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Duchenne muscular dystrophy (DMD) is a fatal, x-linked disease that causes progressive muscle weakness and susceptibility to damage. DMD is caused by a lack of dystrophin, a large muscle protein that performs both structural and signaling functions. A promising treatment currently in clinical trials is microdystrophin gene therapy, which delivers a truncated version of dystrophin to muscle via a viral vector. Preclinical studies have established efficacy of microdystrophin to improve muscle quality and function. With clinical success of this treatment, patients affected by DMD could become more physically active. However, the effect of exercise on both dystrophic and gene therapy-treated muscles is unclear. Recently, we demonstrated that microdystrophin gene therapy with and without 21 weeks of voluntary wheel running (VWR) improved treadmill time to fatigue and in vivo plantarflexor torque output in young mdx mice, a mouse model of DMD. Although treated mice could run well, diaphragm force and power output were blunted by VWR. A subsequent study tested longevity of two different microdystrophin gene therapy constructs in combination with VWR. Versions of each construct are being tested in clinical trials. Construct 1 contained the nNOS-binding site found in full-length dystrophin, which localizes nNOS to the sarcolemma and reduces functional ischemia of exercising limb muscles, while construct 2 lacked the nNOS-binding site and was the same microdystrophin used in the previous study. Gene- therapy treated mice that were sedentary or performed 52 weeks of VWR demonstrated similar outcomes including increased plantarflexor torque and exceptional treadmill endurance capacity. However, ex vivo diaphragm and soleus force, as well as metabolic enzyme and mitochondrial respiration assays were differentially improved, revealing unique physiological adaptations to each microdystrophin construct. Together, the data demonstrated that response to exercise after gene therapy treatment was variable and dependent on age, microdystrophin construct, and muscle type. / Doctor of Philosophy / Duchenne muscular dystrophy (DMD) is a rare, fatal muscle disease that causes progressive muscle weakness and cardiorespiratory failure. Available treatments, such as corticosteroids, slow progression of the disease but do not address the underlying genetic cause. DMD is caused by a genetic mutation that results in the loss of the muscle protein dystrophin. Microdystrophin gene therapy aims to address the genetic cause of the disease by using a non-pathogenic virus to deliver microdystrophin, a small, functional version of dystrophin, to muscle. This gene therapy is in clinical trials, and, if it is successful, treated patients will likely want to engage in more physical activity than previously possible due to muscle weakness. However, the effects of physical activity on muscles treated with gene therapy are unclear. Therefore, we conducted two studies to test the effects of voluntary wheel running on microdystrophin gene therapy in the mdx mouse, a model of DMD. The first study demonstrated that voluntary wheel running was beneficial to whole-body muscle function in mice treated with microdystrophin gene therapy. However, adaptations to the gene therapy and voluntary wheel running were variable in individual muscles. In the second study, we tested two microdystrophin constructs, which each contain different structural components of full-length dystrophin. In addition, mice ran for 52 weeks, more than twice as long as the first study. The results of the second study found that adaptations in individual muscles depended on microdystrophin construct and activity level. Additionally, we confirmed that voluntary wheel running was beneficial to whole-body function of microdystrophin–treated muscles. Together, these studies demonstrated that adaptations of gene therapy-treated muscles were dependent on microdystrophin structure, activity level, and age.

Duchenne muscular dystrophy (DMD) is a severe and fatal muscle wasting disease characterized by a high mutation rate in the gene that encodes the membrane-associated protein dystrophin that results in absence of expressed protein. Although the primary genetic defect for DMD is known, the mechanisms that initiate the onset of DMD are not currently understood. This study tested the hypothesis that pathophysiological processes involved in DMD could be identified by the global expression of mRNA in maturing dystrophin- and utrophin-deficient mouse (mdx:utrn-/-) muscles. Two potential dystrophic onset mechanisms targeted for analysis were (1) disrupted expression of calcium handling proteins; and, (2) increased expression of immune response markers. An mRNA expression profile was developed following isolation of total RNA from control and mdx:utrn-/- triceps surae (TS) muscles at ages 9-10 and 20-21 days using Affymetrix® Mu74Av2 GeneChips®. Compared to control, the mRNA expression profile in mdx:utrn-/- muscles revealed there was a 3-fold increase in the number of gene transcripts differentially expressed more than 2-fold (53 transcripts at ages 9-10 days; 153 at ages 20-21 days). However, there were no changes in the mRNA transcripts for calcium handling proteins. In distinct contrast, there was up-regulation of transcripts that corresponded to an immune response (40 transcripts), extracellular matrix activity (14), and proteolysis (8). Up-regulation of several transcripts corresponded to cytokines and their receptors (11), chemokines and their receptors (5), and lymphoid and myeloid markers (16) suggesting that dystrophic muscle is susceptible to invasion by macrophages, leukocytes, B- and T-cells. These results are consistent with several reports (Spencer et al., 1997; Chen et al., 2000; Porter et al., 2002; Porter et al., 2003a; Porter et al., 2003b; Porter et al., 2004) that indicate the immune system may play an important role in the early pathophysiology of DMD. Understanding the functional aspects of an immune response in DMD onset should lead to more effective therapeutics. / Master of Science

Duchenne Muscular Dystrophy (DMD) is characterized by progressive muscle degeneration and a chronic inflammatory response. Sphingolipid metabolites are associated with the generation or perpetuation of low-grade chronic inflammation critical in atherosclerosis, obesity and cancer. Dietary sphingolipids, however, can suppress intestinal inflammation. We hypothesized that dietary sphingomyelin (SM) from bovine milk can modulate the inflammatory signature and improve muscle function in mdx mice, a model of DMD. C57BL10 (WT) and mdx mice were fed AIN 76A diet ± 0.1% SM for 7 weeks starting at age 4 weeks (n=10/group: WT, WT + S, mdx, mdx + S). At ages 5, 7, and 9 weeks, ankle flexor torque was determined in vivo. Mice were euthanized at 11 wks. Serum creatine kinase and extensor digitorum longus (EDL) contractile properties in vitro were determined; Tibialis Anterior (TA) inflammatory markers were profiled by qRT-PCR; TA sections were stained with H&E and immunohistochemistry for p-Akt was performed. At age 9 weeks, in vivo ankle flexor torque at stimulation frequencies 50-150 Hz was greater in mdx+S vs. mdx (P=0.0160) and WT (P<0.0001). At 11 wks, only WT+S EDL stress in vitro was greater than all other groups at 50-150 Hz. The in vitro relative stress-frequency relationship of mdx+S EDL was left shifted from the other treatment groups. Inflammatory genetic markers were increased in mdx+S mice. These data suggest treatment of mdx mice with 0.1% SM improves ankle flexor torque in vivo, causes a left shift of the stress-frequency relationship in vitro, and modulates the inflammatory gene signature. / Master of Science

Une synchronicité existe entre l’apparition de l’atrophie musculaire et osseuse (ostéoporose) mais, très peu de groupes de recherche se sont intéressés à la possibilité qu’une voie de signalisation commune puisse contrôler simultanément ces tissus dans un contexte pathologique. Le but de cette thèse est de caractériser les rôles du sentier signalétique principal du remodelage osseux soit la voie RANK/RANKL/OPG, sur le muscle squelettique sain ou pathologique. Premièrement, nous avons démontré que RANK est exprimé dans le muscle squelettique et que son absence dans ce tissu induit un effet inotropique sur le muscle rapide extensor digitorum longus (EDL), limitant ainsi la perte de force maximale spécifique, tout en augmentant l'atrophie musculaire, la fatigabilité et la proportion de fibres rapides. Ensuite, nous avons montré qu’un blocage pharmacologique de la voie RANKL/RANK par l’OPG atténue la perte de la force musculaire de manière dose-dépendante et préserve l'intégrité musculaire, en particulier des muscles rapides EDL de souris dystrophiques. Cette étude nous a également permis de démontrer que l’OPG-Fc a un effet intéressant mais plus limité sur la préservation de la force du muscle lent soleus (Sol). Par contre, nous avons découvert que l’OPG-Fc potentialise les effets positifs d'une faible dose de formotérol, un membre de la famille des β2-agonistes, et leur combinaison restaure complètement la fonction du Sol des souris dystrophiques. Finalement, nous avons débuté une étude mécanistique sur l’effet protecteur de l’OPG-Fc sur le muscle squelettique dystrophique. Structurellement, l'OPG-Fc pleine longueur contient quatre domaines TNFR (RANKL), deux domaines de la mort cellulaire par apoptose (TRAIL) et un domaine lié à l'héparine. Nos résultats indiquent que les injections d'anti-RANKL, d’anti-TRAIL et d’OPG-Fc tronquée (possédant seulement les domaines TNFR) ou la suppression génétique de RANK dans le muscle sont nettement moins efficaces sur la préservation de la force des muscles dystrophiques que celles d’OPG-Fc pleine longueur. Étonnamment, l'absence de Ca2+ extracellulaire réduit considérablement les effets de l’OPG-Fc pleine longueur sur la force des muscles dystrophiques dans un modèle de contractilité in vitro. Nos analyses en microscopie confocale ont démontré que l’OPG-Fc pleine longueur pourrait se lier à un récepteur présentement non identifié localisé sur les myotubes et que cette liaison entraîne possiblement une activation d’une kinase liée aux intégrines (ILK) et la surexpression d’une pompe calcique ATPase du réticulum sarcoplasmique appelée SERCA-2a, un déterminant clé de la performance musculaire. Les myotubes traités à l'héparinase, une enzyme connue pour cliver les domaines de l'héparine ou encore l’inhibition de l’ILK réduit significativement la surexpression de SERCA-2a induite par l’OPG-Fc. Cette thèse apporte globalement, une meilleure compréhension des fonctions de RANK/RANKL/OPG dans le muscle squelettique dénervé ou dystrophique et s’inscrit dans la liste des travaux pré-cliniques qui pourrait éventuellement contribuer à l’élaboration de nouveaux traitements pour les maladies musculaires et osseuses. / Although there is an obvious dynamic cross-talk between muscle and bone, a common signalling pathway that efficiently and synchronously controls these tissues has barely been investigated in all forms of muscle diseases. The aim of this thesis is to characterize the roles of RANK/RANKL/OPG, key regulators of bone remodeling, on skeletal muscle atrophy, phenotype and dysfunction. Firstly, we show that RANK is expressed in skeletal muscle and that muscle RANK deletion has inotropic effects in denervated fast-twitch extensor digitorum longus (EDL) muscles, preventing on one side the loss of maximum specific force while promoting muscle atrophy and fatigability, and increasing the proportion of fast-twitch fibers. We next demonstrate that a pharmacological treatment of dystrophic mdx mice with recombinant full-length OPG-Fc mitigates the loss of muscle force in a dose-dependent manner and preserves muscle integrity, particularly in EDL muscles. We also found that the full-length OPG-Fc has limited effects on slow-twitch soleus (Sol) muscles. However OPG-Fc potentiates the positive effects of a low dose of formoterol, a member of β2-agonists, and completely restores the function of the Sol dystrophic muscles. Finally, we investigated the mechanism by which the full-length OPGFc protects the dystrophic muscles. Structurally, the OPG protein contains four TNFR domains (RANKL), two death domains ( TRAIL) and a heparin-binding region. Our results indicate that anti-RANKL or anti-TRAIL or truncated OPG treatments (only TNFR domains) or RANK deletion are much less effective in preserving the strength of dystrophic muscles than full-length OPG-Fc. Surprisingly, the absence of extracellular Ca2+ significantly reduces the effects of full-length OPG-Fc on the force production of dystrophic muscles when incubated in a physiological bath in vitro. Confocal microscopy images showed that the full-length OPG-Fc binds directly to myotubes through a receptor that is currently unidentified activating possibly integrin-linked kinase (ILK) which upregulates sarco/endoplasmic calcium ATPase pump (SERCA-2a) expression in C2C12 myotubes. Heparinase, which cleaves heparin and heparin sulphate proteoglycan, or an inhibitor of ILK activity abrogates OPG-induced SERCA-2a expression, suggesting that OPG through ILK upregulates SERCA-2a expression, a key determinant of muscle performance. Overall, this thesis shed some light on RANK/RANKL/OPG functions in skeletal muscle which will potentially contribute to the development of new treatments for several forms of muscle and bone diseases.

La dystrophie musculaire de Duchenne est une myopathie héréditaire récessive liée au chromosome X. Elle est causée par l’absence de la dystrophine dans les fibres musculaires. La thérapie cellulaire est l’une des approches thérapeutiques possibles, mais son succès dépend du contrôle du rejet des myoblastes greffés et des fibres musculaires hybrides. À présent, le contrôle du rejet est obtenu par l’administration d’immunosuppresseurs puissants. Notre objectif à long terme est de développer un protocole de tolérance immunologique qui permettrait de prévenir le rejet, sans recours à une immunosuppression soutenue. La première étape du protocole de tolérance immunologique que nous souhaitons développer est d’induire la formation de lymphocytes T régulateurs en utilisant le facteur de transcription Foxp3. Nos travaux nous ont permis de produire, dans des bactéries E. coli, la protéine de fusion Tat-Foxp3. Par des essais in vitro nous avons démontré l’augmentation de l’expression du récepteur CD25 sur les lymphocytes T CD4+ naïfs après transduction de la protéine Tat-Foxp3. Cela suggère que la protéine Tat-Foxp3 pourrait convertir les lymphocytes T CD4+ naïfs en lymphocytes T régulateurs. D’autres travaux seront nécessaires pour confirmer que ces cellules exprimant le CD25 sont vraiment des T régulateurs. / The Duchenne muscular dystrophy is the most common hereditary muscular disease. This disease is inherited as an X-linked recessive trait. It is caused by the absence of dystrophin in muscle fibers. Cell therapy is the potential treatment but, its success depends on the control of the rejection of the transplanted myoblasts and of the hybrid fibers that they formed. At present, the control of the graft rejection is achieved by administration of powerful immunosuppressive drug. Our long-term aim is to develop a protocol for immune tolerance that would prevent the graft rejection without sustained immunosuppression. The first step of this tolerance protocol that we want to develop is to induce the formation of regulatory T cells using the transcription factor Foxp3. In this study we generated, in bacteria E. coli, a fusion protein Tat-Foxp3. By in vitro assays, we demonstrated that Tat-Foxp3 protein up-regulated the expression of CD25 in naïve CD4+ T cells. Additional experiments will be required to confirm that these CD25 expressing cells are Treg.

La dystrophie musculaire de Duchenne est une maladie neuromusculaire affectant un garçon sur 3200. Cette maladie se caractérise par l'absence d'une simple protéine, la dystrophine, qui assure normalelnent l'intégrité des fibres musculaires. Sans cette protéine, les fibres musculaires sont plus facilement endommagées, provoquant une dégénérescence musculaire. Rapidement, les facultés locomotrices diminuent, confinant habituellement le patient à un fauteuil roulant vers l'âge de 10 ans. L'utilisation d' une assi stance respiratoire s'avère généralement nécessaire au début de la vingtaine et le décès peut survenir dès l'âge de 17 ans. Les patients atteignent rarement la trentaine. La thérapie cellulaire se situe parmi les traitements curatifs potentiels et consiste à implanter au patient dystrophique des myoblastes contenant le gène normal de la dystrophine. Évidemment, cette thérapie présente quelques inconvénients, dont une réaction immune du patient contre les myoblastes transplantés. C'est ici qu'interviennent les travaux présentés dans ce mémoire avec comme objectif principal d'utiliser les Iymphocytes T régulateurs (Tregs) afin de créer un protocole de tolérance non-toxique pour les patients dystrophiques. Jusqu 'à maintenant, nous avons réussi à isoler efficacement et de manière reproductible ces Tregs en utilisant une armada d'anticorps spécifiques. Plus précisément, nous avons actuellement une pureté moyenne se situant entre 90% et 95%. Par après, en démontrant l'expression du FoxP3 au sein de ces cellules, ces travaux prouvent hors de tout doute que les cellules purifiées sont des Tregs au sens propre du tenne. Finalement, par des essais in vitro, nous avons efficacement démontré que les Tregs isolés conservaient leur capacité immunorégulatrice. En effet, suite à une stimulation artificielle, les lymphocytes T CD4⁺ mis en co-culture avec un ratio de Tregs de 1:4 se divisent deux fois moins que ceux sans Tregs. Afin de conclure ces travaux, il reste deux étapes majeures avant de crier victoire. Premièrement, il faut réussir à multiplier des Tregs spécifiques afin d'en avoir un nombre suffisant et d'avoir une plus grande spécificité. Deuxièmement, il faudra les réintroduire chez un modèle animal afin de tester s' ils empêchent le rejet d' une greffe allogénique.