Optimisation de la stratégie de saut d'exon pour le traitement de la dystrophie musculaire de Duchenne

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder that affects 20 out of 100,000 boys. It is characterized by progressive muscle weakness leading to loss of ambulation between the ages of 8 and 14 years and a decreased life expectancy (mean age of death: 35 years). DMD is caused by mutations in the DMD gene that lead to an absence of the dystrophin protein. There are currently no curative treatment options available. Nevertheless, promising therapy is under development and is based on the exon skipping strategy. Antisense oligonucleotides are used to modulate the splicing of pre-messenger RNA, restore the RNA open reading frame and thus produce functional dystrophin. This strategy has already led to the approval of 4 drugs in the United States but, due to their low efficacy, none of them have been accepted in Europe. These various molecules led only to very low levels of dystrophin restoration (from 1 to 6%), which are not enough to expect a significant therapeutic benefit in patientsMy thesis project therefore aims to optimize the efficacy of the exon-skipping approach by developing combined strategies. To this end, the tricyclo-DNA antisense oligonucleotides developed in my laboratory were co-administered with different molecules.The first therapeutic combination we tested aimed at reducing the urinary elimination of oligonucleotides in order to improve their bioavailability. It has previously been demonstrated that a large proportion of oligonucleotides are eliminated in the urine. A histological study performed in kidney of treated mice revealed a colocalization between the tricyclo-DNA oligonucleotides and the OAT transporters present in the proximal convoluted tubules of the kidney. Unfortunately, their inhibition with Probenecid (a pan OAT transporter inhibitor) did not improve the efficacy of our antisense treatment.The second therapeutic combination we tested aimed at improving the intracellular transport of the oligonucleotides. Oligonucleotides enter in cells by endocytosis. To perform exon skipping, they must leave the endosomes and go into the cell nucleus where the pre-messenger RNA is located. Unfortunately, only a tiny part of them succeeds, while the majority is eliminated by the cell. To help oligonucleotides to exit endosomes, a molecule called UNC7938 was used. This molecule creates pores in the late endosomes membrane which allows the oligonucleotides to come out. Combination of the two treatments increased restoration of dystrophin levels by 31% in the heart and 58% in the diaphragm. In addition, cardiac function was normalized after 12 weeks of treatment.The last therapeutic combination we tested aimed to increase the quantity of pre-messenger RNA produced in order to increase the potential number of targets for our treatment. Three histone deacetylase inhibitors (HDACi) were tested: givinostat (a pan HDACi already used in a clinical trial in DMD), valproic acid (a class I/II HDAC inhibitor) and EX527 (a class III inhibitor). After 4 weeks of co-treatment with the oligonucleotide, valproic acid and givinostat improved the effectiveness of oligonucleotides by increasing dystrophin production by up to +74%. Additionally, co-treatment with valproic acid for 12 weeks significantly improved the muscle function of the animals.In summary, our results establish the proof of principle that combining small molecules to antisense oligonucleotides can improve the efficiency of exon skipping.La dystrophie musculaire de Duchenne (DMD) est une maladie récessive liée à l'X qui touche 20 garçons sur 100 000. Elle se manifeste par une faiblesse musculaire progressive conduisant à une perte de la marche entre 8 et 14 ans puis par le décès des patients autour de 35 ans. La maladie est provoquée par des mutations dans le gène DMD qui conduisent à une absence de la protéine dystrophine. Il n'existe à l'heure actuelle aucun traitement capable de guérir tous les patients. Néanmoins, une stratégie très prometteuse est en développement et repose sur le principe du saut d'exon. Pour ce faire, des oligonucléotides antisens sont utilisés pour moduler l'épissage de l'ARN pré-messager, restaurer le cadre de lecture de l'ARN et ainsi produire une dystrophine fonctionnelle. Cette stratégie a déjà conduit à la mise sur le marché de 4 médicaments aux Etats-Unis mais, en raison de leur faible efficacité, aucun n'a été accepté en Europe. En effet, ces différentes molécules n'ont conduit qu'à des niveaux très faibles de restauration de la dystrophine (de 1 à 6%) ce qui est insuffisants pour espérer un bénéfice thérapeutique significatif chez les patients. Mon projet de thèse a donc pour objectif d'optimiser l'efficacité de l'approche de saut d'exon en développant des stratégies combinées. Pour ce faire, des oligonucléotides antisens de type tricyclo-DNA développés dans le laboratoire ont été co-administrés avec différentes molécules. La première combinaison thérapeutique que nous avons testé avait pour objectif de diminuer l'élimination urinaire des oligonucléotides afin d'améliorer leur biodisponibilité. En effet, la majorité des oligonucléotides sont éliminés dans les urines après une administration systémique. Nous avons réalisé une étude histologique et montré une colocalisation entre les oligonucléotides et les transporteurs OAT présents dans les tubes contournés proximaux du rein. Malheureusement, leur inhibition avec le probénécid (un inhibiteur des transporteurs OAT) n'a pas permis d'améliorer l'efficacité de notre traitement antisens. La seconde combinaison thérapeutique que nous avons testée avait pour objectif d'améliorer le transport intracellulaire des oligonucléotides. Les oligonucléotides entrent en effet dans les cellules par endocytose. Pour pouvoir être actif, ils doivent donc sortir des endosomes et aller dans le noyau cellulaire où se trouve l'ARN pré-messager. Malheureusement, seulement une infime partie d'entre eux y parvient, les autres sont éliminés par la cellule. Afin d'aider les oligonucléotides à sortir des endosomes, une molécule appelée UNC7938 fut utilisée. Cette molécule crée des pores dans les endosomes tardifs permettant ainsi aux oligonucléotides d'en sortir. La combinaison des deux traitements a permis d'augmenter les niveaux de dystrophine restaurée de 31% dans le cœur et de 58% dans le diaphragme. De plus, la fonction cardiaque fut normalisée après 12 semaines de traitement.La dernière combinaison thérapeutique que nous avons testée avait pour objectif d'augmenter les quantités d'ARN pré-messagers produits afin d'augmenter le nombre potentiel de cibles pour notre traitement. Trois inhibiteurs d'histone deacetylase (HDACi) ont été testés : le givinostat (a pan HDACi déjà utilisé en essai clinique dans DMD), l'acide valproique (un inhibiteur des HDAC de classe I/II) et EX527 (un inhibiteur des classe III). Apres 4 semaines de co-traitement avec notre oligonucléotide, l'acide valproique et le givinostat ont permis d'améliorer l'efficacité des oligonucléotides en augmentant jusqu'à +74% la production de dystrophine. De plus, un co-traitement de 12 semaines avec l'acide valproique a significativement amélioré la fonction musculaire des animaux. Tous ces résultats ont permis de démontrer la preuve de principe quant à l'utilisation de petites molécules pour améliorer l'efficacité du saut d'exon

État actuel des connaissance sur l'utilisation des oligonucléotides antisense dans le traitement des maladie neuromusculaire

International audienceNeuromuscular disorders include a wide range of diseases affecting the peripheral nervous system, which are primarily characterized by progressive muscle weakness and wasting. While there were no effective therapies until recently, several therapeutic approaches have advanced to clinical trials in the past few years. Among these, the antisense technology aiming at modifying RNA processing and function has remarkably progressed and a few antisense oligonucleotides (ASOs) have now been approved. Despite these recent clinical successes, several ASOs have also failed and clinical programs have been suspended, in most cases when the route of administration was systemic, highlighting the existing challenges notably with respect to effective ASO delivery. In this review we summarize the recent advances and current status of antisense based-therapies for neuromuscular disorders, using successful as well as unsuccessful examples to highlight the variability of outcomes depending on the target tissue and route of administration. We describe the different ASO-mediated therapeutic approaches, including splice-switching applications, steric-blocking strategies and targeted gene knock-down mediated by ribonuclease H recruitment. In this overview, we discuss the merits and challenges of the current ASO technology, and discuss the future of ASO development

Partial Restoration of Brain Dystrophin and Behavioral Deficits by Exon Skipping in the Muscular Dystrophy X‐Linked ( mdx ) Mouse

International audienceDuchenne muscular dystrophy is associated with various degrees of cognitive impairment and behavioral disturbances. Emotional and memory deficits also constitute reliable outcome measures to assess efficacy of treatments in the mdx mouse lacking the muscle and neuronal full-length dystrophins. The present study aimed to evaluate whether these deficits could be alleviated by the restoration of brain dystrophin

Histone deacetylase inhibitors improve antisense-mediated exon-skipping efficacy in mdx mice

International audienceAntisense-mediated exon skipping is one of the most promising therapeutic strategies for Duchenne muscular dystrophy (DMD), and some antisense oligonucleotide (ASO) drugs have already been approved by the US FDA despite their low efficacy. The potential of this therapy is still limited by several challenges, including the reduced expression of the dystrophin transcript and the strong 5 0-3 0 imbalance in mutated transcripts. We therefore hypothesize that increasing histone acetylation using histone deacetylase inhibitors (HDACi) could correct the transcript imbalance, offering more available pre-mRNA target and ultimately increasing dystrophin rescue. Here, we evaluated the impact of such a combined therapy on the Dmd transcript imbalance phenomenon and on dystrophin restoration levels in mdx mice. Analysis of the Dmd transcript levels at different exon-exon junctions revealed a tendency to correct the 5 0-3 0 imbalance phenomenon following treatment with HDACi. Significantly higher levels of dystrophin restoration (up to 74% increase) were obtained with givinostat and valproic acid compared with mice treated with ASO alone. Additionally, we demonstrate an increase in H3K9 acetylation in human myocytes after treatment with valproic acid. These findings indicate that HDACi can improve the therapeutic potential of exon-skipping approaches, offering promising perspectives for the treatment of DMD

Histone deacetylase inhibitors improve antisense-mediated exon-skipping efficacy in mdx mice

Antisense-mediated exon skipping is one of the most promising therapeutic strategies for Duchenne muscular dystrophy (DMD), and some antisense oligonucleotide (ASO) drugs have already been approved by the US FDA despite their low efficacy. The potential of this therapy is still limited by several challenges, including the reduced expression of the dystrophin transcript and the strong 5′-3′ imbalance in mutated transcripts. We therefore hypothesize that increasing histone acetylation using histone deacetylase inhibitors (HDACi) could correct the transcript imbalance, offering more available pre-mRNA target and ultimately increasing dystrophin rescue. Here, we evaluated the impact of such a combined therapy on the Dmd transcript imbalance phenomenon and on dystrophin restoration levels in mdx mice. Analysis of the Dmd transcript levels at different exon-exon junctions revealed a tendency to correct the 5′-3′ imbalance phenomenon following treatment with HDACi. Significantly higher levels of dystrophin restoration (up to 74% increase) were obtained with givinostat and valproic acid compared with mice treated with ASO alone. Additionally, we demonstrate an increase in H3K9 acetylation in human myocytes after treatment with valproic acid. These findings indicate that HDACi can improve the therapeutic potential of exon-skipping approaches, offering promising perspectives for the treatment of DMD

Oligonucleotide Enhancing Compound Increases Tricyclo-DNA Mediated Exon-Skipping Efficacy in the Mdx Mouse Model

International audienceNucleic acid-based therapeutics hold great promise for the treatment of numerous diseases, including neuromuscular disorders, such as Duchenne muscular dystrophy (DMD). Some antisense oligonucleotide (ASO) drugs have already been approved by the US FDA for DMD, but the potential of this therapy is still limited by several challenges, including the poor distribution of ASOs to target tissues, but also the entrapment of ASO in the endosomal compartment. Endosomal escape is a well recognized limitation that prevents ASO from reaching their target pre-mRNA in the nucleus. Small molecules named oligonucleotide-enhancing compounds (OEC) have been shown to release ASO from endosomal entrapment, thus increasing ASO nuclear concentration and ultimately correcting more pre-mRNA targets. In this study, we evaluated the impact of a therapy combining ASO and OEC on dystrophin restoration in mdx mice. Analysis of exon-skipping levels at different time points after the co-treatment revealed improved efficacy, particularly at early time points, reaching up to 4.4-fold increase at 72 h post treatment in the heart compared to treatment with ASO alone. Significantly higher levels of dystrophin restoration were detected two weeks after the end of the combined therapy, reaching up to 2.7-fold increase in the heart compared to mice treated with ASO alone. Moreover, we demonstrated a normalization of cardiac function in mdx mice after a 12-week-long treatment with the combined ASO + OEC therapy. Altogether, these findings indicate that compounds facilitating endosomal escape can significantly improve the therapeutic potential of exon-skipping approaches offering promising perspectives for the treatment of DMD