Deletion of exons 45 to 55 in the DMD gene: from the therapeutic perspective to the in vitro model.

BACKGROUND: Gene editing therapies in development for correcting out-of-frame DMD mutations in Duchenne muscular dystrophy aim to replicate benign spontaneous deletions. Deletion of 45-55 DMD exons (del45-55) was described in asymptomatic subjects, but recently serious skeletal and cardiac complications have been reported. Uncovering why a single mutation like del45-55 is able to induce diverse phenotypes and grades of severity may impact the strategies of emerging therapies. Cellular models are essential for this purpose, but their availability is compromised by scarce muscle biopsies. METHODS: We introduced, as a proof-of-concept, using CRISPR-Cas9 edition, a del45-55 mimicking the intronic breakpoints harboured by a subset of patients of this form of dystrophinopathy (designing specific gRNAs), into a Duchenne patient's cell line. The edited cell line was characterized evaluating the dystrophin expression and the myogenic status. RESULTS: Dystrophin expression was restored, and the myogenic defects were ameliorated in the edited myoblasts harbouring a specific del45-55. Besides confirming the potential of CRISPR-Cas9 to create tailored mutations (despite the low cleavage efficiency of our gRNAs) as a useful approach to generate in vitro models, we also generated an immortalized myoblast line derived from a patient with a specific del45-55. CONCLUSIONS: Overall, we provide helpful resources to deepen into unknown factors responsible for DMD-pathophysiology.

Correction of exon 2, exon 2-9 and exons 8-9 duplications in DMD patient myogenic cells by a single CRISPR/Cas9 system.

Duchenne Muscular dystrophy (DMD), a yet-incurable X-linked recessive disorder that results in muscle wasting and loss of ambulation is due to mutations in the dystrophin gene. Exonic duplications of dystrophin gene are a common type of mutations found in DMD patients. In this study, we utilized a single guide RNA CRISPR strategy targeting intronic regions to delete the extra duplicated regions in patient myogenic cells carrying duplication of exon 2, exons 2-9, and exons 8-9 in the DMD gene. Immunostaining on CRISPR-corrected derived myotubes demonstrated the rescue of dystrophin protein. Subsequent RNA sequencing of the DMD cells indicated rescue of genes of dystrophin related pathways. Examination of predicted close-match off-targets evidenced no aberrant gene editing at these loci. Here, we further demonstrate the efficiency of a single guide CRISPR strategy capable of deleting multi-exon duplications in the DMD gene without significant off target effect. Our study contributes valuable insights into the safety and efficacy of using single guide CRISPR strategy as a potential therapeutic approach for DMD patients with duplications of variable size.

Musculoskeletal Issues in Children and Adolescents: Genetic Musculoskeletal Disorders.

Duchenne muscular dystrophy (DMD) is an X-linked recessive genetic disorder with progressive proximal weakness as the principal sign. Glucocorticoids and physical therapy are the mainstay of treatment. Exercise intolerance is the hallmark of metabolic myopathies, which require a combination of laboratory testing, electrodiagnostic testing, and muscle biopsy for diagnosis. Joint hypermobility may be an isolated finding or be associated with hypermobility Ehlers-Danlos syndrome (EDS), other variants of EDS, or marfanoid syndromes. The latter conditions are associated with aortic and cardiac valvular abnormalities. Osteogenesis imperfecta encompasses a group of disorders characterized by bone fragility presenting with a low-impact fracture as a result of minimal trauma. Management includes multidiscipline specialists. Down syndrome (DS), or trisomy 21, is the most common chromosome abnormality identified in live births. Routine evaluation of atlantoaxial instability with x-ray is no longer recommended for children with DS without symptoms of atlantoaxial instability; however, clinical evaluation of symptoms is required for sports preparticipation. Achondroplasia is the most common skeletal dysplasia. Clinical signs are macrocephaly, short limb, short stature with disproportionately shorter humerus and femur, along with characteristic findings in pelvis and lumbar spine x-rays. Caregivers should be educated on proper positioning and handling to avoid complications, including car seat-related deaths.

Cell transplantation-mediated dystrophin supplementation efficacy in Duchenne muscular dystrophy mouse motor function improvement demonstrated by enhanced skeletal muscle fatigue tolerance.

BACKGROUND: Duchenne muscular dystrophy (DMD) is an incurable neuromuscular disease leading to progressive skeletal muscle weakness and fatigue. Cell transplantation in murine models has shown promise in supplementing the lack of the dystrophin protein in DMD muscles. However, the establishment of novel, long-term, relevant methods is needed to assess its efficiency on the DMD motor function. By applying newly developed methods, this study aimed to evaluate the functional and molecular effects of cell therapy-mediated dystrophin supplementation on DMD muscles. METHODS: Dystrophin was supplemented in the gastrocnemius of a 5-week-old immunodeficient DMD mouse model (Dmd-null/NSG) by intramuscular xenotransplantation of healthy human immortalized myoblasts (Hu5/KD3). A long-term time-course comparative study was conducted between wild-type, untreated DMD, and dystrophin supplemented-DMD mouse muscle functions and histology. A novel GO-ATeam2 transgenic DMD mouse model was also generated to assess in vivo real-time ATP levels in gastrocnemius muscles during repeated contractions. RESULTS: We found that 10.6% dystrophin supplementation in DMD muscles was sufficient to prevent low values of gastrocnemius maximal isometric contraction torque (MCT) at rest, while muscle fatigue tolerance, assessed by MCT decline after treadmill running, was fully ameliorated in 21-week-old transplanted mice. None of the dystrophin-supplemented fibers were positive for muscle damage markers after treadmill running, with 85.4% demonstrating the utilization of oxidative metabolism. Furthermore, ATP levels in response to repeated muscle contractions tended to improve, and mitochondrial activity was significantly enhanced in dystrophin supplemented-fibers. CONCLUSIONS: Cell therapy-mediated dystrophin supplementation efficiently improved DMD muscle functions, as evaluated using newly developed evaluation methods. The enhanced muscle fatigue tolerance in 21-week-old mice was associated with the preferential regeneration of damage-resistant and oxidative fibers, highlighting increased mitochondrial activity, after cell transplantation. These findings significantly contribute to a more in-depth understanding of DMD pathogenesis.

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

BACKGROUND: The 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. RESULTS: To 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. CONCLUSIONS: Our 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.

LED therapy modulates M1/M2 macrophage phenotypes and mitigates dystrophic features in treadmill-trained mdx mice.

The mdx mouse phenotype, aggravated by chronic exercise on a treadmill, makes this murine model more reliable for the study of Duchenne muscular dystrophy (DMD) and allows the efficacy of therapeutic interventions to be evaluated. This study aims to investigate the effects of photobiomodulation by light-emitting diode (LED) therapy on functional, biochemical and morphological parameters in treadmill-trained adult mdx animals. Mdx mice were trained for 30 min of treadmill running at a speed of 12 m/min, twice a week for 4 weeks. The LED therapy (850 nm) was applied twice a week to the quadriceps muscle throughout the treadmill running period. LED therapy improved behavioral activity (open field) and muscle function (grip strength and four limb hanging test). Functional benefits correlated with reduced muscle damage; a decrease in the inflammatory process; modulation of the regenerative muscular process and calcium signalling pathways; and a decrease in oxidative stress markers. The striking finding of this work is that LED therapy leads to a shift from the M1 to M2 macrophage phenotype in the treadmill-trained mdx mice, enhancing tissue repair and mitigating the dystrophic features. Our data also imply that the beneficial effects of LED therapy in the dystrophic muscle correlate with the interplay between calcium, oxidative stress and inflammation signalling pathways. Together, these results suggest that photobiomodulation could be a potential adjuvant therapy for dystrophinopathies.

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.

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

Background: 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. Objective: This study aimed to understand the perspectives of clinicians about gene therapy for DMD, which has not previously been studied. Methods: 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. Results: 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. Conclusions: 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.

Current Concepts in the Orthopaedic Management of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD), a genetic condition marked by progressive muscle degeneration, presents notable orthopaedic challenges, especially scoliosis, which deteriorates patients' quality of life by affecting sitting balance and complicating cardiac and respiratory functions. Current orthopaedic management strategies emphasize early intervention with corticosteroids to delay disease progression and the use of surgical spinal fusion to address severe scoliosis, aiming to enhance sitting balance, alleviate discomfort, and potentially extend patient lifespan. Despite advancements, optimal management requires ongoing research to refine therapeutic approaches, ensuring improved outcomes for patients with DMD. This review synthesizes recent findings on surgical and nonsurgical interventions, underscoring the importance of a multidisciplinary approach tailored to the dynamic needs of patients with DMD.

Essential components of an effective transition from paediatric to adult neurologist care for adolescents with Duchenne muscular dystrophy; a consensus derived using the Delphi methodology in Eastern Europe, Greece and Israel.

PURPOSE: An increasing number of patients with Duchenne muscular dystrophy (DMD) now have access to improved standard of care and disease modifying treatments, which improve the clinical course of DMD and extend life expectancy beyond 30 years of age. A key issue for adolescent DMD patients is the transition from paediatric- to adult-oriented healthcare. Adolescents and adults with DMD have unique but highly complex healthcare needs associated with long-term steroid use, orthopaedic, respiratory, cardiac, psychological, and gastrointestinal problems meaning that a comprehensive transition process is required. A sub-optimal transition into adult care can have disruptive and deleterious consequences for a patient's long-term care. This paper details the results of a consensus amongst clinicians on transitioning adolescent DMD patients from paediatric to adult neurologists that can act as a guide to best practice to ensure patients have continuous comprehensive care at every stage of their journey. METHODS: The consensus was derived using the Delphi methodology. Fifty-three statements were developed by a Steering Group (the authors of this paper) covering seven topics: Define the goals of transition, Preparing the patient, carers/parents and the adult centre, The transition process at the paediatric centre, The multidisciplinary transition summary - Principles, The multidisciplinary transition summary - Content, First visit in the adult centre, Evaluation of transition. The statements were shared with paediatric and adult neurologists across Central Eastern Europe (CEE) as a survey requesting their level of agreement with each statement. RESULTS: Data from 60 responders (54 full responses and six partial responses) were included in the data set analysis. A consensus was agreed across 100% of the statements. CONCLUSIONS: It is hoped that the findings of this survey which sets out agreed best practice statements, and the transfer template documents developed, will be widely used and so facilitate an effective transition from paediatric to adult care for adolescents with DMD.

Cardiomyopathy in Duchenne Muscular Dystrophy and the Potential for Mitochondrial Therapeutics to Improve Treatment Response.

Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disease caused by mutations to the dystrophin gene, resulting in deficiency of dystrophin protein, loss of myofiber integrity in skeletal and cardiac muscle, and eventual cell death and replacement with fibrotic tissue. Pathologic cardiac manifestations occur in nearly every DMD patient, with the development of cardiomyopathy-the leading cause of death-inevitable by adulthood. As early cardiac abnormalities are difficult to detect, timely diagnosis and appropriate treatment modalities remain a challenge. There is no cure for DMD; treatment is aimed at delaying disease progression and alleviating symptoms. A comprehensive understanding of the pathophysiological mechanisms is crucial to the development of targeted treatments. While established hypotheses of underlying mechanisms include sarcolemmal weakening, upregulation of pro-inflammatory cytokines, and perturbed ion homeostasis, mitochondrial dysfunction is thought to be a potential key contributor. Several experimental compounds targeting the skeletal muscle pathology of DMD are in development, but the effects of such agents on cardiac function remain unclear. The synergistic integration of small molecule- and gene-target-based drugs with metabolic-, immune-, or ion balance-enhancing compounds into a combinatorial therapy offers potential for treating dystrophin deficiency-induced cardiomyopathy, making it crucial to understand the underlying mechanisms driving the disorder.

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.

Split intein-mediated protein trans-splicing to express large dystrophins.

Gene replacement using adeno-associated virus (AAV) vectors is a promising therapeutic approach for many diseases1,2. However, this therapeutic modality is challenged by the packaging capacity of AAVs (approximately 4.7 kilobases)3, limiting its application for disorders involving large coding sequences, such as Duchenne muscular dystrophy, with a 14 kilobase messenger RNA. Here we developed a new method for expressing large dystrophins by utilizing the protein trans-splicing mechanism mediated by split inteins. We identified several split intein pairs that efficiently join two or three fragments to generate a large midi-dystrophin or the full-length protein. We show that delivery of two or three AAVs into dystrophic mice results in robust expression of large dystrophins and significant physiological improvements compared with micro-dystrophins. Moreover, using the potent myotropic AAVMYO4, we demonstrate that low total doses (2 × 1013 viral genomes per kg) are sufficient to express large dystrophins in striated muscles body-wide with significant physiological corrections in dystrophic mice. Our data show a clear functional superiority of large dystrophins over micro-dystrophins that are being tested in clinical trials. This method could benefit many patients with Duchenne or Becker muscular dystrophy, regardless of genotype, and could be adapted to numerous other disorders caused by mutations in large genes that exceed the AAV capacity.

Quantifying the insurance value for rare diseases: Duchenne muscular dystrophy.

OBJECTIVES: To quantify the magnitude of an ISPOR novel value element, insurance value, as applied to new treatments for a rare, severe disease with pediatric onset: Duchenne muscular dystrophy (DMD). STUDY DESIGN: Prospective survey of individuals planning to have children in the future. METHODS: A survey was administered to US adults (aged ≥ 21 years) planning to have a child in the future to elicit willingness to pay (WTP) for insurance coverage for a new hypothetical DMD treatment that improved mortality and morbidity relative to the current standard of care. To identify an indifference point between status quo insurance and insurance with additional cost that would cover the treatment if respondents had a child with DMD, a multiple random staircase design was used. Insurance value-the value individuals receive from a reduction in future health risks-was calculated as the difference between respondent's WTP and what a risk-neutral individual would pay. The risk-neutral value was the product of the (1) probability of having a child with DMD (decision weighted), (2) quality-adjusted life-years (QALYs) gained from the new treatment, and (3) WTP per QALY. RESULTS: Among 207 respondents, 80.2% (n = 166) were aged 25 to 44 years, and 59.9% (n = 124) were women. WTP for insurance coverage of the hypothetical treatment was $973 annually, whereas the decision-weighted risk-neutral value was $452 per year. Thus, insurance value constituted 53.5% ($520) of value for new DMD treatments. CONCLUSIONS: Individuals planning to have children in the future are willing to pay more for insurance coverage of novel DMD treatments than is assumed under risk-neutral, QALY-based frameworks.

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.

mRNA-specific readthrough of nonsense codons by antisense oligonucleotides (R-ASOs).

Nonsense mutations account for >10% of human genetic disorders, including cystic fibrosis, Alagille syndrome, and Duchenne muscular dystrophy. A nonsense mutation results in the expression of a truncated protein, and therapeutic strategies aim to restore full-length protein expression. Most strategies under development, including small-molecule aminoglycosides, suppressor tRNAs, or the targeted degradation of termination factors, lack mRNA target selectivity and may poorly differentiate between nonsense and normal stop codons, resulting in off-target translation errors. Here, we demonstrate that antisense oligonucleotides can stimulate readthrough of disease-causing nonsense codons, resulting in high yields of full-length protein in mammalian cellular lysate. Readthrough efficiency depends on the sequence context near the stop codon and on the precise targeting position of an oligonucleotide, whose interaction with mRNA inhibits peptide release to promote readthrough. Readthrough-inducing antisense oligonucleotides (R-ASOs) enhance the potency of non-specific readthrough agents, including aminoglycoside G418 and suppressor tRNA, enabling a path toward target-specific readthrough of nonsense mutations in CFTR, JAG1, DMD, BRCA1 and other mutant genes. Finally, through systematic chemical engineering, we identify heavily modified fully functional R-ASO variants, enabling future therapeutic development.

Adenine base editing-mediated exon skipping restores dystrophin in humanized Duchenne mouse model.

Duchenne muscular dystrophy (DMD) affecting 1 in 3500-5000 live male newborns is the frequently fatal genetic disease resulted from various mutations in DMD gene encoding dystrophin protein. About 70% of DMD-causing mutations are exon deletion leading to frameshift of open reading frame and dystrophin deficiency. To facilitate translating human DMD-targeting CRISPR therapeutics into patients, we herein establish a genetically humanized mouse model of DMD by replacing exon 50 and 51 of mouse Dmd gene with human exon 50 sequence. This humanized mouse model recapitulats patient's DMD phenotypes of dystrophin deficiency and muscle dysfunction. Furthermore, we target splicing sites in human exon 50 with adenine base editor to induce exon skipping and robustly restored dystrophin expression in heart, tibialis anterior and diaphragm muscles. Importantly, systemic delivery of base editor via adeno-associated virus in the humanized male mouse model improves the muscle function of DMD mice to the similar level of wildtype ones, indicating the therapeutic efficacy of base editing strategy in treating most of DMD types with exon deletion or point mutations via exon-skipping induction.