scholarly journals Effects of Chronic, Maximal Phosphorodiamidate Morpholino Oligomer (PMO) Dosing on Muscle Function and Dystrophin Restoration in a Mouse Model of Duchenne Muscular Dystrophy

2021 ◽  
pp. 1-13
Author(s):  
Margaret E. Benny Klimek ◽  
Maria Candida Vila ◽  
Katie Edwards ◽  
Jessica Boehler ◽  
James Novak ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Muscle Function ◽  
Imaging Techniques ◽  
Exon Skipping ◽  
Becker Muscular Dystrophy ◽  
Mdx Mice ◽  
High Dose ◽  
Combination Treatments ◽  
Dystrophin Expression

Background: Phosphorodiamidate morpholino oligomer (PMO)-mediated exon skipping is currently used in clinical development to treat Duchenne muscular dystrophy (DMD), with four exon-skipping drugs achieving regulatory approval. Exon skipping elicits a truncated, but semi-functional dystrophin protein, similar to the truncated dystrophin expressed in patients with Becker Muscular dystrophy (BMD) where the disease phenotype is less severe than DMD. Despite promising results in both dystrophic animal models and DMD boys, restoration of dystrophin by exon skipping is highly variable, leading to contradictory functional outcomes in clinical trials. Objective: To develop optimal PMO dosing protocols that result in increased dystrophin and improved outcome measures in preclinical models of DMD. Methods: Tested effectiveness of multiple chronic, high dose PMO regimens using biochemical, histological, molecular, and imaging techniques in mdx mice. Results: A chronic, monthly regimen of high dose PMO increased dystrophin rescue in mdx mice and improved specific force in the extensor digitorum longus (EDL) muscle. However, monthly high dose PMO administration still results in variable dystrophin expression localized throughout various muscles. Conclusions: High dose monthly PMO administration restores dystrophin expression and increases muscle force; however, the variability of dystrophin expression at both the inter-and intramuscular level remains. Additional strategies to optimize PMO uptake including increased dosing frequencies or combination treatments with other yet-to-be-defined therapies may be necessary to achieve uniform dystrophin restoration and increases in muscle function.

Eteplirsen treatment for Duchenne muscular dystrophy

Neurology ◽  
2018 ◽  
Vol 90 (24) ◽  
pp. e2146-e2154 ◽  
Author(s):  
Jay S. Charleston ◽  
Frederick J. Schnell ◽  
Johannes Dworzak ◽  
Cas Donoghue ◽  
Sarah Lewis ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Western Blot ◽  
Reverse Transcription ◽  
Exon Skipping ◽  
Fold Increase ◽  
Cell Penetration ◽  
Reverse Transcription Pcr ◽  
Dystrophin Expression ◽  
Immunofluorescence Intensity

ObjectiveTo describe the quantification of novel dystrophin production in patients with Duchenne muscular dystrophy (DMD) after long-term treatment with eteplirsen.MethodsClinical study 202 was an observational, open-label extension of the randomized, controlled study 201 assessing the safety and efficacy of eteplirsen in patients with DMD with a confirmed mutation in the DMD gene amenable to correction by skipping of exon 51. Patients received once-weekly IV doses of eteplirsen 30 or 50 mg/kg. Upper extremity muscle biopsy samples were collected at combined study week 180, blinded, and assessed for dystrophin-related content by Western blot, Bioquant software measurement of dystrophin-associated immunofluorescence intensity, and percent dystrophin-positive fibers (PDPF). Results were contrasted with matched untreated biopsies from patients with DMD. Reverse transcription PCR followed by Sanger sequencing of newly formed slice junctions was used to confirm the mechanism of action of eteplirsen.ResultsReverse transcription PCR analysis and sequencing of the newly formed splice junction confirmed that 100% of treated patients displayed the expected skipped exon 51 sequence. In treated patients vs untreated controls, Western blot analysis of dystrophin content demonstrated an 11.6-fold increase (p = 0.007), and PDPF analysis demonstrated a 7.4-fold increase (p < 0.001). The PDPF findings were confirmed in a re-examination of the sample (15.5-fold increase, p < 0.001). Dystrophin immunofluorescence intensity was 2.4-fold greater in treated patients than in untreated controls (p < 0.001).ConclusionTaken together, the 4 assays, each based on unique evaluation mechanisms, provided evidence of eteplirsen muscle cell penetration, exon skipping, and induction of novel dystrophin expression.Classification of evidenceThis study provides Class II evidence of the muscle cell penetration, exon skipping, and induction of novel dystrophin expression by eteplirsen, as confirmed by 4 assays.

scholarly journals Restoring Dystrophin Expression in Duchenne Muscular Dystrophy: Current Status of Therapeutic Approaches

2019 ◽  
Vol 9 (1) ◽  
pp. 1 ◽  
Author(s):  
Yuko Shimizu-Motohashi ◽  
Hirofumi Komaki ◽  
Norio Motohashi ◽  
Shin’ichi Takeda ◽  
Toshifumi Yokota ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Stop Codon ◽  
Genetic Disorder ◽  
Premature Stop Codon ◽  
Exon Skipping ◽  
Current Status ◽  
Therapeutic Approaches ◽  
Dystrophin Expression

Duchenne muscular dystrophy (DMD), a rare genetic disorder characterized by progressive muscle weakness, is caused by the absence or a decreased amount of the muscle cytoskeletal protein dystrophin. Currently, several therapeutic approaches to cure DMD are being investigated, which can be categorized into two groups: therapies that aim to restore dystrophin expression, and those that aim to compensate for the lack of dystrophin. Therapies that restore dystrophin expression include read-through therapy, exon skipping, vector-mediated gene therapy, and cell therapy. Of these approaches, the most advanced are the read-through and exon skipping therapies. In 2014, ataluren, a drug that can promote ribosomal read-through of mRNA containing a premature stop codon, was conditionally approved in Europe. In 2016, eteplirsen, a morpholino-based chemical capable of skipping exon 51 in premature mRNA, received conditional approval in the USA. Clinical trials on vector-mediated gene therapy carrying micro- and mini- dystrophin are underway. More innovative therapeutic approaches include CRISPR/Cas9-based genome editing and stem cell-based cell therapies. Here we review the current status of therapeutic approaches for DMD, focusing on therapeutic approaches that can restore dystrophin.

scholarly journals Exon skipping induced by nonsense/frameshift mutations in DMD gene results in Becker muscular dystrophy

2020 ◽  
Vol 139 (2) ◽  
pp. 247-255 ◽  
Author(s):  
Mariko Okubo ◽  
Satoru Noguchi ◽  
Shinichiro Hayashi ◽  
Harumasa Nakamura ◽  
Hirofumi Komaki ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Nonsense Mutation ◽  
Frameshift Mutation ◽  
Exon Skipping ◽  
Becker Muscular Dystrophy ◽  
Intronic Sequence ◽  
Frameshift Mutations ◽  
Dmd Gene ◽  
Accurate Quantification

AbstractDuchenne muscular dystrophy (DMD) is caused by a nonsense or frameshift mutation in the DMD gene, while its milder form, Becker muscular dystrophy (BMD) is caused by an in-frame deletion/duplication or a missense mutation. Interestingly, however, some patients with a nonsense mutation exhibit BMD phenotype, which is mostly attributed to the skipping of the exon containing the nonsense mutation, resulting in in-frame deletion. This study aims to find BMD cases with nonsense/frameshift mutations in DMD and to investigate the exon skipping rate of those nonsense/frameshift mutations. We searched for BMD cases with nonsense/frameshift mutations in DMD in the Japanese Registry of Muscular Dystrophy. For each DMD mutation identified, we constructed minigene plasmids containing one exon with/without a mutation and its flanking intronic sequence. We then introduced them into HeLa cells and measured the skipping rate of transcripts of the minigene by RT-qPCR. We found 363 cases with a nonsense/frameshift mutation in DMD gene from a total of 1497 dystrophinopathy cases in the registry. Among them, 14 had BMD phenotype. Exon skipping rates were well correlated with presence or absence of dystrophin, suggesting that 5% exon skipping rate is critical for the presence of dystrophin in the sarcolemma, leading to milder phenotypes. Accurate quantification of the skipping rate is important in understanding the exact functions of the nonsense/frameshift mutations in DMD and for interpreting the phenotypes of the BMD patients.

WED 237 Phosphorodiamidate morpholino oligomers for treatment of duchenne muscular dystrophy

2018 ◽  
Vol 89 (10) ◽  
pp. A34.2-A34
Author(s):  
Maresh Kate ◽  
Tiet May ◽  
Guglieri Michela ◽  
Domingos Joana ◽  
Straub Volker ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Western Blot ◽  
Clinical Studies ◽  
Novel Mutation ◽  
Cumulative Risk ◽  
Exon Skipping ◽  
Dystrophin Expression ◽  
Polymerase Chain ◽  
Phosphorodiamidate Morpholino Oligomers

Exon skipping is a novel, mutation-specific approach to treating patients with Duchenne muscular dystrophy (DMD). Phosphorodiamidate morpholino oligomers are nucleic acid analogues that selectively redirect pre-mRNA splicing to enable production of internally truncated dystrophin.In exon 51 skipping (eteplirsen; n=36) and exon 53 skipping (golodirsen; n=25) clinical studies, internally shortened dystrophin mRNA was observed in all treated patients (per reverse transcription polymerase chain reaction). Eteplirsen increased dystrophin expression 15.5-fold, 11.6-fold, and 2.4-fold vs untreated controls (percent dystrophin-positive fibres, Western blot, and immunohistochemistry intensity, respectively; all, p≤0.007) in a 180 week study, and 2.8-fold (Western blot; p=0.008) in a 48 week study. Golodirsen increased dystrophin expression 10.7-fold (Western blot) over baseline following 48 weeks of treatment. Over 4 years, versus comparable external controls, eteplirsen slowed ambulatory decline (6 min walk test difference, 165 m; p=0.001) and cumulative risk of losing ambulation (83% vs 17%). In 2 clinical studies that included non-ambulatory patients, eteplirsen slowed pulmonary decline versus natural history data (assessed by spirometry).Eteplirsen and golodirsen demonstrated clinical and biochemical effects in patients with DMD; ongoing studies of these compounds are further characterising their effects in various patient populations.

scholarly journals Menstrual Blood-derived Cells Confer Human Dystrophin Expression in the Murine Model of Duchenne Muscular Dystrophy via Cell Fusion and Myogenic Transdifferentiation

2007 ◽  
Vol 18 (5) ◽  
pp. 1586-1594 ◽  
Author(s):  
Chang-Hao Cui ◽  
Taro Uyama ◽  
Kenji Miyado ◽  
Masanori Terai ◽  
Satoru Kyo ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Progenitor Cells ◽  
Cell Fusion ◽  
Menstrual Blood ◽  
Mdx Mice ◽  
Dystrophin Expression ◽  
Human Dystrophin

Duchenne muscular dystrophy (DMD), the most common lethal genetic disorder in children, is an X-linked recessive muscle disease characterized by the absence of dystrophin at the sarcolemma of muscle fibers. We examined a putative endometrial progenitor obtained from endometrial tissue samples to determine whether these cells repair muscular degeneration in a murine mdx model of DMD. Implanted cells conferred human dystrophin in degenerated muscle of immunodeficient mdx mice. We then examined menstrual blood–derived cells to determine whether primarily cultured nontransformed cells also repair dystrophied muscle. In vivo transfer of menstrual blood–derived cells into dystrophic muscles of immunodeficient mdx mice restored sarcolemmal expression of dystrophin. Labeling of implanted cells with enhanced green fluorescent protein and differential staining of human and murine nuclei suggest that human dystrophin expression is due to cell fusion between host myocytes and implanted cells. In vitro analysis revealed that endometrial progenitor cells and menstrual blood–derived cells can efficiently transdifferentiate into myoblasts/myocytes, fuse to C2C12 murine myoblasts by in vitro coculturing, and start to express dystrophin after fusion. These results demonstrate that the endometrial progenitor cells and menstrual blood–derived cells can transfer dystrophin into dystrophied myocytes through cell fusion and transdifferentiation in vitro and in vivo.

scholarly journals Nutraceutical and pharmaceutical cocktails did not improve muscle function or reduce histological damage in D2-mdx mice

2019 ◽  
Vol 127 (4) ◽  
pp. 1058-1066
Author(s):  
Hannah R. Spaulding ◽  
Tiffany Quindry ◽  
Kayleen Hammer ◽  
John C. Quindry ◽  
Joshua T. Selsby
Keyword(s):  
Skeletal Muscle ◽  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Muscle Function ◽  
Sirtuin 1 ◽  
Mdx Mice ◽  
Limb Muscle ◽  
Nicotinamide Riboside ◽  
Histological Damage ◽  
Dystrophin Deficiency

Progressive muscle injury and weakness are hallmarks of Duchenne muscular dystrophy. We showed previously that quercetin (Q) partially protected dystrophic limb muscles from disease-related injury. As quercetin activates PGC-1α through Sirtuin-1, an NAD+-dependent deacetylase, the depleted NAD+ in dystrophic skeletal muscle may limit quercetin efficacy; hence, supplementation with the NAD+ donor, nicotinamide riboside (NR), may facilitate quercetin efficacy. Lisinopril (Lis) protects skeletal muscle and improves cardiac function in dystrophin-deficient mice; therefore, it was included in this study to evaluate the effects of lisinopril used with quercetin and NR. Our purpose was to determine the extent to which Q, NR, and Lis decreased dystrophic injury. We hypothesized that Q, NR, or Lis alone would improve muscle function and decrease histological injury and when used in combination would have additive effects. Muscle function of 11-mo-old DBA (healthy), D2-mdx (dystrophin-deficient), and D2-mdx mice was assessed after treatment with Q, NR, and/or Lis for 7 mo. To mimic typical pharmacology of patients with Duchenne muscular dystrophy, a group was treated with prednisolone (Pred) in combination with Q, NR, and Lis. At 11 mo of age, dystrophin deficiency decreased specific tension and tetanic force in the soleus and extensor digitorum longus muscles and was not corrected by any treatment. Dystrophic muscle was more sensitive to contraction-induced injury, which was partially offset in the QNRLisPred group, whereas fatigue was similar between all groups. Treatments did not decrease histological damage. These data suggest that treatment with Q, NR, Lis, and Pred failed to adequately maintain dystrophic limb muscle function or decrease histological damage. NEW & NOTEWORTHY Despite a compelling rationale and previous evidence to the contrary in short-term investigations, quercetin, nicotinamide riboside, or Lisinopril, alone or in combination, failed to restore muscle function or decrease histological injury in dystrophic limb muscle from D2-mdx mice after long-term administration. Importantly, we also found that in the D2-mdx model, an emerging and relatively understudied model of Duchenne muscular dystrophy dystrophin deficiency caused profound muscle dysfunction and histopathology in skeletal muscle.

scholarly journals Multiple Exon Skipping in the Duchenne Muscular Dystrophy Hot Spots: Prospects and Challenges

2018 ◽  
Vol 8 (4) ◽  
pp. 41 ◽  
Author(s):  
Yusuke Echigoya ◽  
Kenji Rowel Q. Lim ◽  
Akinori Nakamura ◽  
Toshifumi Yokota
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Exon Skipping ◽  
Becker Muscular Dystrophy ◽  
Therapeutic Modality ◽  
Therapeutic Concept ◽  
Patient Registration ◽  
Potential Therapeutic Application ◽  
Dmd Gene ◽  
Retrospective Database

Duchenne muscular dystrophy (DMD), a fatal X-linked recessive disorder, is caused mostly by frame-disrupting, out-of-frame deletions in the dystrophin (DMD) gene. Antisense oligonucleotide-mediated exon skipping is a promising therapy for DMD. Exon skipping aims to convert out-of-frame mRNA to in-frame mRNA and induce the production of internally-deleted dystrophin as seen in the less severe Becker muscular dystrophy. Currently, multiple exon skipping has gained special interest as a new therapeutic modality for this approach. Previous retrospective database studies represented a potential therapeutic application of multiple exon skipping. Since then, public DMD databases have become more useful with an increase in patient registration and advances in molecular diagnosis. Here, we provide an update on DMD genotype-phenotype associations using a global DMD database and further provide the rationale for multiple exon skipping development, particularly for exons 45–55 skipping and an emerging therapeutic concept, exons 3–9 skipping. Importantly, this review highlights the potential of multiple exon skipping for enabling the production of functionally-corrected dystrophin and for treating symptomatic patients not only with out-of-frame deletions but also those with in-frame deletions. We will also discuss prospects and challenges in multiple exon skipping therapy, referring to recent progress in antisense chemistry and design, as well as disease models.

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