Exon 32 Skipping of Dysferlin Rescues Membrane Repair in Patients’ Cells

Barthélémy, Florian; Blouin, Cédric M.; Wein, Nicolas; Mouly, Vincent; Courrier, Sébastien; Dionnet, Eugénie; Kergourlay, Virginie; Mathieu, Yves; Garcı́a, Luis; Butler‐Browne, Gillian; Lamaze, Christophe; Lévy, Nicolas; Krahn, Martin; Bartoli, Marc

doi:10.3233/jnd-150109

Cited by 33 publications

(27 citation statements)

References 46 publications

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“…Not all NMDs will be amenable to NHEJ-mediated gene editing. Based on successful exon skipping studies in limb girdle muscular dystrophies (LGMD) 2B and C (due to mutations in dysferlin or gamma-sarcoglycan respectively) (1,12,73,134), it has been suggested that CRISPR could be used to generate functional proteins by NHEJ. Other NMDs caused by aberrant expression of toxic proteins, such as facioscapulohumeral muscular dystrophy (FSHD), could also in theory be targeted by NHEJ.…”

Section: Nhej For Other Nmdsmentioning

confidence: 99%

CRISPR for Neuromuscular Disorders: Gene Editing and Beyond

Young

Spencer

2019

Physiology

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Section: Nhej For Other Nmdsmentioning

confidence: 99%

CRISPR for Neuromuscular Disorders: Gene Editing and Beyond

Young

Spencer

2019

Physiology

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“… 28 Exon-skipping progress in dysferlinopathy has been limited to the in vitro skipping of DYSF exon 32 in human dysferlinopathy patient cells. 29 To date, no other therapeutic exon-skipping targets have been identified for dysferlinopathy.…”

Section: Introductionmentioning

confidence: 99%

Identification of Novel Antisense-Mediated Exon Skipping Targets in DYSF for Therapeutic Treatment of Dysferlinopathy

Lee

Maruyama

Duddy

et al. 2018

Molecular Therapy - Nucleic Acids

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Dysferlinopathy is a progressive myopathy caused by mutations in the dysferlin (DYSF) gene. Dysferlin protein plays a major role in plasma-membrane resealing. Some patients with DYSF deletion mutations exhibit mild symptoms, suggesting some regions of DYSF can be removed without significantly impacting protein function. Antisense-mediated exon-skipping therapy uses synthetic molecules called antisense oligonucleotides to modulate splicing, allowing exons harboring or near genetic mutations to be removed and the open reading frame corrected. Previous studies have focused on DYSF exon 32 skipping as a potential therapeutic approach, based on the association of a mild phenotype with the in-frame deletion of exon 32. To date, no other DYSF exon-skipping targets have been identified, and the relationship between DYSF exon deletion pattern and protein function remains largely uncharacterized. In this study, we utilized a membrane-wounding assay to evaluate the ability of plasmid constructs carrying mutant DYSF, as well as antisense oligonucleotides, to rescue membrane resealing in patient cells. We report that multi-exon skipping of DYSF exons 26–27 and 28–29 rescues plasma-membrane resealing. Successful translation of these findings into the development of clinical antisense drugs would establish new therapeutic approaches that would be applicable to ∼5%–7% (exons 26–27 skipping) and ∼8% (exons 28–29 skipping) of dysferlinopathy patients worldwide.

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“…Antisense oligonucleotide (AON)-based splice-switching approaches have been developed for therapeutic use in human for Duchenne muscular dystrophy (DMD), spinal muscular atrophy, 15 , 16 , 17 , 18 , 19 , 20 and dysferlinopathy. 21 , 22 However, splice-modulating efficiency remains low in humans, and AONs must be repeatedly applied throughout life using invasive injection protocols, spurring research for more efficient and less invasive strategies. The U7 small nuclear RNA (snRNA)-encoded antisense RNAs differ from DNA-analog based AONs, in that they can easily be packaged into a suitable viral vector (i.e., recombinant adeno-associated virus [rAAV]) to transduce a wide variety of tissues, including skeletal muscle.…”

Section: Introductionmentioning

confidence: 99%

Exon Skipping in a Dysf-Missense Mutant Mouse Model

Malcher

Heidt

Goyenvalle

et al. 2018

Molecular Therapy - Nucleic Acids

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Limb girdle muscular dystrophy 2B (LGMD2B) is without treatment and caused by mutations in the dysferlin gene (DYSF). One-third is missense mutations leading to dysferlin aggregation and amyloid formation, in addition to defects in sarcolemmal repair and progressive muscle wasting. Dysferlin-null mouse models do not allow study of the consequences of missense mutations. We generated a new mouse model (MMex38) carrying a missense mutation in exon 38 in analogy to a clinically relevant human DYSF variant (DYSF p.Leu1341Pro). The targeted mutation induces all characteristics of missense mutant dysferlinopathy, including a progressive dystrophic pattern, amyloid formation, and defects in membrane repair. We chose U7 small nuclear RNA (snRNA)-based splice switching to demonstrate a possible exon-skipping strategy in this new animal model. We show that Dysf exons 37 and 38 can successfully be skipped in vivo. Overall, the MMex38 mouse model provides an ideal tool for preclinical development of treatment strategies for dysferlinopathy.

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Exon 32 Skipping of Dysferlin Rescues Membrane Repair in Patients’ Cells

Cited by 33 publications

References 46 publications

CRISPR for Neuromuscular Disorders: Gene Editing and Beyond

CRISPR for Neuromuscular Disorders: Gene Editing and Beyond

Identification of Novel Antisense-Mediated Exon Skipping Targets in DYSF for Therapeutic Treatment of Dysferlinopathy

Exon Skipping in a Dysf-Missense Mutant Mouse Model

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