Résumé :
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Myotonic Dystrophy type 1 (DM1) also called Steinert disease is one of the most common form of inherited neuromuscular disorders in adults characterized by progressive muscle weakness and wasting, myotonia as well as many other multisystemicdefects. DM1 is an autosomal dominant disease caused by an expanded trinucleotide (CTG)n repeat (n >50) located in the 3' non-coding region of the DMPK gene. The size of the expansion is generally correlated with the clinical severity and the age ofonset of the disease. Expression of pathogenic DMPK transcripts containing expanded CUG repeats (CUGexp-RNAs) results in atoxic RNA gain-of-function mechanism. The CUGexp-RNAs are retained into the nuclei as ribonucleoprotein aggregates or foci,and alter the functions of RNA binding proteins such as MBNL1 and CUG-BP1 involved, at least, in alternative splicing regulation. Thus, mis-splicing of some specific pre-mRNA like CLCN1 and BIN1 contributes to DM1 physiopathology. Several therapeutic strategies are currently under development to reverse trans-dominant effects of the CUGexp-RNAs in DM1. Efficientinhibition of CUGexp-RNA toxicity was obtained by using synthetic antisense oligonucleotides that target the CUG expansion.We have investigated the ability of CAG antisense sequences delivered within a modified hU7-snRNA gene to target the expanded CUG repeat located in the pathogenic DMPK transcripts. In fact, modified human hU7-snRNAs have a nuclear localization and are able to direct RNA maturation machinery. Different antisense sequences were imbedded within a hU7-snRNA-derived lentiviral vector and their efficacy were evaluated in vitro using DM1 primary muscle cell cultures. An efficienttargeting of the CUGexp-RNAs was achieved as indicated by the dramatic silencing of pathogenic DMPK mRNAs in the DM1 cellsexpressing hU7-snRNA-CAG. In contrast, the normal DMPK transcripts were not affected. No cellular toxicity was observed andthe number of DM1 cells without any nuclear foci was significantly increased. The sequestered MBNL1 proteins were releasedand several aberrant pre-mRNA splicing were largely corrected in DM1 myotubes. Finally, the defective myogenic differentiationof the cDM1 muscle cells was normalized confirming that the hU7-snRNA-CAGtool abolishes the cellular toxicity induced by theCUGexp-RNAs. In conclusion, we propose the use of hU7-snRNA-CAG as a new RNA based gene therapy approach for gene silencing in DM1 and possibly other RNA-dominant disorders in which expanded repeats are expressed.
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