title: Dystrophin rescue by trans-splicing: a strategy for DMD genotypes not eligible for exon skipping approaches.
abstract: RNA-based therapeutic approaches using splice-switching oligonucleotides have been successfully applied to rescue dystrophin in Duchenne muscular dystrophy (DMD) preclinical models and are currently being evaluated in DMD patients. Although the modular structure of dystrophin protein tolerates internal deletions, many mutations that affect nondispensable domains of the protein require further strategies. Among these, trans-splicing technology is particularly attractive, as it allows the replacement of any mutated exon by its normal version as well as introducing missing exons or correcting duplication mutations. We have applied such a strategy in vitro by using cotransfection of pre-trans-splicing molecule (PTM) constructs along with a reporter minigene containing part of the dystrophin gene harboring the stop-codon mutation found in the mdx mouse model of DMD. Optimization of the different functional domains of the PTMs allowed achieving accurate and efficient trans-splicing of up to 30% of the transcript encoded by the cotransfected minigene. Optimized parameters included mRNA stabilization, choice of splice site sequence, inclusion of exon splice enhancers and artificial intronic sequence. Intramuscular delivery of adeno-associated virus vectors expressing PTMs allowed detectable levels of dystrophin in mdx and mdx4Cv, illustrating that a given PTM can be suitable for a variety of mutations.
authors.affiliation: Thérapie des maladies du muscle strié, Um76 UPMC - UMR 7215 CNRS - U974 Inserm - Institut de Myologie, 75013 Paris, France and UFR des Sciences de la Santé, Université de Versailles Saint-Quentin-en-Yvelines, 78180 Montigny-le-Bretonneux, France.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Lorain
authors.forename: Cécile
authors.initials: C
authors.lastname: Peccate
authors.forename: Maëva
authors.initials: M
authors.lastname: Le Hir
authors.forename: Graziella
authors.initials: G
authors.lastname: Griffith
authors.forename: Susanne
authors.initials: S
authors.lastname: Philippi
authors.forename: Guillaume
authors.initials: G
authors.lastname: Précigout
authors.forename: Kamel
authors.initials: K
authors.lastname: Mamchaoui
authors.forename: Arnaud
authors.initials: A
authors.lastname: Jollet
authors.forename: Thomas
authors.initials: T
authors.lastname: Voit
authors.forename: Luis
authors.initials: L
authors.lastname: Garcia
mesh_terms: D000818:Animals; D000229:Dependovirus / Q000235:genetics; D016189:Dystrophin / Q000032:analysis / Q000235:genetics*; D005091:Exons; D005822:Genetic Vectors; D005838:Genotype; D006801:Humans; D007438:Introns; D051379:Mice; D018101:Mice, Inbred mdx; D018485:Muscle Fibers, Skeletal / Q000737:chemistry; D009132:Muscles / Q000737:chemistry; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics; D041681:NIH 3T3 Cells; D022821:RNA Splice Sites; D012333:RNA, Messenger / Q000032:analysis; D020040:Trans-Splicing
chemical_list: D016189:Dystrophin; D022821:RNA Splice Sites; D012333:RNA, Messenger