Développer une thérapie génique visée aux cellules souches musculaires de la Dystrophie musculaire de Duchenne de Boulogne

par Romain Domart

Projet de thèse en Sciences de la vie et de la santé

Sous la direction de Helge Amthor et de Markus Schuelke.

Thèses en préparation à Paris Saclay en cotutelle avec l'Université libre de Berlin , dans le cadre de École doctorale Structure et Dynamique des Systèmes Vivants (Gif-sur-Yvette, Essonne ; 2015-....) , en partenariat avec ENDICAP - Handicap Neuromusculaire : Physiopathologie, Biothérapie et Pharmacologie appliquées (laboratoire) et de Université de Versailles-Saint-Quentin-en-Yvelines (établissement de préparation de la thèse) depuis le 30-09-2017 .

  • Titre traduit

    Developing a muscle stem cell targeted gene therapy for Duchenne muscular dystrophy


  • Résumé

    Duchenne muscular dystrophy (DMD) is a severe, progressive and non-curable muscle disease leading to early loss of ambulation and death during the first decencies. It is an X-linked hereditary disease, and its frequency of 1:3.000 males makes it the most frequent hereditary muscle disease in childhood. DMD results from absent dystrophin following mutations in the encoding DMD gene that disrupt the open reading frame. Recent evidence suggests that lack of dystrophin causes abnormal function of skeletal muscle stem cells (mSCs), which likely inflicts on muscle regeneration and the disease progression in DMD. Moreover, the newly discovered role of dystrophin in mSCs underlines the importance of developing a stem cell based therapy for DMD. Recently, different studies have shown that defective exon 23 of mdx mice, the mouse model for DMD, can successfully be removed from the dystrophin gene in postnatal muscle tissue in vivo, using adeno-associated virus-9 (AAV9) to deliver the CRISPR/Cas9 gene editing system. Besides partial recovery of functional dystrophin protein in skeletal myofibers and in cardiac muscle, a pool of endogenously corrected myogenic precursors in mdx mouse muscle was observed in one of the studies. This offers a promise towards a long lasting therapeutic effect in DMD patients. However, such restoration of a truncated dystrophin permits to alleviate the disease towards a less severe Becker phenotype, but no cure. Using the combination of CRISPR/Cas9 technology and gene therapy approaches, for the first time, a regenerative therapy could be developed for DMD by correcting the mutated dystrophin gene at the level of mSCs, thus allowing to replace diseased muscle in time with genetically cured muscle. The laboratory is currently developing a novel gene therapy approach to treat DMD, using AAV-CRISPR/Cas9 vector system that is able to overcome dystrophin mutations in muscle stem cells by exchange of mutated by wildtype exons. A functional vector has already been established. Such therapeutic strategy will restore the full length protein, enabling complete therapeutic effects. However, this requires targeting proliferating mSCs for allowing efficient homologeous recombination. In collaboration with the laboratory of Pr Markus Schuelke, Charité-University Medicine Berlin, we developed a novel mouse model, which permits life cell imaging of dystrophin in vivo and ex vivo using EGFP fluorescence in wildtype as well as in dystrophic mdx mice (DmdEGFP and DmdEGFP-mdx mice). The aim of this project is to develop a novel therapeutic strategy for DMD based on AAV–CRISPR/Cas9 mediated gene editing in mSCs in vivo. What is the dynamics of dystrophin restoration following systemic treatment? Can we observe the transfection efficacy already in activated mSCs, in which dystrophin expression has been corrected? What is the role of dystrophin in mSCs? How many mSCs are transfected and how can the transfection efficacy be improved? Are muscle fibers homogeneously transfected? Do transfected/ corrected mSCs have any selection advantage compared to non/corrected mSCs? To develop such in vivo stem cell therapy, the student will here use our recently developed animal models, DmdEGFP and DmdEGFP-mdx, which offer great advantages to study the expression and dynamics of dystrophin in vivo. The DmdEGFP-mdx reporter mice are the first model that enables the in vivo evaluation and characterization of therapeutic strategies for DMD that aim at restoring dystrophin expression. However, both models are in itself insufficient for muscle stem cell targeted therapeutic approaches as mSCs cannot be traced. Moreover, the expression and role of dystrophin in mSCs remains still debated, and remains entirely unexplored in vivo. The first aim of the project therefore requires to determine the presence and function of dystrophin in mSCs in vivo, before moving to use AAV–CRISPR/Cas9 mediated gene editing to correct mSCs. In the first part of the project the student will determine the function of dystrophin in mSCs in vivo. For this, he/she will generate a new mouse model which permits direct visualization and tracing of dystrophin expressing mSCs. The new model is based on crossing three existing mouse lines: i) Pax7CreERT2 knock-in driver (Lepper et al., 2009); ii) fluorescent linage reporter tdTomato (ROSA26tdTomato) mice (Webster et al., 2016); and iii) our DmdEGFP mice. Resulting mice that harbour triple alleles will be used to trace dystropin expression in mSCs by co-expression of GFP and Tomato, following tamoxifen induced Cre-mediated recombination for stable Tomato expression in Pax7 expressing mSCs and their descendant myogenic progenitors. The newly generated reporter mice will be first characterized and the phenotype confirmed. The student will determine the frequence of dystrophin expressing mSCs at different stages of postnatal development and following injury induced muscle regeneration. He/she will determine the context between dystrophin expression and myogenic lineage progression, the duration of dystrophin expression in mSCs, and will determine the fate of dystrophin expressing mSCs. The student will take advantage of cultures of isolated muscle fibers to quantify dystrophin expression events during mSC lineage progression. He/she will explore the proposed significance of dystrophin for asymmetric cell division in vivo and in vitro. Moreover, the student will compare the molecular signature (using RNA sequencing) of dystrophin positive and negative mSCs (either FACS sorted or after laser capture microdissection), which allows to project more on the function of dystrophin in mSCs. In the second part of the project, the student will repair dystrophin deficient mSCs in vivo using AAV9-mediated CRISPR/Cas9-mediated dystrophin editing. For determining the presence of therapeutic stem cell editing, he will generate DmdEGFP-mdx;Pax7Cre-ERT2;Rosa26tdTomato crosses, which will be AAV and Tamoxifen injected at postnatal stages. The mouse models permit visualize in vivo the dystrophin restoration in mSCs using intra-vital two-photon microscopy. The student will determine the frequency of dystrophin restored mSCs compared to mSCs from DmdEGFP;Pax7Cre-ERT2;Rosa26tdTomato crosses, thus indicating the therapeutic efficacy of the CRISPR/Cas9-mediated reconstitution of full-length dystrophin in mSCs. Intra-vital two-photon microscopy will be performed on skeletal muscle at different intervals following the injections. Using this protocol, the student will construct 3 dimensional images of dystrophin in vivo in relation to transfected satellite cells. Hence these experiments will for the first time establish a therapeutic strategy for a regenerative therapy for Duchenne muscular dystrophy based on gene editing of mSCs and monitor the therapeutic effect in vivo.