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.

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.

scholarly journals Comparison of Long-term Ambulatory Function in Patients with Duchenne Muscular Dystrophy Treated with Eteplirsen and Matched Natural History Controls

2021 ◽  
pp. 1-11
Author(s):  
Jerry R. Mendell ◽  
Navid Khan ◽  
Nanshi Sha ◽  
Helen Eliopoulos ◽  
Craig M. McDonald ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Gene Mutations ◽  
Exon Skipping ◽  
Term Treatment ◽  
Long Term Treatment ◽  
The Difference ◽  
Dmd Gene ◽  
6 Minute Walk Test

Background: Duchenne muscular dystrophy (DMD) is a rare, X-linked, fatal, degenerative neuromuscular disease caused by DMD gene mutations. A relationship between exon skipping and dystrophin production in exon 51-amenable patients treated with eteplirsen (EXONDYS 51 ®) is established. Once-weekly eteplirsen significantly increased dystrophin, with slower decline in ambulatory function compared to baseline. Long-term treatment with eteplirsen leads to accumulation of dystrophin over time and observed functional benefits in patients with DMD. Objective: Compare long-term ambulatory function in eteplirsen-treated patients versus controls. Methods: Study 201/202 included 12 eteplirsen-treated patients assessed twice/year for ambulatory function over 4 years. Ambulatory evaluations (6-minute walk test [6MWT], loss of ambulation, and North Star Ambulatory Assessment [NSAA]) were compared with matched controls from Italian Telethon and Leuven registries. Results: At Years 3 and 4, eteplirsen-treated patients demonstrated markedly greater mean 6MWT than controls (difference in change from baseline of 132 m [95%CI (29, 235), p = 0.015] at Year 3 and 159 m [95%CI (66, 253), p = 0.002] at Year 4). At Year 4, a significantly greater proportion of eteplirsen-treated patients were still ambulant versus controls (10/12 vs 3/11; p = 0.020). At Year 3, eteplirsen-treated patients had milder NSAA decline than controls (difference in change from baseline of 2.6, 95%CI [-6, 11]), however, the difference was not statistically significant; Year 4 control NSAA data were not available. Conclusion: In this retrospective matched control study, eteplirsen treatment resulted in attenuation of ambulatory decline over a 4-year observation period, supporting long-term benefit in patients with DMD.

scholarly journals The lncRNA 44s2 Study Applicability to the Design of 45-55 Exon Skipping Therapeutic Strategy for DMD

Biomedicines ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 219
Author(s):  
Elena Gargaun ◽  
Sestina Falcone ◽  
Guilhem Solé ◽  
Julien Durigneux ◽  
Andoni Urtizberea ◽  
...  
Keyword(s):  
Muscular Dystrophy ◽  
Gene Mutations ◽  
Exon Skipping ◽  
Becker Muscular Dystrophy ◽  
Protein Stabilization ◽  
Progressive Muscular Dystrophy ◽  
Dilatative Cardiomyopathy ◽  
Proliferation And Differentiation ◽  
Dmd Gene ◽  
Dystrophin Protein

In skeletal muscle, long noncoding RNAs (lncRNAs) are involved in dystrophin protein stabilization but also in the regulation of myocytes proliferation and differentiation. Hence, they could represent promising therapeutic targets and/or biomarkers for Duchenne and Becker muscular dystrophy (DMD/BMD). DMD and BMD are X-linked myopathies characterized by a progressive muscular dystrophy with or without dilatative cardiomyopathy. Two-thirds of DMD gene mutations are represented by deletions, and 63% of patients carrying DMD deletions are eligible for 45 to 55 multi-exons skipping (MES), becoming BMD patients (BMDΔ45-55). We analyzed the genomic lncRNA presence in 38 BMDΔ45-55 patients and characterized the lncRNA localized in introns 44 and 55 of the DMD gene. We highlighted that all four lncRNA are differentially expressed during myogenesis in immortalized and primary human myoblasts. In addition, the lncRNA44s2 was pointed out as a possible accelerator of differentiation. Interestingly, lncRNA44s expression was associated with a favorable clinical phenotype. These findings suggest that lncRNA44s2 could be involved in muscle differentiation process and become a potential disease progression biomarker. Based on these results, we support MES45-55 therapy and propose that the design of the CRISPR/Cas9 MES45-55 assay consider the lncRNA sequences bordering the exonic 45 to 55 deletion.

scholarly journals Single Exon Skipping Can Address a Multi-Exon Duplication in the Dystrophin Gene

2020 ◽  
Vol 21 (12) ◽  
pp. 4511 ◽  
Author(s):  
Kane Greer ◽  
Russell Johnsen ◽  
Yoram Nevo ◽  
Yakov Fellig ◽  
Susan Fletcher ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Exon Skipping ◽  
Dystrophin Gene ◽  
Duchenne Muscular Dystrophy Patient ◽  
Exon Duplication ◽  
Dmd Gene ◽  
Muscle Wasting Disease ◽  
Phosphorodiamidate Morpholino Oligomers

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease typically caused by protein-truncating mutations that preclude synthesis of a functional dystrophin. Exonic deletions are the most common type of DMD lesion, however, whole exon duplications account for between 10–15% of all reported mutations. Here, we describe in vitro evaluation of antisense oligonucleotide-induced splice switching strategies to re-frame the transcript disrupted by a multi-exon duplication within the DMD gene. Phosphorodiamidate morpholino oligomers and phosphorodiamidate morpholino oligomers coupled to a cell penetrating peptide were evaluated in a Duchenne muscular dystrophy patient cell strain carrying an exon 14–17 duplication. Two strategies were employed; the conventional approach was to remove both copies of exon 17 in addition to exon 18, and the second strategy was to remove only the first copy of exon 17. Both approaches result in a larger than normal but in-frame DMD transcript, but surprisingly, the removal of only the first exon 17 appeared to be more efficient in restoring dystrophin, as determined using western blotting. The emergence of a normal sized DMD mRNA transcript that was not apparent in untreated samples may have arisen from back splicing and could also account for some of the dystrophin protein being produced.

scholarly journals New Approach for Antisense Oligonucleotide-Mediated Exon Skipping in Duchenne Muscular Dystrophy

2012 ◽  
Vol 16 (4) ◽  
pp. 521-526
Author(s):  
Yoshitsugu Aoki ◽  
◽  
Tetsuya Nagata ◽  
Shin’ichi Takeda
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Antisense Oligonucleotide ◽  
Antisense Oligonucleotides ◽  
Exon Skipping ◽  
New Approach ◽  
Reading Frame ◽  
Promising Therapeutic Approach ◽  
Dmd Gene ◽  
Dystrophin Protein

Duchenne Muscular Dystrophy (DMD) is a lethalmuscle disorder characterized by mutations in the DMD gene. These mutations primarily disrupt the reading frame, resulting in the absence of functional dystrophin protein. Exon skipping, which involves the use of antisense oligonucleotides is a promising therapeutic approach for DMD, and clinical trials on exon skipping are currently underway in DMD patients. Recently, stable and less-toxic antisense oligonucleotides with higher efficacy have been developed in mouse and dog models of DMD. This review highlights a new approach for antisense oligonucleotide-based therapeutics for DMD, particularly for exon skipping-based methods.

scholarly journals A Dystrophin Exon-52 Deleted Miniature Pig Model of Duchenne Muscular Dystrophy and Evaluation of Exon Skipping

2021 ◽  
Vol 22 (23) ◽  
pp. 13065
Author(s):  
Yusuke Echigoya ◽  
Nhu Trieu ◽  
William Duddy ◽  
Hong M. Moulton ◽  
HaiFang Yin ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Exon Skipping ◽  
Pig Model ◽  
Advanced Technology ◽  
Large Animal ◽  
Miniature Pig ◽  
Cardiac Phenotype ◽  
Dmd Gene

Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disorder caused by mutations in the DMD gene and the subsequent lack of dystrophin protein. Recently, phosphorodiamidate morpholino oligomer (PMO)-antisense oligonucleotides (ASOs) targeting exon 51 or 53 to reestablish the DMD reading frame have received regulatory approval as commercially available drugs. However, their applicability and efficacy remain limited to particular patients. Large animal models and exon skipping evaluation are essential to facilitate ASO development together with a deeper understanding of dystrophinopathies. Using recombinant adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer, we generated a Yucatan miniature pig model of DMD with an exon 52 deletion mutation equivalent to one of the most common mutations seen in patients. Exon 52-deleted mRNA expression and dystrophin deficiency were confirmed in the skeletal and cardiac muscles of DMD pigs. Accordingly, dystrophin-associated proteins failed to be recruited to the sarcolemma. The DMD pigs manifested early disease onset with severe bodywide skeletal muscle degeneration and with poor growth accompanied by a physical abnormality, but with no obvious cardiac phenotype. We also demonstrated that in primary DMD pig skeletal muscle cells, the genetically engineered exon-52 deleted pig DMD gene enables the evaluation of exon 51 or 53 skipping with PMO and its advanced technology, peptide-conjugated PMO. The results show that the DMD pigs developed here can be an appropriate large animal model for evaluating in vivo exon skipping efficacy.

scholarly journals Mutation-Based Therapeutic Strategies for Duchenne Muscular Dystrophy: From Genetic Diagnosis to Therapy

2019 ◽  
Vol 9 (1) ◽  
pp. 16 ◽  
Author(s):  
Akinori Nakamura
Keyword(s):  
Muscular Dystrophy ◽  
Single Gene ◽  
Genetic Diagnosis ◽  
Exon Skipping ◽  
Becker Muscular Dystrophy ◽  
Therapeutic Strategies ◽  
Severe Phenotype ◽  
Reading Frame ◽  
Dystrophin Expression ◽  
Dmd Gene

Duchenne and Becker muscular dystrophy (DMD/BMD) are X-linked muscle disorders caused by mutations of the DMD gene, which encodes the subsarcolemmal protein dystrophin. In DMD, dystrophin is not expressed due to a disruption in the reading frame of the DMD gene, resulting in a severe phenotype. Becker muscular dystrophy exhibits a milder phenotype, having mutations that maintain the reading frame and allow for the production of truncated dystrophin. To date, various therapeutic approaches for DMD have been extensively developed. However, the pathomechanism is quite complex despite it being a single gene disorder, and dystrophin is expressed not only in a large amount of skeletal muscle but also in cardiac, vascular, intestinal smooth muscle, and nervous system tissue. Thus, the most appropriate therapy would be complementation or restoration of dystrophin expression, such as gene therapy using viral vectors, readthrough therapy, or exon skipping therapy. Among them, exon skipping therapy with antisense oligonucleotides can restore the reading frame and yield the conversion of a severe phenotype to one that is mild. In this paper, I present the significance of molecular diagnosis and the development of mutation-based therapeutic strategies to complement or restore dystrophin expression.

Duchenne Muscular Dystrophy (DMD) Treatment: Past and Present Perspectives

2020 ◽  
Author(s):  
Nahla O. Mousa ◽  
Ahmed Osman ◽  
Nagia Fahmy ◽  
Ahmed Abdellatif ◽  
Waheed K. Zahra
Keyword(s):  
Stem Cell ◽  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Genetic Defect ◽  
Exon Skipping ◽  
Disease Treatment ◽  
Pulmonary Functions ◽  
Progressive Muscle Weakness ◽  
Dmd Gene ◽  
Dystrophin Protein

Duchenne muscular dystrophy (DMD) is one of the fatal X-linked disorders that are characterized by progressive muscle weakness and occur due to mutation in the largest human gene known as the DMD gene which encodes dystrophin protein that is mandatory for keeping the muscles structurally and functionally intact. The disease always affects boys (1 from every ~5000), and in some cases the female carriers are symptomatic. The disease usually leads to impairment in cardiac and pulmonary functions leading to the death of the patients in very young ages. Understanding DMD through precise molecular diagnosis will aid in determining the suitable therapeutic approach for the cases like designing exon-skipping antisense oligonucleotides (AOs) or stem cell-based therapies in conjunction with gene editing techniques (CRISPR/Cas9). Such therapies can correct the genetic defect in the DMD gene and ameliorate the symptoms. In this chapter, we will illustrate the past and current strategies for DMD disease treatment.

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.

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