title: Exon 45 skipping through U1-snRNA antisense molecules recovers the Dys-nNOS pathway and muscle differentiation in human DMD myoblasts.
abstract: Exon skipping has been demonstrated to be a successful strategy for the gene therapy of Duchenne muscular dystrophy (DMD): the rational being to convert severe Duchenne forms into milder Becker ones. Here, we show the selection of U1 snRNA-antisense constructs able to confer effective rescue of dystrophin synthesis in a Δ44 Duchenne genetic background, through skipping of exon 45; moreover, we demonstrate that the resulting dystrophin is able to recover timing of myogenic marker expression, to relocalize neuronal nitric oxide synthase (nNOS) and to rescue expression of miRNAs previously shown to be sensitive to the Dystrophin-nNOS-HDAC2 pathway. Becker mutations display different phenotypes, likely depending on whether the shorter protein is able to reconstitute the wide range of wild-type functions. Among them, efficient assembly of the dystrophin-associated protein complex (DAPC) and nNOS localization are important. Comparing different Becker deletions we demonstrate the correlation between the ability of the mutant dystrophin to relocalize nNOS and the expression levels of two miRNAs, miR-1 and miR29c, known to be involved in muscle homeostasis and to be controlled by the Dys-nNOS-HDAC2 pathway.
authors.affiliation: Department of Biology and Biotechnology "Charles Darwin," Institut Pasteur Fondazione Cenci-Bolognetti and IBPM, Rome, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Cazzella
authors.forename: Julie
authors.initials: J
authors.lastname: Martone
authors.forename: Chiara
authors.initials: C
authors.lastname: Pinnarò
authors.forename: Tiziana
authors.initials: T
authors.lastname: Santini
authors.forename: Shyam Sundar
authors.initials: SS
authors.lastname: Twayana
authors.forename: Olga
authors.initials: O
authors.lastname: Sthandier
authors.forename: Adele
authors.initials: A
authors.lastname: D'Amico
authors.forename: Valeria
authors.initials: V
authors.lastname: Ricotti
authors.forename: Enrico
authors.initials: E
authors.lastname: Bertini
authors.forename: Francesco
authors.initials: F
authors.lastname: Muntoni
authors.forename: Irene
authors.initials: I
authors.lastname: Bozzoni
mesh_terms: D000293:Adolescent; D017398:Alternative Splicing; D002454:Cell Differentiation; D002478:Cells, Cultured; D002648:Child; D002675:Child, Preschool; D003001:Cloning, Molecular; D016189:Dystrophin / Q000235:genetics* / Q000378:metabolism; D005091:Exons; D015316:Genetic Therapy; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism; D024510:Muscle Development; D020388:Muscular Dystrophy, Duchenne / Q000473:pathology / Q000503:physiopathology* / Q000628:therapy; D032448:Myoblasts, Skeletal / Q000378:metabolism / Q000502:physiology*; D052248:Nitric Oxide Synthase Type I / Q000378:metabolism*; D020320:Oligoribonucleotides, Antisense / Q000235:genetics; D061251:Primary Cell Culture; D021381:Protein Transport; D034622:RNA Interference; D012342:RNA, Small Nuclear / Q000235:genetics*; D015398:Signal Transduction
chemical_list: D016189:Dystrophin; D035683:MicroRNAs; D020320:Oligoribonucleotides, Antisense; D012342:RNA, Small Nuclear; C405483:U1 small nuclear RNA; D052248:Nitric Oxide Synthase Type I