Dysferlinopathies are a family of disabling muscular dystrophies with LGMD2B and Miyoshi myopathy as the main phenotypes. They are associated with molecular defects in DYSF, which encodes dysferlin, a key player in sarcolemmal homeostasis. Previous investigations have suggested that exon skipping may be a promising therapy for a subset of patients with dysferlinopathies. Such an approach aims to rescue functional proteins when targeting modular proteins and specific tissues. We sought to evaluate the dysferlin functional recovery following exon 32 skipping in the cells of affected patients. Exon skipping efficacy was characterized at several levels by use of in vitro myotube formation assays and quantitative membrane repair and recovery tests. Data obtained from these assessments confirmed that dysferlin function is rescued by quasi-dysferlin expression in treated patient cells, supporting the case for a therapeutic antisense-based trial in a subset of dysferlin-deficient patients.
Last. Luis Garcia(French Institute of Health and Medical Research)H-Index: 28
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The modification of the pre-mRNA cis-splicing process employing a pre-mRNA trans-splicing molecule (PTM) is an attractive strategy for the in situ correction of genes whose careful transcription regulation and full-length expression is determinative for protein function, as it is the case for the dysferlin (DYSF, Dysf) gene. Loss-of-function mutations of DYSF result in different types of muscular dystrophy mainly manifesting as limb girdle muscular dystrophy 2B (LGMD2B) and Miyoshi muscular dyst...
Objective Dysferlinopathies are a family of untreatable muscle disorders caused by mutations in the dysferlin gene. Lack of dysferlin protein results in progressive dystrophy with chronic muscle fiber loss, inflammation, fat replacement, and fibrosis; leading to deteriorating muscle weakness. The objective of this work is to demonstrate efficient and safe restoration of dysferlin expression following gene therapy treatment. Methods Traditional gene therapy is restricted by the packaging capacity...
Last. Marc Bartoli(AMU: Aix-Marseille University)H-Index: 30
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Missense, iso-semantic, and intronic muta- tions are challenging for interpretation, in particular for their impact in mRNA. Various tools such as the Human Splicing Finder (HSF) system could be used to predict the impact on splicing; however, no diagnosis result could rely on predictions alone, but requires functional testing. Here, we report an in vitro approach to study the im- pact of DYSF mutations on splicing. It was evaluated on a series of 45 DYSF mutations, both intronic and exonic. We ...
#2Natalie Woolger(Children's Hospital at Westmead)H-Index: 3
Last. St T. Cooper(USYD: University of Sydney)H-Index: 1
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Dysferlin and calpain are important mediators of the emergency response to repair plasma membrane injury. Our previous research revealed that membrane injury induces cleavage of dysferlin to release a synaptotagmin-like C-terminal module we termed mini-dysferlinC72. Here we show that injury-activated cleavage of dysferlin is mediated by the ubiquitous calpains via a cleavage motif encoded by alternately spliced exon 40a. An exon 40a–specific antibody recognizing cleaved mini-dysferlinC72 intense...
Summary Background Duchenne muscular dystrophy is caused by dystrophin deficiency and muscle deterioration and preferentially affects boys. Antisense-oligonucleotide-induced exon skipping allows synthesis of partially functional dystrophin. We investigated the efficacy and safety of drisapersen, a 2′-O-methyl-phosphorothioate antisense oligonucleotide, given for 48 weeks. Methods In this exploratory, double-blind, placebo-controlled study we recruited male patients (≥5 years of age; time to rise...
#1Janice A. Dominov(UMMS: University of Massachusetts Medical School)H-Index: 15
#2Özgün Uyan(UMMS: University of Massachusetts Medical School)H-Index: 6
Last. Robert H. Brown(UMMS: University of Massachusetts Medical School)H-Index: 141
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Objective Mutations in dysferlin (DYSF), a Ca2+-sensitive ferlin family protein important for membrane repair, vesicle trafficking, and T-tubule function, cause Miyoshi myopathy, limb-girdle muscular dystrophy type 2B, and distal myopathy. More than 330 pathogenic DYSF mutations have been identified within exons or near exon–intron junctions. In ~17% of patients who lack normal DYSF, only a single disease-causing mutation has been identified. We studied one family with one known mutant allele to...
Deficits in membrane repair may contribute to disease progression in dysferlin-deficient muscular dystrophy. Dysferlin, a type-II transmembrane phospholipid-binding protein, is hypothesized to regulate fusion of repair vesicles with the sarcolemma to facilitate membrane repair, but the dysferlin-containing compartments involved in membrane repair and the mechanism by which these compartments contribute to resealing are unclear. A dysferlin-pHluorin [dysf-pH-sensitive green fluorescent protein (p...
Previously, we identified family with sequence similarity 65, member B (Fam65b), as a protein transiently up-regulated during differentiation and fusion of human myogenic cells. Silencing of Fam65b expression results in severe reduction of myogenin expression and consequent lack of myoblast fusion. The molecular function of Fam65b and whether misregulation of its expression could be causative of muscle diseases are unknown. Protein pulldowns were used to identify Fam65b-interacting proteins in d...
Last. Jyoti K. Jaiswal(GW: George Washington University)H-Index: 32
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Dysferlin deficiency compromises the repair of injured muscle, but the underlying cellular mechanism remains elusive. To study this phenomenon, we have developed mouse and human myoblast models for dysferlinopathy. These dysferlinopathic myoblasts undergo normal differentiation but have a deficit in their ability to repair focal injury to their cell membrane. Imaging cells undergoing repair showed that dysferlin-deficit decreased the number of lysosomes present at the cell membrane, resulting in...
Dysferlin is a calcium-binding transmembrane protein involved in membrane fusion and membrane repair. In humans, mutations in the dysferlin gene are associated with muscular dystrophy. In this study, we isolated plasma membrane-enriched fractions from full-grown immature oocytes of the sea star, and identified dysferlin by mass spectrometry analysis. The full-length dysferlin sequence is highly conserved between human and the sea star. We learned that in the sea star Patiria miniata, dysferlin R...
In 1995, we were the first to propose antisense oligonucleotide (ASO)-mediated exon-skipping therapy for the treatment of Duchenne muscular dystrophy (DMD), a noncurable, progressive muscle-wasting disease. DMD is caused by deletion mutations in one or more exons of the DMD gene that shift the translational reading frame and create a premature stop codon, thus prohibiting dystrophin production. The therapy aims to correct out-of-frame mRNAs to produce in-frame transcripts by removing an exon dur...
#2Robert C. Griggs(URMC: University of Rochester Medical Center)H-Index: 85
Increased understanding of disease pathophysiology and advances in gene therapies and drug technologies are revolutionizing treatment of muscular dystrophies and motor neuron disorders (MNDs). New drugs have been approved for Duchenne muscular dystrophy, spinal muscular atrophy, and amyotrophic lateral sclerosis. For other diseases, new targets have been identified, and new therapies are in clinical trials. The impact of such therapies will be fully understood only in the next decades. Cost burd...
Caveolae are plasma membrane organelles that are, among many other features, involved in mechanosensing and mechanoprotection. Different tools have been developed to study caveolae-dependent mechanoprotection and had to be adapted to the tissue or cells studied, as these structures are found in almost every type of cells. This chapter focuses on a protocol combining the use of live-cell imaging, micropatterning, hypo-osmotic shock as a mechanical stress, and dyes such as calcein-AM and propidium...
Muscular dystrophy is a group of genetic disorders characterised by degeneration of muscles. Different forms of muscular dystrophy can show varying phenotypes with a wide range of age, severity and location of muscle deterioration. Many palliative care options are available for muscular dystrophy patients, but no curative treatment is available. Exon-skipping therapy aims to induce skipping of exons with disease-causing mutations and/or nearby exons to restore the reading frame, which results in...
: Muscular dystrophy (MD) is a group of progressive genetic diseases affecting the musculature that are characterized by inflammatory infiltrates, necrosis and connective tissue and fat replacement of the affected muscles. Unfortunately, treatments do not exist for the vast majority of MD patients. Adeno-associated viral vector (AAV)-based gene therapy is thus emerging as a potential treatment for many types of MD. Treatments strategies based on AAV are being adapted for replacement of mutant di...
#1Elizabeth M. Gibbs(UCLA: University of California, Los Angeles)H-Index: 10
#2Florian Barthélémy(UCLA: University of California, Los Angeles)H-Index: 8
Last. M. Carrie Miceli(UCLA: University of California, Los Angeles)H-Index: 22
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Abstract Duchenne muscular dystrophy is caused by mutations in the dystrophin-encoding DMD gene. While Duchenne is most commonly caused by large intragenic deletions that cause frameshift and complete loss of dystrophin expression, in-frame deletions in DMD can result in the expression of internally truncated dystrophin proteins and may be associated with a milder phenotype known as Becker muscular dystrophy. In this study, we describe two individuals with large in-frame 5’ deletions (exon 3-23 ...
#1Courtney S. Young(UCLA: University of California, Los Angeles)H-Index: 8
#2April D. Pyle(UCLA: University of California, Los Angeles)H-Index: 26
Last. Melissa J. Spencer(UCLA: University of California, Los Angeles)H-Index: 51
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This is a review describing advances in CRISPR/Cas-mediated therapies for neuromuscular disorders (NMDs). We explore both CRISPR-mediated editing and dead Cas approaches as potential therapeutic st...
Glucocorticoids are candidates for the pharmacological treatment of dysferlinopathy. Deflazacort, however, showed a worse effect on muscle strength than placebo. Alternate-day low-dose prednisone may have beneficial effects with fewer adverse effects. The outcomes for a female patient with dysferlinopathy (limb-girdle muscular dystrophy type 2B) were assessed by maximal voluntary isometric contraction (MVIC) using a newly devised chair and arm table with push-pull type strain gauges. Grip streng...
#1William Lostal(French Institute of Health and Medical Research)H-Index: 9
Last. Isabelle Richard(French Institute of Health and Medical Research)H-Index: 22
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Dysferlin deficiencies are responsible for muscular dystrophies with different presentations with the most frequent being limb-girdle muscular dystrophy type 2B and Miyoshi myopathy. The dysferlin (DYSF) gene is expressed through a 6.2 kb coding sequence, a size that impedes classical gene transfer approach using adeno-associated vectors (AAVs). There is no treatment for dysferlin deficiencies, but in the last decade, a large spectrum of strategies has been evaluated in different setups. In this...
#2Rika Maruyama(U of A: University of Alberta)H-Index: 14
Last. Toshifumi Yokota(U of A: University of Alberta)H-Index: 34
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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...