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2013-04-22 - Colloque/Présentation - communication orale - Anglais - 1 page(s)

Ansseau Eugénie , Vanderplanck Céline, Wallace Lindsay M, Tassin Alexandra , Domire Jacqueline S, Guckes Susan M, Yip Cassandre, Laoudj-Chenivesse Dalila, Coppée Frédérique , Wilton D. Steve, Harper Scott Q., Belayew Alexandra , "Evaluation of new antisense oligomers targeting the DUX4 mRNA as a therapeutic strategy for FSHD " in MDA Scientific Conference, Washington DC, USA, 2013

  • Codes CREF : Pathologies particulières (DI3370)
  • Unités de recherche UMONS : Biologie moléculaire (M122)
  • Instituts UMONS : Institut des Sciences et Technologies de la Santé (Santé)

Abstract(s) :

(Anglais) Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common hereditary muscle disorders. Its molecular mechanism involves DNA hypomethylation and chromatin de-repression at the D4Z4 repeat array of chromosome 4q35. This FSHD-permissive chromatin structure can arise from reduction in D4Z4 repeat copy number (FSHD1) or mutations causing haploinsufficiency of the SMCHD1 gene, which is involved in epigenetically regulating the 4q35 locus (FSHD2). Ultimately, either change contributes to an environment that allows expression of the myotoxic double homeobox 4 (DUX4) gene in FSHD muscles. We initially characterized DUX4 in the D4Z4 element and found its only stable mRNA was transcribed from the most distal D4Z4 unit, which is located adjacent to the pLAM region with two untranslated exons that include a polyadenylation signal. DUX4 encodes a transcription factor expressed at very low levels in healthy muscle cells and induced 10 to 50 fold in FSHD myonuclei, where it initiates a gene dysregulation cascade causing differentiation defects, muscle atrophy and oxidative stress. Our hypothesis on the causal role of DUX4 expression in FSHD pathogenesis is now supported by compelling evidence from several laboratories. The expertise of Prof. Wilton's group in antisense oligomer (AO) design and induced exon skipping in Duchenne muscular dystrophy (DMD) has led to a collaboration with our group to knockdown DUX4 expression in an in vitro model of FSHD. Several AOs designed to target exons 2 and 3 of pLAM for exclusion from the DUX4 mRNA, synthesized as 2'-O-methyl on a phosphorothioate backbone, were able to suppress DUX4 protein expression in primary human myoblast cultures. The effect was specific, although some of the AOs were more efficient than others, in that they suppressed endogenous DUX4 mRNA and protein expression at lower transfection concentrations and, more importantly the AOs did not alter expression of the homologous DUX4c protein. While all the AOs suppressed DUX4 expression, the ones targeted to exon 3 appeared to be more effective. Moreover, the DUX4 inhibition suppressed expression of FSHD markers in FSHD primary myotubes and prevented development of the atrophic myotube phenotype. The 3 most effective AO sequences were prepared as “vivo-morpholino” to test them in a mouse model. In collaboration with Prof. Scott Harper’s group, we performed a preliminary experiment to test those AOs in an alternative FSHD model in which recombinant AAV (adeno-associated virus) vectors expressing DUX4 were injected in the tibialis anterior muscle of mice. After co-injection with one of these AOs we observed a decrease in the amount of DUX4 mRNA. We are now further testing the efficiency of these AOs in vivo to both inhibit DUX4 and prevent downstream myopathic events related to DUX4 expression.