Efficient Skipping of Single Exon Duplications in <i>DMD</i> Patient-Derived Cell Lines Using an Antisense Oligonucleotide Approach

Wein, Nicolas; Vulin, Adeline; Findlay, Andrew; Gumienny, Felecia; Huang, Nianyuan; Wilton, Steve D.; Flanigan, Kevin M.

doi:10.3233/jnd-170233

Cited by 28 publications

(17 citation statements)

References 28 publications

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“…Among our Saudi patients with DMD gene deletions, the exons most frequently skipped were exons 51 (42.9%, 9/21), 53 (14.3%, 3/21), 44 (9.5%, 2/21), 45 (4.8%, 1/21), 43 (4.8%, 1/21), and 50 (4.8%, 1/21). Wein et al have recently reported the efficiency of exon skipping in the DMD gene, with each duplicated exon expressing a wild-type, full-length mRNA [ 66 ]. For more than one duplicated exon, several antisense oligomers can be delivered as a cocktail of drugs to skip larger regions of the transcript.…”

Section: Discussionmentioning

confidence: 99%

Molecular characterization of exonic rearrangements and frame shifts in the dystrophin gene in Duchenne muscular dystrophy patients in a Saudi community

Elhawary

Jiffri

Jambi³

et al. 2018

Hum Genomics

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BackgroundIn individuals with Duchenne muscular dystrophy (DMD), exon skipping treatment to restore a wild-type phenotype or correct the frame shift of the mRNA transcript of the dystrophin (DMD) gene are mutation-specific. To explore the molecular characterization of DMD rearrangements and predict the reading frame, we simultaneously screened all 79 DMD gene exons of 45 unrelated male DMD patients using a multiplex ligation-dependent probe amplification (MLPA) assay for deletion/duplication patterns. Multiplex PCR was used to confirm single deletions detected by the MLPA.ResultsThere was an obvious diagnostic delay, with an extremely statistically significant difference between the age at initial symptoms and the age of clinical evaluation of DMD cases (t value, 10.3; 95% confidence interval 5.95–8.80, P < 0.0001); the mean difference between the two groups was 7.4 years. Overall, we identified 147 intragenic rearrangements: 46.3% deletions and 53.7% duplications. Most of the deletions (92.5%) were between exons 44 and 56, with exon 50 being the most frequently involved (19.1%). Eight new rearrangements, including a mixed deletion/duplication and double duplications, were linked to seven cases with DMD. Of all the cases, 17.8% had duplications with no hot spots. In addition, confirmation of the reading frame hypothesis helped account for new DMD rearrangements in this study. We found that 81% of our Saudi patients would potentially benefit from exon skipping, of which 42.9% had a mutation amenable to skipping of exon 51.ConclusionsOur study could generate considerable data on mutational rearrangements that may promote future experimental therapies in Saudi Arabia.Electronic supplementary materialThe online version of this article (10.1186/s40246-018-0152-8) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Molecular characterization of exonic rearrangements and frame shifts in the dystrophin gene in Duchenne muscular dystrophy patients in a Saudi community

Elhawary

Jiffri

Jambi³

et al. 2018

Hum Genomics

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“…However, when skipping is too efficient and both exons are skipped, the reading frame will be disrupted. In cell cultures, generally both exons are skipped [43,44], although for some exons using lower AON doses can increase the amount of single exon skipping [44,45]. It is anticipated that in vivo the efficiency of AON delivery is much more limited and that it is very unlikely that two AONs target the same dystrophin transcript in one nucleus [8].…”

Section: Clinical Development For Small Mutations and Duplicationsmentioning

confidence: 99%

Developments in reading frame restoring therapy approaches for Duchenne muscular dystrophy

Schneider

Aartsma‐Rus

2020

Expert Opinion on Biological Therapy

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Introduction: Exon skipping compounds restoring the dystrophin transcript reading frame have received regulatory approval for Duchenne muscular dystrophy (DMD). Recently, focus shifted to developing compounds to skip additional exons, improving delivery to skeletal muscle, and to genome editing, to restore the reading frame on DNA level. Areas covered: We outline developments for reading frame restoring approaches, challenges of mutation specificity, and optimizing delivery. Also, we highlight ongoing efforts to better detect exon skipping therapeutic effects in clinical trials. Searches on relevant terms were performed, focusing on recent publications (<3 years). Expert opinion: Currently, 3 AONS are approved. Whether dystrophin levels are sufficient to slowdown disease progression needs to be confirmed. Enhancing AON uptake by muscles is currently under investigation. Gene editing is an alternative, but one that involves practical and ethical concerns. Given the field's momentum, we believe the efficiency of frame-restoring approaches will improve.

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“…AO-mediated exon skipping can correct the reading frame by removing the mutated exon and/or its flanking exon(s) from the DMD pre-mRNA, leading to a truncated but partly functional dystrophin protein, thus producing a milder phenotype as in the case of BMD patients [ 25 ]. In animal and cell models of DMD, exon skipping has been demonstrated to correct deletion, duplication, nonsense, and splice site mutations [ 26 , 27 , 28 , 29 ]. An antisense phosphorodiamidate morpholino oligomer (PMO) targeting exon 51, called eteplirsen or Exondys 51 (Sarepta Therapeutics, Cambridge, MA, USA), was conditionally approved by the Food and Drug Administration (FDA) in 2016, and several PMOs targeting other DMD exons, including golodirsen (SRP-4053) and NS-065/NCNP-01 (NS Pharma, Paramus, NJ, USA) are currently under clinical trials.…”

Section: Exon Skipping Therapy For Dmdmentioning

confidence: 99%

Immortalized Muscle Cell Model to Test the Exon Skipping Efficacy for Duchenne Muscular Dystrophy

Nguyen

Yokota

2017

JPM

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Duchenne muscular dystrophy (DMD) is a lethal genetic disorder that most commonly results from mutations disrupting the reading frame of the dystrophin (DMD) gene. Among the therapeutic approaches employed, exon skipping using antisense oligonucleotides (AOs) is one of the most promising strategies. This strategy aims to restore the reading frame, thus producing a truncated, yet functioning dystrophin protein. In 2016, the Food and Drug Administration (FDA) conditionally approved the first AO-based drug, eteplirsen (Exondys 51), developed for DMD exon 51 skipping. An accurate and reproducible method to quantify exon skipping efficacy is essential for evaluating the therapeutic potential of different AOs sequences. However, previous in vitro screening studies have been hampered by the limited proliferative capacity and insufficient amounts of dystrophin expressed by primary muscle cell lines that have been the main system used to evaluate AOs sequences. In this paper, we illustrate the challenges associated with primary muscle cell lines and describe a novel approach that utilizes immortalized cell lines to quantitatively evaluate the exon skipping efficacy in in vitro studies.

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Efficient Skipping of Single Exon Duplications in DMD Patient-Derived Cell Lines Using an Antisense Oligonucleotide Approach

Cited by 28 publications

References 28 publications

Molecular characterization of exonic rearrangements and frame shifts in the dystrophin gene in Duchenne muscular dystrophy patients in a Saudi community

Molecular characterization of exonic rearrangements and frame shifts in the dystrophin gene in Duchenne muscular dystrophy patients in a Saudi community

Developments in reading frame restoring therapy approaches for Duchenne muscular dystrophy

Immortalized Muscle Cell Model to Test the Exon Skipping Efficacy for Duchenne Muscular Dystrophy

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