Résumé :
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The analysis of DMD/BMD databases, which include patient records have allowed to assess the proportion of DMD patients that is eligible for exon skipping strategies as well as to appraise the impact of various exon skipping events. Among them, the delta45-55 deletion shows a very high potential. Indeed, a few individuals carrying this deletion have been identified worldwide displaying mild symptoms, telling us that the resulting truncated dystrophin is quite suitable provided it is produced in satisfactory amounts. In addition, statistical analysis shows that about 2/3 of the DMD population, whatever the very nature of the mutation (i.e. deletions, insertions, point mutations), could be eligible for such a truncation.These years, we have successfully designed a significant number of constructs derived from the U7 small nuclear RNA for skipping exons in many genes. A retrospective analysis of operational and non-effective constructs has allowed us to theorize that the stem loop of U7 needed to interact with the antisense (through a kiss domain) in order to allow either maximal interaction between the antisense and its target or alternatively to be efficiently processed into active snRNPs. Consistently, the need for a kiss domain was verified by rescuing non-operative constructs as well as slaughtering valuable ones after customizing either the antisense sequence or the very end of the stem. These studies have provided insights for the optimal design of exon skipping U7-derived snRNAs. Our ultimate objective was to develop U7 constructs for multi-skipping from exon 45 to exon 55 over more than 300,000 nucleotides surrounded by two giant introns. Here, we present a library of U7 constructs targeting crucial sites in the 45-55 gap. These constructs were cloned in both AAV and LV backbones. We show promising multi-skipping in cell lines from patients with different DMD genotypes.
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