Antisense Modulation of Both Exonic and Intronic Splicing Motifs Induces Skipping of a DMD Pseudo-Exon Responsible for X-Linked Dilated Cardiomyopathy
| Autor: | Francesca Gualandi, Daniela Perrone, Sofia Falzarano, Elena Bassi, Fabio Coccolo, Matteo Bovolenta, M. Fabris, Paola Rimessi, Alessandro Medici, Alessandra Ferlini, Claudio Rapezzi |
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| Přispěvatelé: | Rimessi P, Fabris M, Bovolenta M, Bassi E, Falzarano S, Gualandi F, Rapezzi C, Coccolo F, Perrone D, Medici A, Ferlini A. |
| Rok vydání: | 2010 |
| Předmět: |
Cardiomyopathy
Dilated RNA Splicing Genetic enhancement Duchenne muscular dystrophy Molecular Sequence Data Exonic splicing enhancer Fluorescent Antibody Technique Regulatory Sequences Nucleic Acid Biology NO Dystrophin Exon Transformation Genetic Genetics medicine Humans Molecular Biology X chromosome MyoD Protein Muscle Cells Base Sequence Cell-Free System Reverse Transcriptase Polymerase Chain Reaction Genetic Diseases X-Linked Exons Fibroblasts Oligonucleotides Antisense medicine.disease Introns Exon skipping X-linked dilated cardiomyopathy RNA splicing Molecular Medicine Biological Assay |
| Zdroj: | Human Gene Therapy. 21:1137-1146 |
| ISSN: | 1557-7422 1043-0342 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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