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International audience; Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder, characterised by cognitive and behavioural impairment, in addition to the typical muscle pathology. Imaging studies revealed widespread white matter lesions in DM1 patients, which may be partially explained by local demyelination. DM1 is caused by the abnormal expansion of non-coding CTG trinucleotide repeat, which is transcribed into toxic CUG RNA that accumulates in nuclear RNA foci and disrupts critical RNA-binding proteins. To investigate the impact of RNA toxicity on myelin biology, we used a transgenic mouse model of DM1, known as the DMSXL mice, which express expanded CUG transcripts in multiple tissues and cell types.We found delayed axon myelination of the corpus callosum at two weeks of age, which later recovered at four months. Early myelin defects were linked to an overall reduction in the number of oligodendroglia cells, specifically myelinating oligodendrocytes (OL), with a concurrent increase in oligodendrocyte progenitor cells (OPC). These phenotypes were accompanied by abundant RNA foci and splicing dysregulation in oligodendroglia, which were more pronounced at two weeks. Taking advantage of primary cell models, we investigated the mechanisms triggered by toxic CUG RNA in oligodendroglia. OPC isolated from DMSXL newborns exhibited impaired differentiation into fully ramified OLs in culture, a defect we confirmed in human oligodendroglia derived from DM1 patients. RNA sequencing revealed expression and splicing changes in DMSXL OL that affect transcripts related to the cytoskeleton and cell differentiation. Importantly, the transcriptomic defects of DMSXL OL recreated expression and splicing profiles typical of immature OPC.In conclusion, toxic CUG RNA disrupts the molecular program of oligodendroglia differentiation, impairing the transcriptome changes occurring during the OPC-OL transition and leading to transient hypomyelination in mice.

Monogenic disorder are inherited mutation(s) in a single gene that result in abnormal protein synthesis impairing normal cellular homeostasis and organ function that reduces patient life-pan. Recently autologous transplantation of genetically corrected patient's hematopoietic stem cells (HSCs) defined as ex-vivo gene therapy (GT) has emerged as a promising curative approach for such genetic diseases. In ex-vivo GT, targeted integration of a therapeutic transgene into a tolerant and safe locus appears to be a powerful approach to produce the therapeutic protein above physiological level in a broad type of hematopoietic cells using endogenous promoter. Such genome editing strategy relies on DNA double strand break and delivery of a functional copy of the mutated gene in the locus of interest. Here, we exploited the α-globin locus (HBA) as a safe harbor locus for transgene integration to develop a universal platform for expression and secretion of therapeutic proteins into erythroid cells. In HSCs, we induced double strand breaks (DSBs) in HBA locus with CRISPR-Cas9 and delivered a therapeutic transgene using AAV-6 to target integration by homology directed repair. Due to the abundance of erythroid cells in human (5X10^6 RBCs/microliter of blood) and the high HBA expression in erythroid population (around 1,5g/day), the HBA platform would require minimal transgene integration events in HBA locus to provide sufficient amount of therapeutic proteins reducing as consequence the potential off-target and toxicity due to editing and DNA donor delivery. First, we applied the HBA platform for β-thalassemia : a genetic condition impairing the hemoglobin formation and triggering toxic α-globin chain precipitation. The genetic correction of β-thalassemia using our HBA platform results in 1 copy of A-globin gene deletion decreasing the α-globin chain precipitation while α-globin gene integration into HBA locus restore the normal hemoglobin formation. Then, we evaluated the HBA platform for erythroid-specific secretion of therapeutic proteins into bloodstream and, in particular,we focused on a genetic metabolic disorder: lysosomal acid lipase (LAL) deficiency caused by LIPA gene mutation(s) that lead to toxic lipid accumulation especially in liver. We achieved efficient expression and secretion of LAL enzyme that retained enzymatic activity and cross-corrected the lipid accumulation in patients' fibroblasts. We then focused on the optimization of LIPA cDNA transgene using lentiviral vectors (LVVs) to maximize the LAL expression and secretion. The optimized LIPA transgene will serve for LVV-based ex-vivo GT and for our HBA targeted platform in the purpose of enhancing the LAL expression while minimizing the targeting integration events required to fully correct the disorder. Finally, to better characterize LAL-D phenotype and to evaluate the therapeutic potency of our GT strategy, we set-up a multimodal cytometry approach using flow cytometry and image flow cytometry. We evaluated this pipeline on mainly patient's fibroblasts and on peripheral mononuclear blood mononuclear cells (PBMCs) for LAL-D mouse model.; Les maladies monogéniques sont des mutations héréditaires d'un seul gène qui entraînent une synthèse anormale de protéines, altérant l'homéostasie cellulaire normale et la fonction des organes et réduisant la durée de vie des patients. Récemment, la greffe autologue de cellules souches hématopoïétiques (HSCs) de patients génétiquement corrigés appelée thérapie génique (TG) ex-vivo, est apparue comme une approche curative prometteuse pour ces maladies. Dans la TG ex-vivo, l'intégration ciblée d'un transgène thérapeutique dans un locus tolérant apparaît comme une approche puissante pour produire les protéines thérapeutiques à un niveau supérieur au niveau physiologique dans un large type de cellules hématopoïétiques en utilisant un promoteur endogène. Une telle stratégie d'édition du génome repose sur la cassure double brin d'ADN et la livraison d'une copie fonctionnelle du gène muté dans le locus d'intérêt. Ici, nous avons exploité le locus de l'α-globine (HBA) comme un locus tolérant pour l'intégration de transgènes afin de développer une plateforme universelle pour l'expression et la sécrétion de protéines thérapeutiques par les globules rouges (GR). Dans les HSCs, nous avons induit des cassures double brin dans le locus HBA avec CRISPR-Cas9 et délivré un transgène thérapeutique en utilisant un AAV-6 pour cibler l'intégration par réparation dirigée par l'homologie. En raison de l'abondance des globules rouges chez l'homme (5X10^6 globules rouges/microlitre de sang) et de la forte expression de HBA dans cette population (environ 1,5g/jour), la plateforme HBA nécessiterait un minimum d'événement d'intégration du transgène pour fournir une quantité suffisante de protéines thérapeutiques, réduisant ainsi les risques de toxicité et d'édition non spécifique induit par CRISPR-Cas9 ou l'AAV6. Dans un premier temps, nous avons appliqué la plateforme HBA à la β-thalassémie : une condition génétique altérant la formation de l'hémoglobine (composé de 2 α-globine et 2 β-globine) et déclenchant la précipitation toxique de la chaîne α-globine. La correction génétique de la β-thalassémie à l'aide de notre plateforme HBA entraîne la délétion d'une copie du gène de l'α-globine, ce qui diminue la précipitation de cette chaîne, tandis que l'intégration du gène de la β-globine dans le locus HBA rétablit la formation normale d'hémoglobine. Ensuite, nous avons évalué la plateforme HBA pour la sécrétion spécifique de protéines thérapeutiques par les GRs dans la circulation sanguine et, en particulier, nous nous sommes concentrés sur un trouble métabolique génétique : la déficience en lipase acide lysosomale (LAL) causée par une ou plusieurs mutations du gène LIPA qui entraînent une accumulation toxique de lipides, notamment dans le foie. Nous avons obtenu une expression et une sécrétion efficaces de l'enzyme LAL qui a conservé son activité enzymatique et corrigé l'accumulation de lipides dans les fibroblastes des patients. Nous nous sommes ensuite concentrés sur l'optimisation du transgène LIPA en utilisant des vecteurs lentiviraux (LVVs) pour maximiser l'expression et la sécrétion de la LAL. Le transgène LIPA optimisé servira à développer une TG ex-vivo basée sur les LVVs et à notre plateforme ciblée HBA dans le but d'améliorer l'expression de LAL tout en minimisant les événements d'intégration nécessaires pour corriger complètement la maladie. Enfin, pour mieux caractériser le phénotype la déficience en LAL et évaluer le pouvoir thérapeutique de notre stratégie de GT, nous avons mis en place une approche de cytométrie multimodale utilisant la cytométrie en flux et la cytométrie en flux d'images. Nous avons évalué ces techniques principalement sur les fibroblastes du patient et sur les cellules mononucléaires du sang périphérique (PBMCs) provenant de souris déficiente en LAL.

Monogenic disorder are inherited mutation(s) in a single gene that result in abnormal protein synthesis impairing normal cellular homeostasis and organ function that reduces patient life-pan. Recently autologous transplantation of genetically corrected patient's hematopoietic stem cells (HSCs) defined as ex-vivo gene therapy (GT) has emerged as a promising curative approach for such genetic diseases. In ex-vivo GT, targeted integration of a therapeutic transgene into a tolerant and safe locus appears to be a powerful approach to produce the therapeutic protein above physiological level in a broad type of hematopoietic cells using endogenous promoter. Such genome editing strategy relies on DNA double strand break and delivery of a functional copy of the mutated gene in the locus of interest. Here, we exploited the α-globin locus (HBA) as a safe harbor locus for transgene integration to develop a universal platform for expression and secretion of therapeutic proteins into erythroid cells. In HSCs, we induced double strand breaks (DSBs) in HBA locus with CRISPR-Cas9 and delivered a therapeutic transgene using AAV-6 to target integration by homology directed repair. Due to the abundance of erythroid cells in human (5X10^6 RBCs/microliter of blood) and the high HBA expression in erythroid population (around 1,5g/day), the HBA platform would require minimal transgene integration events in HBA locus to provide sufficient amount of therapeutic proteins reducing as consequence the potential off-target and toxicity due to editing and DNA donor delivery. First, we applied the HBA platform for β-thalassemia : a genetic condition impairing the hemoglobin formation and triggering toxic α-globin chain precipitation. The genetic correction of β-thalassemia using our HBA platform results in 1 copy of A-globin gene deletion decreasing the α-globin chain precipitation while α-globin gene integration into HBA locus restore the normal hemoglobin formation. Then, we evaluated the HBA platform for erythroid-specific secretion of therapeutic proteins into bloodstream and, in particular,we focused on a genetic metabolic disorder: lysosomal acid lipase (LAL) deficiency caused by LIPA gene mutation(s) that lead to toxic lipid accumulation especially in liver. We achieved efficient expression and secretion of LAL enzyme that retained enzymatic activity and cross-corrected the lipid accumulation in patients' fibroblasts. We then focused on the optimization of LIPA cDNA transgene using lentiviral vectors (LVVs) to maximize the LAL expression and secretion. The optimized LIPA transgene will serve for LVV-based ex-vivo GT and for our HBA targeted platform in the purpose of enhancing the LAL expression while minimizing the targeting integration events required to fully correct the disorder. Finally, to better characterize LAL-D phenotype and to evaluate the therapeutic potency of our GT strategy, we set-up a multimodal cytometry approach using flow cytometry and image flow cytometry. We evaluated this pipeline on mainly patient's fibroblasts and on peripheral mononuclear blood mononuclear cells (PBMCs) for LAL-D mouse model., Les maladies monogéniques sont des mutations héréditaires d'un seul gène qui entraînent une synthèse anormale de protéines, altérant l'homéostasie cellulaire normale et la fonction des organes et réduisant la durée de vie des patients. Récemment, la greffe autologue de cellules souches hématopoïétiques (HSCs) de patients génétiquement corrigés appelée thérapie génique (TG) ex-vivo, est apparue comme une approche curative prometteuse pour ces maladies. Dans la TG ex-vivo, l'intégration ciblée d'un transgène thérapeutique dans un locus tolérant apparaît comme une approche puissante pour produire les protéines thérapeutiques à un niveau supérieur au niveau physiologique dans un large type de cellules hématopoïétiques en utilisant un promoteur endogène. Une telle stratégie d'édition du génome repose sur la cassure double brin d'ADN et la livraison d'une copie fonctionnelle du gène muté dans le locus d'intérêt. Ici, nous avons exploité le locus de l'α-globine (HBA) comme un locus tolérant pour l'intégration de transgènes afin de développer une plateforme universelle pour l'expression et la sécrétion de protéines thérapeutiques par les globules rouges (GR). Dans les HSCs, nous avons induit des cassures double brin dans le locus HBA avec CRISPR-Cas9 et délivré un transgène thérapeutique en utilisant un AAV-6 pour cibler l'intégration par réparation dirigée par l'homologie. En raison de l'abondance des globules rouges chez l'homme (5X10^6 globules rouges/microlitre de sang) et de la forte expression de HBA dans cette population (environ 1,5g/jour), la plateforme HBA nécessiterait un minimum d'événement d'intégration du transgène pour fournir une quantité suffisante de protéines thérapeutiques, réduisant ainsi les risques de toxicité et d'édition non spécifique induit par CRISPR-Cas9 ou l'AAV6. Dans un premier temps, nous avons appliqué la plateforme HBA à la β-thalassémie : une condition génétique altérant la formation de l'hémoglobine (composé de 2 α-globine et 2 β-globine) et déclenchant la précipitation toxique de la chaîne α-globine. La correction génétique de la β-thalassémie à l'aide de notre plateforme HBA entraîne la délétion d'une copie du gène de l'α-globine, ce qui diminue la précipitation de cette chaîne, tandis que l'intégration du gène de la β-globine dans le locus HBA rétablit la formation normale d'hémoglobine. Ensuite, nous avons évalué la plateforme HBA pour la sécrétion spécifique de protéines thérapeutiques par les GRs dans la circulation sanguine et, en particulier, nous nous sommes concentrés sur un trouble métabolique génétique : la déficience en lipase acide lysosomale (LAL) causée par une ou plusieurs mutations du gène LIPA qui entraînent une accumulation toxique de lipides, notamment dans le foie. Nous avons obtenu une expression et une sécrétion efficaces de l'enzyme LAL qui a conservé son activité enzymatique et corrigé l'accumulation de lipides dans les fibroblastes des patients. Nous nous sommes ensuite concentrés sur l'optimisation du transgène LIPA en utilisant des vecteurs lentiviraux (LVVs) pour maximiser l'expression et la sécrétion de la LAL. Le transgène LIPA optimisé servira à développer une TG ex-vivo basée sur les LVVs et à notre plateforme ciblée HBA dans le but d'améliorer l'expression de LAL tout en minimisant les événements d'intégration nécessaires pour corriger complètement la maladie. Enfin, pour mieux caractériser le phénotype la déficience en LAL et évaluer le pouvoir thérapeutique de notre stratégie de GT, nous avons mis en place une approche de cytométrie multimodale utilisant la cytométrie en flux et la cytométrie en flux d'images. Nous avons évalué ces techniques principalement sur les fibroblastes du patient et sur les cellules mononucléaires du sang périphérique (PBMCs) provenant de souris déficiente en LAL.

Myotonic dystrophy type I (DM1) is one the most frequent muscular dystrophies in adults. Although DM1 has long been considered mainly a muscle disorder, growing evidence suggests the involvement of peripheral nerves in the pathogenicity of DM1 raising the question of whether motoneurons (MNs) actively contribute to neuromuscular defects in DM1.By using micropatterned 96-well plates as a co-culture platform, we generated a functional neuromuscular model combining DM1 and MBNL-knock-out human induced pluripotent stem cells-derived MNs and human healthy skeletal muscle cells.This approach led to the identification of pre-synaptic defects which affect the formation or stability of the neuromuscular junction at an early developmental stage. These neuropathological defects could be reproduced by the loss of RNA-binding MBNL proteins, whose loss of function in vivo is associated with muscular defects associated with DM1. These experiments indicate that the functional defects associated with MNs can be directly attributed to MBNL family proteins. Comparative transcriptomic analyses also revealed specific neuronal-related processes regulated by these proteins that are commonly misregulated in DM1.Beyond the application to DM1, our approach to generating a robust and reliable human neuromuscular system should facilitate disease modelling studies and drug screening assays.

International audience; Recent studies indicate that neurodegenerative processes that appear during childhood and adolescence in individuals with Wolfram syndrome (WS) occur in addition to early brain development alteration, which is clinically silent. Underlying pathological mechanisms are still unknown. We have used induced pluripotent stem cell-derived neural cells from individuals affected by WS in order to reveal their phenotypic and molecular correlates. We have observed that a subpopulation of Wolfram neurons displayed aberrant neurite outgrowth associated with altered expression of axon guidance genes. Selective inhibition of the ATF6α arm of the unfolded protein response prevented the altered phenotype, although acute endoplasmic reticulum stress response—which is activated in late Wolfram degenerative processes—was not detected. Among the drugs currently tried in individuals with WS, valproic acid was the one that prevented the pathological phenotypes. These results suggest that early defects in axon guidance may contribute to the loss of neurons in individuals with WS.

International audience; Retinal degeneration is an increasing global burden without cure for the majority of patients. Once retinal cells have degenerated, vision is permanently lost. Different strategies have been developed in the recent years to prevent retinal degeneration or to restore sight (e.g., gene therapy, cell therapy and electronic implants). Herein, we present current treatment strategies with a focus on cell therapy for photoreceptor replacement using human pluripotent stem cells. We will describe the state of the art and discuss obstacles and limitations observed in preclinical animal models as well as future directions to improve graft integration and functionality.

Brain dysfunction in myotonic dystrophy type 1 (DM1), the prototype of toxic RNA disorders, has been mainly attributed to neuronal RNA misprocessing, while little attention has been given to non-neuronal brain cells. Using a transgenic mouse model of DM1 that expresses mutant RNA in various brain cell types, we demonstrate that astrocytes exhibit impaired ramification and polarization in vivo and defects in adhesion, spreading and migration. RNA-dependent toxicity and phenotypes was also found in human transfected glial cells. In line with the cell phenotypes, molecular analyses revealed extensive expression and accumulation of toxic RNA in astrocytes, which resulted in RNA spliceopathy that was remarkably more severe than in neurons. Astrocyte missplicing affected primarily transcripts that regulate cell adhesion, cytoskeleton and morphogenesis, and it was confirmed in human brain tissue. We demonstrate for the first time that DM1 impacts astrocyte cell biology, possibly compromising their support and regulation of synaptic function.

International audience; Abstract Cell motility is critical for tumor malignancy. Metabolism being an obligatory step in shaping cell behavior, we looked for metabolic weaknesses shared by motile cells across the diverse genetic contexts of patients’ glioblastoma. Computational analyses of single-cell transcriptomes from thirty patients’ tumors isolated cells with high motile potential and highlighted their metabolic specificities. These cells were characterized by enhanced mitochondrial load and oxidative stress coupled with mobilization of the cysteine metabolism enzyme 3-Mercaptopyruvate sulfurtransferase (MPST). Functional assays with patients’ tumor-derived cells and -tissue organoids, and genetic and pharmacological manipulations confirmed that the cells depend on enhanced ROS production and MPST activity for their motility. MPST action involved protection of protein cysteine residues from damaging hyperoxidation. Its knockdown translated in reduced tumor burden, and a robust increase in mice survival. Starting from cell-by-cell analyses of the patients’ tumors, our work unravels metabolic dependencies of cell malignancy maintained across heterogeneous genomic landscapes.

Congenital myasthenic syndromes (CMS) are predominantly characterized by muscle weakness and fatigability and can be caused by a variety of mutations in genes required for neuromuscular junction formation and maintenance. Among them, AGRN encodes agrin, an essential synaptic protein secreted by motoneurons. We have identified severe CMS patients with uncharacterized p.R1671Q, p.R1698P and p.L1664P mutations in the LG2 domain of agrin. Overexpression in primary motoneurons cultures in vitro and in chick spinal motoneurons in vivo revealed that the mutations modified agrin trafficking, leading to its accumulation in the soma and/or in the axon. Expression of mutant agrins in cultured cells demonstrated accumulation of agrin in the endoplasmic reticulum associated with induction of unfolded protein response (UPR) and impaired secretion in the culture medium. Interestingly, evaluation of the specific activity of individual agrins on AChR cluster formation indicated that when secreted, mutant agrins retained a normal capacity to trigger the formation of AChR clusters. To confirm agrin accumulation and secretion defect, iPS cells were derived from a patient and differentiated into motoneurons. Patient iPS-derived motoneurons accumulated mutant agrin in the soma and increased XBP1 mRNA splicing, suggesting UPR activation. Moreover, co-cultures of patient iPS-derived motoneurons with myotubes confirmed the deficit in agrin secretion and revealed a reduction in motoneuron survival. Altogether, we report the first mutations in AGRN gene that specifically affect agrin secretion by motoneurons. Interestingly, the three patients carrying these mutations were initially suspected of spinal muscular atrophy (SMA). Therefore, in the presence of patients with a clinical presentation of SMA but without mutation in the SMN1 gene, it can be worth to look for mutations in AGRN.

International audience; Although placental small extracellular vesicles (sEVs) are extensively studied in the context of pregnancy, little is known about their role during viral congenital infection, especially at the beginning of pregnancy. In this study, we examined the consequences of human cytomegalovirus (hCMV) infection on sEVs production, composition, and function using an immortalized human cytotrophoblast cell line derived from first trimester placenta. By combining complementary approaches of biochemistry, electron microscopy, and quantitative proteomic analysis, we showed that hCMV infection increases the yield of sEVs produced by cytotrophoblasts and modifies their protein content towards a potential proviral phenotype. We further demonstrate that sEVs secreted by hCMV-infected cytotrophoblasts potentiate infection in naive recipient cells of fetal origin, including human neural stem cells. Importantly, these functional consequences are also observed with sEVs prepared from an ex vivo model of infected histocultures from early placenta. Based on these findings, we propose that placental sEVs could be important actors favoring viral dissemination to the fetal brain during hCMV congenital infection.

International audience

International audience

DDX5 and DDX17 are DEAD-box RNA helicase paralogs which regulate several aspects of gene expression, especially transcription and splicing, through incompletely understood mechanisms. A transcriptome analysis of DDX5/DDX17-depleted human cells confirmed the large impact of these RNA helicases on splicing and revealed a widespread deregulation of 3′ end processing. In silico analyses and experiments in cultured cells showed the binding and functional contribution of the genome organizing factor CTCF to chromatin sites at or near a subset of DDX5/DDX17-dependent exons that are characterized by a high GC content and a high density of RNA Polymerase II. We propose the existence of an RNA helicase-dependent relationship between CTCF and the dynamics of transcription across DNA and/or RNA structured regions, that contributes to the processing of internal and terminal exons. Moreover, local DDX5/DDX17-dependent chromatin loops spatially connect RNA helicase-regulated exons with their cognate promoter, and we provide the first direct evidence that de novo gene looping modifies alternative splicing and polyadenylation. Overall our findings uncover the impact of DDX5/DDX17-dependent chromatin folding on pre-messenger RNA processing.

International audience; Inherited retinal diseases (IRDs) are a leading cause of blindness in industrialized countries, and gene therapy is quickly becoming a viable option to treat this group of diseases. Gene replacement using a viral vector has been successfully applied and advanced to commercial use for a rare group of diseases. This, and the advances in gene editing, are paving the way for the emergence of a new generation of therapies that use CRISPR-Cas9 to edit mutated genes in situ. These CRISPR-based agents can be delivered to the retina as transgenes in a viral vector, unpackaged transgenes or as proteins or messenger RNA using non-viral vectors. Although the eye is considered to be an immune-privileged organ, studies in animals, as well as evidence from clinics, have concluded that ocular gene therapies elicit an immune response that can under certain circumstances result in inflammation. In this review, we evaluate studies that have reported on pre-existing immunity, and discuss both innate and adaptive immune responses with a specific focus on immune responses to gene editing, both with non-viral and viral delivery in the ocular space. Lastly, we discuss approaches to prevent and manage the immune responses to ensure safe and efficient gene editing in the retina.

International audience; Introduction: Brain dysfunction in neurological diseases is frequently mediated by the impairment of neuronal and non-neuronal cells. Although DMPK gene expression is higher in cortical astrocytes than in neurons isolated from adult human and mouse brains, the contribution of astroglia to DM1 brain disease has been poorly investigated. Methods: Transgenic DMSXL mice express expanded human DMPK transcripts in multiple cell types of the brain, providing a good model to investigate the impact of RNA toxicity on astroglia.Results: DMSXL astrocytes exhibit impaired ramification and polarization in vivo, as well as defects in adhesion, spreading and migration in culture. In line with these pronounced phenotypes, DMSXL astrocytes express high levels of toxic RNA and accumulate abundant RNA foci, relative to neurons. RNA sequencing revealed MBNL-dependent RNA spliceopathy, which affects primarily transcripts that regulate cell adhesion, cytoskeleton and morphogenesis. To study the impact of defective astrocytes on neurons, we used co-culture cell systems, and found that DMSXL astrocytes impair neuritogenesis.Conclusions: We demonstrate that DM1 impacts astrocyte cell biology, possibly compromising the support and regulation of synaptic function through defective neuroglia interplay.

International audience; Introduction: Myotonic dystrophy type I (DM1) is one the most frequent muscular dystrophy in adults. Although DM1 has long been considered mainly as a muscle disorder, growing evidence suggests the involvement in peripheral nerves in the pathogenicity of DM1 raising the question whether motoneurons actively contribute to neuromuscular defects in DM1.Methods: By using a micropatterned 96-well plate as a co-culture platform, we generated a functional humanized cellular model combining DM1 hiPSC-derived MNs and healthy skeletal muscle cells.Results: Such approaches led to the identification of pre-synaptic defects which affect development or stability of the neuromuscular junction at an early developmental stage. These neuropathological defects could be reproduced by the loss of RNA-binding MBNL proteins, whose loss of function is associated with muscular defects associated with DM1.Conclusions: These experiments suggested that the functional defects associated to MNs can be directly attributed to the MBNL family proteins. Altogether, these findings hold several new implications for DM1 pathogenesis.

Maple syrup urine disease (MSUD) is a rare recessively inherited metabolic disorder causing accumulation of branched chain amino acids leading to neonatal death, if untreated. Treatment for MSUD represents an unmet need because the current treatment with life-long low-protein diet is challenging to maintain, and despite treatment the risk of acute decompensations and neuropsychiatric symptoms remains. Here, based on significant liver contribution to the catabolism of the branched chain amino acid leucine, we develop a liver-directed adeno-associated virus (AAV8) gene therapy for MSUD. We establish and characterize the Bckdha (branched chain keto acid dehydrogenase a)−/− mouse that exhibits a lethal neonatal phenotype mimicking human MSUD. Animals were treated at P0 with intravenous human BCKDHA AAV8 vectors under the control of either a ubiquitous or a liver-specific promoter. BCKDHA gene transfer rescued the lethal phenotype. While the use of a ubiquitous promoter fully and sustainably rescued the disease (long-term survival, normal phenotype and correction of biochemical abnormalities), liver-specific expression of BCKDHA led to partial, though sustained rescue. Here we show efficacy of gene therapy for MSUD demonstrating its potential for clinical translation.

Limb girdle muscular dystrophies (LGMD), caused by mutations in 29 different genes, are the fourth most prevalent group of genetic muscle diseases, leading to progressive weakness and atrophy of the skeletal muscles. Although the link between LGMD and their genetic origins has been determined, LGMD still represent an unmet medical need. In this paper, we describe a platform for modeling LGMD based on the use of human induced pluripotent stem cells (hiPSC). Thanks to the self-renewing and pluripotency properties of hiPSC, this platform provides an alternative and renewable source of skeletal muscle cells (skMC) to primary, immortalized or overexpressing cells. We report that skMC derived from hiPSC express the majority of the genes and proteins causing LGMD. As a proof of concept, we demonstrate the importance of this cellular model for studying LGMDR9 by evaluating disease-specific phenotypes in skMC derived from hiPSC obtained from four patients.

Evaluation of immune responses to adeno-associated virus (AAV)-mediated gene therapies prior to and following dose administration plays a key role in determining therapeutic safety and efficacy. This report describes up to 3 years of immunogenicity data following administration of valoctocogene roxaparvovec (BMN 270), an AAV5-mediated gene therapy encoding human B domain-deleted FVIII (hFVIII-SQ) in a phase 1/2 clinical study of adult males with severe hemophilia A. Patients with pre-existing humoral immunity to AAV5 or with a history of FVIII inhibitors were excluded from the trial. Blood plasma and peripheral blood mononuclear cell (PBMC) samples were collected at regular intervals following dose administration for assessment of humoral and cellular immune responses to both the AAV5 vector and transgene-expressed hFVIII-SQ. The predominant immune response elicited by BMN 270 administration was largely limited to the development of antibodies against the AAV5 capsid that were cross-reactive with other common AAV serotypes. No FVIII inhibitor responses were observed within 3 years following dose administration. In a context of prophylactic or on-demand corticosteroid immunosuppression given after vector infusion, AAV5 and hFVIII-SQ peptide-specific cellular immune responses were intermittently detected by an interferon (IFN)-γ and tumor necrosis factor (TNF)-α FluoroSpot assay, but they were not clearly associated with detrimental safety events or changes in efficacy measures., Graphical Abstract, This report describes up to 3 years of immunogenicity data following administration of valoctocogene roxaparvovec, an AAV5-mediated gene therapy encoding hFVIII-SQ, in a phase 1/2 clinical study of adult males with severe hemophilia A. Immunogenicity elicited by BMN 270 administration was predominantly a humoral antibody response against the AAV5 capsid. No FVIII inhibitor responses were observed within 3 years following dose administration. No immunogenicity measures were consistently associated with detrimental safety or efficacy parameters.

International audience; Glycogen storage disorder type III (GSDIII) is a rare inborn error of metabolism due to loss of glycogen debranching enzyme activity, causing inability to fully mobilize glycogen stores and its consequent accumulation in various tissues, notably liver, cardiac and skeletal muscle. In the pediatric population, it classically presents as hepatomegaly with or without ketotic hypoglycemia and failure to thrive. In the adult population, it should also be considered in the differential diagnosis of left ventricular hypertrophy or hypertrophic cardiomyopathy, myopathy, exercise intolerance, as well as liver cirrhosis or fibrosis with subsequent liver failure. In this review article, we first present an overview of the biochemical and clinical aspects of GSDIII. We then focus on the recent findings regarding cardiac and neuromuscular impairment associated with the disease. We review new insights into the pathophysiology and clinical picture of this disorder, including symptomatology, imaging and electrophysiology. Finally, we discuss current and upcoming treatment strategies such as gene therapy aimed at the replacement of the malfunctioning enzyme to provide a stable and long-term therapeutic option for this debilitating disease.

Limb-girdle muscular dystrophy type R3 (LGMD R3) is a rare genetic disorder characterized by a progressive proximal muscle weakness and caused by mutations in the SGCA gene encoding alpha-sarcoglycan (α-SG). Here, we report the results of a mechanistic screening ascertaining the molecular mechanisms involved in the degradation of the most prevalent misfolded R77C-α-SG protein. We performed a combinatorial study to identify drugs potentializing the effect of a low dose of the proteasome inhibitor bortezomib on the R77C-α-SG degradation inhibition. Analysis of the screening associated to artificial intelligence-based predictive ADMET characterization of the hits led to identification of the HDAC inhibitor givinostat as potential therapeutical candidate. Functional characterization revealed that givinostat effect was related to autophagic pathway inhibition, unveiling new theories concerning degradation pathways of misfolded SG proteins. Beyond the identification of a new therapeutic option for LGMD R3 patients, our results shed light on the potential repurposing of givinostat for the treatment of other genetic diseases sharing similar protein degradation defects such as LGMD R5 and cystic fibrosis.

One of the major goals of in vivo gene transfer is to achieve long-term expression of therapeutic transgenes in terminally differentiated cells. The extensive clinical experience and the recent approval of Luxturna® (Spark Therapeutics, now Roche) and Zolgensma® (AveXis, now Novartis) place vectors derived from adeno-associated viruses (AAV) among the best options for gene transfer in multiple tissues. Despite these successes, limitations remain to the application of this therapeutic modality in a wider population. AAV was originally identified as a promising virus to derive gene therapy vectors because, despite infecting humans, it was not associated with any evident disease. Thee large proportion of AAV infections in the human population is now revealing as a limitation because after exposure to wild-type AAV, anti-AAV antibodies develops and may neutralize the vectors derived from the virus. Injection of AAV in humans is generally well-tolerated although the immune system can activate after the recognition of AAV vectors capsid and genome. The formation of high-titer neutralizing antibodies to AAV after the first injection precludes vector re-administration. Thus, both pre-existing and post-treatment humoral responses to AAV vectors greatly limit a wider application of this gene transfer modality. Different methods were suggested to overcome this limitation. The extensive preclinical data available and the large clinical experience in the control of AAV vectors immunogenicity are key to clinical translation and to demonstrate the safety and efficacy of these methods and ultimately bring a curative treatment to patients.

International audience; Mutations leading to haploinsufficiency in SCN5A, the gene encoding the cardiac sodium channel Na v 1.5 α-subunit, are involved in life-threatening cardiac disorders. Using CRISPR/Cas9-mediated genome edition, we generated here a human induced-pluripotent stem cell (hiPSC) line carrying a heterozygous mutation in exon 2 of SCN5A, which leads to apparition of a premature stop codon. SCN5A-clone 5 line maintained normal karyotype, morphology and pluripotency and differentiated into three germ layers. Cardiomyocytes derived from these hiPSCs would be a useful model for investigating channelopathies related to SCN5A heterozygous deficiency.

International audience; Positive clinical outcomes in adeno-associated virus (AAV)mediated retinal gene therapy have often been attributed to the low immunogenicity of AAVs and immune privilege of the eye. However, several recent studies have shown potential for inflammatory responses. The current understanding of the factors contributing to inflammation, such as the preexistence of serum antibodies against AAVs and their contribution to increases in antibody levels post-injection, is incomplete. The parameters that regulate the generation of new antibodies in response to the AAV capsid or transgene after intraocular injections are also insufficiently described. This study is a retrospective analysis of the pre-existing serum antibodies in correlation with changes in antibody levels after intraocular injections of AAV in non-human primates (NHPs) of the species Macaca fascicularis. In NHP serums, we analyzed the binding antibody (BAB) levels and a subset of these called neutralizing antibodies (NABs) that impede AAV transduction. We observed significantly higher pre-existing serum BABs against AAV8 compared with other serotypes and a dose-dependent increase in BABs and NABs in the serums collected post-injection, irrespective of the serotype or the mode of injection. Lastly, we were able to demonstrate a correlation between the serum BAB levels with clinical grading of inflammation and levels of transgene expression.

Spinal muscular atrophy (SMA) is a genetic disease resulting in the loss of α-motoneurons followed by muscle atrophy. It is caused by knock-out mutations in the survival of motor neuron 1 (SMN1) gene, which has an unaffected, but due to preferential exon 7 skipping, only partially functional human-specific SMN2 copy. We previously described a Drosophila-based screening of FDA-approved drugs that led us to discover moxifloxacin. We showed its positive effect on the SMN2 exon 7 splicing in SMA patient-derived skin cells and its ability to increase the SMN protein level. Here, we focus on moxifloxacin's therapeutic potential in additional SMA cellular and animal models. We demonstrate that moxifloxacin rescues the SMA-related molecular and phenotypical defects in muscle cells and motoneurons by improving the SMN2 splicing. The consequent increase of SMN levels was higher than in case of risdiplam, a potent exon 7 splicing modifier, and exceeded the threshold necessary for a survival improvement. We also demonstrate that daily subcutaneous injections of moxifloxacin in a severe SMA murine model reduces its characteristic neuroinflammation and increases the SMN levels in various tissues, leading to improved motor skills and extended lifespan. We show that moxifloxacin, originally used as an antibiotic, can be potentially repositioned for the SMA treatment.

Human glioblastoma (GBM) is the most common primary malignant brain tumor. A minor subpopulation of cancer cells, known as glioma stem-like cells (GSCs), are thought to play a major role in tumor relapse due to their stem cell-like properties, their high resistance to conventional treatments and their high invasion capacity. We show that ionizing radiation specifically enhances the motility and invasiveness of human GSCs through the stabilization and nuclear accumulation of the hypoxia-inducible factor 1α (HIF1α), which in turn transcriptionally activates the Junction-mediating and regulatory protein (JMY). Finally, JMY accumulates in the cytoplasm where it stimulates GSC migration via its actin nucleation-promoting activity. Targeting JMY could thus open the way to the development of new therapeutic strategies to improve the efficacy of radiotherapy and prevent glioma recurrence. The authors thank members of the LRP for helpful discussions and are indebted to V. Barroca and the staff of the animal facilities and to N. Deschamps and J. Baijer for cell sorting. We also thanks I. Naguibneva for the gift of the pTRIP shHIF1α plasmid. MS is the recipient of a doctoral fellowship from the Ministère de la Recherche. This work was supported by grants from CEA (Segment Radiobiologie), La Ligue contre le Cancer (Comité d’Ile de France), Electricité de France (EDF), Fondation de France (N° Engt: 2013-00042632) and Ramón y Cajal program (RYC-2013-13450).

International audience; Targeted genome editing has a great therapeutic potential to treat disorders that require protein replacement therapy. To develop a platform independent of specific patient mutations, therapeutic transgenes can be inserted in a safe and highly transcribed locus to maximize protein expression. Here, we describe an ex vivo editing approach to achieve efficient gene targeting in human hematopoietic stem/progenitor cells (HSPCs) and robust expression of clinically relevant proteins by the erythroid lineage. Using CRISPR-Cas9, we integrate different transgenes under the transcriptional control of the endogenous α-globin promoter, recapitulating its high and erythroid-specific expression. Erythroblasts derived from targeted HSPCs secrete different therapeutic proteins, which retain enzymatic activity and cross-correct patients' cells. Moreover, modified HSPCs maintain long-term repopulation and multilineage differentiation potential in transplanted mice. Overall, we establish a safe and versatile CRISPR-Cas9-based HSPC platform for different therapeutic applications, including hemophilia and inherited metabolic disorders.

T cell receptor (TCR) activation is modulated by mechanisms such as TCR endocytosis, which is thought to terminate TCR signalling. Here we show that, upon internalization, TCR continues to signal from a set of specialized endosomes that are crucial for T cell functions. Mechanistically, TCR ligation leads to clathrin-mediated internalization of the TCR-CD3ζ complex, while maintaining CD3ζ signalling, in endosomal vesicles that contain the insulin responsive aminopeptidase (IRAP) and the SNARE protein Syntaxin 6. Destabilization of this compartment through IRAP deletion enhances plasma membrane expression of the TCR-CD3ζ complex, yet compromises overall CD3ζ signalling; moreover, the integrity of this compartment is also crucial for T cell activation and survival after suboptimal TCR activation, as mice engineered with a T cell-specific deletion of IRAP fail to develop efficient polyclonal anti-tumour responses. Our results thus reveal a previously unappreciated function of IRAP-dependent endosomal TCR signalling in T cell activation., T cell receptors (TCR) are internalized when activated by their ligands. Here the authors show that the internalized TCRs are localized to endosomes expressing IRAP and Syntaxin 6 to maintain intracellular signalling capacity, whose importance is shown by the absence of efficient polyclonal anti-tumour response in mice with T-specific conditional deletion of IRAP.

International audience; Congenital titinopathies are myopathies with variable phenotypes and inheritance modes. Here, we fully characterized, using an integrated approach (deep phenotyping, muscle morphology, mRNA and protein evaluation in muscle biopsies), two siblings with congenital multicore myopathy harboring three TTN variants predicted to affect titin stability and titin-myosin interactions. Muscle biopsies showed multicores, type 1 fiber uniformity and sarcomeric structure disruption with some thick filament loss. Immunohistochemistry and Western blotting revealed a marked reduction of fast myosin heavy chain iso-forms. This is the first observation of a titinopathy suggesting that titin defect leads to secondary loss of fast myosin heavy chain isoforms.

International audience; Generation of relevant and robust models for neurological disorders is of main importance for both target identification and drug discovery. The non-cell autonomous effects of glial cells on neurons have been described in a broad range of neurodegenerative and neurodevelopmental disorders, pointing to neuroglial interactions as novel alternative targets for therapeutics development. Interestingly, the recent breakthrough discovery of human induced pluripotent stem cells (hiPSCs) has opened a new road for studying neurological and neurodevelopmental disorders “in a dish”. Here, we provide an overview of the generation and modeling of both neuronal and glial cells from human iPSCs and a brief synthesis of recent work investigating neuroglial interactions using hiPSCs in a pathophysiological context.

Wolfram syndrome type 1 (WS1) is a rare autosomal recessive neurodegenerative disorder characterized by a combination of devastating clinical symptoms, namely: diabetes insipidus (DI), insulin-dependent diabetes mellitus (DM), optic atrophy (OA) and deafness (D), also known as DIDMOAD. Other impairments appear over time including various neurological complications, frequently causing premature death of patients around 40 years old, usually due to respiratory failure induced by brainstem atrophy. To date, and despite scientific advances, there is no cure. Recently, several MRI studies of young patients have shown important early damage including an overall reduction of brain volume and hypomyelination. These findings pointed out the need to reconsider the syndrome not only as a neurodegenerative disease but also as a neurodevelopmental disorder. Wolfram syndrome type 1 is caused by mutations in the WFS1 gene which encodes a protein located in the endoplasmic reticulum membrane, wolframin. The development of cellular and animal models of the disease has identified certain physiological roles for the protein. Thus, an increase in the activation of the unfolded protein response (UPR) and defects in calcium homeostasis have been described in its absence. Recently, the localization of the protein to mitochondria-associated membranes (MAMs) and mitochondrial defects have been described. These mechanisms were mainly observed in pancreatic β cells, but to date they have not been studied in the context of neurological impairments. This can be explained by the complexity regarding the development of a neurological model genetically and physiologically relevant. Thus, the objective of my thesis was to exploit the unique properties of induced pluripotent stem cells (iPSCs) in order to develop a human cellular model to study neurological damage reported in patients. Using this approach, we were able to generate the cells of interest, neural stem cells (NSC) and cortical neurons. Then, we analyzed the consequences of wolframin loss for the neuronal differentiation process and identified, in neurons derived from patient's iPSC lines, a yet unknown major neurodevelopmental defect. This defect is characterized by an alteration of neuritic outgrowth with increase neuritic bundling. It was correlated to the absence of the protein thanks to the use of a WS iPSC rescue line. Transcriptomic analyses revealed the deregulation of genes known to be involved in neurogenesis, nervous system development and axonal guidance. By studying the mechanisms underlying this abnormal neuronal network, we were able to identify the involvement of the ATF6α pathway of the UPR. Finally, the potential effect of several molecules currently in clinical trials was also evaluated. We found that, among all these molecules, only valproic acid (VPA) prevented the appearance of the phenotype. The use of this anti-epileptic molecule also allowed the correction of the expression of several genes in the mutated neurons. These results contributed to the launch of a clinical trial in WS1 patient complementary to the Treatwolfram one. In conclusion, this work has allowed the development of a new model using patient cells and the identification of a neuritic growth defect strengthening the hypothesis of a neurodevelopmental component in Wolfram syndrome., Le syndrome de Wolfram de type 1 (SW1) est une maladie neurodégénérative autosomique récessive rare caractérisée par l'association de quatre symptômes principaux : un diabète insipide (DI), un diabète sucré insulino-dépendant (DM), une atrophie optique (OA) et une surdité (D). D'autres atteintes apparaissent avec le temps dont des complications neurologiques variées principalement associées à une atrophie du cervelet et du tronc cérébral. Récemment, plusieurs études réalisées par IRM sur de jeunes patients ont mis en évidence la présence d'atteintes précoces majeures dont une diminution du volume cérébral et une hypomyélinisation. Ces résultats ont soulevé la nécessité de requalifier le syndrome non seulement en maladie neurodégénérative mais également en maladie neurodéveloppementale. La maladie, d'évolution lente, progresse généralement vers un décès précoce, le plus souvent dû à une détresse respiratoire consécutive aux atteintes neurologiques. A ce jour, aucun traitement curatif n'existe et les patients décèdent généralement avant 40 ans. Le SW1 est causé par des mutations au sein du gène WFS1 qui code pour une protéine située à la membrane du réticulum endoplasmique, la wolframine. L'élaboration de modèles cellulaires et animaux de la pathologie a permis d'identifier certains rôles physiologiques de la protéine. Ainsi, une augmentation de l'activation de la réponse aux protéines mal repliées (UPR), des défauts de l'homéostasie calcique, ou encore un dysfonctionnement des mitochondries, liés aux membranes associées aux mitochondries (MAMs), ont été identifiés. Principalement observés dans les atteintes pancréatiques, ces mécanismes restent à ce jour peu étudiés dans le contexte des atteintes neurologiques. Cela peut s'expliquer par la complexité et les limites liées à la mise en place d'un modèle neurologique génétiquement et physiologiquement proche des patients. Ainsi, l'objectif de ma thèse a consisté à exploiter les propriétés uniques des cellules souches pluripotentes induites (CSPi) dans le but de développer un modèle cellulaire humain pour étudier les atteintes neurologiques observées chez les patients. Par cette approche, nous sommes parvenus à générer, à partir de CSPi contrôles et de CSPi issues de patients, les cellules d'intérêt, à savoir des cellules souches neurales (NSC) et des neurones corticaux. Puis, nous avons étudié les conséquences de la perte de wolframine au cours de la différenciation neuronale, et avons ainsi pu identifier un défaut neurodéveloppemental encore méconnu à ce jour. Ce défaut se traduit par une altération de la pousse neuritique et l'apparition de fasciculations axonales anormalement épaisses au cours du développement des neurones issus de CSPi mutées. Cette altération de la pousse neuritique a été corrélée à l'absence de la protéine grâce à l'utilisation d'une lignée rescue. Des analyses transcriptomiques ont permis de mettre en évidence dans les NSC et les neurones corticaux une dérégulation de gènes connus pour être impliqués dans la neurogenèse, le développement du système nerveux et le guidage axonal. En étudiant les mécanismes à l'origine de ce réseau neuronal anormal, nous avons pu identifier l'implication de la voie ATF6α de l'UPR. Enfin, l'effet potentiel de plusieurs molécules actuellement en essai clinique a également été évalué. Nous avons mis en évidence que, parmi toutes ces molécules, seul l'acide valproïque (VPA) permettait de prévenir l'apparition du phénotype. L'utilisation de cette molécule antiépileptique a également permis de corriger certains défauts d'expression génique dans les neurones mutés. Ces résultats ont contribué au lancement d'un nouvel essai clinique dans le cadre du SW1. Pour conclure, ces travaux ont permis l'élaboration d'un nouveau modèle à partir de cellules de patients ainsi que l'identification d'un défaut de pousse neuritique renforçant l'hypothèse d'une composante neurodéveloppementale dans le syndrome de Wolfram de type 1.

International audience; To investigate which cytokines, chemokines and growth factors are involved in the immunopathogenesis of idiopathic uveitis, and whether cytokine profiles are associated with. Serum and aqueous humor (AH) samples of 75 patients with idiopathic uveitis were analyzed by multiplex immunoassay. Infectious controls consisted of 16 patients with ocular toxoplasmosis all confirmed by intraocular fluid analyses. Noninfectious controls consisted of 7 patients with Behçet disease related uveitis and 15 patients with sarcoidosis related uveitis. The control group consisted of AH and serum samples from 47 noninflammatory control patients with age-related cataract. In each sample, 27 immune mediators ± IL-21 and IL-23 were measured. In idiopathic uveitis, 13 of the 29 mediators, including most proinflammatory and vascular mediators such as IL-6, IL-8, IL-12, G-CSF, GM-CSF, MCP-1, IP-10, TNF-α and VEGF, were significantly elevated in the aqueous humor when compared to all controls. Moreover, IL-17, IP-10, and IL-21, were significantly elevated in the serum when compared to all controls. We clustered 4 subgroups of idiopathic uveitis using a statistical analysis of hierarchical unsupervised classification, characterized by the order of magnitude of concentrations of intraocular cytokines. The pathogenesis of idiopathic uveitis is characterized by the presence of predominantly proinflammatory cytokines and chemokines and vascular endothelial growth factor with high expression levels as compared to other causes of uveitis. There are indications for obvious Th-1/ IL21-Th17 pathways but also IL9-Th9 and increased IFN-γ-inducing cytokine (IL12) and IFN-γ-inducible CXC chemokine (IP-10). The combined data suggest that immune mediator expression is different among idiopathic uveitis. This study suggests various clusters among the idiopathic uveitis group rather than one specific uveitis entity.

Sickle cell disease and β-thalassemia affect the production of the adult β-hemoglobin chain. The clinical severity is lessened by mutations that cause fetal γ-globin expression in adult life (i.e., the hereditary persistence of fetal hemoglobin). Mutations clustering ~200 nucleotides upstream of the HBG transcriptional start sites either reduce binding of the LRF repressor or recruit the KLF1 activator. Here, we use base editing to generate a variety of mutations in the −200 region of the HBG promoters, including potent combinations of four to eight γ-globin-inducing mutations. Editing of patient hematopoietic stem/progenitor cells is safe, leads to fetal hemoglobin reactivation and rescues the pathological phenotype. Creation of a KLF1 activator binding site is the most potent strategy – even in long-term repopulating hematopoietic stem/progenitor cells. Compared with a Cas9-nuclease approach, base editing avoids the generation of insertions, deletions and large genomic rearrangements and results in higher γ-globin levels. Our results demonstrate that base editing of HBG promoters is a safe, universal strategy for treating β-hemoglobinopathies.

Brain dysfunction in myotonic dystrophy type 1 (DM1), the prototype of toxic RNA disorders, has been mainly attributed to neuronal RNA misprocessing, while little attention has been given to non-neuronal brain cells. Here, using a transgenic mouse model of DM1 that expresses mutant RNA in various brain cell types (neurons, astroglia, and oligodendroglia), we demonstrate that astrocytes exhibit impaired ramification and polarization in vivo and defects in adhesion, spreading, and migration. RNA-dependent toxicity and phenotypes are also found in human transfected glial cells. In line with the cell phenotypes, molecular analyses reveal extensive expression and accumulation of toxic RNA in astrocytes, which result in RNA spliceopathy that is more severe than in neurons. Astrocyte missplicing affects primarily transcripts that regulate cell adhesion, cytoskeleton, and morphogenesis, and it is confirmed in human brain tissue. Our findings demonstrate that DM1 impacts astrocyte cell biology, possibly compromising their support and regulation of synaptic function.

Pompe disease (PD) is a severe neuromuscular disorder caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). PD is currently treated with enzyme replacement therapy (ERT) with intravenous infusions of recombinant human GAA (rhGAA). Although the introduction of ERT represents a breakthrough in the management of PD, the approach suffers from several shortcomings. Here, we developed a mouse model of PD to compare the efficacy of hepatic gene transfer with adeno-associated virus (AAV) vectors expressing secretable GAA with long-term ERT. Liver expression of GAA results in enhanced pharmacokinetics and uptake of the enzyme in peripheral tissues compared to ERT. Combination of gene transfer with pharmacological chaperones boosts GAA bioavailability, resulting in improved rescue of the PD phenotype. Scale-up of hepatic gene transfer to non-human primates also successfully results in enzyme secretion in blood and uptake in key target tissues, supporting the ongoing clinical translation of the approach., Pompe disease is currently treated with enzyme replacement therapy (ERT) with recombinant human acid alpha-glucosidase (GAA). Here, the authors show hepatic-directed gene therapy with AAV vectors enhances GAA bioavailability compared with ERT, resulting in improved rescue of the disease phenotype in mice and broad enzyme distribution in mice and non-human primates.

International audience; Dans la souris mdx, l’injection d’exosomes permet d’améliorer les phénotypes pathologiques musculaires, cardiaques et squelettiques, et ce grâce à plusieurs mécanismes non exclusifs. Les exosomes, qui font partie des vésicules extracellulaires, mesurent une centaine de nanomètres et sont produits par de très nombreux types cellulaires. Leurs marqueurs de surface et leurs contenus sont variables en fonction des cellules productrices et des conditions environnementales. Libérés par exocytose, les exosomes sont ensuite internalisés par endocytose ou fusion membranaire. Ils présentent une action pléiotropique et jouent un rôle primordial de messagers intercellulaires en conditions normales et pathologiques. Dans la souris mdx, l’absence de dystrophine entraîne une perte de l’intégrité membranaire, un déséquilibre de l’homéostasie, une dégénérescence progressive et une inflammation musculaire, le tout se traduisant par une baisse de force, une fatigabilité, et le développement d’une insuffisance cardiaque.Dans une première étude [1], l’injection intrapéritonéale d’exosomes produits à partir de cellules souches mésenchymateuses humaines et murines, de cellules dendritiques immortalisées murines, de myotubes murins, ou d’extraits de sérum murin, permet de limiter l’afflux de calcium intracellulaire, de diminuer l’activation de protéases, et de stabiliser les complexes existants de protéines associées à la dystrophine (sarcoglycans, dystroglycans). En restaurant partiellement la myo-architecture et l’intégrité membranaire des cellules musculaires squelettiques des souris mdx, les exosomes bloquent l’influx de marqueurs exogènes (tel le colorant bleu Evans) et la fuite de protéines endogènes (comme les CPK et la LDH). Leur activité immunomodulatrice diminue l’infiltration leucocytaire et l’inflammation, et réduit l’expansion ultérieure de la fibrose. Ces effets sur des cibles multiples se traduisent par une augmentation de la force et de l’endurance des souris.Dans une autre étude sur le même sujet [2], une injection intraveineuse unique d’exosomes produits par des cellules dérivées de progéniteurs cardiaques (cardiosphères) améliore les fonctions musculaires cardiaques et squelettiques des souris mdx. Les exosomes font régresser les lésions cardiaques, augmentent la fraction d’éjection myocardique, améliorant la capacité maximale d’exercice et diminuant la fibrose myocardique. Ils stimulent la régénération musculaire squelettique, augmentent la prolifération des cellules satellites, diminuent les réponses inflammatoires, et augmentent in fine la force isométrique des muscles squelettiques. Les bénéfices sont liés aux modifications transcriptomiques exercées par les micro-ARN (miR) contenus dans les exosomes, en particulier miR148a, ainsi qu’aux mécanismes d’immunomodulation. Les exosomes sont aussi efficaces que les cellules productrices elles-mêmes et constituent probablement leur principal mécanisme d’action.

Description, A new therapeutic approach for the treatment of a skeletal disorder with AAVs bypasses bone resistance to gene transfer., Adeno-associated virus (AAV) vectors are a well-established gene transfer approach for rare genetic diseases. Nonetheless, some tissues, such as bone, remain refractory to AAV. X-linked hypophosphatemia (XLH) is a rare skeletal disorder associated with increased levels of fibroblast growth factor 23 (FGF23), resulting in skeletal deformities and short stature. The conventional treatment for XLH, lifelong phosphate and active vitamin D analogs supplementation, partially improves quality of life and is associated with severe long-term side effects. Recently, a monoclonal antibody against FGF23 has been approved for XLH but remains a high-cost lifelong therapy. We developed a liver-targeting AAV vector to inhibit FGF23 signaling. We showed that hepatic expression of the C-terminal tail of FGF23 corrected skeletal manifestations and osteomalacia in a XLH mouse model. Our data provide proof of concept for AAV gene transfer to treat XLH, a prototypical bone disease, further expanding the use of this modality to treat skeletal disorders.

International audience; Recent advances in genome editing tools, especially novel developments in the clustered regularly interspaced short palindromic repeats associated to Cas9 nucleases (CRISPR/Cas9)-derived editing machinery, have revolutionized not only basic science but, importantly, also the gene therapy field. Their flexibility and ability to introduce precise modifications in the genome to disrupt or correct genes or insert expression cassettes in safe harbors in the genome underline their potential applications as a medicine of the future to cure many genetic diseases. In this review, we give an overview of the recent progress made by French researchers in the field of therapeutic genome editing, while putting their work in the general context of advances made in the field. We focus on recent hematopoietic stem cell gene editing strategies for blood diseases affecting the red blood cells or blood coagulation as well as lysosomal storage diseases. We report on a genome editing-based therapy for muscular dystrophy and the potency of T cell gene editing to increase anticancer activity of chimeric antigen receptor T cells to combat cancer. We will also discuss technical obstacles and side effects such as unwanted editing activity that need to be surmounted on the way toward a clinical implementation of genome editing. We propose here improvements developed today, including by French researchers to overcome the editing-related genotoxicity and improve editing precision by the use of novel recombinant nuclease-based systems such as nickases, base editors, and prime editors. Finally, a solution is proposed to resolve the cellular toxicity induced by the systems employed for gene editing machinery delivery.

International audience

International audience; We recently identified a signaling pathway that links the upregulation of miR-379 with a mitochondrial response in dystrophic muscle. In the present commentary, we explain the significance that this pathway may have in mitochondrial dysfunction in Duchenne muscular dystrophy (DMD). We identified the upregulation of miR-379 in the serum and muscles of DMD animal models and patients. We found that miR-379 is one of very few miRNAs whose expression was normalized in DMD patients treated with glucocorticoid. We identified EIF4G2 as a miR-379 target, which may promote mitochondrial oxidative phosphorylation (OxPhos) in the skeletal muscle. We found enriched EIF4G2 expression in oxidative fibers, and identified the mitochondrial ATP synthase subunit DAPIT as a translational target of EIF4G2. The identified signaling cascade, which comprises miR-379, EIF4G2 and DAPIT, may link the glucocorticoid treatment in DMD to a recovered mitochondrial ATP synthesis rate. We propose an updated model of mitochondrial dysfunction in DMD.

Titin is a giant sarcomeric protein that is involved in a large number of functions, with a primary role in skeletal and cardiac sarcomere organization and stiffness. The titin gene (TTN) is subject to various alternative splicing events, but in the region that is present at the M-line, the only exon that can be spliced out is Mex5, which encodes for the insertion sequence 7 (is7). Interestingly, in the heart, the majority of titin isoforms are Mex5+, suggesting a cardiac role for is7. Here, we performed comprehensive functional, histological, transcriptomic, microscopic and molecular analyses of a mouse model lacking the Ttn Mex5 exon (ΔMex5), and revealed that the absence of the is7 is causative for dilated cardiomyopathy. ΔMex5 mice showed altered cardiac function accompanied by increased fibrosis and ultrastructural alterations. Abnormal expression of excitation–contraction coupling proteins was also observed. The results reported here confirm the importance of the C-terminal region of titin in cardiac function and are the first to suggest a possible relationship between the is7 and excitation–contraction coupling. Finally, these findings give important insights for the identification of new targets in the treatment of titinopathies.

The study of human development is essential to further our knowledge and to improve our therapeutic strategies in the fields of reproductive and regenerative medicine. Given the limited access to supernumerary embryos and the prohibition on creating new ones for research, two alternative strategies can be proposed to study human embryonic development. The first is to create pseudo-embryos or blastoids. The second is to create human/animal chimeric embryos by injecting pluripotent stem cells, ES or iPS, into animal embryos. We explain herein the importance of these new experimental paradigms for studying human development and their complementarity.Des embryons chimères et des pseudo-embryons comme alternatives pour la recherche sur l’embryon humain.L’étude du développement humain est indispensable afin d’approfondir nos connaissances et, à long terme, perfectionner nos stratégies thérapeutiques dans les domaines de la médecine de la reproduction et de la médecine régénératrice. Face à la limite d’accès aux embryons surnuméraires et à l’interdiction d’en créer de nouveaux seulement à des fins de recherche, deux stratégies alternatives peuvent être proposées pour étudier le développement embryonnaire humain. La première consiste à fabriquer des pseudo-embryons ou blastoïdes. La seconde consiste à créer des embryons chimères homme/animal par injection de cellules souches pluripotentes, ES ou iPS, dans des embryons d’animaux. Nous expliquons ici l’importance de ces nouveaux paradigmes expérimentaux pour étudier le développement humain, et leur complémentarité.

The study of human development is essential to further our knowledge and to improve our therapeutic strategies in the fields of reproductive and regenerative medicine. Given the limited access to supernumerary embryos and the prohibition on creating new ones for research, two alternative strategies can be proposed to study human embryonic development. The first is to create pseudo-embryos or blastoids. The second is to create human/animal chimeric embryos by injecting pluripotent stem cells, ES or iPS, into animal embryos. We explain herein the importance of these new experimental paradigms for studying human development and their complementarity., L’étude du développement humain est indispensable afin d’approfondir nos connaissances et, à long terme, perfectionner nos stratégies thérapeutiques dans les domaines de la médecine de la reproduction et de la médecine régénératrice. Face à la limite d’accès aux embryons surnuméraires et à l’interdiction d’en créer de nouveaux seulement à des fins de recherche, deux stratégies alternatives peuvent être proposées pour étudier le développement embryonnaire humain. La première consiste à fabriquer des pseudo-embryons ou blastoïdes. La seconde consiste à créer des embryons chimères homme/animal par injection de cellules souches pluripotentes, ES ou iPS, dans des embryons d’animaux. Nous expliquons ici l’importance de ces nouveaux paradigmes expérimentaux pour étudier le développement humain, et leur complémentarité.

International audience; While lactate shuttle theory states that glial cells metabolize glucose into lactate to shuttle it to neurons, how glial cells support axonal metabolism and function remains unclear. Lactate production is a common occurrence following anaerobic glycolysis in muscles. However, several other cell types, including some stem cells, activated macrophages and tumor cells, can produce lactate in presence of oxygen and cellular respiration, using Pyruvate Kinase 2 (PKM2) to divert pyruvate to lactate dehydrogenase. We show here that PKM2 is also upregulated in myelinating Schwann cells (mSC) of mature mouse sciatic nerve versus postnatal immature nerve. Deletion of this isoform in PLP-expressing cells in mice leads to a deficit of lactate in mSC and in peripheral nerves. While the structure of myelin sheath was preserved, mutant mice developed a peripheral neuropathy. Peripheral nerve axons of mutant mice failed to maintain lactate homeostasis upon activity, resulting in an impaired production of mitochondrial ATP. Action potential propagation was not altered but axonal mitochondria transport was slowed down, muscle axon terminals retracted and motor neurons displayed cellular stress. Additional reduction of lactate availability through dichloroacetate treatment, which diverts pyruvate to mitochondrial oxidative phosphorylation, further aggravated motor dysfunction in mutant mice. Thus, lactate production through PKM2 enzyme and aerobic glycolysis is essential in mSC for the long-term maintenance of peripheral nerve axon physiology and function.

Duchenne Muscular Dystrophy (DMD) is a lethal muscle disease with no curative treatment. It is caused by mutations in the DMD gene, that encodes a protein participating in the linkage between the cytoskeleton and the extracellular matrix in skeletal and cardiac muscles. In this thesis, I am presenting 3 studies that led to the discovery of new pathophysiological aspects in DMD related to metabolism.The first study was entitled “Cholesterol metabolism is a potential therapeutic target in DMD”. We sequenced plasma miRNA in a DMD cohort comprising 54 DMD patients compared to 27 healthy controls. We identified 96 dysregulated miRNAs with many of them not previously reported in DMD. We developed an bioinformatics approach based on both targets and host genes of miRNAs for the interpretation of miRNA dysregulation. This analysis predicted that dysregulation of lipid metabolism has a central role in DMD. Further investigation in the mdx mouse revealed dysregulation of transcription factors of fatty acid (SREBP1) and cholesterol metabolism (SREBP2), perturbation of the mevalonate pathway, and accumulation of cholesterol in skeletal muscles. Elevated cholesterol was also found in biopsies of DMD patients. Treatment of mdx mice with Simvastatin normalized these perturbations and partially restored dystrophic parameters. This investigation supports the hypothesis that cholesterol metabolism and the mevalonate pathway are potential therapeutic targets in DMD.The second and third studies focussed on mitochondrial perturbations in DMD. We noticed the dysregulation in DMD patients of many miRNAs residing in the DLK1-DIO3 locus (DD-miRNAs). In the study entitled “miR-379 links glucocorticoid treatment with the mitochondrial response in DMD”, we selected one specific DD-miRNAs, miR-379. Using an in vitro model system, we identified a novel glucocorticoid-responsive signaling cascade: miR-379 represses the expression of EIF4G2, a translation initiation factor that promotes the expression of DAPIT protein. DAPIT is a component of Oxidative Phosphorylation (OxPhos) complex V. We demonstrated that in DMD, DAPIT inhibition by the identified signaling pathway results in reduced OxPhos capacity.In the last study, entitled “The DLK1-DIO3 cluster miRNAs regulate mitochondrial functions in the dystrophic muscle in DMD”, we undertook a more global approach. The combination of bioinformatics analysis and the in vivo simultaneous overexpression of 14 DD-miRNAs, in the healthy muscle suggested that DD-miRNAs may cooperatively modulate OxPhos capacity. Transcriptomic analysis revealed a high degree of similarity between the dystrophic muscle and a normal muscle that ectopically overexpressed the 14-DD-miRNAs, supporting that DD-miRNAs mediate mitochondrial adaptation in DMD. Knocking down the entire DD-miRNA cluster in iPS-derived myotubes resulted in increased mitochondrial activity and OxPhos.Together, the two last studies provide evidence for the modulation of mitochondrial activity in the dystrophic muscle by the upregulated DD-miRNAs and support an updated model for understanding mitochondrial dysfunction in DMD.The prevailing hypothesis for the dominant molecular mechanisms in DMD is of mechanical destabilization of the myofibers in the dystrophic muscle. Yet, accumulating evidence supports a critical role for metabolic and mitochondrial perturbations in DMD, in addition to mechanical destabilization. However, the nature of these metabolic perturbations has remained poorly understood. The studies reported here contribute to a better understanding of these metabolic aspects and, therefore, promote translational development and improved therapeutic care in DMD.; La dystrophie musculaire de Duchenne est une myopathie conduisnat à une réduction de la durée de vie et pour laquelle il n'existe pas de traitement curatif. Elle résulte de mutations dans le gène DMD, qui code une protéine participant à la liaison entre le cytosquelette et la matrice extracellulaire dans les muscles squelettiques et cardiaques. Dans cette thèse, je présente trois études qui ont conduit à la découverte de nouveaux aspects pathophysiologiques dans DMD reliés au métabolisme.La première étude s'intitule “Cholesterol metabolism is a potential therapeutic target in DMD”. Nous avons séquencé les miRNA plasmatiques dans une cohorte comprenant 54 patients DMD comparés à 27 contrôles sains. Nous avons identifié 96 miARNs dérégulés, dont beaucoup n'avaient jamais été rapportés dans DMD. Nous avons développé une approche bioinformatique basée à la fois sur les cibles et les gènes hôtes des miARNs pour l'interprétation de leur dysrégulation. Cette analyse a prédit que la dysrégulation du métabolisme lipidique a un rôle central dans DMD. Des investigations chez la souris mdx ont montré une dérégulation des facteurs de transcription lié aux acides gras (SREBP1) ou au métabolisme du cholestérol (SREBP2), une perturbation de la voie de mévalonate, et une accumulation du cholestérol dans les muscles squelettiques. Un taux de cholestérol élevé a été également montré dans des biopsies des patients de DMD. Un traitement de souris mdx avec de la Simvastatine a normalisé ces perturbations et partiellement restauré des paramètres dystrophiques. Cette étude suggère que le métabolisme de cholestérol et la voie de mevalonate puissent être des cibles thérapeutiques potentielles dans DMD.Les deuxième et troisième études incluses dans cette thèse sont orientés sur l'étude des perturbations mitochondriales dans DMD suite à la démonstration d'une dérégulation de nombreux miRNAs résidant dans le locus DLK1-DIO3 (DD-miRNAs) chez les patients DMD. Dans l'étude intitulée “miR-379 links glucocorticoid treatment with the mitochondrial response in DMD”, nous nous sommes concentrés sur un DD-miRNAs particulier, le miR-379. En utilisant un modèle in vitro, nous avons identifié une nouvelle cascade de signalisation: miR-379 réprime l'expression d'EIF4G2, un facteur d'initiation de traduction qui favorise l'expression de protéine DAPIT. DAPIT est un composant de phosphorylation oxydative (OxPhos). Nous avons démontré que dans DMD, l'inhibition de DAPIT par la voie de signalisation identifiée aboutit à une réduction de la capacité d'OxPhos.Dans la dernière étude, intitulée “The DLK1-DIO3 cluster miRNAs regulate mitochondrial functions in the dystrophic muscle in DMD”, nous avons entrepris une approche plus globale. La combinaison d'une analyse bioinformatique et de la surexpression simultanée in vivo de 14 DD-miRNAs dans le muscle sain a suggéré que les DD-miRNAs peuvent moduler en coopération la capacité d'OxPhos. Une analyse transcriptomique a montré une importante similitude entre le muscle dystrophique et un muscle normal surexprmant ectopiquement les DD-miRNAs, suggèrant que les DD-miRNAs puissent médier l'adaptation mitochondriale dans DMD. L'inactivation de l'ensemble du cluster DD-miRNA dans des myotubes dérivés d'iPS a entraîné une augmentation de l'OxPhos.Ensemble, les deux études apportent des évidences de la modulation de l'activité mitochondriale dans le muscle dystrophique par les DD-miRNAs et soutiennent un modèle actualisé pour comprendre le dysfonctionnement mitochondriale dans DMD.Les études rapportées ici contribuent à une meilleure compréhension de ces aspects métaboliques et, par conséquent, pourraient favoriser le développement translationnel et l'amélioration des soins thérapeutiques dans le DMD.

Duchenne Muscular Dystrophy (DMD) is a lethal muscle disease with no curative treatment. It is caused by mutations in the DMD gene, that encodes a protein participating in the linkage between the cytoskeleton and the extracellular matrix in skeletal and cardiac muscles. In this thesis, I am presenting 3 studies that led to the discovery of new pathophysiological aspects in DMD related to metabolism.The first study was entitled “Cholesterol metabolism is a potential therapeutic target in DMD”. We sequenced plasma miRNA in a DMD cohort comprising 54 DMD patients compared to 27 healthy controls. We identified 96 dysregulated miRNAs with many of them not previously reported in DMD. We developed an bioinformatics approach based on both targets and host genes of miRNAs for the interpretation of miRNA dysregulation. This analysis predicted that dysregulation of lipid metabolism has a central role in DMD. Further investigation in the mdx mouse revealed dysregulation of transcription factors of fatty acid (SREBP1) and cholesterol metabolism (SREBP2), perturbation of the mevalonate pathway, and accumulation of cholesterol in skeletal muscles. Elevated cholesterol was also found in biopsies of DMD patients. Treatment of mdx mice with Simvastatin normalized these perturbations and partially restored dystrophic parameters. This investigation supports the hypothesis that cholesterol metabolism and the mevalonate pathway are potential therapeutic targets in DMD.The second and third studies focussed on mitochondrial perturbations in DMD. We noticed the dysregulation in DMD patients of many miRNAs residing in the DLK1-DIO3 locus (DD-miRNAs). In the study entitled “miR-379 links glucocorticoid treatment with the mitochondrial response in DMD”, we selected one specific DD-miRNAs, miR-379. Using an in vitro model system, we identified a novel glucocorticoid-responsive signaling cascade: miR-379 represses the expression of EIF4G2, a translation initiation factor that promotes the expression of DAPIT protein. DAPIT is a component of Oxidative Phosphorylation (OxPhos) complex V. We demonstrated that in DMD, DAPIT inhibition by the identified signaling pathway results in reduced OxPhos capacity.In the last study, entitled “The DLK1-DIO3 cluster miRNAs regulate mitochondrial functions in the dystrophic muscle in DMD”, we undertook a more global approach. The combination of bioinformatics analysis and the in vivo simultaneous overexpression of 14 DD-miRNAs, in the healthy muscle suggested that DD-miRNAs may cooperatively modulate OxPhos capacity. Transcriptomic analysis revealed a high degree of similarity between the dystrophic muscle and a normal muscle that ectopically overexpressed the 14-DD-miRNAs, supporting that DD-miRNAs mediate mitochondrial adaptation in DMD. Knocking down the entire DD-miRNA cluster in iPS-derived myotubes resulted in increased mitochondrial activity and OxPhos.Together, the two last studies provide evidence for the modulation of mitochondrial activity in the dystrophic muscle by the upregulated DD-miRNAs and support an updated model for understanding mitochondrial dysfunction in DMD.The prevailing hypothesis for the dominant molecular mechanisms in DMD is of mechanical destabilization of the myofibers in the dystrophic muscle. Yet, accumulating evidence supports a critical role for metabolic and mitochondrial perturbations in DMD, in addition to mechanical destabilization. However, the nature of these metabolic perturbations has remained poorly understood. The studies reported here contribute to a better understanding of these metabolic aspects and, therefore, promote translational development and improved therapeutic care in DMD., La dystrophie musculaire de Duchenne est une myopathie conduisnat à une réduction de la durée de vie et pour laquelle il n'existe pas de traitement curatif. Elle résulte de mutations dans le gène DMD, qui code une protéine participant à la liaison entre le cytosquelette et la matrice extracellulaire dans les muscles squelettiques et cardiaques. Dans cette thèse, je présente trois études qui ont conduit à la découverte de nouveaux aspects pathophysiologiques dans DMD reliés au métabolisme.La première étude s'intitule “Cholesterol metabolism is a potential therapeutic target in DMD”. Nous avons séquencé les miRNA plasmatiques dans une cohorte comprenant 54 patients DMD comparés à 27 contrôles sains. Nous avons identifié 96 miARNs dérégulés, dont beaucoup n'avaient jamais été rapportés dans DMD. Nous avons développé une approche bioinformatique basée à la fois sur les cibles et les gènes hôtes des miARNs pour l'interprétation de leur dysrégulation. Cette analyse a prédit que la dysrégulation du métabolisme lipidique a un rôle central dans DMD. Des investigations chez la souris mdx ont montré une dérégulation des facteurs de transcription lié aux acides gras (SREBP1) ou au métabolisme du cholestérol (SREBP2), une perturbation de la voie de mévalonate, et une accumulation du cholestérol dans les muscles squelettiques. Un taux de cholestérol élevé a été également montré dans des biopsies des patients de DMD. Un traitement de souris mdx avec de la Simvastatine a normalisé ces perturbations et partiellement restauré des paramètres dystrophiques. Cette étude suggère que le métabolisme de cholestérol et la voie de mevalonate puissent être des cibles thérapeutiques potentielles dans DMD.Les deuxième et troisième études incluses dans cette thèse sont orientés sur l'étude des perturbations mitochondriales dans DMD suite à la démonstration d'une dérégulation de nombreux miRNAs résidant dans le locus DLK1-DIO3 (DD-miRNAs) chez les patients DMD. Dans l'étude intitulée “miR-379 links glucocorticoid treatment with the mitochondrial response in DMD”, nous nous sommes concentrés sur un DD-miRNAs particulier, le miR-379. En utilisant un modèle in vitro, nous avons identifié une nouvelle cascade de signalisation: miR-379 réprime l'expression d'EIF4G2, un facteur d'initiation de traduction qui favorise l'expression de protéine DAPIT. DAPIT est un composant de phosphorylation oxydative (OxPhos). Nous avons démontré que dans DMD, l'inhibition de DAPIT par la voie de signalisation identifiée aboutit à une réduction de la capacité d'OxPhos.Dans la dernière étude, intitulée “The DLK1-DIO3 cluster miRNAs regulate mitochondrial functions in the dystrophic muscle in DMD”, nous avons entrepris une approche plus globale. La combinaison d'une analyse bioinformatique et de la surexpression simultanée in vivo de 14 DD-miRNAs dans le muscle sain a suggéré que les DD-miRNAs peuvent moduler en coopération la capacité d'OxPhos. Une analyse transcriptomique a montré une importante similitude entre le muscle dystrophique et un muscle normal surexprmant ectopiquement les DD-miRNAs, suggèrant que les DD-miRNAs puissent médier l'adaptation mitochondriale dans DMD. L'inactivation de l'ensemble du cluster DD-miRNA dans des myotubes dérivés d'iPS a entraîné une augmentation de l'OxPhos.Ensemble, les deux études apportent des évidences de la modulation de l'activité mitochondriale dans le muscle dystrophique par les DD-miRNAs et soutiennent un modèle actualisé pour comprendre le dysfonctionnement mitochondriale dans DMD.Les études rapportées ici contribuent à une meilleure compréhension de ces aspects métaboliques et, par conséquent, pourraient favoriser le développement translationnel et l'amélioration des soins thérapeutiques dans le DMD.

Alternative splicing has emerged as a fundamental mechanism for the spatiotemporal control of development. A better understanding of how this mechanism is regulated has the potential not only to elucidate fundamental biological principles, but also to decipher pathological mechanisms implicated in diseases where normal splicing networks are misregulated. Here, we took advantage of human pluripotent stem cells to decipher during human myogenesis the role of muscleblind-like (MBNL) proteins, a family of tissue-specific splicing regulators whose loss of function is associated with myotonic dystrophy type 1 (DM1), an inherited neuromuscular disease. Thanks to the CRISPR/Cas9 technology, we generated human-induced pluripotent stem cells (hiPSCs) depleted in MBNL proteins and evaluated the consequences of their losses on the generation of skeletal muscle cells. Our results suggested that MBNL proteins are required for the late myogenic maturation. In addition, loss of MBNL1 and MBNL2 recapitulated the main features of DM1 observed in hiPSC-derived skeletal muscle cells. Comparative transcriptomic analyses also revealed the muscle-related processes regulated by these proteins that are commonly misregulated in DM1. Together, our study reveals the temporal requirement of MBNL proteins in human myogenesis and should facilitate the identification of new therapeutic strategies capable to cope with the loss of function of these MBNL proteins.

BackgroundDuchenne Muscular Dystrophy (DMD) is a lethal muscle disease detected in approximately 1:5000 male births. DMD is caused by mutations in the DMD gene, encoding a critical protein that link the cytoskeleton and the extracellular matrix in skeletal and cardiac muscles. The primary consequence of the disrupted link between the extracellular matrix and the myofiber actin cytoskeleton is thought to involve sarcolemma destabilization, perturbation of Ca+2 homeostasis, activation of proteases, mitochondrial damage and tissue degeneration. A recently emphasized secondary aspect of the dystrophic process is a progressive metabolic change of the dystrophic tissue; however, the mechanism and nature of the metabolic dysregulation is yet poorly understood. In this study, we characterized a molecular mechanism of metabolic perturbation in DMD.MethodsWe sequenced plasma miRNA in a DMD cohort, comprising of 54 DMD patients treated or not by glucocorticoid, compared to 27 healthy controls, in three age groups. We developed an original approach for the biological interpretation of miRNA dysregulation, and produced a novel hypothesis concerning metabolic perturbation in DMD. We then used the mdx mouse model for DMD for the investigation of this hypothesis.ResultsWe identified 96 dysregulated miRNAs, of which 74 were up- and 22 down-regulated in DMD. We confirmed the dysregulation in DMD of Dystro-miRs, Cardio-miRs and a large number of the DLK1-DIO3 miRNAs. We also identified numerous dysregulated miRNAs, yet unreported in DMD. Bioinformatics analysis of both target and host genes for dysregulated miRNAs predicted that lipid metabolism might be a critical metabolic perturbation in DMD. Investigation of skeletal muscles of the mdx mouse uncovered dysregulation of transcription factors of cholesterol and fatty acid metabolism (SREBP1 and SREBP2), perturbation of the mevalonate pathway, and accumulation of cholesterol. Elevated cholesterol level was also found in muscle biopsies of DMD patients. Treatment of mdx mice with Simvastatin, a cholesterol-reducing agent, normalized these perturbations and partially restored the dystrophic parameters.ConclusionThis investigation supports that cholesterol metabolism and the mevalonate pathway are potential therapeutic targets in DMD.