend a step-wise approach for design and evaluation to support broader applicability of gene therapy.Expert opinion: The gene therapy development approach should intentionally design the therapeutic construct and the clinical study to systematically evaluate agent delivery, safety, and efficacy. Rigorous preclinical work is essential for establishing an effective gene delivery platform and determining the efficacious dose. Clinical studies should thoroughly evaluate transduction, on-target transgene expression at the tissue and cellular level, and functional efficacy.

CRISPR-Induced Deletion with SaCas9 Restores Dystrophin Expression in Dystrophic Models In Vitro and In Vivo.

Duchenne muscular dystrophy (DMD), a severe hereditary disease affecting 1 in 3,500 boys, mainly results from the deletion of exon(s), leading to a reading frameshift of the DMD gene that abrogates dystrophin protein synthesis. Pairs of sgRNAs for the Cas9 of Staphylococcus aureus were meticulously chosen to restore a normal reading frame and also produce a dystrophin protein with normally phased spectrin-like repeats (SLRs), which is not usually obtained by skipping or by deletion of complete exons. This can, however, be obtained in rare instances where the exon and intron borders of the beginning and the end of the complete deletion (patient deletion plus CRISPR-induced deletion) are at similar positions in the SLR. We used pairs of sgRNAs targeting exons 47 and 58, and a normal reading frame was restored in myoblasts derived from muscle biopsies of 4 DMD patients with different exon deletions. Restoration of the DMD reading frame and restoration of dystrophin expression were also obtained in vivo in the heart of the del52hDMD/mdx. Our results provide a proof of principle that SaCas9 could be used to edit the human DMD gene and could be considered for further development of a therapy for DMD.

Systemic AAV Micro-dystrophin Gene Therapy for Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by dystrophin gene mutation. Conceptually, replacing the mutated gene with a normal one would cure the disease. However, this task has encountered significant challenges due to the enormous size of the gene and the distribution of muscle throughout the body. The former creates a hurdle for viral vector packaging and the latter begs for whole-body therapy. To address these obstacles, investigators have invented the highly abbreviated micro-dystrophin gene and developed body-wide systemic gene transfer with adeno-associated virus (AAV). Numerous microgene configurations and various AAV serotypes have been explored in animal models in many laboratories. Preclinical data suggests that intravascular AAV micro-dystrophin delivery can significantly ameliorate muscle pathology, enhance muscle force, and attenuate dystrophic cardiomyopathy in animals. Against this backdrop, several clinical trials have been initiated to test the safety and tolerability of this promising therapy in DMD patients. While these trials are not powered to reach a conclusion on clinical efficacy, findings will inform the field on the prospects of body-wide DMD therapy with a synthetic micro-dystrophin AAV vector. This review discusses the history, current status, and future directions of systemic AAV micro-dystrophin therapy.

Micro-Dystrophin Gene Therapy Goes Systemic in Duchenne Muscular Dystrophy Patients.

Whole-body systemic gene therapy is likely the most effective way to reduce greatly the disease burden of Duchenne muscular dystrophy (DMD), an X-linked inherited muscle disease that leads to premature death in early adulthood. Genetically, DMD is due to null mutation of the dystrophin gene, one of the largest genes in the genome. Recent studies have shown highly promising improvements in animal models with intravascular delivery of the engineered micro-dystrophin gene by adeno-associated virus (AAV). Several human trials are now started to advance AAV micro-dystrophin therapy to DMD patients. This is a historical moment for the entire field. Results from these trials will shape the future of neuromuscular disease gene therapy.

An Overview of Recent Therapeutics Advances for Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy in childhood. Mutations of the DMD gene destabilize the dystrophin associated glycoprotein complex in the sarcolemma. Ongoing mechanical stress leads to unregulated influx of calcium ions into the sarcoplasm, with activation of proteases, release of proinflammatory cytokines, and mitochondrial dysfunction. Cumulative damage and reparative failure leads to progressive muscle necrosis, fibrosis, and fatty replacement. Although there is presently no cure for DMD, scientific advances have led to many potential disease-modifying treatments, including dystrophin replacement therapies, upregulation of compensatory proteins, anti-inflammatory agents, and other cellular targets. Recently approved therapies include ataluren for stop codon read-through and eteplirsen for exon 51 skipping of eligible individuals. The purpose of this chapter is to summarize the clinical features of DMD, to describe current outcome measures used in clinical studies, and to highlight new emerging therapies for affected individuals.

Designing Effective Antisense Oligonucleotides for Exon Skipping.

During the past 10 years, antisense oligonucleotide-mediated exon skipping and splice modulation have proven to be powerful tools for correction of mRNA splicing in genetic diseases. In 2016, the US Food and Drug Administration (FDA)-approved Exondys 51 (eteplirsen) and Spinraza (nusinersen), the first exon skipping and exon inclusion drugs, to treat patients with Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA), respectively. The exon skipping of DMD mRNA aims to restore the disrupted reading frame using antisense oligonucleotides (AONs), allowing the production of truncated but partly functional dystrophin proteins, and slow down the progression of the disease. This approach has also been explored in several other genetic disorders, including laminin α2 chain-deficient congenital muscular dystrophy, dysferlin-deficient muscular dystrophy (e.g., Miyoshi myopathy and limb-girdle muscular dystrophy type 2B), sarcoglycanopathy (limb-girdle muscular dystrophy type 2C), and Fukuyama congenital muscular dystrophy. Antisense-mediated exon skipping is also a powerful tool to examine the function of genes and exons. A significant challenge in exon skipping is how to design effective AONs. The mechanism of mRNA splicing is highly complex with many factors involved. The selection of target sites, the length of AONs, the AON chemistry, and the melting temperature versus the RNA strand play important roles. A cocktail of AONs can be employed to skip multiples exons. In this chapter, we discuss the design of effective AONs for exon skipping.

The Use of Antisense Oligonucleotides for the Treatment of Duchenne Muscular Dystrophy.

Antisense oligonucleotides (AONs) hold great promise for therapeutic splice-switching correction in many genetic diseases and in particular for Duchenne muscular dystrophy (DMD), where AONs can be used to reframe the dystrophin transcript and give rise to a partially deleted but yet functional dystrophin protein. Many different chemistries of AONs can be used for splice switching modulation, and some of them have been evaluated in clinical trials for DMD. However, despite advances in AON chemistry and design, systemic use of AONs is limited due to poor tissue uptake, and sufficient therapeutic efficacy is difficult to achieve. Therefore, there is still a critical need to develop efficient AONs able to restore the expression of dystrophin in all relevant tissues and international efforts are currently on going to develop new compounds or alternative chemistries with higher therapeutic potential. Here, we describe the methods to evaluate the potency of antisense oligonucleotides, and in particular of tricyclo-DNA (tcDNA)-AONs, a novel class of AONs which displays unique pharmacological properties and unprecedented uptake in many tissues after systemic administration. We focus on the most widely used mouse model for DMD, the mdx mouse and detail methods to analyze the skipping of the mouse exon 23 both in vitro in H2K mdx cells and in vivo in the mdx mouse model.

Identification of Splicing Factors Involved in DMD Exon Skipping Events Using an In Vitro RNA Binding Assay.

Mutation-induced exon skipping in the DMD gene can modulate the severity of the phenotype in patients with Duchenne or Becker Muscular Dystrophy. These alternative splicing events are most likely the result of changes in recruitment of splicing factors at cis-acting elements in the mutated DMD pre-mRNA. The identification of proteins involved can be achieved by an affinity purification procedure. Here, we provide a detailed protocol for the in vitro RNA binding assay that we routinely apply to explore molecular mechanisms underlying splicing defects in the DMD gene. In vitro transcribed RNA probes containing either the wild type or mutated sequence are oxidized and bound to adipic acid dihydrazide-agarose beads. Incubation with a nuclear extract allows the binding of nuclear proteins to the RNA probes. The unbound proteins are washed off and then the specifically RNA-bound proteins are released from the beads by an RNase treatment. After separation by SDS-PAGE, proteins that display differential binding affinities for the wild type and mutant RNA probes are identified by mass spectrometry.

PMO Delivery System Using Bubble Liposomes and Ultrasound Exposure for Duchenne Muscular Dystrophy Treatment.

Duchenne muscular dystrophy (DMD) is a genetic disorder characterized by progressive muscle degeneration, caused by nonsense or frameshift mutations in the dystrophin (DMD) gene. Antisense oligonucleotides can be used to induce specific exon skipping; recently, a phosphorodiamidate morpholino oligomer (PMO) has been approved for clinical use in DMD. However, an efficient PMO delivery strategy is required to improve the therapeutic efficacy in DMD patients. We previously developed polyethylene glycol (PEG)-modified liposomes containing ultrasound contrast gas, "Bubble liposomes" (BLs), and found that the combination of BLs with ultrasound exposure is a useful gene delivery tool. Here, we describe an efficient PMO delivery strategy using the combination of BLs and ultrasound exposure to treat muscles in a DMD mouse model (mdx). This ultrasound-mediated BL technique can increase the PMO-mediated exon-skipping efficiency, leading to significantly increased dystrophin expression. Thus, the combination of BLs and ultrasound exposure may be a feasible PMO delivery method to improve therapeutic efficacy and reduce the PMO dosage for DMD treatment.

Lentiviral vectors can be used for full-length dystrophin gene therapy.

Duchenne Muscular Dystrophy (DMD) is caused by a lack of dystrophin expression in patient muscle fibres. Current DMD gene therapy strategies rely on the expression of internally deleted forms of dystrophin, missing important functional domains. Viral gene transfer of full-length dystrophin could restore wild-type functionality, although this approach is restricted by the limited capacity of recombinant viral vectors. Lentiviral vectors can package larger transgenes than adeno-associated viruses, yet lentiviral vectors remain largely unexplored for full-length dystrophin delivery. In our work, we have demonstrated that lentiviral vectors can package and deliver inserts of a similar size to dystrophin. We report a novel approach for delivering large transgenes in lentiviruses, in which we demonstrate proof-of-concept for a 'template-switching' lentiviral vector that harnesses recombination events during reverse-transcription. During this work, we discovered that a standard, unmodified lentiviral vector was efficient in delivering full-length dystrophin to target cells, within a total genomic load of more than 15,000 base pairs. We have demonstrated gene therapy with this vector by restoring dystrophin expression in DMD myoblasts, where dystrophin was expressed at the sarcolemma of myotubes after myogenic differentiation. Ultimately, our work demonstrates proof-of-concept that lentiviruses can be used for permanent full-length dystrophin gene therapy, which presents a significant advancement in developing an effective treatment for DMD.

Optimization of Internally Deleted Dystrophin Constructs.

Duchenne muscular dystrophy (DMD) is a severe, genetic muscle disease caused by the absence of the sarcolemmal protein dystrophin. Gene replacement therapy is considered a potential strategy for the treatment of DMD, aiming to restore the missing protein. Although the elements of the dystrophin molecule have been identified and studies in transgenic mdx mice have explored the importance of a number of these structural domains, the resulting modified dystrophin protein products that have been developed so far are only partially characterized in relation to their structure and function in vivo. To optimize a dystrophin cDNA construct for therapeutic application we designed and produced four human minidystrophins within the packaging capacity of lentiviral vectors. Two novel minidystrophins retained the centrally located neuronal nitric oxide synthase (nNOS)-anchoring domain in order to achieve sarcolemmal nNOS restoration, which is lost in most internally deleted dystrophin constructs. Functionality of the resulting truncated dystrophin proteins was investigated in muscle of adult dystrophin-deficient mdx mice followed by a battery of detailed immunohistochemical and morphometric tests. This initial assessment aimed to determine the overall suitability of various constructs for cloning into lentiviral vectors for ex vivo gene delivery to stem cells for future preclinical studies.

AAV-mediated gene therapy in Dystrophin-Dp71 deficient mouse leads to blood-retinal barrier restoration and oedema reabsorption.

Dystrophin-Dp71 being a key membrane cytoskeletal protein, expressed mainly in Müller cells that provide a mechanical link at the Müller cell membrane by direct binding to actin and a transmembrane protein complex. Its absence has been related to blood-retinal barrier (BRB) permeability through delocalization and down-regulation of the AQP4 and Kir4.1 channels (1). We have previously shown that the adeno-associated virus (AAV) variant, ShH10, transduces Müller cells in the Dp71-null mouse retina efficiently and specifically (2,3). Here, we use ShH10 to restore Dp71 expression in Müller cells of Dp71 deficient mouse to study molecular and functional effects of this restoration in an adult mouse displaying retinal permeability. We show that strong and specific expression of exogenous Dp71 in Müller cells leads to correct localization of Dp71 protein restoring all protein interactions in order to re-establish a proper functional BRB and retina homeostasis thus preventing retina from oedema. This study is the basis for the development of new therapeutic strategies in dealing with diseases with BRB breakdown and macular oedema such as diabetic retinopathy (DR).

Safe and bodywide muscle transduction in young adult Duchenne muscular dystrophy dogs with adeno-associated virus.

The ultimate goal of muscular dystrophy gene therapy is to treat all muscles in the body. Global gene delivery was demonstrated in dystrophic mice more than a decade ago using adeno-associated virus (AAV). However, translation to affected large mammals has been challenging. The only reported attempt was performed in newborn Duchenne muscular dystrophy (DMD) dogs. Unfortunately, AAV injection resulted in growth delay, muscle atrophy and contracture. Here we report safe and bodywide AAV delivery in juvenile DMD dogs. Three ∼2-m-old affected dogs received intravenous injection of a tyrosine-engineered AAV-9 reporter or micro-dystrophin (µDys) vector at the doses of 1.92-6.24 × 10(14) viral genome particles/kg under transient or sustained immune suppression. DMD dogs tolerated injection well and their growth was not altered. Hematology and blood biochemistry were unremarkable. No adverse reactions were observed. Widespread muscle transduction was seen in skeletal muscle, the diaphragm and heart for at least 4 months (the end of the study). Nominal expression was detected in internal organs. Improvement in muscle histology was observed in µDys-treated dogs. In summary, systemic AAV gene transfer is safe and efficient in young adult dystrophic large mammals. This may translate to bodywide gene therapy in pediatric patients in the future.

Exon-skipping therapy: a roadblock, detour, or bump in the road?

Exon skipping is a promising therapeutic for Duchenne muscular dystrophy patients, but the road to drug approvals is foggy and may require more early-stage derisking and regulatory guidance.

Antisense oligonucleotide-mediated exon skipping for Duchenne muscular dystrophy: progress and challenges.

Duchenne muscular dystrophy (DMD) is the most common childhood neuromuscular disorder. It is caused by mutations in the DMD gene that disrupt the open reading frame (ORF) preventing the production of functional dystrophin protein. The loss of dystrophin ultimately leads to the degeneration of muscle fibres, progressive weakness and premature death. Antisense oligonucleotides (AOs) targeted to splicing elements within DMD pre-mRNA can induce the skipping of targeted exons, restoring the ORF and the consequent production of a shorter but functional dystrophin protein. This approach may lead to an effective disease modifying treatment for DMD and progress towards clinical application has been rapid. Less than a decade has passed between the first studies published in 1998 describing the use of AOs to modify the DMD gene in mice and the results of the first intramuscular proof of concept clinical trials. Whilst phase II and III trials are now underway, the heterogeneity of DMD mutations, efficient systemic delivery and targeting of AOs to cardiac muscle remain significant challenges. Here we review the current status of AO-mediated therapy for DMD, discussing the preclinical, clinical and regulatory hurdles and their possible solutions to expedite the translation of AO-mediated exon skipping therapy to clinic.