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Utilisation Des Cellules Souches Pluripotentes Pour Le Criblage À Haut Débit De Molécules Thérapeutiques

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Utilisation Des Cellules Souches Pluripotentes Pour Le Criblage À Haut Débit De Molécules Thérapeutiques Utilisation des cellules souches pluripotentes pour le criblage à haut débit de molécules thérapeutiques dans la maladie de Lesch-Nyhan : 2019SACLE011 NNT Thèse de doctorat de l'Université Paris-Saclay, préparée à l’Université d’Evry Val-d’Essonne École doctorale n°569 Innovation thérapeutique : du fondamental à l’appliqué (ITFA) Spécialité de doctorat: Immunologie Thèse présentée et soutenue à Corbeil-Essonnes, le 01 juillet 2019, par Valentin Ruillier Composition du Jury : Dr. Olivier Goureau Institut de la Vision, Université Sorbonne, Paris Président Pr. Odile Boespflug-Tanguy Université Paris Diderot, Hôpital Robert Debré, Paris Rapporteur Dr. Amélie Piton IGBMC, Hôpitaux Universitaires de Strasbourg Rapporteur Dr. Terence Beghyn APTEEUS, Lille Examinateur Pr. Christelle Monville UEVE/INSERM U861, I-STEM, Corbeil-Essonnes Directeur de thèse Dr. Alexandra Benchoua CECS, I-STEM, Corbeil-Essonnes Co-Encadrant Résumé - Abstract Mots clés : Maladie de Lesch-Nyhan, cellules souches pluripotentes, iPSC, criblage à haut débit, HGPRT, purines Résumé : Les mutations affectant la fonction identifier, par une approche de criblage à haut d’enzymes impliquées dans le cycle des purines débit, de nouvelles molécules chimiques capables sont responsables d’une multitude de syndromes de corriger ces défauts. Plus de 3000 molécules pédiatriques, caractérisés par des atteintes ont été testées et 6 composés, tous dérivés de neurologiques et comportementales. A ce jour, l’adénosine, ont pu être identifiés comme aucune stratégie thérapeutique n’a été compensant le métabolisme par un mécanisme réellement efficace pour contrôler ces d’action indépendant de l’HGPRT. De manière symptômes. La maladie de Lesch-Nyhan (MLN), intéressante, un des composés, la S- associée à la perte de fonction de l’enzyme de adenosylmethionine (SAM) a par le passé déjà recyclage HGPRT, constitue un bon modèle démontré des effets bénéfiques sur les d’étude. Mon travail a consisté à utiliser la symptômes comportementaux typiques de la technologie des cellules souches induites à la MLN dans plusieurs études de cas. Cela démontre pluripotence, reprogrammées à partir de que la stratégie abordée ici a permis fibroblastes de patients atteints des formes l’identification de cibles thérapeutiques sévères de la MLN, pour identifier des permettant d’améliorer les symptômes phénotypes neuronaux associés à la perte de neurospychiatriques de cette pathologie et fonction de l’HGPRT. Ces marqueurs constitue un modèle réplicable pour différentes phénotypiques ont ensuite été utilisés pour pathologies touchant le métabolisme cérébral. Keywords : Lesch-Nyhan disease, pluripotent stem cells, iPSC, High-throughput screening, HGPRT, purines Abstract : Mutations in genes coding for identified 6 compounds, all possessing an enzymes involved in purine synthesis or recycling adenosine moiety, that corrected LND related lead to dramatic neurological conditions with neuronal phenotypes by promoting metabolism poor pharmacological options. Lesch–Nyhan compensations in a HGPRT-independent manner. disease (LND) is caused by deficiency of the One of these compound, S-adenosylmethionine salvage pathway enzyme HGPRT that (SAM), has already been reported as providing compromises recycling of guanine and amelioration of behavioral symptoms in some hypoxanthine into GMP and IMP. LND is LND cases, demonstrating that our screening characterized by severe neuropsychiatric allowed the identification of pathways that can symptoms that are out of reach of be relevant therapeutic targets to ease the pharmacological treatments. Here we use human devastating neuropsychiatric symptoms cortical neural stem cells and neurons derived associated with this pathology. Interestingly, from iPSC of children affected by severe forms of these pathways can be activated in LND patients LND to identify neural phenotypes associated via simple food supplementation. This with HGPRT-deficiency and of interest to develop experimental paradigm can also be easily a target-agnostic based drug screening system. adapted to other purine associated neurological We screened more than 3000 molecules and disorders affecting normal brain development. 1 REMERCIEMENTS Mes premiers remerciements vont à Marc Peschanski, Cécile Martinat et Raymond Zakhia, pour m’avoir accueilli à I-Stem et permis d’évoluer dans un environnement scientifique de qualité. Je remercie également Christelle Monville, qui a accepté de diriger ma thèse, ainsi que Mathilde Girard, qui a cru en mon parcours et avec qui j’ai pu débuter mon aventure dans la recherche. Mon projet de recherche n’aurait pas été possible sans le soutien de l’AFM Téléthon et de l’association Lesch-Nyhan Action. Merci aux membres de ces deux associations pour leur implication auprès des malades et leur réel soutien pour faire avancer la recherche scientifique. Je tiens ensuite à remercier Odile Boespflug-Tanguy, Amélie Piton, Sylvain Fisson, Olivier Goureau et Terence Beghyn, pour avoir accepté de participer à mon jury de thèse. Merci également à Guillaume Pinna et Jérôme Denis, pour leurs conseils avisés lors de mon comité de mi-thèse. Merci à tous pour l’intérêt porté à mon travail. Une pensée particulière à Alexandra Benchoua pour m’avoir ouvert les portes de l’équipe Neuroplasticité. Merci de m’avoir donné l’opportunité de travailler sur ce projet passionnant et de m’avoir accompagné tout au long de ces trois années. Merci également à Marie, pour ton aide pendant ces quelques mois. Je te souhaite une bonne continuation, que ce soit à I-Stem ou ailleurs. J’en profite aussi pour dire un grand merci aux filles de l’équipe, pour leur accueil si chaleureux, leur bonne humeur, leurs attentions, et l’excellente ambiance du secteur 3. Merci à toutes pour votre aide et j’espère que l’on pourra ouvrir rapidement ta bouteille de champagne, Claire ! J’aimerai également remercier l’ensemble de l’équipe HTS, pour leur implication dans le projet Lesch- Nyhan et pour tous ces moments passés avec vous au milieu des robots. Prenez soin du Benchcel, qui m’aura accompagné tout au long de mon passage à I-Stem. Un grand merci à Johana et Benjamin, avec qui j’ai débuté cette aventure et qui ont largement participé à ma formation. Merci pour votre aide et surtout pour votre sympathie. Je tiens bien sûr à remercier les personnes que j’ai rencontrées au cours de ces quatre années, et qui sont aujourd’hui devenues des amies. Merci à mes voisins du bureau des thésards, Vincent, Déborah, Julie, Sylvain et Florian, mais aussi Gurvan, Marie et Margot, pour tous ces moments inoubliables passés avec vous, et sans qui cette expérience n’aurait pas été la même. Grâce à vous je suis devenu incollable sur les guerres de Vendée et un « expert » en escalade. Je n’oublierai pas non plus Océane, Julie P, Camille, Sandra, Antoine, Julie T, Manon, Florine et Alexandre, pour tous ces moments de rire et de partage passés avec vous. Je tiens également à remercier ma famille et tous mes proches, pour avoir toujours été présents. Merci à toi, Claire, pour m’avoir soutenu tout au long de ce travail. Ça n’a pas dû être de tout repos. Le bon déroulement d’un travail de recherche ne pouvant pas se faire tout seul, j’aimerai pour finir remercier l’ensemble des membres du laboratoire, qui ont tous, à un moment, participé de près ou de loin à faire avancer mon projet. Merci à vous tous ! 2 Table des matières Index des illustrations ............................................................................................................................. 6 Index des tableaux................................................................................................................................... 7 Abréviations ............................................................................................................................................ 8 I. Les troubles liés au métabolisme purinergique .............................................................................. 9 1. Les purines : rôles biologiques .................................................................................................... 9 2. Le cycle des purines ................................................................................................................... 10 3. Les troubles liés au métabolisme des purines : un équilibre fragile du cycle ........................... 13 II. La maladie de Lesch-Nyhan ........................................................................................................... 15 1. Présentation clinique de la maladie de Lesch-Nyhan ............................................................... 16 a) Les anomalies métaboliques ................................................................................................. 16 b) Les symptômes moteurs ....................................................................................................... 17 c) Les symptômes cognitifs et neuropsychiatriques ................................................................. 19 d) Autres .................................................................................................................................... 20 2. Epidémiologie et génétique ...................................................................................................... 21 a) Une maladie de transmission récessive liée à l’X .................................................................. 21 b) Le gène HGPRT et sa distribution .......................................................................................... 21 c) Les mutations
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