title: Efficient bypass of mutations in dysferlin deficient patient cells by antisense-induced exon skipping.
abstract: Mutations in DYSF encoding dysferlin cause primary dysferlinopathies, autosomal recessive diseases that mainly present clinically as Limb Girdle Muscular Dystrophy type 2B and Miyoshi myopathy. More than 350 different sequence variants have been reported in DYSF. Like dystrophin, the size of the dysferlin mRNA is above the limited packaging size of AAV vectors. Alternative strategies to AAV gene transfer in muscle cells must then be addressed for patients. A gene therapy approach for Duchenne muscular dystrophy was recently developed, based on exon-skipping strategy. Numerous sequences are recognized by splicing protein complexes and, when specifically blocked by antisense oligoucleotides (AON), the corresponding exon is skipped. We hypothesized that this approach could be useful for patients affected with dysferlinopathies. To confirm this assumption, exon 32 was selected as a prioritary target for exon skipping strategy. This option was initially driven by the report from Sinnreich and colleagues of a patient with a very mild and late-onset phenotype associated to a natural skipping of exon 32. Three different antisense oligonucleotides were tested in myoblasts generated from control and patient MyoD transduced fibroblasts, either as oligonucleotides or after incorporation into lentiviral vectors. These approaches led to a high efficiency of exon 32 skipping. Therefore, these results seem promising, and could be applied to several other exons in the DYSF gene. Patients carrying mutations in exons whose the in-frame suppression has been proven to have no major consequences on the protein function, might benefit of exon-skipping based gene correction.
authors: Université de la Méditerranée, Inserm UMR_S 910 Génétique Médicale et Génomique Fonctionnelle, Faculté de Médecine de Marseille, France.
authors: Nicolas
authors: N
authors: Wein
authors: Aurélie
authors: A
authors: Avril
authors: Marc
authors: M
authors: Bartoli
authors: Cyriaque
authors: C
authors: Beley
authors: Soraya
authors: S
authors: Chaouch
authors: Pascal
authors: P
authors: Laforêt
authors: Anthony
authors: A
authors: Behin
authors: Gillian
authors: G
authors: Butler-Browne
authors: Vincent
authors: V
authors: Mouly
authors: Martin
authors: M
authors: Krahn
authors: Luis
authors: L
authors: Garcia
authors: Nicolas
authors: N
authors: Lévy
mesh_terms: D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D018389:Codon, Nonsense / Q000235:genetics; D004252:DNA Mutational Analysis; D000073939:Dysferlin; D005091:Exons / Q000235:genetics*; D005347:Fibroblasts / Q000378:metabolism* / Q000473:pathology*; D016368:Frameshift Mutation / Q000235:genetics; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008565:Membrane Proteins / Q000172:deficiency* / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000172:deficiency* / Q000235:genetics*; D009154:Mutation / Q000235:genetics*; D016376:Oligonucleotides, Antisense / Q000378:metabolism*
chemical_list: D018389:Codon, Nonsense; C114839:DYSF protein, human; D000073939:Dysferlin; D008565:Membrane Proteins; D009124:Muscle Proteins; D016376:Oligonucleotides, Antisense