title: Antisense modulation of both exonic and intronic splicing motifs induces skipping of a DMD pseudo-exon responsible for x-linked dilated cardiomyopathy.
abstract: Antisense-mediated exon skipping has proven to be efficacious for subsets of Duchenne muscular dystrophy mutations. This approach is based on targeting specific splicing motifs that interfere with the spliceosome assembly by steric hindrance. Proper exon recognition by the splicing machinery is thought to depend on exonic splicing enhancer sequences, often characterized by purine-rich stretches, representing potential targets for antisense-mediated exon skipping. We identified and functionally characterized two purine-rich regions located within dystrophin intron 11 and involved in splicing regulation of a pseudo-exon. A functional role for these sequences was suggested by a pure intronic DMD deletion causing X-linked dilated cardiomyopathy through the prevalent cardiac incorporation of the aberrant pseudo-exon, marked as Alu-exon, into the dystrophin transcript. The first splicing sequence is contained within the pseudo-exon, whereas the second is localized within its 3' intron. We demonstrated that the two sequences actually behave as splicing enhancers in cell-free splicing assays because their deletion strongly interferes with the pseudo-exon inclusion. Cell-free results were then confirmed in myogenic cells derived from the patient with X-linked dilated cardiomyopathy, by targeting the identified motifs with antisense molecules and obtaining a reduction in dystrophin pseudo-exon recognition. The splicing motifs identified could represent target sequences for a personalized molecular therapy in this particular DMD mutation. Our results demonstrated for the first time the role of intronic splicing sequences in antisense modulation with implications in exon skipping-mediated therapeutic approaches.
authors.affiliation: Department of Experimental and Diagnostic Medicine, University of Ferrara, Italy. rsp@unife.it
authors.forename: Paola
authors.initials: P
authors.lastname: Rimessi
authors.forename: Marina
authors.initials: M
authors.lastname: Fabris
authors.forename: Matteo
authors.initials: M
authors.lastname: Bovolenta
authors.forename: Elena
authors.initials: E
authors.lastname: Bassi
authors.forename: Sofia
authors.initials: S
authors.lastname: Falzarano
authors.forename: Francesca
authors.initials: F
authors.lastname: Gualandi
authors.forename: Claudio
authors.initials: C
authors.lastname: Rapezzi
authors.forename: Fabio
authors.initials: F
authors.lastname: Coccolo
authors.forename: Daniela
authors.initials: D
authors.lastname: Perrone
authors.forename: Alessandro
authors.initials: A
authors.lastname: Medici
authors.forename: Alessandra
authors.initials: A
authors.lastname: Ferlini
mesh_terms: D001483:Base Sequence; D001681:Biological Assay; D002311:Cardiomyopathy, Dilated / Q000235:genetics*; D002474:Cell-Free System; D016189:Dystrophin / Q000235:genetics*; D005091:Exons / Q000235:genetics*; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D040181:Genetic Diseases, X-Linked / Q000235:genetics*; D006801:Humans; D007438:Introns / Q000235:genetics*; D008969:Molecular Sequence Data; D032342:Muscle Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D017570:MyoD Protein / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D012326:RNA Splicing / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014170:Transformation, Genetic / Q000187:drug effects
chemical_list: D016189:Dystrophin; D017570:MyoD Protein; D016376:Oligonucleotides, Antisense