Résumé :
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Exon skipping by antisense oligonucleotides (AONs) represents a promising tool successfully used for reframing dystrophin as demonstrated in a recent pilot trial in DMD patients. Since all the effort has been focused on favourable exons skipping in the commonest deletion mutations, we focused our research on the identification of optimal AONs for modulating small mutations. These account for 20-30% of all reported mutations and, if occurring within skippable exons, are eligible for AONs modulation. Among 50 patients characterised by us and carrying small mutations in the dystrophin gene we have selected 5 patients (c.1132_1135dup in exon 10, c.1912delC in exon 16, c.3447_3448delinsTT in exon 26, c.4565delT in exon 33, c.4780delTins37 in exon 34) with mutations occurring in skippable exons, and we have designed for each exon one AON on the wild type sequence and one on the mutated sequence. In order to avoid the usage of patients' cells we have set up an in vitro cell-free splicing assay for testing the designed AONs. We have therefore validated all the AONs designed on MyoD transformed patients' fibroblasts. Our experiments in cells resulted both in a re-framing of the dystrophin transcript lacking the skipped mutated exon and in a protein product. Although both wild type and mutated AONs were able to induce a specific exon-skipping, their efficiency varied if measured by qRT-PCR in patients' cells and densitometry analysis on cell free splicing assay. In conclusion, we have developed a cell-free splicing assay able to reproduce the splicing of dystrophin exons with private small mutations useful to search for optimal antisense without using patients' material. These data highlight the complexity in identifying the optimal AON for exons with private small mutations with possible implications on therapeutic designing.
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