Selected abstracts presented in Myology 2008, 3rd International Congress Of Myology Marseille, France, May 2008

LONG-TERM BLINDED PLACEBO-CONTROLLED STUDY SHOWING PHENOTYPIC CORRECTION OF DYSTROPHIN DEFICIENCY IN THE MDX MOUSE BY SNT-MC17/IDEBENONE

BUYSE G1, VAN DER MIEREN G1, ERB M2, D'HOOGE J1, HERIJGERS P1,

VERBEKEN E1, JARA A3, VAN DEN BERGH A1, MERTENS L1, COURDIER-FRUH I2, BARZAGHI P2, MEIER T2

(1) University Hospitals K.U. Leuven, Leuven, BELGIUM. (2) Santhera Pharmaceuticals, Liestal, SWITZERLAND. (3) Biostatistical Center K.U. Leuven, Leuven, BELGIUM.

Background - Duchenne muscular dystrophy (DMD) is a severe and still incurable disease, with heart failure as a major cause of death. The identification of a disease modifying therapy may require early-initiated and long-term administration, but such type of therapeutic trial is not evident in humans. We have performed such a trial of SNT-MC17/idebenone in the mdx mouse model of DMD, based on the drug’s potential to improve mitochondrial respiratory chain function and reduce oxidative stress. The mouse model allowed presymptomatic initiation and veritable long-term administration of treatment, as well as the use of gold standard in vivo invasive pressure-volume measurements for assessing cardiac contractility. Methods & Results - 200 mg/kg bodyweight of either SNT-MC17/idebenone or placebo was given from age 4 weeks until 10 months in mdx and wild-type mice. All evaluators were blinded to mouse type and treatment groups. Compared to wild-type mice, placebo-treated mdx mice showed cardiac hypertrophy, diastolic dysfunction, reduced contractile reserve with systolic failure and 58% mortality during low-dose dobutamine stress, cardiac inflammation and fibrosis, and reduced voluntary wheel running performance. Idebenone treatment significantly corrected cardiac diastolic dysfunction and significantly prevented mortality from cardiac pump failure induced by dobutamine stress testing, significantly reduced cardiac inflammation and fibrosis, and significantly improved voluntary running performance in mdx mice. Conclusions - We have identified a novel potential therapeutic strategy for human DMD, as SNT-MC17/idebenone was cardioprotective and improved exercise performance in the dystrophin-deficient mdx mouse. These animal data encourage investigation of SNT-MC17/idebenone in human DMD. Our data also illustrate that the mdx mouse provides unique opportunities for long-term controlled prehuman therapeutic studies.

SNT-MC17/IDEBENONE IMPROVES CARDIAC AND RESPIRATORY  FUNCTION IN DUCHENNE MUSCULAR DYSTROPHY: RESULTS OF A 12 MONTH DOUBLE BLIND RANDOMIZED CONTROLLED TRIAL

BUYSE G1, MERTENS L1, VAN DEN HAUWE M1, THIJS D1, DE GROOT I2, SCHARA U3, CEULEMANS B4, MEIER T5, GOEMANS N1

(1) University Hospitals K.U. Leuven, Leuven, BELGIUM. (2) UMC St Radboud, Nijmegen, THE NETHERLANDS. (3) Lukaskrankenhaus GmbH, Neuss,  GERMANY.(4) University Hospital Antwerp, Antwerp, BELGIUM. (5) Santhera Pharmaceuticals Ltd, Liestal, SWITZERLAND

Objective - To evaluate efficacy and tolerability of treatment with SNTMC17/ idebenone compared to placebo in children with Duchenne muscular dystrophy (DMD). Background - Idebenone supports mitochondrial respiratory chain function

and reduces oxidative stress, pathways that are involved in DMD pathogenesis. In a controlled study we have shown that presymptomatic-initiated and long-term treatment with SNT-MC17/idebenone is cardioprotective and improves exercise performance in the homologous dystrophin-deficient mdx mouse. Design/Methods - 21 DMD patients (8-16 yr) with cardiac dysfunction were enrolled in a phase II double-blind randomized placebo-controlled trial. Comedication with glucocorticoids was allowed at stable dosage; use of ACE-inhibitors was excluded. Thirteen patients received SNT-MC17 (450 mg daily) for 52 weeks, 8 patients were randomized to the placebo group. The primary endpoint was the change from baseline in peak systolic radial strain of the left ventricular (LV) inferolateral wall, the region of the heart which is most affected in DMD. Secondary outcome measures included other cardiac parameters, respiratory function and upper limb muscle strength tests. Results - All subjects completed the study, and SNT-MC17 showed good safety and tolerability. Compared to patients on placebo, patients on SNT-MC17 showed significant improvement in peak systolic radial strain of the LV inferolateral wall (p= 0.030). Peak systolic longitudinal strain of the LV lateral wall improved in patients on SNT-MC17 and deteriorated in patients on placebo (p=0.039). Whereas SNT-MC17 treatment was associated with improvement in respiratory peak flow, patients on placebo deteriorated (p= 0.039). Changes in

forced vital capacity and maximal inspiratory pressures were not statistically significant between the groups. No significant differences were detected for quantitative upper limb muscle strength. Conclusions - This is the first indication of

clinical efficacy with SNT-MC17/idebenone on functional cardiac and respiratory parameters in DMD. The results provide the basis and guidance for phase II/III studies with SNT-MC17/idebenone in DMD.

THERAPEUTIC EFFICACY AND MECHANISM OF ACTION OF NITRIC OXIDE RELEASING NON STEROIDAL ANTI-INFLAMMATORY DRUGS IN MUSCULAR DYSTROPHY

SCIORATI C1, AZZONI E1, ONGINI E2, MONOPOLI A2, BRUNELLI S1, COSSU G1,CLEMENTI E3

(1) Stem Cell Research Institute-San Raffaele Scientific Institute, Milano, ITALY. (2)NicOx Research Institute, Bresso, ITALY. (3) E. Medea Scientific Institute and Department of Preclinical Sciences, University of Milano, Milano, ITALY.

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Duchenne muscular dystrophy is a relatively common disease that affects skeletal muscle leading to progressive paralysis and death. There is currently no resolutive therapy. We have developed a novel strategy based on the combination of nitric oxide (NO), which has beneficial effects in skeletal muscle, with non steroidal anti-inflammatory drugs (NSAIDs). To this end we used a new class of NO-releasing NSAIDs (NO-NSAIDs). We report the results of long term (one-year) oral treatment in the mouse model for limb girdle muscular dystrophy (-sarcoglycan null mice) with two such NO-NSAIDs, nitroibuprofen and nitroparacetamol. Both drugs significantly ameliorated the morphological, biochemical and functional phenotype in the absence of secondary effects, efficiently slowing down disease progression and were significantly more effective than the corticosteroid prednisolone analyzed in parallel. NO-NSAIDs acted by reducing inflammation, preventing muscle damage and preserving the number and function of satellite cells. To assess the mechanisms of NO-NSAID action we dissected the contribution of NO and NSAID activities, by analyzing the effects of the NSAID ibuprofen and of isosorbide dinitrate that release NO with a pharmacokinetic profile similar to that of NO-NSAIDs. The NO-NSAIDs were significantly more effective than either isosorbide dinitrate or ibuprofen. Of importance, the NO-NSAIDs were more effective therapeutic agents also with respect to the combination of isosorbide dinitrate and ibuprofen, suggesting that NO-NSAIDs have properties additional to NO release and NSAID activity. The new therapeutic strategy we propose is not selective for a subset of mutations, provides ground for immediate clinical experimentation with NO-NSAIDS, which are approved for use in humans.

GREEN TEA POLYPHENOLS DISPLAY POTENT ANTI-FIBROTIC ACTIVITY ON PRIMARY CULTURES OF DYSTROPHIC MUSCLE CELLS

DORCHIES O1, COMYN S1, RUEGG U1

(1) University of Geneva, Geneva, SWITZERLAND.

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Muscular dystrophies are characterized by fibrosis, a process leading to abnormal accumulation of materials of fibroblastic origin in the skeletal muscles as necrosis regeneration cycles take place. Fibrosis results from alterations in a multifactorial balance involving cytokines and growth factors, expression of their receptors at the cells’ surface, oxidative stress, production of extracellular matrix components, release of matrix-metalloproteinases (MMPs) and of their endogenous inhibitors TIMPs (tissue inhibitors of MMPs), all collectively regulating extracellular matrix turnover and fibroblast invasiveness. Fibrosis severely impairs skeletal and cardiac muscle functions both in patients suffering from Duchenne muscular dystrophy (DMD) and in the mdx mouse, the most commonly used model for DMD. Published data indicate that green tea polyphenols (GTP) exhibit anti-fibrotic properties in several cell types and tissues, and pentoxifylline (PTX), a well-known inhibitor of TNFrelease, has been proposed to reduce fibrosis in the dystrophic mdx mouse. Treatment of primary cultures of mouse muscle at the myotube stage with GTP or PTX (2.5 to 25ıM) inhibited the expression and/or the release of the fibrosis modulators TNFı, TGF1, and PDGF-BB. When needed, fibrosis was induced in the cultures with TNFor TGB1 (30ng/mL). GTP dose-dependently decreased both basal and TNFor TGB1-induced expression of ı-smooth muscle actin (ıSMA, a marker for activated fibroblasts), collagen, TGF receptor type 2, and CTGF (connective tissue growth factor, a key mediator of TGFı1 actions). As determined by zymography, the expression of pro- and active forms of MMP-2 and MMP-9 were not changed with GTP and PTX. However, the MMP-2 and MMP-9 gelatinase activity in the supernatants was significantly decreased and correlated with a marked elevation of TIMP-1 levels. Overall, PTX was less potent than GTP on these endpoints. Our results indicate that GTP profoundly alter the expression and/or the activity of proteins involved in the fibrogenic process.

OXIDATIVE INJURY OF MYOBLASTS: SEARCH FOR A PROTECTIVE EFFECT BY A GLYCOSAMINOGLYCAN MIMETIC (OTR4120)

VAN ZOGGEL J1, ALABANESE P1, JACOBS MS1, COURTY J1, PAPY-GARCIA D1,MORIN C1, MARTELLY I1

(1) CRRET Laboratory, Université Paris-Est, Faculté de Sciences et Technologie, Paris 12, Créteil, FRANCE.

Overproduction of reactive oxygen species has been implicated in cellular defect that may lead to apoptosis. Skeletal muscle cells are frequently submitted to oxidative stress upon excessive exercise or in disease. Although cells withstand oxidative exposure by developing antioxidant defences, their resistance capacity could be sometimes overcome, resulting in cell entering apoptosis. It is therefore important to investigate mechanisms occurring in skeletal muscle cells in response to oxidative stress. Among defence mechanisms, it has been suggested that glycosaminoglycans (GAGs) could exert a protective effect. Therefore, we have explored how myoblasts react in vitro to oxidative stress and whether treatment with a synthetic GAG mimetic (namely OTR4120) would protect them against oxidative injury. An oxidative stress induced by oxygen peroxide (H202, 10pmol/cell, 30min) was applied on proliferating myoblasts (C2.7 cell line). Cell survival was decreased by about 70% within 24 hrs. Compared to untreated cells, oxidative stress induced apoptosis assessed either by the presence of apoptotic features of nuclei stained with diaminophenyl indol, or by the presence of subG1-DNA observed by Flow cytometry or by an increase in caspases-3, 9 and 8 activities at 48hrs. This was accompanied by a two times increase in the amount of total sulphated GAGs produced by stressed cells compared to untreated cells, without change in the overall GAG composition. However, as shown by immunocytology, these GAGs seemed to remain in the intracellular compartment instead of being addressed to the cellular membrane. Treatment with the GAG mimetic OTR4120 (1.0 μg/ml) added just after the oxidative stress period partially prevented such features associated with apoptosis, in part restored GAG localisation at membranes and allowed cells to differentiate after 72hrs. Possible mechanisms of action of OTR4120 are being further explored. These data support the working hypothesis that GAG mimetics could protect myoblasts from oxidative stress injury

TREATMENT WITH A SOLUBLE ACTIVIN RECEPTOR TYPE IIB ATTENUATES MUSCLE WEAKNESS IN YOUNG MDX MICE.

LACHEY J1, PULLEN A1, WONG V1, PEARSALL RS1, SEEHRA J1

(1) Acceleron Pharma, Cambridge, USA.

Dystrophin-deficient, or mdx, mice contain a mutation in the dystrophin gene and are therefore the genetic homolog of Duchenne muscular dystrophy. While the consequences of dystrophin deficiency in mice are less severe than what is seen in DMD patients, mdx mice exhibit a period of muscle degeneration that manifests as muscle weakness before subsequent regeneration restores strength to wild-type levels. Activin type llB receptors (ActRllB) are one of the receptors responsible for TGFı signaling and are capable of binding myostatin as well as other negative muscle mass regulators in the TGFı superfamily. Treatment with a soluble ActRllB molecule blocks the ability of these ligands to signal and results in increased lean tissue mass. Here we describe the ability of a soluble ActRIIB fusion protein (RAP- 031) to increase lean tissue mass and restore strength in young mdx mice. RAP-031 treatment significantly increases body weight in both C57BL/10 and mdx mice. NMR scans revealed increased lean tissue gain accompanied the higher body weights. RAP-031 treated C57BL/10 mice gained 35.2% and the RAP-031 treated mdx group gained 48.3% more lean tissue mass than their respective control cohorts. Further, the effect of RAP031 treatment on strength was assessed. Vehicle treated mdx mice grip strength scores were 15.7% lower than the vehicle C57BL/10 cohort thereby illustrating the muscle weakness associated with dystrophin deficiency. In contrast, the RAP-031 mdx mice improved their grip strength compared to the mdx vehicle group, and attained grip strength measurements which surpassed C57BL/10 vehicle mice and reached the level of the RAP-031 C57BL/10 grip strength scores (vehicle mdx: 0.140 ± 0.01 KgF; RAP-031 mdx: 0.199 ± 0.02 KgF; vehicle C57BL/10: 0.166 ± 0.03; RAP-031 C57BL/10: 0.205 ± 0.02 KgF). These data support the idea that inhibition of ActRllB signaling could have important clinical applications in Duchenne

muscular dystrophy.

CREATINE AND L-ARGININE TREATMENTS IMPROVE SARCOPLASMIC RETICULUM FUNCTION AND CONTRACTILE PERFORMANCE OF DIAPHRAGME FROM MDX MICE.

LAFOUX A1, BERTRAND J2, GERVIER P1, HUCHET-CADIOU C1

(1) UMR CNRS 6204 "Biocatalyse, Biotechnologie et Biorégulation", Nantes, FRANCE. (2) UMR 6187 "Institut de Physiologie et

Biologie Cellulaires", Poitiers, FRANCE.

In duchenne’s muscular dystrophy, that is characterized by a progressive skeletal muscle fiber necrosis, the membrane-stabilizing protein dystrophin is missing, and this leads to altered total Ca2+ content in muscle fibers. This study investigated the

potential therapeutic benefits of pharmacological tools in reversing the Ca2+ sequestration function of the sarcoplasmic reticulum and improving the functionnal capacity of skeletal muscle of mdx mice. Creatine (CrM: 2,15 mg/mL/day) and Larginine (L-Arg: 3,75 mg/mL/day) were administred per os to male mdx mice (12 weeks) during 4 weeks. Then, the effectiveness of the treatment was investigated on the contractile protein properties, the sarcoplasmic reticulum Ca2+ uptake and the expression of SERCA1 and calsequestrin in diaphragm muscle of mdx mice. The data demonstrate that after treatments, the maximal Ca2+ activated tension developped by skinned fibers were increased approxymately by 30% (Tmax mdx= 46.8 ± 4.5 mN.mm- 2; CrM= 60.3 ± 5.7 mN.mm-2; L-Arg= 61.0 ± 2.8 mN.mm-2). The study of sarcoplasmic function, assessed in skinned fibers and vesicle preparations, shows that the Ca2+ uptake was improved in mdx diaphragm muscle after both pharmacological treatments (Ca2+ uptake mdx= 20.3 ± 1.1 nmole Ca2+.s-1.mg-1; CrM= 22.1 ± 0.6 nmole Ca2+.s- 1.mg-1; L-Arg= 26.4 ± 1.1 nmole Ca2+.s-1.mg-1). Furthemore, the overall force of the musculature was tested in living animals using the grip strenght test, the wire test and the rotarod. The results show that the locomotor activity was enhanced and that mdx mice were more resistant to fatigue. During the grip test, the force developped by the four limbs was ameliorated in mdx mice after both treatments (Strength mdx= 4.0 ± 0.1 g.g-1; CrM= 4.2 ± 0.2 g.g-1; L-Arg= 4.7 ± 0.2 g.g-1). In conclusion, the data show that creatine and L-arginine treatments significantly normalized many functionnal and biochemical parameters by acting on events that are related to Ca2+ homeostasis

TAMOXIFEN IMPROVES THE STRUCTURE AND THE FUNCTION OF SKELETAL MUSCLE IN MDX MICE

DORCHIES O1, REUTENAUER J1, VUADENS O1, COMYN S1, RUEGG U1

(1)   University of Geneva, Geneva, SWITZERLAND.

Data from the literature indicate that tamoxifen (Tam), a selective estrogen receptor modulator (SERM) used in the treatment of certain types of breast cancers, displays antioxidant, anti-apoptotic and anti-fibrotic properties and stabilises biological membranes. We hypothetised that Tam might improve both the structure and the function of dystrophic muscles. When applied to primary cultures of dystrophic myotubes, Tam and 4-hydroxy-Tam (10-8 to 10-6M) were not toxic, reduced collagen content slightly, and exhibited a limited antioxidant activity against hydrogen peroxide. Mdx dystrophic mice, the most common model for Duchenne muscular dystrophy, were treated for about 15 months following weaning with 0.01% tamoxifen mixed into the food pellets (Tam intake approx. 10mg/kg/day). Tam-treated mdx mice showed a significant decrease in body weight, likely due to the important reduction in white adipose tissue compared to untreated mice. The mass of the triceps surae (composed of soleus, plantaris and gastrocnemius muscles) was lowered to normal values, while the EDL and the diaphragm showed significant hypertrophy compared with both normal and untreated dystrophic animals. Using the horizontal wire test, the Tamtreated mdx performed as well as the normal mice and 3 times better than theuntreated mdx mice. Isometric muscle force of the triceps surae was recorded.

Remarkably, specific phasic and tetanic twitch tensions were increased by 100% and 70%, respectively, compared with untreated mdx, resulting in values higher than those of normal animals. The rates of contraction and relaxation were much slower than in the untreated mdx, and the force-frequency curve was significantly shifted to the left, suggesting a marked change toward a slower phenotype. In the Tam-treated mdx, the resistance to repetitive tetanisations was improved by 44% and creatine kinase levels were lowered by 50% compared with the untreated mdx. On-going histological analysis suggests a normalisation of the mean fiber diameter.

MEDICAL FOOD IN MDX MICE: GENISTEIN AND FLAVOCOXID AMELIORATE MUSCLE PATHOLOGY AND FUNCTION.

MESSINA S1, MAZZEO A1, BITTO A2, AGUENNOUZ M1, MIGLIORATO A1, DEPASQUALE MG1, SQUADRITO F2, VITA G1

(1) Department of Neuroscience, Psychiatry and Anaesthesiology, University of Messina, Messina, ITALY. (2) Department of Clinical and Experimental Medicine and Pharmacology, University of Messina, Messina, ITALY.

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Abstract

Soy isoflavones have been reported to have antioxidant bioactivities, scavenging free radicals and increasing antioxidant protein expression, and also to inhibit the transcription factor NF-kB. We showed in previous studies that the inhibition of the

transcription factor NF-kB throught drugs with also antioxidant properties, have beneficial effects in mdx mice. The drugs used are not available for clinical studies. We tested whether genistein and flavocoxid, supplements with known antioxidant and antinflammatory properties readily available for clinical use, could have a beneficial effect on muscle function, morphology and biochemical pattern in mdx mice. Five-week old mdx mice received for five weeks either genistein (2 mg/kg i.p. daily), flavocoxid (5 mg/kg i.p. daily) or vehicle. Flavonoids treatment 1) increased forelimb strength ( p<0.05) and strength normalized to weight (p<0.05) and decreased fatigue (p<0.05); 2) reduced serum creatine-kinase levels (p<0.01); 3) increased GPX activity and reduced markers of oxidative stress (p<0.05); 4) blunted NF-B DNA-binding activity (p<0.05); 5) reduces muscle necrosis (p<0.01) and enhances regeneration (p<0.05) with an augmented number of myogenin-positive satellite cells and myonuclei, and of developmental myosin heavy chain-positive fibers. Our results suggest that these flavonoids might have a beneficial effect on muscle function and morphology in mdx mice. Further studies are needed to investigate the biochemical substrates of such encouraging preliminary results taking into account that these supplements could be easily introduced in the daily diet of patients with DMD.

IMIPRAMINE TREATMENT INCREASES THE MUSCLE STRENGTH OF MDX MICE

HUCHET-CADIOU C1, CARRE-PIERRAT M2, LAFOUX A1, TANNIOU G2, GERVIER P1, FOUGEROUSSE F3, SEGALAT L2

(1) UMR CNRS 6204 Universite de Nantes, NANTES, FRANCE. (2) UMR CNRS 5534 Universite de Lyon, LYON, FRANCE. (3) Laboratoire d’Evaluation Fonctionnelle, Departement Recherche et Developpement, Genethon, EVRY,FRANCE.

Several bioactive molecules, including antidepressants, efficiently prevent muscle necrosis in dystrophin-deficient C. elegans. We tested the effect of the antidepressant, Imipramine, on the muscle degeneration of mdx mice. The mdx 5cv strain was preferred to the classical mdx Scsn strain because it has almost no revertant fibers, and is easier to breed. Groups of 6-8 mice (sex matched) were fed from 2 to 6 weeks of age with a diet containing 10 and 40 mg/kg/day of Imipramine, and tested at 6 weeks. The analysis included both histological and functional assays. The histology was performed on the extensor digitorum longus (EDL), a fast-twitch muscle, and the diaphragm. Histological parameters (% centrally nucleated fibers, fiber size variance, necrosis index) were improved 20-40% in treated versus nontreated animals. Treated animals also performed significantly better in the wire test behavioral assay. Moreover, the analysis of mechanical properties showed that the force developed by the whole EDL muscle in situ was 25% higher in treated versus non-treated animals. Surprisingly, the force developed by the EDLs of Imipramine treated mdx mice was also larger than that of wild-type mice. Finally, using Tritonskinned fibers, diaphragm muscle from treated mice exhibited 30% higher maximal tension than mdx mice. Thus, Imipramine treatment of young mdx mice reduces the first wave of muscle necrosis and improves muscle strength of fast-twitch muscles

RESTORATION OF THE CALCIUM SIGNALING IN MDX MOUSE DUODENUM MYOCYTES BY GENTAMYCIN SULFATE TREATMENT AND DYSTROPHIN EXON SKIPPING.

DABERTRAND F1, MACREZ N1, MIRONNEAU J2, HENAFF M1, MOREL JL1

(1) umr5228 CNRS université Bordeaux, talence, FRANCE. (2) retired, umr5017CNRS université bordeaux, bordeaux, FRANCE.

We have previously described that the loss of dystrophin expression in duodenum myocytes from mdx mouse induces the decrease of ryanodine receptor subtype 2 (RYR2)-dependent calcium signalling. This effect is due to a lower level of RYR2 expression in mdx duodenum myocytes [Morel et al., 2004]. Both aminoglycoside treatment and dystrophin exon skipping are proposed as therapeutic ways for the Duchenne muscular dystrophy. After 14 days of gentamycin treatment, the activation of RYR2 by caffeine in mdx duodenum myocytes induced the same calcium responses than those observed in wild-type mice and an increase of the expression of RYR2. In addition, mdx mice were treated by antisense oligo-2’ OMethyl (O-Me) nucleotides directed against the dystrophin exon23 (asDYS) to induce exon skipping and the expression of truncated dystrophin. The Cy5 labelling of asDYS enables to follow its presence in duodenum myocytes. As observed in gentamycin treated mdx mice, the treatment with asDYS was able to increase the RYR2 expression and restore the calcium signals encoded by RYR activation. These results suggest that gentamycin as well as antisense strategy could be used to restore the Ca2+ signalling in smooth muscle from mdx mice. Both of these therapeutic ways have benefits and inconvenience but are possibly usable to restore the smooth muscle function for patients affected by Duchenne muscular dystrophy

BENEFICIAL EFFECT OF MOLSIDOMINE ON MDX MICE MUSCLE MEMBRANE EVIDENCED BY IN SITU MALDI PROFILING

BENABDELLAH F1, TAHALLAH N1, TOUBOUL D1, YU H2, BRUNELLE A1, LAPRÉVOTE O1, DE LA PORTE S2

(1) Institut de Chimie des Substances Naturelles, Laboratoire de Spectrométrie de Masse, CNRS - UPR 2301, Gif-sur-Yvette, FRANCE. (2) Institut de Neurobiologie Alfred Fessard - FRC2118, Laboratoire de Neurobiologie Cellulaire et Moléculaire,

CNRS -UPR 9040, Gif-sur-Yvette, FRANCE

Duchenne muscular dystrophy (DMD) is the most common genetic disorder characterized by the lack of dystrophin, a sub-sarcolemmal protein necessary for normal muscle functions. Among therapeutic approaches, the upregulation of utrophin to replace defectious dystrophin is developed. Previous studies demonstrate an increase of utrophin labelling, a decrease of necrotic surface, a diminution of creatine kinase release in the serum, and an improvement of isometric strength as consequences of improved NO production via the injection of L-arginine, the substrat for NO synthase or of molsidomine, an NO donor commercialized as Corvasal®. While histology indicates that molsidomine can reverse the degenerating process induced in mdx mice, the mechanism of action on the muscle structure is not really known. We examine the part of the phospholipid composition in membrane myofibers by addressing the phospholipid intensity ratio PC34:2(PC C16:0/C18:2)/PC34:1(PC C16:0/C18:1). This ratio has been demonstrated to be different in normal and dystrophic mice models by in situ MALDI-MS profiling. This variation was recently confirmed by cluster-TOF-SIMS (Time-of-flight Secondary Ion Mass Spectrometry) imaging on human dystrophic tissue sections. On the other hand, cell cultures were performed and the same intensity ratio inversion was shown during the differentiation from myoblasts to myotubes.It was thus suggested that this ratio could be a marker of the regenerating process of muscle cell lines. After the treatment of mdx mice with molsidomine, we evidenced by MALDI-TOF MS profiling a restoration of a membrane lipid composition equivalent to those of wild-type mice. This restoration was associated with an increase of the regeneration process in the mice. This indicates that NO donors restore a normal membrane structure mainly via a regeneration process although the eventuality of a simple compensation of the membrane structure by incorporation of specific class of fatty acids cannot be excluded.

UPGRADING U7SNRNA TO COMPLETE EFFICIENT RESCUE OF DYSTROPHIN BY EXON-SKIPPING IN DMD PATIENTS

GOYENVALLE A1, BABBS A1, GARCIA L2, DAVIES KE1

(1) Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UNITED-KINGDOM. (2) INSERM, S UMR 787, Groupe myologie, Paris, FRANCE.

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Most cases of Duchenne muscular dystrophy (DMD) are caused by mutations that disrupt the dystrophin mRNA reading frame. In many cases, skipping of a single exon could supposedly restore the reading frame, giving rise to a shorter but still functional quasi-dystrophin protein. It has previously been proposed to use small nuclear RNAs, especially U7snRNA, to shuttle antisense sequences designed to mask key elements involved in the splicing of targeted exons. Our present project focuses on the upgrading of U7snRNA to complete rescue of dystrophin by exon-skipping in DMD patients. We indeed developed bifunctional U7snRNAs carrying a complementary sequence to the targeted exon and a free tail harbouring canonical binding sites for the heterogeneous nuclear ribonucleoproteins A1/A2 (hnRNP) that are powerful splicing repressors. The presence of this generic strong silencer tail could indeed circumvent the always tricky and time-consuming specific optimization required for each new exon-target.

We first focused on the exon 51 of the dystrophin gene and therefore designed new tailed U7snRNA constructs. Each construct has been inserted into lentiviral vectors for in vitro analysis on myoblasts from DMD patients. After transduction of these cells with lentiviral vectors encoding the tailed U7-ex51, we confirmed the skipping of the exon 51 by nested RT-PCR and dystrophin restoration by Western blot. By Comparison with their controls with a mutated tail, we could show that the skipping efficiency of these constructs was due to the tail carrying silencer motifs, therefore confirming its splicing repressor action, and not the annealing sequence to the exon. These very encouraging results on exon 51 provide evidence that bifunctional U7snRNA can achieve efficient exon-skipping in myoblasts from DMD patients. Since these tailed U7 could be very useful for the development of the exonskipping strategy for the other dystrophin exons, the confirmation of their efficiency shown in this study offers very promising tools for clinical treatment of DMD

THE USE OF IMMORTALISED HUMAN FIBROBLASTS FROM A DMD PATIENT TO TEST EXON SKIPPING IN VIVO

CHAOUCH S1, GOYENVALLE A1, MAMCHAOUI K1, ; BUTLER-BROWNE G1, DI SANTO J2, TORRENTE Y3, MOULY V1, GARCIA L1, FURLING D1

(1) UMR S 787 Groupe Myologie INSERM/UPMC-ParisVI, Institut de Myologie, Paris, FRANCE. (2) Cytokines and Lymphoid Development Unit, Immunology Department, Institut Pasteur, Paris, FRANCE. (3) Fondazione IRCCS Ospedale Maggiore Policlinico of Milan, Department of Neurological Sciences, Dino Ferrari Center, University of Milan, Milan, ITALY.

We are now at a point in time where gene therapy is becoming a reality. However, in order to validate these strategies, it is essential to have in vitro human cell culture models. For the study of neuro-muscular diseases, the use of patient myoblasts is not always possible due to their drastically decreased proliferative capacity induced by the repeated cycles of degeneration and regeneration. Therefore it is neccessary to envisage new in vitro models. In the pioneering studies of Weintraub et al, it had been shown that the forced expression of the myogenic transcription factor myoD was able to convert fibroblasts into myogenic cells. Based on this, we have developed a universal in vitro model from skin fibroblasts which have been immortalised using hTERT and then converted into myoblasts by a lentivirus containing an inducible myoD contruct. We have then used this model to validate a strategy for exon skipping using fibroblasts isolated from a DMD patient. These fibroblasts were immortalized and then transduced using an inducible myoD construct. We first confirmed the expression of myoD in vitro and the potential of these cells to form differentiated myotubes. These cells were then transfected with an U7 construct that promote exon skipping in the mutated dystrophin transcript. In order to test if these cells could reconstitue muscle fibres in vivo expressing human dystrophin, they were injected into cryodamaged TA muscles of immunodeficient RAG-/- gammaC-/- C5-/- mice. Muscle were analysed after 27 days of regeneration and fibres expressing human dystrophin were observed. Therefore this cellular model provides us with an alternative model system to test different therapeutic strategies for various neuromuscular diseases when patient myoblasts are not available.

SYSTEMIC DELIVERY OF ANTISENSE OLIGONUCLEOTIDES RESTORES DYSTROPHIN EXPRESSION AND FUNCTIONALITY IN THE MDX MOUSE

VAN PUTTEN M1, DE WINTER C1, HEEMSKERK H1, DE KIMPE S2, VAN DEUTEKOM J2, VAN OMMEN G-J1, AARTSMA-RUS A1

(1) Leiden University Medical Center, Leiden, THE NETHERLANDS. (2) Prosensa BV, Leiden, THE NETHERLANDS.

Duchenne Muscular Dystrophy (DMD) is a chromosome X-inherited disorder, characterised by the absence of the protein dystrophin which plays a role in the

protection of muscle fibers against damage. Patients display progressive degeneration of skeletal muscles leading to functional impairment and premature death. One of the most promising therapeutic approaches for DMD involves the

restoration of the disrupted reading frame through exon skipping with antisense

oligonucleotides (AON). In cultured cells from patients and in the mdx mouse model AON treatment resulted in the generation of dystrophin proteins similar to those seen in Becker Muscular Dystrophy patients, who generally display a milder phenotype than Duchenne patients. Recently, we showed local restoration of dystrophin expression after intramuscular injection of an exon 51 specific 2’-O-methyl phosphorothioate AON in four Duchenne patients in a first-in-man clinical trial. For future clinical studies on full body treatment we are currently optimizing systemic AON delivery. We here compared different doses (25 mg/kg to 100 mg/kg) and treatment periods (4 to 8 weeks) with murine exon 23 specific AONs in mdx mice applying subcutaneous administration. The exon skip levels varied between specific muscle groups from 8% in the diaphragm to 26% in the extensor muscles of the lower forelimb and up to 2% in the heart, after twice weekly repeated injections with a 100 mg/kg AON dose during 8 weeks treatment. Dystrophin expression was observed in all samples analysed. This was accompanied by trends of decreased CK and improved rota-rod running times of treated mice compared to saline injected control mice. Markers for kidney and liver function remained unaffected, indicating that repeated AON treatment was well tolerated even when performed over longer time periods. Our results show that systemic injections of 2’-O-methyl phosphorothioate AON lead to restoration of dystrophin and an improved functionality without apparent toxicity.

IN VIVO DELIVERY OF NAKED AND LIPID-COMPLEXED ANTISENSE OLIGOS IN MDX MICE: EFFECTS ON SKELETAL AND CARDIAC MUSCLE

PIGOZZO S1, REPELE A1, REGGIANI C2, AUSONI S3, ZAGLIA T3, BARONI MD1, AMBEGIA E4, MCLACHLAN I4, VITIELLO L1

(1) Department of Biology, University of Padova, Padova, ITALY. (2) Department of Anatomy and Physiology, University of Padova, Padova, ITALY. (3) Department of Biomedical Sciences, University of Padova, Padova, ITALY. (4) Protiva Biotherapeutics Inc., Burnaby, CANADA.

Antisense-mediated exon skipping holds great potential for the treatment of DMD. In mdx mice, functional recovery of skeletal muscle has been reported upon systemic delivery of “naked” oligonucleotides or viral vectors encoding for antisense snRNAs. However, only one study achieved dystrophin restoration in cardiac muscle (using an adeno-associated vector). Here we report the in vivo delivery of morpholino oligos in aged mdx mice, both in skeletal muscle, via intra-arterial injection, and in cardiac muscle, via intramuscular injection. Intra-arterial delivery yielded levels of dystrophin restoration comparable to those reported in the literature with the intra-venous approach, but with smaller amounts of oligonucleotides. Intra-cardiac injections, on the other hand, showed that the level and duration of the skipping effect found in cardiac muscle were greatly decreased compared to skeletal muscle. This latter finding provides the first direct evidence that antisense-mediated dystrophin restoration in cardiac muscle might suffers from limitations that do not exist in skeletal muscle. All data published so far have indicated that systemic delivery via the vasculature requires large amount of naked oligos to achieve therapeutically significant results. Here we also report that the use of lipid carriers has the potential to greatly improvethe delivery efficiency; in particular, we found that the use of lipid-encapsulated oligo RNA allowed to detect dystrophin re-expression with a single dose of ~40 μg of oligos per adult mdx mouse. Importantly, dystrophin restoration could be seen not only in skeletal and but also (albeit to a smaller extent) in cardiac muscle.

CLINICAL DEVELOPMENT OF A GENE THERAPY PRODUCT FOR THE TREATMENT OF LIMB GIRDLE MUSCULAR DYSTROPHY TYPE 2C

HADDAD H1, RIGOLET A2, CAIZERGUES D1, DOUAR AM1, MILLOT L1, LAMBERT I1, LIABEUF M1, LAFORÊT P2, LETURCQ F3, VOIT T4, BÉHIN A2, EYMARD B2, CARLIER P4, BENVENISTE O2, LEMOINE F2, HOGREL JY4, ROSIER-MONTUS M1, DENÈFLE P1, HERSON S2, MASQUELIER AM1

(1) Généthon, Evry, FRANCE. (2) Hôpital Pitié-Salpêtrière, Paris, FRANCE. (3) Hôpital Cochin, Paris, FRANCE. (4) Institut de Myologie, Paris, FRANCE.

In late November 2006, GENETHON initiated its first gene therapy clinical trial in gamma-sarcoglycanopathy (LGMD2C), a rare autosomal recessive muscular disorder caused by mutations in the gamma-sarcoglycan gene. Patients commonly present with proximal and progressive muscular weakness before the age of 10 and loose ambulance by age 12 on average. Cardiomyopathy and respiratory insufficiency may develop during the course of the disease, leading to poor prognosis and premature death. Earlier during the clinical development, GENETHON had obtained an Orphan Drug Designation by EMEA. Approval from the French Agencies was obtained in November 2006 to initiate the phase I/IIa trial, which is being held at Pitié–Salpêtrière Hospital in Paris. The investigational product, a serotype 1 adeno-associated virus (AAV1) vector harboring the human gamma-sarcoglycan gene, is administered by a single intramuscular injection into the carpi radialis muscle. The primary objective of this trial is to evaluate the clinical safety of local intramuscular injection of the gene therapy product. Secondary objectives are to monitor local and systemic immune responses, assess histological modifications and gene transfer into injected muscles. 9 patients, aged above 15, will be enrolled sequentially in the study, assigned to 3 cohorts with a single dose-escalation and followed-up for 6 months. Evaluation will address clinical, histological, biological, immunological and functional parameters as well as MRI. As of today, four patients have been treated. The first cohort has been completed and the first patient of the second cohort has been treated. Encouraging data will be presented on the status of the product activity, immunologic response and patient compliance to the treatment. As the clinical trial is halfway, a next phase IIb clinical trial is being planned, in which systemic administration will be performed. This implies important preclinical and clinical points to consider, as well as a series of preclinical studies to achieve.

EFFICIENT IN VIVO DYSFERLIN EXPRESSION BY DUAL AAV VECTORS RECONSTRUCTION.

LOSTAL W1, BOURG N1, ROUDAUT C1, MIYAKE K2, MCNEIL P2, BARTOLI M1, RICHARD I1

(1) GENETHON, EVRY, FRANCE. (2) Medical College of Georgia, Augusta, USA.

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Dysferlinopathies are recessive muscular disorders caused by defects in dysferlin. Genetic mutations are responsible for two major phenotypes: Limb Girdle Muscular Dystrophy type 2B and Distal Miyoshi Myopathy. These skeletal muscle diseases are characterized by progressive loss of muscle integrity and strength. Recently, dysferlin was demonstrated to be involved in membrane repair process, providing a preliminary understanding of the pathophysiological mechanism in these diseases.

Currently, no treatment is available. Considering the recessive nature of dysferlinopathies, a possible therapeutic strategy is gene transfer. To date, the best vector for gene transfer in muscle is Adeno Associated Virus (AAV). However, the human dysferlin cDNA size approximates 7 kb, preventing its direct incorporation into a single AAV vector, since the encapsidation limit is around 4.7 kb. In order to bypass this limitation, we set up a strategy taking advantage of the concatemerization ability of AAV vectors. The dysferlin cDNA was separated into two AAV2/1 vectors, one carrying a muscle specific promoter followed by the 5’ half of dysferlin cDNA and a 5’ splicing signal and another one carrying a 3’ splicing signal, the remaining dysferlin sequence and a polyadenylation signal. We demonstrated the ability of these vectors to produce a human full-length dysferlin mRNA in a cellular model. To test this approach in vivo, intramuscular injection of both vectors was performed into dysferlin deficient mice. Dysferlin mRNA was correctly spliced, expressed at a level close to the endogenous one and was stable for an entire year. At the protein level, dysferlin was detected at 237 kDa, the size of the expected full-length protein. The functionality of dysferlin was demonstrated by a membrane repair assay based on 2-photon injury on isolated muscle fibers. Taken together, all these data prove that gene transfer based on AAV concatemerization allows the expression of a full-length functional dysferlin.

DUCHENNE AND AUTISM: HIGHER THAN EXPECTED ASSOCIATION?

BERARDINELLI A1, ORCESI S1, ROSSI M1, MOTTA C1, GORNI K1, BALOTTIN U1

(1) Deaprtment of Child NeuropsychiatryDepartment , IRCCS “C. Mondino” Foundation, University of Pavia, Pavia, ITALY.

Duchenne muscular dystrophy (DMD) is an X-linked progressive neuromuscular disorder due to lack of Dystrophin protein in muscle. Dystrophin is mainly concentrated in skeletal and cardiac muscle and less in smooth muscle, its deficiency causing severe progressive motor, respiratory and cardiac impairment. Dystrophin is also in the brain. Boys with Duchenne muscular dystrophy often have also central nervous system involvement such as mental retardation and learning disabilities. Autism is a brain development disorder impairing social interaction and communication and causing restricted and repetitive behavioural patterns, with all symptoms starting before three years of age.This set of signs distinguishes autism from milder autism spectrum disorders (ASDs), including Asperger syndrome. Genetic susceptibility to this condition has been documented. In the past only a single case report has been found about autism and DMD association In the last few years the hypothesis of a significative association between DMD andAutism has been proposed. Based on “ad hoc” chosen items of ADI-R and ADOS scales, both currently being considered the gold standard for autism diagnosis, we retrospectively evaluated the possible relational and  communicational impairment and related risk factors in 43 DMD children very well known to our Centre . Only one child ( 2.3%) was found to be affected by autism , even though we have about 20% children with low IQ and some with depressive traits, speech and emotional problems

 EFFECT OF AGE AT INITIATION OF CORTICOSTEROIDS ON AGE AT LOSS OF AMBULATION IN PATIENTS WITH DUCHENNE MUSCULAR DYSTROPHY (DMD)

PANDYA S1, FOX D2, CIAFALONI E1, DRUSCHEL C2, MOXLEY R1

(1) University of Rochester, Rochester, USA. (2) New York State Department of Health, Troy, USA.

Objective: To determine the effect of age at initiation of corticosteroids on age at loss of ambulation in patients with Duchenne muscular dystrophy (DMD) Background: The hypothesis among clinicians is that earlier the initiation of corticosteroids, the greater the benefit on prolongation of ambulation. No data is available to provide support for this hypothesis. Methods: We retrospectively analyzed data on all boys followed at our site since 1991 to look at the effect of age at initiation of corticosteroids on the age at loss of ambulation. We categorized the boys as follows: 1) Never treated with corticosteroids and 2) treated with corticosteroids. 2) Treated group was subdivided into 3 categories based on age at initiation of steroids as follows: Group a) age 4 to <7 years; b) age 7 to <9 years: c) 9 to <13 years. Results: Complete follow up data was available on 68 boys. 21 boys had never been treated with corticosteroid and ceased ambulation at an average age of 8 years. Of the 47 treated boys: Group a) N =24 initiated treatment at age 6.1( sd 0.7 yrs) and ceased ambulation at age 13.5 (sd 3.3 yrs) Group b) N=14 initiated treatment at age 7.8 (sd 0.6 yrs) and ceased ambulation at age 13.5 (sd 2.5. yrs) Group c) N=9 initiated treatment at age 10.3 (sd 0.6 yrs) and

ceased ambulation at age 12.8 (sd 2.5 yrs) The results between the 3 groups categorized by age at initiation of treatment were not statistically significant. This may be due to the small sample sizes. Conclusions: Retrospective data from our site demonstrates a trend that earlier the age (<9yrs) at initiation of corticosteroid the greater the prolongation of ambulation.

COMBINED NON-INVASIVE VENTILATION AND MECHANICAL INSUFFLATIONEXSUFFLATION ASSISTANCE IN PEDIATRIC NEUROMUSCULAR DISORDERS WITH HYPERCAPNIC RESPIRATORY INSUFFICIENCY

PEDEMONTE M1, OTTONELLO G1, SCAPOLAN S1, DOGLIO L1, BRUNO C1, MINETTI C1

(1) Istututo Giannina Gaslini, Genova, ITALY.

Neuromuscular disorders (NMD) are the most important cause of hypercapnic respiratory insufficiency in childhood and respiratory failure is the major cause of morbidity and mortality in NMD. The use of non-invasive ventilation (NIV) in NMD patients can improve the prognosis by correction of hypoventilation and reduction of respiratory work. However, it does not prevent acute respiratory failure during recurrent episodes of pneumonia or atelectasis due to inability to clear airways from secretions. Combined non-invasive ventilation with mechanical insufflation-exsufflation (MI-E), by means of mechanical cough-assistance, can ameliorate these conditions in neuromuscular patients. We have performed a retrospective evaluation of the safety, tolerance and effectiveness of this combined treatment in 27 patients affected by NMD (Duchenne and Becker Muscular Dystrophy=5, Limb Girdle Muscular Dystrophy=1, Spinal Muscular Atrophy=8, Congenital Myopathy=8, Ullrich’s Syndrome=4, Congenital Myotonic Dystrophy=1), aging 2 months to 18 years. The follow-up of these patients has been developed through the guideline of the European Neuromuscular Centre and of the American Thoracic Society. All patients used nocturnal NIV and during broncopulmonary acute episodes were treated with MI-E cough-assistance, with a complete resolution. In particular, fourteen patients, who presented severe exacerbations of respiratory conditions with development of pulmonary acute infections and atelectasia, showed a significant improvement after MI-E treatment. Seven patients needed invasive ventilation, only one received tracheotomy, while six were extubated by Bach’s protocol. In conclusion, our data indicate the good tolerance and the effectiveness of the combined management of NIV and MI-E in pediatric neuromuscular patients with respiratory insufficiency.

DIAGNOSTIC UTILITY OF SKIN BIOPSY IN DYSTROPHINOPATHIES

SHARMA M1, TANVEER N2, SARKAR C3, GULATI G4, KALRA K5, SINGH S6,BHATIA R7

(1) AIIMS, DEPARTMENT OF PATHOLOGY, New DELHI, INDIA. (2) AIIMS,DEPARTMENT OF PATHOLOGY, New DELHI, INDIA. (3) AIIMS, DEPARTMENTOF PATHOLOGY, NEW DELHI, INDIA. (4) AIIMS,DEPARTMENT OF PEDIATRICS, NEW DELHI, INDIA. (5) AIIMS,DEPARTMENT OF PEDIATRICS, NEW DELHI, INDIA. (6) AIIMS, DEPARTMENT OF NEUROLOGY, NEW DELHI, INDIA. (7) AIIMS, DEPARTMENT OF NEUROLOGY, NEW DELHI, INDIA.

Objectives: To elucidate the role of skin biopsy in the diagnosis of dystrophinopathies. Study design: Paired skin and muscle biopsies from 39 cases of Duchenne muscular dystrophy, 4 cases of Becker muscular dystrophy and 37 controls were studied. Immunostaining for dystrophin (Dys1, 2, 3) and utrophin was done on frozen sections of the cases and controls and their staining pattern in skin biopsies was compared with corresponding muscle biopsies. Results: Immubostaining for Dys1, 2 and 3 was negative in the skin biopsies of all patients (39/39, 100%) who were diagnosed as DMD and was weakly expressed in BMD patients (4/4,100%). Dys1, 2, 3 were strongly expressed in the arrector pili muscles of 35 controls patients (94.6%) but was weakly expressed in 2 controls. Utrophin was expressed on the arrector pili muscles of all test patients (39/39,100%) but was also expressed in controls (30/37, 81.1%). Conclusion: Our study suggests that skin biopsy is very useful for the diagnosis of Duchenne/ Becker’s muscular dystrophy. It can be a useful adjunct/replacement for muscle biopsy in the diagnosis of these cases especially in end stage muscle diseases where muscle is replaced by fat infiltration, and as a screening test for the diagnosis before genetic studies. In future it can be used as a routine test in the follow up of these cases after gene therapy, as it is easy to perform and can be repeated frequently. Our findings also suggest that punch biopsy is better than open skin biopsy.

TEST-RETEST RELIABILITY OF DUAL ENERGY XRAY ABSORPTIOMETRY (DEXA) MEASUREMENTS IN PATIENTS WITH DUCHENNE MUSCULARDYSTROPHY (DMD)

PANDYA S1, DILEK N1, MARTENS W1, MOXLEY R1

(1) University of Rochester, Rochester, USA.

Objective: To establish the test- retest reliability of DEXA measurements in patients with DMD. Background: DEXA measurements are frequently used as an outcome measure in therapeutic trials of DMD to document changes in lean body mass. There is no data available in the literature documenting the reliability of these measurements. Methods: We performed repeated DEXA measurements on a single day on two separate visits on 12 boys with DMD participating in clinical trials at our site. The measurements were made with instrumentation and software from the Lunar corporation, Madison WI. Results: Demographic data: Age 6.6 (sd 0.9), Height 114cm (sd 7.4) Weight 20.4 kg (sd 3.0) All patients were ambulatory at the time of testing. The Intra class correlations (ICC) for the measurements of DEXA total, DEXA Bone Mineral Content (BMC), DEXA Fat, and DEXA Lean Body Mass (LBM) were: Visit 1: DEXA Total 0.99, DEXA BMC 0.97, DEXA Fat 0.99 and DEXA LBM 0.97 Visit 2: DEXA Total 0.97, DEXA BMC 0.98, DEXA Fat 0.99 and DEXA LBM 0.91. Conclusions: All the ICC’s were greater than .90 which denotes excellent reliability. This also allows for the calculations of the minimal clinically important difference for natural history studies and therapeutic trials.

EFFECT OF SPINAL SURGERY ON THE LUNG FUNCTION IN DUCHENNE MUSCULAR DYSTROPHY

RAUSCENT H1, BERARD C3, HUMBERTCLAUDE V2, GAUTHERON V4, RICHELMEC1

(1) Maladies Neuromusculaires Pédiatriques, centre Hospitalier Archet 2, 06202 Nice Cedex 3, Nice, FRANCE. (2) Neuropédiatrie, Institut Saint Pierre, 34250 Palavas les Flots, Palavas les Flots, FRANCE. (3) Rééducation Pédiatrique L'ESCALE Centre Hospitalier Lyon-Sud, 69495 PIERRE BENITE Cedex, Lyon, FRANCE. (4) Rééducation Pédiatrique Centre Hospitalier Nord, 42055 Saint-Etienne Cedex 2, Saint Etienne, FRANCE.

Concerning multidisciplinary care of Duchenne Muscular Dystophy (DMD), the prevention of scoliosis remains a priority. Spinal surgery is considered as the treatment of choice and early instrumentation and fusion are widely proposed. The objectives of surgery are to improve the comfort and quality of life of the patient and to prevent the degradation of their respiratory function.

Nevertheless, the effect of spinal surgery on respiratory function is still controversial. We intended then to study, retrospectively, the loss of forced vital capacity in 64 DMD patients born between 1970 and 1990, and we followed up on average 9 years after they lost deambulation. The average time before the onset of arthrodesis was 4 years for the surgery group comprising 52 patients. Our study (which covered 4 french departments) did not show any improvement in the lung function arising from surgery. The annual forced vital capacity decrease was the same in the 52 patients operated before (5,12%/year) and after the spinal fusion (5,89%/year) on as for the 12 patients not operated on. Consequently, our study showed that no improvement in respiratory function could be expected from surgery. There is therefore no argument in favour of advising patients to accept surgery. Our results are consistent with other recent studies, which consider the question of whether systematic and early spinal surgery should still be widely proposed. Furthermore, recent studies report that 25% of patients with DMD would suffer from a moderate and non progressive scoliosis, that is likely to be improved by the early introduction of corticosteroids.

DUCHENNE MUSCLE ACTIVITY EVALUATION AND MUSCLE FUNCTION PRESERVATION: IS A PROPHYLACTIC STRATEGY FEASIBLE?

SBLENDORIO V1, PALMIERI B1, FERRARI A2, PIETROBELLI A3

(1) University of Modena, Modena, ITALY. (2) S. Maria Nuova Hospital, Reggio Emilia, ITALY. (3) Verona University Medical School, Verona, ITALY.

Abstract. Duchenne muscular dystrophy yields pervasive and progressive muscle mass loss. In the current measures relating to the monitoring of disease progression is relevant: 1) the type of scale used; 2) the clinical significance of the attribute being measured and 3) the mathematical properties of the data provided. The high prevalence of obesity at an early stage of this pathology could result not only from reduced physical activity, but may also involve low resting energy expenditure (REE), abnormal nutrient utilization, or overfeeding. This muscle weakness may be attenuated by regular, low-intensity exercise. However, there is a critical lack of data to support appropriate exercise prescription. Because inappropriate activity may exacerbate the dystrophic process, a systematic analysis of muscle function to determine potential exercise load thresholds to avoid injury in dystrophic mice and dogs, and then in human is recommended.

EVALUATION OF PATHOLOGICAL PATTERN IN EARLY STAGE OF DUCHENNE MUSCULAR DYSTROPHY (DMD) BY GAIT ANALYSIS

DOGLIO L1, PERNIGOTTI I1, TACCHINO C1, PEDEMONTE M1, SCAPOLAN S1, BRUNO C1, MINETTI C1

(1) Istituto Giannina Gaslini, Genova, ITALY.

DMD patients walk with a non-physiological pattern showing a initially weakness of antigravitary muscles, followed by loss of walking capability. Initially, DMD patients do not show clinical signs of pathological pattern, that become manifest with the grown and the progression of illness. Aim of our study was to analyze the pathological pattern in early stage of DMD patients by Gait Analysis, a technique which analyses the human walking, allowing the comparison of kinematics and kinetics data in the same and among different patients. We have performed Gait Analysis in 10 DMD patients (aged from 5 to 7 years) and in 10 healthy matched control age group. All patients have been evaluated according to the following items: range of motion analysis, Gowers test, capability of run for 10 meters, walking on tip-toe and heel, and functional HAMA test. All patients were not taking any drug. All patients were able to perform all the items requested, but by Gait Analysis we showed that in the DMD group early pathological signs were present. In all patients, pelvis showed the most important modification in term of improvement of movements on the coronal and transversal plane, probably due to a compensation of gluteus weakness. In 4 patients our data showed an increase of drop-foot. Hip and knee did not show any significant alteration in early stage. Our study indicates that Gait Analysis is a useful tool in detecting early modification of the walking pattern of DMD patients, and may help us to choose further clinical and rehabilitation programs.

COGNITIVE EVOKED POTENTIALS AND NEUROPSICOPHYCOLOGY TEST IN DUCHENNE MUSCULAR DYSTROPHY PATIENTS

ESQUITIN N1, ESCOBAR RE1, MIRANDA A1, ESCOBAR MG1, CORAL R2, RODRIGUEZ M2, VELASQUEZ AC2

(1) Instituto Nacional de Rehabilitacion, México, MEXICO. (2) Centro Medico Nacional Siglo XXI, México, MEXICO.

K

Duchenne muscular dystrophy is a neuromuscular disease genetically inherited X-linked condition with progressive physical disability. The exact functional role of the dystrophin in the brain is unknown, but their absence is associated with a downward shift in intelligence quotients (IQ). P300 makes an objective estimation of the cognition disorders related with central nervous system Objective: To examine the relation between cognitive evoked potentials (P300 potentials) and neuropsychological dysfunction in patients with Duchenne muscular dystrophy (DMD). Method: this was a prospective, comparative and descriptive study. P300 potentials and neuropsychological test (Standford- Binet ) results were obtained from 31 DMD patients and 30 healthy control boys. Mean age was 9.44 (2.25). Full Intelligence Quotients (IQ) were estimated for patients and control group.Statistic Analysis were made with Man Whitney Test and Chi square to compare IQ and P300, significance (p>0.05). Results: The mean IQ values were 93.21 (10.39) DMD patient group and 107.54 (9.24) in the control group (p=0.000) both are in the normal IQ. Mean P300 values in Pz were 314.75 in the DMD group and 311.50 in the control group (p=0. 730). There was no significant correlation between parameters in each group. The most afected area in the was numeric reasoning (p=0.001), conceptual thinking (p=0.002), memory (p=0.003), reasoning (p=0.005), social intelligence (p=0.033) and visuomotor area (p=0.051). Conclusion: there is no correlation statistically significant between the P300 component and the IQ. The values were normal in both groups. The mayority of the patients in the Duchenne group had normal intelligence. Systematic alterations in neuropsychological test results were found to be statistically significant.

ABUNDANCE OF CIRCULATING PROGENITORS WITH MYO-ENDOTHELIALPOTENTIAL CORRELATES WITH A MILD PHENOTYPE IN PATIENTS AFFECTED BY DUCHENNE MUSCULAR DYSTROPHY

FARINI A1, BELICCHI M1, MEREGALLI M1, MARCHESI C1, LOPA R2, PORRETTI L2, PAROLINI D1, D'ANGELO MG3, BRESOLIN N1, COSSU G4, TORRENTE Y1

(1) 1Stem cell Laboratory, Department of Neurological Science, Fondazione IRCCS Ospedale Maggiore Policlinico, Centro Dino Ferrari, University of Milan, Milan, ITALY. (2) 2Centro Trasfusionale e di Immunologia dei Trapianti, Fondazione IRCCS Ospedale Maggiore Policlinico, Milan, ITALY. (3) 3IRCCS E. Medea, La Nostra Famiglia, Bosisio Parini, Bosisio Parini, ITALY. (4) 4Stem Cell Research Institute, San Raffaele Hospital, Milan, ITALY.

Although the natural history of patients with Duchenne Muscular dystrophy (DMD) is characterized by a progressive impairment of muscle function leading to death for cardio-pulmonary failure, there is a clinical variability in these patients regarding age of onset, patterns of skeletal muscle involvement, heart damage, and rate of progression. Most therapeutic strategies for DMD have been palliative rather than curative. Experimental treatments in DMD are difficult due the absence of reliable biomarkers that could be prognostic of the progression of the disease or response to the treatment. Recent works from several laboratories support the idea that increased circulating endothelial progenitors predict cardiovascular and vascular diseases. We hypothesized that the levels of circulating stem cells expressing the CD133 antigen which possess myo/endothelial potential would predict the progression of DMD. The count of circulating CD133+ stem cells was similar in DMD patients and healthy subjects. We found a subpopulation of CD133+ stem cells also expressing the CXCR4 receptor but not CD34 that was significantly higher in DMD patients compared with healthy controls and positively correlated with the clinical score. DMD patients exhibiting mild phenotype have higher levels of this subpopulation of circulatingCD133+ stem cells than patients exhibiting severe phenotype. Linear regression analysis showed a direct correlation between the levels of these cells and the clinical condition of the DMD patients. The circulating AC133+CXCR4+CD34- cells isolated from DMD and healthy subjects express early myogenic and endothelial markers in vitro and differentiate into muscle and endothelial cells in vivo after their transplantation into scid/mdx dystrophic mice. Based on these data we believe that the levels of a subpopulation of circulating CD133+ stem cells in DMD patients may be a promising new prognostic clinical marker of the progression of the disease with practical significance to allow any beneficial effect in future clinical trials

EX VIVO EXPANSION OF HUMAN CIRCULATING CD133+ PROGENITOR CELLS: PROMISING TOOL FOR CELL-BASED THERAPEUTIC APPROACHES IN MUSCULAR DYSTROPHY

BELICCHI M1, MEREGALLI M1, RAZINI P1, CATTANEO A2, FARINI A1, IACCHETTI E3, PORRETTI L2, MILANI P3, BRESOLIN N1, TORRENTE Y1

(1) Stem Cell Laboratory, department of neurological Sciences, Fondazione IRCCS Ospedale Maggiore Policlinico, Centro Dino Ferrari, University of Milan, Milan, ITALY. (2) Centro Trasfusionale e di Immunologia dei Trapianti, Fondazione IRCCS Ospedale Maggiore Policlinico, Milan, ITALY. (3) Centro Interdisciplinare Materiali e Interfacce Nanostrutturati (CIMAINA), Dipartimento di Fisica, Università di Milan, Milan, ITALY.

K

The use of stem cells in regenerative medicine and cell-based therapies offersimmense potential in diseases witch have currently no treatment such as Duchenne muscular dystrophy. A limitation to the use of CD133+ for a therapeutic application is the relatively low number of cells that can be recovered from peripheral blood mononuclear cells. The goal of ex vivo expansion is to induce proliferation of blood derived (circulating) CD133+ cells while maintaining their primary functional characteristic. In this work we explored an alternative methods for the expansion of  blood-derived stem cells we investigated the interactions between CD133+ cells and smooth surfaces of titanium (TiO2). Our results indicate that cluster-assembled nanostructured TiO2 is biocompatible surface for cell culturing directly supporting normal growth and proliferation of hematopoietic stem cells. In these experiments we also identified a cocktail of cytokines SCF, bFGF, EGF, VEGF, LIF, TEPA, IL6 witch supported the growth of blood CD133+ stem cells. In this condition the cells can be expanded for more than 50 and we observed no indication of replicative senescence or significant changes in cellular division time. The proliferating cells still had the capacity to form hematopoietic and endothelial colonies in semisolid media and differentiate into myogenic cells. Human circulating CD133+ cells were also cultured at 5-percent or 20- percent oxygen in liquid culture in presence of the better cocktail of cytokines and we analysed and compared their expansion capacity and their vitality. The total number of cells increased 6-fold at 5-percent oxygen and could result in a better maintenance of the balance between primitive progenitor cell renewal and clonogenic progenitor expansion, thus representing a tool of remarkable therapeutic interest

MUSCLE FUNCTION RECOVERY IN DYSTROPHIC DOG AFTER EXON SKIPPING GENE THERAPY

VULIN A1, BARTHÉLÉMY I2, DREYFUS P1, BLOT S2, GARCIA L1

(1) Association Institut de Myologie - UMR787, Paris, FRANCE. (2) Laboratoire de Neurobiologie ENVA, Maisons-Alfort, FRANCE.

Duchenne Muscular Dystrophy (DMD) is an X-linked recessive disorder due to mutations in the gene that encodes dystrophin. Most of these mutations consist in large genomic deletions, although their extent is not directly correlated with the severity of the phenotype. Out-of-frame deletions lead to abortion of translation, dystrophin deficiency and severe DMD phenotypes, while internal deletions that produce in frame mRNAs leading to shorter proteins are responsible for a milder myopathy known as Becker Muscular Dystrophy (BMD). About 80% of the out-offrame mutations could be theoretically rescued after restoring the translational frame by using exon skipping strategies. Here we used gene transfer in a large animal model of DMD, the Golden Retriever Muscular Dystrophy (GRMD) dog, to achieve the precise skipping of multiple exons spaced over 125,000 bp of the dystrophin premRNA and the re-expression of a functional protein. This led to sustained correction of the dystrophic phenotype in extended muscle areas and muscle strength recovery. Exon skipping was obtained with U7snRNAs (U7smOPT) carrying antisense sequences designed to mask determinants of exon 6 and 8 definition. These U7smOPT were introduced into skeletal muscle fibres by using Adeno Associated Viral (AAV2/1) vectors. After two months, levels of dystrophin were almost normal in transduced fibres. Histological examination revealed that the dystrophin glycoprotein complex was restored and that spontaneous muscle damages were stopped. Muscle architecture was fully corrected and fibres displayed the hallmarks of mature and functional units. Muscle force reflecting fibers functional integrity were improved. Our study documents for the first time the recovery of dystrophin at the scale of entire limbs in a large animal and thus represents a critical milestone for the development of clinical trials in human patients.

SYSTEMIC RESTORATION OF DYSTROPHIN EXPRESSION IN DMD BY PEPTIDE-CONJUGATED ANTISENSE OLIGONUCLEOTIDES

YIN H1, SEOW Y1, MOULTON HM2, IVERSEN PL2, BOUTILIER JK2, WOOD MJA1

(1) Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UNITED-KINGDOM. (2) AVI BioPharma Inc, Corvallis, USA.

Duchene Muscular Dystrophy (DMD) is a severe muscle disorder caused by mutations in the dystrophin gene. The efficacy of antisense oligonucleotide (AO)- mediated exon skipping for the restoration of dystrophin has been established in animal models and in DMD patients. However there remain significant limitations to this therapeutic approach due to the lack of effective systemic AO delivery to target tissues of muscle and heart. Here we investigate systemic tissue-specific AO delivery by testing AOs directly conjugated to cell penetrating peptides (CPPs) alone or in combination with tissue-specific homing peptides (e.g. muscle-specific peptide, MSP). Morpholino (PMO) chemistry AOs were directly conjugated to CPPs alone or in combination with homing peptides and evaluated in mdx mice following systemic delivery. Effective exon skipping and dystrophin expression were induced in bodywide skeletal muscles at extremely low AO doses of 3mg/kg and also in heart. This is the first time that targeted AO delivery to muscle and successful body-wide restoration of dystrophin expression have been achieved at such low AO doses. In parallel we also report the discovery and characterization of a novel delivery formulation which facilitates AO uptake in muscle. A series of studies have shown that this delivery formulation enhances the delivery of AOs of different chemistries (e.g. 2-OMeRNA, PNA and PMO), depends on the activity of specific muscle membrane transporters, and that it induces significant restoration of dystrophin expression in muscle compared with commonly used delivery formulations. In summary, we report data demonstrating the potential of tissue-specific homing peptides, CPPs and novel delivery formulations for the targeted restoration of dystrophin in DMD.

DIRECT COMPARISON OF 2’O-METHYL AND PMO AONS FOR EXON SKIPPING IN DMD

HEEMSKERK H1, DE WINTER C1, DE KIMPE S2, VAN KUIK-ROMEIJN P2, HEUVELMANS N2, PLATENBURG G2, VAN OMMEN G-J1, VAN DEUTEKOM J2,AARTSMA-RUS A1

(1) DMD Genetic Therapy Group, Department of Human Genetics, Leiden University Medical Center, Leiden, THE NETHERLANDS. (2) Prosensa B.V., Leiden, THE NETHERLANDS.

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Antisense-mediated exon skipping is a potential treatment for Duchenne muscular dystrophy (DMD). Using antisense oligonucleotides (AONs) the disrupted DMD reading frame is restored, allowing the generation of partially functional dystrophin and conversion of a severe Duchenne into a milder Becker muscular dystrophy phenotype. This strategy has been studied in cultured patient cells and in mouse and dog models. Further, clinical proof of concept was obtained through an exploratory study in which four DMD patients received a local dose of a 2’O-methyl phosphorothioate (2OMePS) AON targeting exon 51. Recent studies in mdx mice show relatively high exon 23 skipping efficiencies with another type of AON chemistry, morpholino AONs (PMOs). We here directly compared both chemistries and assessed the effects of length (PMOs are typically 25 nucleotides and 2OMePS 20 nucleotides), cellular uptake and sequence. The study included short 2OMePS, long 2OMePS and PMO AONs targeting mouse exon 23 and human exons 44, 45, 46 and 51. Exon 23 AONs were tested intramuscularly and intravenously in mdx mice, human AONs only intramuscularly in the hDMD mouse, which has an integrated full-length copy of the human DMD gene. For mouse exon 23, PMO was confirmed to be more efficient than 2OMePS, however for the human exons PMO and 2OMePS skipping efficiencies were more comparable, which suggests sequence-dependence. Notably two mismatches rendered 2OMePS but not PMO AONs ineffective, implying PMOs may be less sequence-specific. Increasing the length of 2OMePS AONs enhanced skipping efficiencies of human exon 45, but decreased efficiency for mouse exon 23. After intravenous administration, exon skipping and novel protein was shown in the heart for the first time with both AON chemistries. Further, PMO showed lower intramuscular concentrations with higher exon 23 skipping levels compared to 2OMePS AONs. Possibly, the charged 2OMePS is sequestered in the interstitial space.

ANTISENSE OLIGOMER (AO) INDUCED EXON SKIPPING IN THE MDX4CV MOUSE MODEL

MITRPANT C1, FLETCHER S1, WILTON S1

(1) Australian Neuromuscular Research Institute (ANRI), Perth, AUSTRALIA.

Duchenne muscular dystrophy (DMD), a relentless progressive muscular dystrophy is caused by protein truncating mutations in the dystrophin gene that result in the absence of functional dystrophin. Loss of dystrophin leads to irreparable membrane damage, thus promoting calcium ion influx and cell death. Antisense Oligomer (AO) induced exon skipping is a molecular intervention

whereby AOs are targeted to motifs involving in pre-mRNA splicing. This has been used to induce specific exon removal and by-pass the disease-causing gene lesion in the mdx mouse model of muscular dystrophy. We are investigating exon skipping in the B6Ros.Cg-Dmdmdx-4Cv/J (mdx 4CV) muscular dystrophy mouse, which carries a nonsense mutation in exon 53 of the dystrophin gene. To restore the reading frame, both exons 52 and 53 must be excised from the mature dystrophin gene transcript to by-pass the primary gene lesion and maintain the reading frame. 2'-O-Methyl AOs, on a phosphorothioate backbone have been designed to mask these exons from the splicing machinery and lead to their excision. Initial AO combinations induced skipping of exons 52/53 and restored some protein expression. However the predominant transcript was missing only exon 53 as determined by RNA studies. We designed additional AOs to enhance exon skipping of both exons 52 and 53. We highlight the importance of AO design to enhance efficiency of single and multi-exon removal.

IDENTIFICATION AND CHARACTERIZATION OF SMALL MOLECULES FOR THE TREATMENT OF DUCHENNE MUSCULAR DYSTROPHY

ACHARJEE S1, FRIESEN W1, TOMIZAWA Y1, BAIAZITOV R1, LEE S1, NADARAJAN T1, MOON YC1, SWEENEY L2, WELCH EM1

(1) PTC Therapeutics, South Plainsfield, USA. (2) University of Pennsylvania,

Philadelphia, USA.

PTC Therapeutics, Inc. (PTC) and Parent Project Muscular Dystrophy (PPMD) are collaborating to discover new drugs to treat Duchenne muscular dystrophy (DMD). Several targets were selected to enter the drug discovery program based on functional validation from animal studies. The targets selected for high throughput screening (HTS) included: utrophin (UTRN), muscle-specific insulin-like growth factor (mIGF1), and 7 integrin (ITGA7). Using a proprietary drug discovery platform technology, referred to as GEMS (Gene Expression Modulation by Small-molecules), we sought to identify small molecules that upregulate the production of these protein targets to identify potential treatments for DMD. Constructs containing the firefly luciferase (fLuc) reporter gene flanked by the 5’ and 3’ untranslated regions (UTR) specific for each of the targets were stably transfected in human muscle (RD) or kidney (293H) cells and used in HTS. We identified hits that demonstrate concentration dependent activities in cell-based reporter assays and in assays that measure protein levels. Further, a number of molecules exhibit good pharmacological properties (e. g., low cytotoxicity and microsome metabolic stability). Presently, we are focused on optimizing the activity, potency and pharmacological properties of 2 chemical scaffolds for the mIGF1 program which exhibit up to 5 fold upregulation of mIGF1 and demonstrate structure-activity relationships. For UTRN and ITGA7, we have identified 2 chemical scaffolds for each target and are in the process of establishing structure-activity relationships for these chemical classes. The ultimate goal of this drug discovery and development effort is to identify small molecules that can specifically modulate the production of a number of proteins that can be used as monotherapy or as part of a combination therapy to treat Duchenne muscular dystrophy.

RHOA INDUCES EXON SKIPPING AND DYSTROPHIN RE-EXPRESSION IN MDX MICE.

DURAND S1, POMIÈS P2, FABBRIZIO E1, LEJEUNE F1, BONET-KERRACHE A2

(1) Institut de génétique Moléculaire, Montpellier, FRANCE. (2) Centre de Recherche en Biochimie Macromoléculaire, Montpellier, FRANCE.

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Duchenne muscular dystrophy (DMD) is a severe X-linked muscle degenerative disease caused by mutations or deletions in the dystrophin gene leading to absence of the protein. The members of the Rho family of small GTPases are molecular switches that control a wide variety of signal transduction pathways linked to the regulation of the actin cytoskeleton, cell polarity, microtubule dynamics, membrane transport pathways and transcription factor activity. Here, we show that in mdx mice (which possess a stop codon within exon 23 of dystrophin and, thus, do not produce the protein), exogenous expression of activated RhoA in muscle fibers induces dystrophin reexpression. Full length dystrophin could be detected with different antibodies at the sarcolemma of fibers in which RhoA was electro-transferred and this re-expression led to re-appearance of proteins of the DGC complex. Moreover electroporated fibers presented 2 times more peripherical nucleated fibers than controls and consequently, a reduction in the severity of the dystrophic phenotype. We then demonstrate that RhoA-induced re-expression of dystrophin is not due to an activation of ribosomal read-through by RhoA. Moreover, we show than RhoA can activate the dystrophin promoter and inhibit NMD (Nonsense Mediated mRNA Decay) thus helping to stabilize dystrophin mRNA. Finally, we reveal by PCR amplification that RhoA induces the exon skipping of exon 23 in the dystrophin gene of mdx mice, allowing the translation of dystrophin mRNA and re-expression of the protein. In this study we demonstrate that in mdx mice dystrophin re-expression following exogenous expression of active form of RhoA is due to skipping of dystrophin exon 23. This finding suggests that RhoA can regulate exon skipping in the dystrophin gene. Understanding the underlying mechanism will be important for the discovery of new therapeutic targets able to lead to or to amplify exon skipping in DMD patients.

THE SEVERELY DYSTROPHIC MDX TRIANGULARIS STERNI (TS) MUSCLE IS A USEFUL PREPARATION FOR EXAMINING DRUG EFFICACY IN THE MDX MOUSE MODEL.

SIEGEL A1, ZIMMERMAN A1, HENLEY S1, RHODES J1, MILES M1, SHIN G2, BECK B1, HOFF H1, KURZ J1, BALCH F1, CARLSON CG1

(1) AT Still University, Kirksville, USA. (2) Truman State University, Kirksville, USA.

The TS is an expiratory muscle that is passively stretched with each inspiration and concentrically activated with each expiration. The degree of passive stretch of TS muscle fibers depends upon the location of the fiber within the muscle, with caudal fibers stretched up to 10% more than cephalad fibers (DeTroyer et al., J. Physiol. (Lond.) 513.3, 915-925, 1998). Cross sections of adult nondystrophic TS indicate roughly uniform fiber density in the caudal, middle, and cephalad thirds of the muscle and fiber diameters that increase significantly in the cephalad to caudal direction. In comparison to age-matched nondystrophic mice, the density of muscle fibers in mdx TS muscles at 1 to 2 years of age was significantly reduced in the middle and cephalad regions of the TS muscle, and the cross-sectional diameter significantly reduced in all 3 regions. The total working diameter and working cross sectional area of mdx TS muscle was also significantly reduced relative to nondystrophic values. Increased centronucleation was observed in all 3 regions of the mdx TS muscle. Transmission electron micrographs from mdx TS indicate substantial Z line streaming, sarcomere disorganization, and numerous hypercontracted areas where dense myofilamentous material was observed adjacent to empty sarcoplasm containing numerous mitochondria. Long term treatment of mature adult mdx mice with pyrrolidine dithiocarbamate (PDTC; 50 mg/kg, intraperitoneal) produced a significant increase in fiber diameter in the middle TS and reduced centronucleation throughout the TS. Daily treatment with N-acetylcysteine (NAC; 100 mg/kg, ip) for a period of 3.5 months also significantly increased the fiber diameter and fiber cross-sectional area in the cephalad region of the mdx TS. These results provide evidence supporting the utility of the mdx TS preparation in assessing the morphological benefits of a variety of potential treatments for Duchenne and related muscular dystrophies. (Supported by AFM, Charley’s Fund, Strategic Research Grant from ATSU

DIRECT EFFECT OF PYRROLIDINE DITHIOCARBAMATE ON THE RESTING MEMBRANE POTENTIAL OF FRESHLY ISOLATED MDX MUSCLE FIBERS.

MILES M1, CARLSON CG1

(1) AT Still University, Kirksville, USA.

Daily treatment of adult mdx mice with intraperitoneal injections of pyrrolidine dithiocarbamate (PDTC) substantially improved the resting membrane potential in severely dystrophic (mdx) triangularis sterni (TS) muscle fibers (Carlson et al., Neurobiology of Disease , 20 (3), 719-730, 2005). To determine whether this effect was mediated by circulating factors exogenous to skeletal muscle, the effect of PDTC on the resting potential of freshly excised triangularis sterni (TS) muscle fibers was examined in vitro. Although PDTC had no effect on the resting potential of nondystrophic fibers, 100 ıM PDTC produced a significant -6.9 mV increase in the average resting potential of mdx TS fibers (7.5 month, caudal region) over a 90 minute interval. Pre-treatment with ouabain abolished this restorative effect of PDTC on the resting potential of mdx fibers. To determine the contribution of the Na+ - K+ ATPase on the resting membrane potential, nondystrophic and mdx fibers were exposed to 10- 3 M ouabain for 30 minutes. The reduction in resting potential produced by ouabain in mdx TS fibers (+4.3 mV) was significantly and substantially less than that observed in nondystrophic TS fibers (+14.7 mV). Fluorimetric determinations (F340/F380) in SBFI loaded muscle fibers also suggested that intracellular Na+ is elevated in mdx TS fibers. Consistent with the resting potential measurements, the increase in F340/F380 induced by ouabain in nondystrophic fibers was substantially and significantly greater than that observed in mdx fibers. These results suggest that: (1) the reduced resting potentials in dystrophic muscle are associated with altered Na+-K+ pump activity, increases in intracellular Na+, and a reduction in the outward Na+ pump current; and (2) PDTC improves the resting potential by a direct or indirect effect on Na+-K+ pump activity. (Supported by AFM, Charley’s Fund, Strategic Research Grant from ATSU

EFFECTS OF TREATMENT WITH URSODEOXYCHOLIC ACID (UDCA) ON P65 NUCLEAR ACTIVATION AND WHOLE BODY TENSION (WBT) DEVELOPMENT IN THE MDX MOUSE

TURIN E1, HOFF H1, GATTI F1, WINDERS T1, SINGH R1, STARKE J1, RUTTER J1, BLEDSOE C1, LAVIN J1, PALMIERI B2, CARLSON CG1

(1) AT Still University, Kirksville, USA. (2) University of Modena, Modena, ITALY.

Ursodeoxycholic acid (UDCA) is in current clinical use for the treatment of biliary cirrhosis and has been shown to reduce nuclear p65 activation in HeLa cells expressing elevated glucocorticoid receptor (Miura et al., J. Biol. Chem., 276(50), 47371-47378, 2001). Nuclear extracts obtained from mdx diaphragms exposed to 100 or 200 ıM UDCA for 2 hours exhibited a significant 65% reduction in p65 activation in comparison to mdx preparations bathed in HEPES Ringer for the same period. Preparations exposed to 200 ıM UDCA for 4 hours also exhibited significant reductions in nuclear p65 activation to approximately 50% of the levels seen in preparations exposed to HEPES Ringer. To examine the efficacy of UDCA administration, several adult mdx mice were administered a single dose of 5, 10, or 20 mg/kg intraperitoneally and euthanized at 3 and 5 hours after injection (vehicle treated mdx served as controls). UDCA exposure did not reduce nuclear p65 activation at 3 hours after the injection but significantly reduced activation at all 3 doses to approximately 33% of vehicle-treated levels at 5 hours after the injection. Daily treatment of 1 month old mdx mice at 40 mg/kg for a period of 30 days significantly reduced nuclear p65 activation to approximately 60% of vehicle–treated levels. Whole body tension determinations obtained after 30 days of UDCA treatment indicated significant, 24 to 27%, increases in the WBT5 and WBT10 measures (Carlson and Makiejus, Muscle and Nerve, 13:480-484, 1990) corresponding to 31 and 26% recovery in WBT5 and WBT10, respectively. UDCA treatment had no effect on the WBT10/WBT5 ratio or on 4 limb hang time using a wire grid. These experiments suggest that UDCA may be effective in the treatment of Duchenne and related muscular dystrophies. Experiments examining the efficacy of oral administration of UDCA in the mdx mouse are underway. (Supported by AFM, Charley’s Fund, Strategic Research Grant from ATSU)

ROLE OF TRPC1 AN TRPC3 IN THE EXCESS OF CA2+ CONCENTRATION OBSERVED IN DUCHENNE MUSCULAR DYSTROPHY

DHAMANE E1, GALLO C1, PETERMANN O1, RUEGG U1, ROULET E1

(1) Laboratory of Pharmacology, section of Pharmaceutical Sciences, University of Geneva, Geneva, SWITZERLAND.

Calcium dysbalance is expected to be one of the triggering events causing muscular degeneration in Duchenne muscular dystrophy (DMD). It has been proposed that the increased Ca2+ influx could result from transient membrane lesions (Menke et al., 1995; Petrof et al., 1993) or from influx through plasma membrane Ca2+ channels. Recent results indicate that the increased permeability of the sarcolemma of dystrophic fibres may be due to increased activity of cationic channels belonging to the TRP family which could result in an increase of Ca2+ concentration. Both, stretchactivated channels (SAC) and store-operated channels (SOC) have been proposed to be involved in the enhanced Ca2+ entry occurring in dystrophic skeletal muscle cell. In our study we focused mainly on TRPC family. We and others have shown that TRPC1 as well as TRPC 2,3,4 and 6 are expressed in skeletal muscle and were proposed to be candidates for these Ca2+ channels (Vandebrouck et al., 2002). In a series of experiments we have shown by quantitative RT-PCR that TRPC3 mRNA is overexpressed in mdx muscles compared to wt. This prompted us to consider TRPC3 as a potential candidate for the increase of Ca2+ influx described in mdx muscle. TRPC1 has been described as important and necessary for (SOC) and (SAC). Therefore we have cloned a series of shRNAs directed against TRPC1 an TRPC3 into the adeno-associated viral vector type 2 and 6 (pAAV) together with the Pol III promoter H1. We have validated the efficiency of our constructions in heterologous systems and we have obtained an inhibition of TRPC3 and TRPC1 mRNAs from 50% up to 90%. We are currently producing high titer viruses in order to transduce our myotubes from EDL-mdx cell line as well as mdx FDB fibres and we will analyse the effect of their inhibition on calcium

BRAIN-DERIVED NEUROTROPHIC FACTOR (BDNF) REGULATES SKELETAL MUSCLE REGENERATION AND IS MIS-REGULATED IN DYSTROPHIC MUSCLE

CLOW C1, MOUSAVI K1, RAVEL-CHAPUIS A1, JASMIN B1

(1) University of Ottawa, Center for Neuromuscular Disease, Ottawa, CANADA.

Muscle-derived BDNF has long been thought to serve as a retrograde trophic factor for innervating motor neurons throughout their lifespan. However, our recent studies have shown that BDNF is not enriched at the neuromuscular junction in adult skeletal muscle, and instead, is highly expressed in satellite cells (J. Neurosci., 26: 5739, 2006). Furthermore, we have shown that BDNF depletion results in precocious differentiation of myoblasts in culture. These findings suggest a role for BDNF in myogenic differentiation. In order to expand on these findings, and determine whether BDNF plays a similar role in vivo, we designed a series of complementary experiments to elucidate the role of BDNF in muscle development, regeneration and disease. First, we generated a mouse in which BDNF is specifically depleted in skeletal muscle. Characterization of the muscle-specific BDNF knockout mouse showed abnormal neuromuscular junction formation, and fiber-type switching. Next, we compared regeneration of wild-type and BDNF-depleted muscle following cardiotoxin injury. BDNF-depleted muscle showed delayed regeneration, as indicated by delayed appearance of centrally nucleated fibers and developmental myosin heavy chains (MyHC). Finally, given the therapeutic importance of regeneration for treating various muscle diseases, we examined the expression profile of BDNF in dystrophic muscles from mdx mice. Compared to wild-type, aged mdx muscles consistently displayed greater levels of BDNF. Interestingly, high levels of BDNF correlate with the age at which regeneration becomes defective in mdx muscle, resulting in rapid deterioration and early death compared to wild-type. These findings demonstrate that muscle-derived BDNF is important for regulating: i) the organization of the postsynaptic compartment of muscle fibers; ii) the pattern of MyHC isoform expressed; and iii) the regenerative potential of muscle. Based on these findings, BDNF manipulation may represent an important theraputic tool to alleviate the dystrophic muscle pathology. This work is supported by CIHR, AFM and MDA (USA).

SUBCUTANEOUS INJECTION FROM BIRTH OF EPIGALLOCATECHIN-3- GALLATE, A COMPONENT OF GREEN TEA, LIMITS THE ONSET OF MUSCULAR DYSTROPHY IN MDX MICE: QUANTITATIVE HISTOLOGICAL AND ELECTROPHYSIOLOGICAL STUDY

NAKAE Y1, HIRASAKA K2, GOTO J2, NIKAWA T2, SHONO M3, YOSHIDA M4,STOWARD PJ5

(1) Department of Oral and Maxillofacial Anatomy, The University of Tokushima Graduate School, Tokushima, JAPAN. (2) Department of Nutritional Physiology, The University of Tokushima Graduate School, Tokushima, JAPAN. (3) Support Centre for Advanced Medical Sciences, The University of Tokushima Graduate School, Tokushima, JAPAN. (4) Department of Degenerative Neurological Diseases, National Institute of Neuroscience, National Centre of Neurology and Psychiatry, Kodaira, JAPAN. (5) College of Life Sciences, University of Dundee, Dundee, UNITEDKINGDOM.

Dystrophic muscles suffer from enhanced oxidative stress. We have investigated whether administration of an antioxidant, epigallocatechin-3-gallate (EGCG), a major polyphenol of green tea, reduces their oxidative stress and pathophysiology in mdx mice, a mild phenotype model of human Duchenne-type muscular dystrophy. EGCG (5mg/kg body weight in saline) was injected subcutaneously four times a week into the backs of C57BL/10 normal mice and dystrophin-deficient mdx mice for 8 weeks from either the day of birth or a day after birth. Saline was injected into normal and mdx controls. At the end of the treatment EGCG had almost no observable effects on normal mice or on the body weights of mdx mice. In contrast, it produced the following improvements in the blood chemistry, muscle histology and electrophysiology of the treated mdx mice. First, the activities of serum creatine kinase, an index of muscle damage, were reduced to near normal levels. Second, the numbers per unit volume of an oxidative stress marker, autofluorescent lipofuscin granules, in soleus and diaphragm muscles were significantly decreased by about 50 % compared to the numbers in the corresponding saline-treated controls. Third, in sections of diaphragm muscles, the relative area of histologically normal muscle fibres increased significantly about 2-fold whereas the relative areas of connective tissue and necrotic muscle fibres were significantly reduced by about 40 and 90% respectively. In sections of soleus muscles the relative area of normal muscle fibres significantly increased about 1.5-fold but that of necrotic muscle fibres decreased by 95 %. Fourth, the times for the maximum tetanic force of soleus muscles to fall by a half increased to almost normal values. Our study corroborates other recent studies that EGCG is effective for limiting the onset of muscular dystrophy in mdx mice without causing side effects.

THE DECREASE OF EXPRESSION OF RYANODINE RECEPTOR SUBTYPE 2 IS REVERSED BY GENTAMYCIN SULFATE IN VASCULAR MYOCYTES FROM MDX MOUSE.

MOREL JL1, FRITZ N2, DABERTRAND F1, MACREZ N1, HENAFF M1, MIRONNEAU J3, MIRONNEAU C3

(1) umr5228 CNRS université Bordeaux, talence, FRANCE. (2) karolinska institut, molecular neurobiology, stockolm, SWEDEN. (3) retired umr5017 CNRS université de bordeaux, Bordeaux, FRANCE.

The mdx mouse, a model of the human Duchenne muscular dystrophy displays incompletely understood impaired contractile function of skeletal, cardiac and smooth muscles. We explored the possibility that ryanodine receptor (RYR) expression could be altered in vascular muscle. The three RYR subtypes are expressed in portal vein myocytes. As observed on the mRNA and protein levels, RYR2 expression was strongly decreased in mdx myocytes whereas RYR3 and RYR1 expression were unaltered. The use of antisense oligonucleotide directed against RYR subtypes indicated that caffeine-induced Ca2+ response depended on RYR1 and RYR2. In mdx mouse, caffeine-induced Ca2+ responses was decreased in both amplitude and maximal rate of rise and the frequency of Ca2+ sparks was also strongly decreased. The gentamycin treatment of mdx mice was able to restore both the expression of RYR2 and the caffeine-induced Ca2+ response at the same level that observed in wildtype mice. Taken together, these results confirm that both RYR1 and RYR2 are required for vascular Ca2+ signalling and indicate that inhibition of RYR2 expression may account for the decreased Ca2+ release from the SR in mdx vascular myocytes. These results may help to explain the reduced efficacy of contraction in vascular myocytes of mdx mice and possibly Duchenne muscular dystrophy-afflicted patients. Finally, we suggest that gentamycin treatment of mdx mice could restore the Ca2+ signalling in smooth muscle and possibly the vascular function

NEW INSIGTH ON THE BENIFICAL EFFECT OF L-ARGININE ON MDX MUSCLE: IMPACT ON INFLAMMATION, CALCIUM HOMEOSTASIS AND SARCOLEMMA INTEGRITY.

HNIA K1, GAYRAUD G1, LACAMPAGNE A2, KOECHLIN C3, HUGON G1, RIVIER F1, DE LA PORTE S4, MORNET D1, MATECKI S1

(1) INSERM ERI 25, Montpellier, FRANCE. (2) INSERM U 637, Montpellier, FRANCE. (3) INRA remodelage musculaire, Montpellier, FRANCE. (4) CNRS UPR 9040, Gif sur Yvette, FRANCE.

L-Arginine was proposed as a potential pharmacological tool in Duchenne muscular dystrophy (DMD), a progressive-muscle wasting disease due to mutations in the dystrophin gene. Despite the beneficial effect on L-arginine on muscle weakness and force, mechanisms by each this molecule acts on muscle remain poorly investigated. Here, we showed that L-Arginine administration to 5 week-old mdx mice improves Ca2+-sparks’s properties with a significant increase in spark amplitude, a shorter risetime and increase spatial spread. In the other hand we demonstrate that L-arginine treatment decreases inflammatory secreted cytokine IL-1, IL-6 and TNF-targeted to dystrophic muscle fibres. This leads to decrease level and activity of NF-kB and its targeted proteins such as the muscle specific metalloproteinases MMP-2 and MMP-9. The -dystroglycan (a key transmembrane glycoprotein of the dystrophin glycoprotein complex) which anchors utrophin to sarcolemma is a target protein of the MMP-2 and MMP-9 in mdx muscle. Increase activiy of MMPs promotes an abnormal cleavage of the ı-dystroglycan into a ~30 kDa form. Acting on NF-kB/MMPs cascade L-arginine promotes a better membrane stability of -dystroglycan/utrophin couple and relocalizes nNOS in subsarcolemmal compartment of the dystrophic fibres which could explain the beneficial effect on resistance to contraction-induced mechanical stress observed in treated muscle. In conclusion our study points on the involved signaling cascades targeted by Larginine in dystrophin-deficient muscle and strengthens the use of L-arginine as a potential pharmacological tool in Duchenne muscular dystrophies and also enlarges its use in other muscle-inflammation mediated myopathies.

EFFECT OF CALPAIN/PROTEASOME INHIBITION ON CALCIUM DEPENDENT PROTEOLYSIS AND MUSCLE HISTOPATHOLOGY IN THE MDX MOUSE

BRIGUET A1, ERB M1, COURDIER-FRUH I1, BARZAGHI P1, SANTOS G1,HERZNER H1, LESCOP C1, SIENDT H1, HENNEBOEHLE M1, WEYERMANN P1, MAGYAR J1, DUBACH-POWELL J1, METZ G1, MEIER T1

(1) Santhera Pharmaceuticals, Liestal, SWITZERLAND.

Dystrophin deficiency is the underlying molecular cause of progressive muscle weakness observed in Duchenne muscular dystrophy (DMD). Loss of functional dystrophin leads to elevated levels of intracellular Ca2+, a key step in the cellular

pathology of DMD. Calpains are activated in dystrophin-deficient muscle, and their inhibition is regarded as a potential therapeutic approach. The contribution of the ubiquitin-proteasome system in muscle wasting conditions and the finding that its

pharmacological inhibition mitigates the dystrophic phenotype in mdx mice qualify the proteasome as a potential target for therapeutic intervention in DMD. Here we present evidence that uncontrolled Ca2+ influx into muscle cells similar to that observed in dystrophin-deficient muscle not only initiates calpain- but also proteasome-mediated proteolysis. Based on this finding we set out to optimize novel dual small-molecule inhibitors that inhibit both calpain as well as the proteasome in a cellular system with impaired Ca2+ homeostasis. Such dual calpain/proteasome inhibitors, when administered to mdx mice, significantly improve quantitative histological parameters. For further assessment of the role of calpain inhibition, we crossed mdx mice with transgenic mice overexpressing the endogenous calpain inhibitor calpastatin. While our data show that proteolysis by calpain is strongly inhibited in the trangenic mdx mouse, this calpain inhibition alone does not ameliorate muscle histology. In conclusion, these results indicate that inhibition of the proteasome rather than calpain inhibition alone is required to show histological improvement in dystrophin deficient muscles.

EARLY ENDOMYSIAL FIBROSIS IS UNIQUELY ASSOCIATED WITH SEVERE FUTURE MOTOR OUTCOME IN DUCHENNE MUSCULAR DYSTROPHY (DMD)

DESGUERRE I1, PORON F1, BARBET P2, GHERARDI R1, CHRISTOV C1 (1) INSERM U841-E10 (Institut Mondor de Recherche Biomédicale); Paris 12University, Créteil, FRANCE. (2) Service d'Histologie, Hôpital Saint-Vincent de Paul, Paris, FRANCE.

In DMD, repeated cycles of acute myofiber necrosis and regeneration progressively lead to severe terminal myofiber degeneration and extensive fibrosis. A preliminary analysis of 39 muscle biopsies from DMD patients at different ages allowed rough time-course characterization of both interstitial and cellular alterations in DMD. It includes initial prominence of edema and fiber hypercontraction (2-4 years), followed by prominence of myonecrosis and regeneration (5-7 years), and then by fibrosis and fatty degeneration (8-10 years). However, there were marked interindividual variations at both the myopathological and clinical level in patients of similar age. As DMD is more clinically heterogeneous than classically described and may involve fibrosis as a central pathogenic player (see the other abstracts by the same group), we carried out a mulpiparametric analysis of myopathological alterations and all available clinical variables in a series of 35 DMD steroid-free patients with both quadriceps muscle biopsy performed from 3 to 6 years and a long term follow-up (>10 years). Seven myopathological alterations (endomysial and perimysial fibrosis, myofiber size, myonecrosis, hypercontraction, endomysial oedema, and fatty degeneration) were assessed using Gomori trichrome and Collagen-1 immunostaining, stereological methods and color image segmentation applied to digitized images. Analysis was done with the Categories module of SPSS 11.0 software using non linear Categorical Principal Component Analysis, an approach allowing one to handle together nominal, ordinal, and interval variables, and suitable for data recorded with uncertain units (e.g. MMT scores). Plotting of myopathologic variables detected in the quadriceps muscle with motor scores obtained in the same muscle group (mean score, minimal score, score at 10 years, deterioration score), allowed us to point out endomysial fibrosis as the sole myopathologic alteration correlated with motor scores. We conclude that early muscle fibrosis represents a sign of poor future motor outcome.

ENDOMYSIAL FIBROSIS IN DUCHENNE MUSCULAR DYSTROPHY (DMD) IS ASSOCIATED WITH CAPILLARY ARTERIOLIZATION,TORTUOSITY, AND INCREASED CAPILLARY-TO-FIBER DISTANCE

DESGUERRE I1, BARBET P2, GHERARDI R1, CHRISTOV C1

(1) INSERM U841-E10 (Institut Mondor de Recherche Biomédicale); Paris 12University, Créteil, FRANCE. (2) Service d'Histologie, Hôpital Saint-Vincent-de-Paul, Paris, FRANCE.

The primary muscle fiber injury in DMD is due to total absence of dystrophin. With evolution of the disease, an increasing extent of myofibrosis, the precise mechanisms of which remain to be fully determined, likely accelerates myofiber death. Ischaemia has long been considered a possible factor of myoinjury. It is currently attributed to functional vascular alterations resulting from smooth muscle cell dystrophin deficiency and from ectopic sarcoplasmic nNOS location impeding paracrine NO release that normally regulates vasoconstrictive responses to exercise. We examined here the possible relationships between fibrosis and myofiber ischaemia on an anatomical basis. Frozen transversal sections from 15 DMD patients (6–10 years) and normal age-matched controls, immunostained for endothelial (CD31) and smooth muscle (alpha-SMA) cells, were used to set-up large scale reconstructions containing hundreds of microvessels. Vessels, myofiber sections and endomysium were segmented out of the images, and the following parameters measured: 1) arteriolarization index (percentage of arteriolar to total

vascular area), 2) number of capillaries/mm2, 3) distance separating capillaries from their nearest neighbors, 4) size/size variation of capillary domains defined as part of the muscle section closer to one capillary than to any other, 5) capillary tortuosity (length of capillaries) and individual myofiber vascularization indices: (i) the individual capillary-to-fiber ratio which takes into account the sharing factor, (ii) capillary-to-fiber perimeter exchange index, (iii) capillary-to-fiber distance. On the one hand DMD muscle showed conspicuous arteriolization and marked increase of the capillary-tofiber distance with intersposed collagen deposits, forming. two major obstacles to gas exchanges. On the other hand, there was marked increase of both capillary tortuosity and capillary-to-fiber perimeter exchange index, suggesting an adaptative neoangiogenic phenomenon associated with injury and/or hypoxia-induced myofiber regeneration (see Christov et al, Mol Biol Cell 2007). Apparently, microvascular hyperplasia is likely to precede capillary depletion seen in the terminal stages of the disease.

AN EFFICIENT WAY TO DIFFERENTIATE HUMAN EMBRYONIC STEM CELLS INTO SKELETAL MUSCLE CELLS

B HUOT N1, LAPOINTE E1, FUJII I2, MAKOTO M2, P. TREMBLAY J1

(1) Centre de Recherche du CHUL, Québec, CANADA. (2) Laboratory of Clinical Pharmacology and Therapeutics, Kumamoto, JAPAN.

Human embryonic stem cells (hESC) have a self-renewal capacity and can differentiate into all cells find in the body. For this reason, they represent an unlimited source of cells for the treatment of degenerative disease, such as Duchenne Muscular Dystrophy (DMD). Previous studies have reported the derivation of skeletal muscle cells from hESC but the techniques used in these studies were long and had a low efficiency. Here we report a new method to differentiate hESC into skeletal muscle cells using an adenovirus expressing the master gene MyoD under the CAG promoter (Ad.CAGMyoD). This adenovirus is very efficient and five days after the infection nearly 50% of the cells stained positive for desmin, a well know myogenic marker. Immunochemistry also confirmed that these cells expressed MyoD. Thereafter, we tested the in vitro functionality of the resulting myogenic precursors by measuring their fusion potential. When these cells were cultured in a low serum medium, they fused and formed myotubes. This differentiation was confirmed by immunostaining for the myosin heavy chain, a myotube marker. These preliminary results indicate that the Ad.CAGMyoD is an efficient way to differentiate hESC into skeletal muscle cells that are functional in vitro. Further in vivo experiments are underway to determine if these cells can be use for cellular therapy.

OVER-EXPRESSION OF FOLLISTATIN IN MYOBLASTS INCREASES THEIR PROLIFERATION AND DIFFERENTIATION, AND IMPROVES THE GRAFT SUCCESS

BENABDALLAH BF1, BOUCHENTOUF M2, ROUSSEAU J3, TREMBLAY JP4 (1) CHUL-Université Laval, Quebec, CANADA. (2) CHUL-Université Laval, Quebec, CANADA. (3) CHUL-Université Laval, Quebec, CANADA. (4) CHUL-Université Laval, Quebec, CANADA.

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Duchenne muscular dystrophy is caused by the absence of functional dystrophin protein, leading to the myofiber membrane instability and progressive muscle atrophy. Myoblast transplantation in dystrophic muscles is a potential therapy for the disease, as it permits the long term restoration of dystrophin expression in transplanted muscles. However, the success of this approach is limited by the short period of muscle repair which follows myoblast transplantation. Myostatin, known as a powerful inhibitor of muscle growth, is involved in terminating the period of muscle repair following injury by blocking myoblast proliferation and delaying myoblast differentiation. Follistatin forms a complex with myostatin preventing its interaction with its receptor and thus blocking the myostatin signal. Here, we used a lentivirus to overexpress the follistatin protein in normal myoblasts to block the myostatin signaling. Our results first confirmed the over-expression of the human follistatin into lentivirus transduced myoblasts, and second showed that the over-expression of the follistatin protein in normal human myoblasts improved in vitro their proliferation rate by about 1,5 folds after 96 h and also their differentiation rate by about 1,6 and 1,8 folds respectively in the absence and in the presence of recombinant myostatin. Finally, our data demonstrated that the engrafment of those transduced human normal myoblasts with the follistatin lentivirus into SCID mouse muscles was enhanced by 2 folds

PROTECTIVE EFFECT OF CYCLOSPORINE A AGAINST CALCIUM-DEPENDENT CELL DEATH IN DYSTROPHIN-DEFICIENT CELLS.

MONDIN L1, SEBILLE S1, BALGHI H1, CONSTANTIN B1, RAYMOND G1, COGNARD C1

(1) IPBC, UMR CNRS 6187, Université de Poitiers, Poitiers, FRANCE.

Evidence is presented for the involvement of IP3-dependent release calcium pathway in the contribution of calcium overload in dystrophin-deficient cells. Our previous results presented that global calcium release during stimulation (K+-evoked calcium increase) was found higher in dystrophin-deficient SolC1(-) myotubes than in minidystrophin transfected SolD(+) myotubes. SolC1(-) myotubes also displayed higher activity of spontaneous calcium release as compared to SolD(+) myotubes. Moreover, dystrophin-deficient myotubes were naturally dying faster than mini-dystrophin transfected myotubes. These features prompted us to conduct experiments with cyclosporine A (CsA), known to, among other effects, modulate IP3 pathway, in order to investigate its effects on both cell death and calcium signaling. Cell survival assays, performed with MTT test, revealed a protective effect of both 2-APB (IP3R inhibitor) and CsA against natural cell death occurring in mature dystrophin-deficient myotubes. The area under the curve and kinetics parameters of K+-evoked calcium increase were significantly reduced with the incubation of SolC1(-) myotubes with 2-APB. In the same way, 2-APB exposure was significantly decreasing spontaneous calcium release at rest in SolC1(-) myotubes. Interestingly, for both K+-evoked calcium increases and spontaneous release events, similar results were obtained with CsA

exposure with SolC1(-) myotubes. Furthermore, CsA reduced IP3-R1 mRNA levels in SolC1(-), leading to levels measured in mini-dystrophin transfected myotubes

(SolD(+)) in control conditions. Taken together, these data suggest that CsA could

modulate calcium releases in dystrophin-deficient cells by direct IP3Rs inhibition

and/or by reduction of IP3R-1 mRNA expression via the calcineurin pathway, leading to the regulation of the calcium overload in dystrophin-deficient cells. On that account, these data underline a strong involvement of IP3 pathway in calcium-dependent cell death in dystrophin deficiency, and that a modulation of calcineurin pathway could be a therapeutic approach to counteract the calcium overload in dystrophin-deficient myopathies.

UROCORTINS IMPROVE SKELETAL MUSCLE STRUCTURE AND FUNCTION OF DYSTROPHIC MDX MOUSE VIA A CYCLIC AMP/PKA DEPENDENT PATHWAY

REUTENAUER J1, DORCHIES O1, BOITTIN FX2, PATTHEY-VUADENS O1, RUEGG U1

(1) Laboratory of Pharmacology, Geneva Lausanne School of Pharmaceutical Sciences, University of Geneva, Geneva, SWITZERLAND. (2) Laboratory of Vascular Cell Physiology, Department of Zoology & Animal Biology, University of Geneva, Geneva, SWITZERLAND.

Urocortins are structurally related peptides of the corticotrophin-releasing factor (CRF), which has been widely implicated in responses to physical, emotional andenvironmental stress. Recently, it has been reported that a CRF-receptor-2 (CRFR2) agonist increased muscle mass and slowed disease progression in the mdx mouse model. Here, we report on our investigations on urocortins in protecting mdx muscle from necrosis and on the mechanism involved. Three week-old mdx5Cv mice were treated daily by sub-cutaneous injection for 2 weeks with either urocortin 1 at 300 µg/kg, urocortin 2 (30, 100 or 300 µg/kg), or vehicle. Isometric force recordings of the triceps surae (comprising soleus, plantaris and gastrocnemius muscles) showed that contraction and relaxation kinetics were shorter than in untreated mice. Moreover, the force-frequency curve was shifted to the right, suggesting a change in the calcium homeostasis or in the distribution of fibres toward a faster phenotype. Interestingly, hindlimb muscles from mice receiving urocortins displayed a higher resistance to mechanical stress and muscle mass was significantly increased. Histology demonstrated that urocortins remarkably protected diaphragm, EDL and soleus muscles from necrosis. Finally, urocortins administration lowered plasma creatine kinase levels up to 49% compared with vehicle. In addition, basal calcium influx was measured in whole dystrophic muscle (EDL and soleus) and in diaphragm strips. Urocortin 2 concentration-dependently decreased the permeability of the dystrophic muscle to calcium by 20-40%. This effect was completely abolished by the selective CRFR2 antagonist astressin2-B or an inhibitor of protein kinase A (PKA) while addition of forskolin, an activator of adenylate cyclase, mimicked the effect of urocortin 2. We conclude that urocortins improve both the structure and the function of dystrophic skeletal muscle via the stimulation of a cAMP/PKA-dependent signal transduction pathway. Thus, urocortins should be considered as potential candidates to counteract the impairment of calcium homeostasis observed in Duchenne Muscular Dystrophy.

THE 6-MINUTE WALK TEST AS A CLINICAL TRIAL OUTCOME MEASURE IN DUCHENNE MUSCULAR DYSTROPHY: RELIABILITY AND CORRELATION WITH OTHER MEASURES OF DISEASE SEVERITY

MCDONALD CM1, HENRICSON EK1, HAN JJ1, NICORICI AR1, ABRESCH RT1,

ATKINSON LA2, REHA AL2, ELFRING GL2, MILLER LL2

(1) University of California, Davis Medical Center, Sacramento CA, USA. (2) PTC

Therapeutics, South Plainfield NJ, USA.

Background: The 6-minute walk test (6MWT) is a commonly used measure of cardiorespiratory endurance. Researchers have begun to use it as a strength-related outcome measure in clinical trials in neuromuscular disease. We evaluated the 6MWT differentiation between boys with Duchenne muscular dystrophy (DMD) and healthy controls, test-retest variability in boys with DMD, and correlation of the 6MWT with timed functional measures. Methods: We enrolled ambulatory boys 5-12 years old with DMD (n=15) and without (n=20). Boys with DMD were tested 7 days apart using a modified American Thoracic Society 6MWT and standard clinical timed function testing. Healthy controls underwent testing at a single time point. Results: Across all ages, distance traveled differed between boys with DMD and healthy controls. In boys aged 5-6, those with DMD averaged 367 ± 74 m compared to 574 ± 35 m (p<.01) for the healthy controls. In boys aged 7-9, those with DMD averaged 354 +/- 31 m compared to 622 ± 50 m (p<.001) for the healthy controls. In boys aged 10-12, those with DMD averaged 265 ± 146 m compared to 646 ± 49 m (p<.001) for the healthy controls. As age increased in the DMD group, percent predicted scores decreased from 82% at age 5 to 56% at age 12. 6MWT test-retest correlation was high (r=.92). In the DMD group, the 6MWT correlated well with time to walk 10 m (r=.80), time to walk 25 m (r=.80), time to climb 4 standard stairs (r=.77), and time to stand (r=.64). Conclusion: A modified 6MWT in ambulatory boys with DMD is reproducible, differentiates boys with DMD from healthy controls at all ages, and correlates with other measures of disease severity.

QUALITY OF LIFE OF ADOLESCENTS WITH NEUROMUSCULAR DISEASES: HERE’S WHAT THEY SAY

VUILLEROT C1, HODGKINSON I1, BERARD C1, ECOCHARD R2, D'ANJOU MC3, COMMARE MC4

(1) Hospices Civils de Lyon, CHU Lyon Sud, L'Escale, Service de Médecine Physique et de Réadaptation Pédiatrique, Pierre Benite, FRANCE. (2) Hospices Civils de Lyon, Service de Biostatistique, LYON, FRANCE. (3) CHU St Etienne, Service de MPR pédiatrique, St Etienne, FRANCE. (4) CHU Grenoble, service MPR Pédiatrique, Grenoble, FRANCE.

Little is known about quality of life of adolescents with neuromuscular diseases or the factors that influence it. We searched whether physical impairment, physical disability, and medical complications were predictors of low quality of life. Motor function, health, orthopaedic status, and rehabilitation were assessed in 43 adolescents with neuromuscular diseases (age: 13.8±1.7; sex ratio 2.9/1). Quality of life was measured with the VSP-A (Vécu Santé Perçu par l’Adolescent), a validated health-related quality of life self-perception test. A multiple linear mixed regression related quality of life to impairment, disability, and respiratory status.

Comparisons were made with data from an age/sex-matched healthy population. On the average, the VSP-A scores in diseased adolescents were: i) similar to those of the healthy population as regards vitality, body image, relationships with parents and friends, and physical and psychological well-being ; ii) higher as regards school performance (68 vs. 52.8%) and relationships with teachers (67.4 vs. 43.2%); iii) lower as regards leisure activities (43.9 vs. 60.9%). Physical disability and physical impairment were not significantly associated with seven out of the nine domains but scores for leisure activities and vitality were significantly

associated with physical impairment (p=0,001 and p=0,006 respectively). Adolescents with ventilatory support did not express lower scores than non-ventilated ones (67.7% +/- 11 vs. 62.9%+/-15, p=0.39). These surprising results should question our medical, educational, and rehabilitation practices. Already well-managed disabled adolescents should benefit from a less compassionate and more daring and dynamic interpersonal contacts. Conclusions - These surprising results should question our medical, educational, and rehabilitation practices. Already well-managed disabled adolescents should benefit from a less compassionate and more daring and dynamic interpersonal contacts.

THE MOTOR FUNCTION MEASURE (MFM) SCALE WAS USED TO EVALUATE PATIENTS WITH DUCHENNE MUSCULAR DYSTROPHY (DMD).

BERARD C1, VUILLEROT C1, PAYAN C2, GIRARDOT F1

(1) Hospices Civils de Lyon, CHU Lyon Sud, L'Escale, Service de Médecine Physique et de Réadaptation Pédiatrique, Pierre Benite, FRANCE. (2) Institut de myologie, Paris, FRANCE.

Study participants and setting: Patients without medication were evaluated at 3 months for 13 patients (mean age 11y 7 mo; SD 1y 10mo) and at one year for 41 patients (mean age 14y 1 mo; SD 5y 5mo) in a referral center. Twelve patients treated with steroids (mean age 10 y 2 mo; SD 2y 2 mo) were evaluated after one year of treatment and compared with 12 age-matched DMD controls. Results: In a 3-month period, subscore D1 showed a significant average decrease of 4.7% (p<0,01). In a 1-year period, all scores showed significant decreases: D1 4.9% (p<0,01), D2 7.7% (p<0,01), D3 4.3% (p<0,03) and Total Score 5.8% (p<0,01). For the 11 walking patients at the beginning of the study, the average value of D1 annual decrease is 26.1%. For the non-ambulant patients, the annual average decrease was 11.8% for D2 and 6.3% for Total Score. A sensitive threshold value for the loss of the ability to walk and a predictive value a year before loss of ambulation could be estimated. In the group of patients treated with steroids, compared to controls, it was evidenced a stabilization of the total score (-0.59 vs. -5.87, p<0,02 ) and dimension D2 (0.98 vs. -8.50, p<0,01). Conclusions: Our preliminary results are promising for the use of the MFM in clinical trials to evidence either deterioration of disease or lack of deterioration induced by

therapeutics.

ASSESSMENT OF GRIP STRENGTH USING PRECISION DYNAMOMETRY

HOGREL JY1, LI K2, DUCHENE J2, HEWSON D2

(1) Institut de Myologie, Paris, FRANCE. (2) Institut Charles Delaunay - Université de Technologie de Troyes, Troyes, FRANCE.

Grip dynamometry is an important and easy-gathered method to evaluate hand function. Reliable and valid evaluation of grip strength depends on the quantified measurements and standardised testing procedures. A major drawback of all

commercially available grip handles is that they are not adapted to weak patients, particularly during therapeutic trials.To this end, the Myogrip was designed for the assessment of grip strength of weak patients suffering from various neuromuscular disorders such as muscle dystrophies. The device was built around a precision force sensor (full scale: 89 daN; accuracy:0.05 daN; sensitivity: 0.01 daN). The Myogrip can complete several functions: real time display, maximal force recording, wireless communication with a computer, RS232 or BNC connections. The handle size is finely adjustable. Several works are undergone using the Myogrip, aiming at:

- testing the effect of elbow positioning and grip handle size on maximal grip

strength

- comparing the Myogrip with the gold standard (represented by the Jamar)

and another available device (Martin Vigorimeter)

- developing a normative database on 450 subjects aged from 5 to 80 years

- assessing the reproducibility and repeatability of the measure on 120 subjects

- proposing a predictive model of grip strength to compute patient deficit

- testing the validity of the Myogrip for the evaluation of weak patients

Prelimlinary results showed no effect of elbow positioning. However when the elbow is fully extended, patient evaluation was more reproducible, probably due to less possibility of compensation and better patient maintaining by the evaluator. A

significant effect of grip handle size was observed. Results obtained with Myogrip and Jamar were highly correlated (0.93). Test-retest reliability of all grip devices was excellent (ICC higher than 0.96). The Myogrip is a new device devoted to the measure of weak patients. Its validation is ongoing and preliminary results show excellent reliability.

GAIT ANALYSIS OF NEUROMUSCULAR PATIENTS USING ACCELEROMETRY

HOGREL JY1, CANAL A1, BARREY E2, OLLIVIER G1

(1) Institut de Myologie, Paris, FRANCE. (2) LEPHE-INRA - Genopole, Evry,

FRANCE.

Quality of gait is a fundamental outcome measurement for the follow-up of neuromuscular patients either during the natural history of their disorder or during a therapeutic trial. Precise gait analysis can be performed using kinematic systems in lab conditions. Unless very useful, the measurement and analysis procedures are long and complex and not always suitable for clinical routine depending on the patients. Accelerometry can bring much useful information on gait without many constraints for the patient or the medical staff. This has been considered in the present work during six-minute walk tests (6MWT). The gait analysis system used in this study (Locometrix-2™) includes three accelerometers in a small (20 x 40 x 80 mm) and light (50 g) box and a data logger. The apparatus is incorporated into a semi-elastic belt, which is fastened around the subject's waist, close to the centre of gravity. Signals were recorded with a sampling frequency of 100 Hz. The recorded signals were transferred to a computer and analyzed by a specific software. Several gait variables were computed on a sample of 10 s of stationary gait: walking velocity, stride frequency and length, symmetry and regularity indexes, mechanical power and high frequency shocks. As a pilot study, the measurements were performed on 12 patients suffering from acid maltase deficiency during 6MWT. Their results were compared to normative data available from more than 400 healthy subjects and show deep modifications of their gait depending on the severity of their disorder. Accelerometry is a very simple tool to assess gait quality. Measurements will also be considered at home in further studies.