Etude de la physiopathologie de la dystrophie musculaire tibiale et de la dystrophie des ceintures 2J et stratégies thérapeutiques

La titine est une protéine géante exprimée dans les muscles squelettiques et cardiaque. Un certain nombre de mutations pathogéniques ont été identifiées dans son dernier exon codant. La mutation la plus fréquente, FINmaj, conduit au remplacement de 4 acides aminés et est retrouvée chez de nombreux patients en Finlande. Cette mutation cause une dystrophiemusculaire tibiale (TMD) à l état hétérozygote et une dystrophie des ceintures de type 2J(LGMD2J) à l état homozygote.Pour obtenir une modèle d étude de la physiopathologie de ces maladies et évaluer des stratégiesthérapeutiques, nous avons introduit la mutation FINmaj dans le génome murin par une stratégiede Knock-In par recombinaison homologue. Ce modèle a été caractérisé et a permis de montrer qu il reproduit en grande partie les symptômes de la TMD et de la LGMD2J aux niveaux histologique et moléculaire. L étude de ce modèle murin a permis une meilleure compréhension de la physiopathologie de ces deux maladies et nous amené à étudier plus attentivement les interactions de la titine en C-ter avec ses partenaires afin de mieux comprendre l implication de la ligne M dans la vie du sarcomère. L étude de la physiopathologie de la TMD et de la LGM2J a permis de montrer que la calpaïne 3 (une protéase à l origine d une autre dystrophie des ceintures), jouait un rôle majeur dans laTMD. Cette constatation nous a permis d envisager une approche thérapeutique pour cette dernière visant à diminuer les symptômes en régulant négativement la calpaïne 3. Une approche de thérapie génique a aussi été testée dans le but de traiter ces deux pathologies: le trans-épissage des derniers exons de la titine. En effet, étant donné la grande taille de l'ADNc de la titine (~100 kb), des stratégies classiques de transfert de gène n étaient pas envisageables. Pour s'affranchir de ce problème, nous avons testé une approche de trans-épissage de l ARN pour remplacer le ou les derniers exons du messager de la titine. Nous avons pu ainsi démontrer la faisabilité de la correction de la titine in vitro.Titin is a giant protein expressed in both skeletal muscles and heart. Several pathogenic mutations were identified in its last coding exon. The most frequent mutation commonly referred to as FINmaj, results in the replacement of 4 amino acids and affects a subset of patients in Finland. The mutation causes a Tibial Muscular Dystrophy (TMD) when present on one allele and a Limb Girdle Muscular Dystrophy phenotype 2J (LGMD2J) when present on both alleles.To obtain a model for studying the physiopathology of these diseases and evaluating therapeutic strategies, we introduced the FINmaj mutation in the murine genome by a knock-in strategy by homologous recombination. This model was characterized and showed that it reproduces mainly the symptoms of both the human TMD and LGMD2J at histological and molecular levels.The study of this mouse model has allowed a better understanding of the pathophysiology of these two diseases and we have to study more carefully the interactions beyond titin C-ter with its partners to better understand the involvement of the M line in life of the sarcomere.The study of the pathophysiology of TMD and LGM2J showed that calpain 3 (a protease thatlind to an other limb-girdle muscular dystrophy), played a major role in TMD. This finding allowed us to consider a treatment approach for TMD to reduce symptoms by regulating negatively calpain 3. A gene therapy approach was also tested: the trans-splicing of the last exon of titin.Indeed, given the large size of the cDNA of titin (~ 100 kb), conventional strategies of genetransfer were not envisaged. To overcome this problem, we tested an approach to exchange the last exon or the last exons of the titin messenger. We were able to demonstrate the correction of titin in vitro.EVRY-Bib. électronique (912289901) / SudocSudocFranceF

Evaluation de trois approches de thérapie génique pour le traitement des dysferlinopathies (miniprotéine, compensation et trans-épissage)

Les dysferlinopathies sont des maladies musculaires dues à une déficience en protéine dysferline, codée par le gène DYSF. Dans ce travail de thèse, trois approches thérapeutiques ont été évaluées pour ces pathologies, sur des modèles cellulaires et murins. Un variant transcriptionnel court de la dysferline a été vectorisé dans un AAV8r et injecté dans le modèle murin Bla/J, déficient en dysferline. L analyse des muscles des animaux traités montre une augmentation de la résistance des fibres musculaires au stress mécanique, mais n apporte pas de correction histologique. Cette étude souligne également la toxicité de cette miniprotéine. L anoctamine 5, impliquée dans des pathologies et des activités similaires à la dysferline, a été testée en tant que protéine compensatrice. L anoctamine 5 surexprimée dans le modèle Bla/J ne permet pas la restauration d un phénotype normal. La compensation de DYSF par ANO5 n est donc pas une voie thérapeutique à exploiter pour les dysferlinopathies. Enfin, une thérapie génique par chirurgie de l ARN dysferline a été évaluée en utilisant le trans-épissage médié par le splicéosome (SMaRT). La preuve de principe de la reprogrammation d un minigène dysferline a été faite (ARN et protéine trans-épissée obtenus in vitro). L efficacité du SMaRT dans un contexte endogène s est en revanche révélée faible, et n a pas permis la restauration d une protéine dysferline fonctionnelle dans des myoblastes humains. De plus, l observation de l expression de protéines directement à partir du RTM (RNA-trans-splicing molecule) a fait apparaître des limites à l utilisation du SMaRT pour la thérapie génique, et en particulier pour les dysferlinopathies.Dysferlinopathies are muscular diseases due to mutations in DYSF gene, inducing dysferlin protein deficiency. In this thesis, three therapeutic approaches have been investigated for these pathologies, on cell or mice models. A short transcriptional dysferlin variant has been injected into Bla/J dysferlin deficient mouse model, using AAV8r vector. Muscle fibers of treated animals displayed an increased resistance to mechanical stress without therapeutic benefit. These experiments also pointed out the toxicity of this strategy. A protein compensation approach has been tested using anoctamin 5, known to be involved in pathologies and activities similar to dysferlin s ones. AAVr mediated Anoctamin 5 overexpression in Bla/J model does not rescue their muscle phenotype. Overexpression of ANO5 does not seem to be a valuable therapeutic strategy for dysferlin deficiency. Dysferlin RNA surgery was evaluated as a possible genetic therapy using Spliceosome-Mediated RNA Trans-splicing (SMaRT). On a Minigene target, SMaRT is able to induce RNA reprogramming by trans-splicing, and produce the corresponding protein. But efficiency is by far decreased in endogenous context and not good enough to restore functional dysferlin in human myoblasts. Moreover, we described proteins resulting from RNA-trans-splicing molecule (RTM) self-expression, limiting the value of SMaRT as therapeutic strategy, especially for dysferlinopathies.EVRY-Bib. électronique (912289901) / SudocSudocFranceF

Pysiopathology and therapeutic approaches for TMD and LGMD2J

La titine est une protéine géante exprimée dans les muscles squelettiques et cardiaque. Un certain nombre de mutations pathogéniques ont été identifiées dans son dernier exon codant. La mutation la plus fréquente, FINmaj, conduit au remplacement de 4 acides aminés et est retrouvée chez de nombreux patients en Finlande. Cette mutation cause une dystrophiemusculaire tibiale (TMD) à l‟état hétérozygote et une dystrophie des ceintures de type 2J(LGMD2J) à l‟état homozygote.Pour obtenir une modèle d‟étude de la physiopathologie de ces maladies et évaluer des stratégiesthérapeutiques, nous avons introduit la mutation FINmaj dans le génome murin par une stratégiede Knock-In par recombinaison homologue. Ce modèle a été caractérisé et a permis de montrer qu‟il reproduit en grande partie les symptômes de la TMD et de la LGMD2J aux niveaux histologique et moléculaire. L‟étude de ce modèle murin a permis une meilleure compréhension de la physiopathologie de ces deux maladies et nous amené à étudier plus attentivement les interactions de la titine en C-ter avec ses partenaires afin de mieux comprendre l‟implication de la ligne M dans la vie du sarcomère. L‟ étude de la physiopathologie de la TMD et de la LGM2J a permis de montrer que la calpaïne 3 (une protéase à l‟origine d‟une autre dystrophie des ceintures), jouait un rôle majeur dans laTMD. Cette constatation nous a permis d‟envisager une approche thérapeutique pour cette dernière visant à diminuer les symptômes en régulant négativement la calpaïne 3. Une approche de thérapie génique a aussi été testée dans le but de traiter ces deux pathologies: le trans-épissage des derniers exons de la titine. En effet, étant donné la grande taille de l'ADNc de la titine (~100 kb), des stratégies classiques de transfert de gène n‟étaient pas envisageables. Pour s'affranchir de ce problème, nous avons testé une approche de trans-épissage de l‟ARN pour remplacer le ou les derniers exons du messager de la titine. Nous avons pu ainsi démontrer la faisabilité de la correction de la titine in vitro.Titin is a giant protein expressed in both skeletal muscles and heart. Several pathogenic mutations were identified in its last coding exon. The most frequent mutation commonly referred to as FINmaj, results in the replacement of 4 amino acids and affects a subset of patients in Finland. The mutation causes a Tibial Muscular Dystrophy (TMD) when present on one allele and a Limb Girdle Muscular Dystrophy phenotype 2J (LGMD2J) when present on both alleles.To obtain a model for studying the physiopathology of these diseases and evaluating therapeutic strategies, we introduced the FINmaj mutation in the murine genome by a knock-in strategy by homologous recombination. This model was characterized and showed that it reproduces mainly the symptoms of both the human TMD and LGMD2J at histological and molecular levels.The study of this mouse model has allowed a better understanding of the pathophysiology of these two diseases and we have to study more carefully the interactions beyond titin C-ter with its partners to better understand the involvement of the M line in life of the sarcomere.The study of the pathophysiology of TMD and LGM2J showed that calpain 3 (a protease thatlind to an other limb-girdle muscular dystrophy), played a major role in TMD. This finding allowed us to consider a treatment approach for TMD to reduce symptoms by regulating negatively calpain 3. A gene therapy approach was also tested: the trans-splicing of the last exon of titin.Indeed, given the large size of the cDNA of titin (~ 100 kb), conventional strategies of genetransfer were not envisaged. To overcome this problem, we tested an approach to exchange the last exon or the last exons of the titin messenger. We were able to demonstrate the correction of titin in vitro

Exploiting The CRISPR/CAS9 System to Study Alternative Splicing In Vivo: Application to Titin

The giant protein titin is the third most abundant protein in striated muscle. Mutations in its gene are responsible for diseases affecting the cardiac and/or the skeletal muscle. Titin has been reported to be expressed in multiple isoforms with considerable variability in the I-band, ensuring the modulation of the passive mechanical properties of the sarcomere. In the M-line, only the penultimate Mex5 exon coding for the specific is7 domain has been reported to be subjected to alternative splicing. Using the CRISPR-Cas9 editing technology, we generated a mouse model where we stably prevent the expression of alternative spliced variant(s) carrying the corresponding domain. Interestingly, the suppression of the domain induces a phenotype mostly in tissues usually expressing the isoform that has been suppressed, indicating that it fulfills (a) specific function(s) in these tissues allowing a perfect adaptation of the M-line to physiological demands of different muscles

A comparison of AAV strategies distinguishes overlapping vectors for efficient systemic delivery of the 6.2 kb Dysferlin coding sequence

International audienceRecombinant adeno-associated virus (rAAV) is currently the best vector for gene delivery into the skeletal muscle. However, the 5-kb packaging size of this virus is a major obstacle for large gene transfer. This past decade, many different strategies were developed to circumvent this issue (concatemerization-splicing, overlapping vectors, hybrid dual or fragmented AAV). Loss of function mutations in the DYSF gene whose coding sequence is 6.2kb lead to progressive muscular dystrophies (LGMD2B: OMIM_253601; MM: OMIM_254130; DMAT: OMIM_606768). In this study, we compared large gene transfer techniques to deliver the DYSF gene into the skeletal muscle. After rAAV8s intramuscular injection into dysferlin deficient mice, we showed that the overlap strategy is the most effective approach to reconstitute a full-length messenger. After systemic administration, the level of dysferlin obtained on different muscles corresponded to 0.5- to 2-fold compared to the normal level. We further demonstrated that the overlapping vector set was efficient to correct the histopathology, resistance to eccentric contractions and whole body force in the dysferlin deficient mice. Altogether, these data indicate that using overlapping vectors could be a promising approach for a potential clinical treatment of dysferlinopathies

Circulating miRNAs are generic and versatile therapeutic monitoring biomarkers in muscular dystrophies

International audienceThe development of medical approaches requires preclinical and clinical trials for assessment of therapeutic efficacy. Such evaluation entails the use of biomarkers, which provide information on the response to the therapeutic intervention. One newly-proposed class of biomarkers is the microRNA (miRNA) molecules. In muscular dystrophies (MD), the dysregulation of miRNAs was initially observed in muscle biopsy and later extended to plasma samples, suggesting that they may be of interest as biomarkers. First, we demonstrated that dystromiRs dysregulation occurs in MD with either preserved or disrupted expression of the dystrophin-associated glycoprotein complex, supporting the utilization of dystromiRs as generic biomarkers in MD. Then, we aimed at evaluation of the capacity of miRNAs as monitoring biomarkers for experimental therapeutic approach in MD. To this end, we took advantage of our previously characterized gene therapy approach in a mouse model for α-sarcoglycanopathy. We identified a dose-response correlation between the expression of miRNAs on both muscle tissue and blood serum and the therapeutic benefit as evaluated by a set of new and classically-used evaluation methods. This study supports the utility of profiling circulating miRNAs for the evaluation of therapeutic outcome in medical approaches for MD

Restriction of Calpain3 Expression to the Skeletal Muscle Prevents Cardiac Toxicity and Corrects Pathology in a Murine Model of Limb-Girdle Muscular Dystrophy

International audienceBackground Genetic defects in calpain3 (CAPN3) lead to limb-girdle muscular dystrophy type 2A, a disease of the skeletal muscle that affects predominantly the proximal limb muscles. We previously demonstrated the potential of adeno-associated virus-mediated transfer of the CAPN3 gene to correct the pathological signs in a murine model for limb-girdle muscular dystrophy type 2A after intramuscular and locoregional administrations. Methods and Results Here, we showed that intravenous injection of calpain3-expressing vector in mice can induce mortality in a dose-dependent manner. An anatomopathological investigation revealed large areas of fibrosis in the heart that we related to unregulated proteolytic activity of calpain3. To circumvent this toxicity, we developed new adeno-associated virus vectors with skeletal muscle-restricted expression by using new muscle-specific promoters that include the CAPN3 promoter itself and by introducing a target sequence of the cardiac-specific microRNA-208a in the cassette. Our results show that CAPN3 transgene expression can be successfully suppressed in the cardiac tissue, preventing the cardiac toxicity, whereas expression of the transgene in skeletal muscle reverts the pathological signs of calpain3 deficiency. Conclusions The molecular strategies used in this study may be useful for any gene transfer strategy with potential toxicity in the heart

Cis -splicing and Translation of the Pre- Trans -splicing Molecule Combine With Efficiency in Spliceosome-mediated RNA Trans -splicing

International audienceMuscular dystrophies are a group of genetically distinct diseases for which no treatment exists. While gene transfer approach is being tested for several of these diseases, such strategies can be hampered when the size of the corresponding complementary DNA (cDNA) exceeds the packaging capacity of adeno-associated virus vectors. This issue concerns, in particular, dysferlinopathies and titinopathies that are due to mutations in the dysferlin (DYSF) and titin (TTN) genes. We investigated the efficacy of RNA trans-splicing as a mode of RNA therapy for these two types of diseases. Results obtained with RNA trans-splicing molecules designed to target the 3' end of mouse titin and human dysferlin pre-mRNA transcripts indicated that trans-splicing of pre-mRNA generated from minigene constructs or from the endogenous genes was achieved. Collectively, these results provide the first demonstration of DYSF and TTN trans-splicing reprogramming in vitro and in vivo. However, in addition to these positive results, we uncovered a possible issue of the technique in the form of undesirable translation of RNA pre-trans-splicing molecules, directly from open reading frames present on the molecule or associated with internal alternative cis-splicing. These events may hamper the efficiency of the trans-splicing process and/or lead to toxicity

Endoplasmic reticulum maintains ion homeostasis required for plasma membrane repair

International audienceOf the many crucial functions of the ER, homeostasis of physiological calcium increase is critical for signaling. Plasma membrane (PM) injury causes a pathological calcium influx. Here, we show that the ER helps clear this surge in cytoplasmic calcium through an ER-resident calcium pump, SERCA, and a calcium-activated ion channel, Anoctamin 5 (ANO5). SERCA imports calcium into the ER, and ANO5 supports this by maintaining electroneutrality of the ER lumen through anion import. Preventing either of these transporter activities causes cytosolic calcium overload and disrupts PM repair (PMR). ANO5 deficit in limb girdle muscular dystrophy 2L (LGMD2L) patient cells compromises their cytosolic and ER calcium homeostasis. By generating a mouse model of LGMD2L, we find that PM injury causes cytosolic calcium overload and compromises the ability of ANO5-deficient myofibers to repair. Addressing calcium overload in ANO5-deficient myofibers enables them to repair, supporting the requirement of the ER in calcium homeostasis in injured cells and facilitating PMR

AAV-mediated transfer of FKRP shows therapeutic efficacy in a murine model but requires control of gene expression

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