La médecine régénérative porte un grand intérêt aux hiPSCs en raison de leurs caractéristiques de pluripotence et d’auto-renouvellement. Pour qu’elles soient utilisées dans le traitement de myopathies héréditaires, elles doivent d’abord être corrigées génétiquement, puis différenciées efficacement en cellules myogéniques.
Ce mémoire démontre qu’il est possible de corriger génétiquement des MSCs provenant d’hiPSCs dérivées de fibroblastes de patient DMD et de permettre une expression d’une micro-dystrophine, une version tronquée de la dystrophine, dans les muscles de souris, et ce, à l’aide d’un vecteur lentiviral. Le protocole de transformation myogénique utilisé lors de ce projet requerrait l’emploi d’un milieu myogénique développé au laboratoire, le MB-1, ainsi qu’un vecteur adénoviral codant pour MyoD. Toutefois, ce protocole demande d’être optimisé pour augmenter l’efficacité et la reproductibilité et réduire les dangers associés au vecteur viral lors d’essais cliniques.
De plus, des outils de correction génique sont toujours en cours de développement au laboratoire. La version actuelle de la micro-dystrophine sera éventuellement remplacée par une version permettant l’échafaudage de tout le complexe glycoprotéique associé. Dans un contexte clinique utilisant ce type de thérapie cellulaire, il sera plus facile et sécuritaire de corriger les fibroblastes du patient DMD avant de les induire à la pluripotence. Ainsi, les cellules qui seront greffées au patient proviendront d’un clone dont le site d’insertion de la micro-dystrophine sera connu, réduisant les risques associés à la mutagenèse d’insertion.
La deuxième partie de ma recherche décrit la production de protéines recombinantes afin d’induire la myogenèse à partir d’hiPSCs. Il a été démontré que MyoD, Pax3 et Pax7, trois régulateurs de la myogenèse, pouvaient être couplés à Tat, un CPP permettant l’entrée des protéines dans les cellules. Ces facteurs de transcription ont été produits et purifiés d'un système bactérien avec succès. Toutefois, dû aux faibles quantités de protéines obtenues, seulement 6x[His]-Tat-MyoD a pu être testée in vitro à ce jour. Il a finalement été démontré que cette protéine pouvait entrer dans les
La prochaine étape de ces études se concentra sur l’optimisation du protocole de différenciation des hiPSCs en myoblastes. Les protéines recombinantes seront produites à plus grande échelle afin de permettre différentes analyses in vitro, notamment leur impact sur la dynamique de l’expression des gènes myogéniques. De plus, il sera impératif de développer un protocole de purification des cellules myogéniques afin d’éviter que des cellules à un stade de différenciation trop précoce n’engendrent le développement de tumeurs in vivo. Cette étape sera essentielle lorsque des myoblastes dérivés d’hiPSCs seront utilisés pour traiter un patient DMD.
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