G.P.90

Abstracts / Neuromuscular Disorders 24 (2014) 791–924 mutations however are ultra-rare with only few patients worldwide, and require the skipping of an exon outside the hotspot region. Combining these ultra-rare mutations by applying multiple exon skipping is therefore attractive. The feasibility of multiple exon skipping using a cocktail of AONs has been demonstrated, but a drawback is the increased complexity of the development pathway and manufacturing costs. We therefore studied the possibility of designing a single AON capable of inducing simultaneous skipping of multiple exons. The repetitive nature of the central rod domain in dystrophin implies the occurrence of homologous stretches within the transcript. Indeed, in the exon 10 to 40 region several exon pairs were identified sharing such a homologous stretch, which we explored as a potential dual target for a single AON. In-frame exon combinations that would mimic a Becker-like mutation associated with a relatively mild phenotype and that would apply to a considerable patient subpopulation (up to 15%) were selected. In vitro proof-of-concept was obtained for healthy donor as well as for DMD patient-derived muscle cells and resulted in novel dystrophin expression. Results demonstrating the applicability in the mdx mouse model in vitro and in vivo will be presented. The data support further (pre) clinical development of such multiple exon skipping AONs, not only for more rare mutations that are not targeted by current single skip programs but also for mutations in the hotspot region. http://dx.doi:10.1016/j.nmd.2014.06.101

G.P.88 Novel inhibitor of hematopoietic prostaglandin D synthase improves the muscle disorder in an experimental model of Duchenne muscular dystrophy K. Tanaka 1, K. Aritake 2, M. Tayama 1, E. Sasaki 1, T. Utsugi 1, T. Sasaoka 3, Y. Urade 2 1 Taiho Pharmaceutical Co., Ltd., Tsukuba, Japan; 2 Osaka Bioscience Institute, Suita, Japan; 3 Niigata University, Niigata, Japan Duchenne muscular dystrophy (DMD) is a fatal genetic disease. The fibers of muscle in DMD patients easily suffer necrotic change, because of the loss of dystrophin. Thus the patients present with a weakening motor activity. There is still no complete cure for the disease. Recently, we have reported that prostaglandin D2 (PGD2) synthesized by hematopoietic PGD synthase (HPGDS) may play an important role in the pathology of DMD. In this study, we evaluated the beneficial effects of a highly selective HPGDS inhibitor (TAS-205 which is found in our laboratory) in dystrophin-deficient mice. We used dystrophin-deficient mdx mice (C57BL/6 back ground) and the wild-type mice (C57BL/6). Mice were treated with TAS-205 daily by supplied the diet including 0.01% or 0.1% TAS-205 from 5 to 9 weeks of age. Mice of control group and normal group were supplied the same food without drug. At 9 weeks of age, we measured the locomotor activity during night-time, collected the mouse urine of at night-time to determine the urinary concentration of tetranor-PGDM, a metabolite of PGD2, and evaluated the rate of necrotic fibers area in cross sections of the diaphragm muscle. The locomotor activity at the night-time was significantly lower in mdx mice than that in wild-type mice. In histological evaluation, many necrotic fibers were detected in diaphragm muscle of mdx mice but hardly in wild-type mice. The urinary tetranor-PGDM concentration was significantly higher in mdx mice than that in wild-type mice. TAS-205 dose-dependently suppressed the urinary tetranor-PGDM amount in mdx mice. TAS-205 at high dose significantly reduced the necrotic muscle fibers and recovered the locomotor activity in mdx mice. These results indicate that PGD2 synthesized by HPGDS is involved in the progression of muscular necrosis in DMD and a HPGDS inhibitor, such as TAS-205, would be an effective therapy for DMD. http://dx.doi:10.1016/j.nmd.2014.06.102

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G.P.89 New orally available compounds which modulate utrophin expression for the therapy of Duchenne muscular dystrophy (DMD) R.J. Fairclough 1, S. Guiraud 1, S.E. Squire 1, A. Babbs 1, B. Edward 1, N. Shah 1, A. Bracchi 2, F.X. Wilson 2, G. Horne 2, N. Robinson 2, N. Araujo 3, D.S. Hewings 3, A. Vuorinen 3, S.G. Davies 3, G.M. Wynne 3, A.J. Russell 3, J. Tinsley 2, K.E. Davies 1 1 MRC Functional Genomics Unit – University of Oxford, Oxford, UK; 2 Summit plc, Abingdon, UK; 3 University of Oxford, Oxford, UK DMD is a devastating X-linked muscle-wasting disease caused by lack of the cytoskeletal protein dystrophin. There is currently no cure for DMD although various promising approaches (e.g. exon skipping, read through of stop codons, gene therapy) are being developed. By pharmacologically modulating the dystrophin-related protein utrophin, we aim to develop a therapy applicable to all DMD patients by targeting the primary defect and restoring sarcolemmal stability. In partnership with Summit plc we previously developed SMT C1100; a small molecule utrophin modulator that reduced dystrophic symptoms in the mdx mouse. As a potential first-in-class molecule, SMT C1100 recently successfully completed a Phase 1a trial. DMD patients are currently being dosed in an ongoing Phase 1b trial for which a multicomponent biomarker strategy has been implemented with a quantification of utrophin level, regeneration, fibrosis, inflammation and analysis of specific miRNAs. The successful clinical progression to date provides crucial proof-of-concept for the strategy we developed. We are now optimising next generation utrophin modulator molecule which were discovered using an improved drug screening assay based on immortalised myoblasts from the utrophin luciferase knock-in mouse. This enables us to screen utrophin in its genomic context, better mimicking the in vivo situation and enabling identification of compounds which modulate utrophin through regulatory pathways outside of the 8.4 kb promoter fragment used in our previous screen. Thus, we identified multiple structural classes which significantly modulate utrophin expression in both mouse and human DMD myoblasts. New compounds exhibit favourable solubility, stability, oral absorption and are well tolerated in the mouse and structure–activity studies are underway, with the objective to improve compound effectiveness. The two of the best new utrophin modulators are currently being studied in pre-clinical in vivo studies in mdx mice. http://dx.doi:10.1016/j.nmd.2014.06.103

G.P.90 Effects of S48168/Arm210, a new rycalÒ compound, on pathology related signs of exercised dystrophic mdx mouse R.F. Capogrosso, A. Cozzoli, A. Giustino, P. Mantuano, A.M. Massari, E. Conte, M. Cannone, M. De Bellis, A. Liantonio, G.M. Camerino, A. De Luca University of Bari, Bari, Italy The progressive myofiber degeneration in Duchenne muscular dystrophy (DMD) is caused by the complex cascade of events triggered by the absence of dystrophin. Studies in the mdx mouse model of DMD have shown that pathology-related post-translational modifications of the Ryanodine receptor subtype 1 (RyR1) result in the dissociation of the stabilizing subunit calstabin1, leading to “leaky” channels and altered Ca2 + homeostasis, a key event in DMD (Bellinger, Nat Med 2009). S48168, also known as ARM210, is a small molecule stabilizer of the calstabin-RyR channel complex that prevents Ca2+ leak without altering RyR1 post-translational modifications. We assessed the effects of 4 weeks oral administration of S48168 (10 and 50 mg/kg/day) on treadmill-exercised mdx mice, starting at 4–5 weeks of age. A functional improvement was observed in vivo in S48168 -treated mdx mice, with a

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Abstracts / Neuromuscular Disorders 24 (2014) 791–924

50% recovery score of maximal forelimb force at 50 mg/kg. Both doses prevented the decline in running performance, observed in vehicle-treated mdx mice, as assessed in an exhaustion test. Ex vivo assessment showed a dose-dependent improvement of diaphragm specific force, with a significant 30% increase of maximal force (100–140 Hz) at 50 mg/kg, while minor, if any, effects were observed in hind-limb EDL muscle. However, and consistently with the S48168 mechanism of action, the mechanical threshold EDL myofibers, an index of calcium handling during contraction, was significantly shifted toward the wild type (wt) values at the highest dose. In parallel, the resting cytosolic calcium level in FDB myofibers was significantly reduced by 25% in the S48168-treated mice. S48168 did not lower CK or LDH plasma levels. However a reduction of damaged area was detected in diaphragm and in gastrocnemius muscle of 50 mg/kg treated mdx mice. Dose-proportional increases in plasma and muscle drug content were also observed, without any signs of toxicity. These results support S48168 as a potential therapy for DMD. http://dx.doi:10.1016/j.nmd.2014.06.104

G.P.91 Diapocynin, a putative NADPH oxidase inhibitor, ameliorates the phenotype of a mouse model of Duchenne muscular dystrophy H.M. Ismail, L. Scapozza, U.T. Ruegg, O. Dorchies University of Geneva, 1211, Switzerland Duchenne muscular dystrophy (DMD) is a severe X-linked muscular disease that causes premature death and for which no cure exists. We have shown previously that in vitro treatment of dystrophic myotubes and excised muscles with diapocynin, a dimer of the classically used NADPH oxidase inhibitor apocynin, ameliorated several molecular events involved in DMD pathogenesis, of which ROS production, phospholipase A2 activity, Ca2+ influx and sarcolemmal integrity. Here, we report on the in vivo effects of diapocynin and apocynin in mdx5Cv dystrophic mice, a model of DMD. Apocynin (50 mg/kg/day) and diapocynin (10 and 100 mg/kg/day) were given orally to mdx5Cv mouse pups, first via the lactating mothers from post-natal day 14 to 28 and subsequently directly to the weaned pups till post-natal day 35 ± 1 or 60 ± 3. Diapocynin but not apocynin enhanced spontaneous locomotor activity, rescued voluntary wheel running capabilities, and ameliorated diaphragm structure of dystrophic mice. Diapocynin and apocynin were equally potent at increasing the resistance to fatigue of triceps surae muscles exposed to repeated isometric contractions in situ and at preserving sarcolemmal integrity as evidenced by Evans blue dye uptake. Furthermore, microarray analyses showed a tendency of the treatments to correct gene expression in dystrophic mice towards wild type controls. Although apocynin and diapocynin had beneficial effects in dystrophic mice, diapocynin was superior in improving locomotion. http://dx.doi:10.1016/j.nmd.2014.06.105

G.P.92 Persistence and stability at 6 months of AAV genome and dystrophin expression in GRMD dogs after forelimb delivery of a recombinant AAV carrying an exon skipping sequence L. Servais 1, C. Le Guiner 2, M. Montus 3, Y. Cherel 4, V. Francois 2, J.L. Thibaud 5, C. Wary 1, B. Matot 1, T. Larche 4, L. Guigand 4, M. Dutilleul 4, H. Goubin 4, J.Y. Deschamps 4, C. Domenger 2, M. Allais 2, J. Le Duff 2, M. Devaux 2, N. Jaulin 2, M. Guilbaud 2, O. Adjali 2, V. Latournerie 3, P. Veron 3, S. Boutin 3, C. Leborgne 3, D. Desgue 3, S. Moullec 6,

F. Barnay-Toutain 3, C. Riviere 3, N. Delaunay 3, G. Bonne 1, C. Masurier 3, J.Y. Hogrel 1, P. Carlier 1, P. Moullier 7, T. Voit 1 1 Institute of Myology, Paris, France; 2 Atlantic Gene Therapies, INSERM UMR 1089, Universite´ de Nantes, CHU de Nantes, Nantes, France; 3 Genethon, Evry, France; 4 Atlantic Gene Therapies, INRA UMR 703, ONIRIS, Nantes, France; 5 UPR de Neurobiologie, Ecole Nationale Ve´te´rinaire d’Alfort, Maisons Alfort, France; 6 Atlantic Gene Therapies, Centre de Boisbonne, ONIRIS, Nantes, France; 7 Gene Therapies, INSERM UMR 1089, Universite´ de Nantes, CHU de Nantes, Nantes, France We have previously demonstrated that a recombinant adenoassociated virus vector, serotype 8 (rAAV8) carrying a modified U7snRNA sequence promoting exon skipping injected in GRMD by locoregional transvenous perfusion of the forelimb, restores up to 80% dystrophin expression and improves histological, Nuclear Magnetic Resonance (NMR) pathological indices and strength in a dose-dependent manner at 3 months post injection (PInj). A recent paper suggested that dystrophin-positive fibers and rAAV genome drastically decrease with time, especially between 3 and 6 months PInj. This may compromise the clinical development of an AAV-mediated gene transfer approach. To assess this point, we realized a 6 to 7 months follow-up in a group of 3 GRMD aged 3–4 months when injected in both forelimbs with 2.5E13 vg/kg of a rAAV8 U7snRNA vector promoting exon skipping. Dogs were injected, followed up and evaluated (NMR, strength assessment, Dystrophin expression, vector biodistribution) in the same way as the previously reported large cohort of dogs that were followed for 3 months. Muscle biopsies were performed at 3 months and complete autopsy at 6 to 7 months PInj. Compared to previously reported dogs injected with the same dose, evaluation of dystrophin expression, histological aspect, AAV genome, NMR parameters and strength showed not differences indicating that the treatment effect was fully maintained over 6 months. No loss in AAV genome quantification and dystrophin expression was observed between the 3 months muscle biopsy specimens and 6 months muscle autopsy in the 3 dogs. A decrement in transgene copy numbers at 6 months PInj was observed in non muscular tissues. These data show, for the first time, that AAV-mediated exon skipping fully maintains dystrophin expression and functional recovery over a six months period. We suggest that long-term rAAV genome stability is obtained in dystrophic muscles when a substantial reversion of the muscular pathological pattern is reached. http://dx.doi:10.1016/j.nmd.2014.06.106

G.P.93 Entry and intracellular trafficking of adeno-associated viral 8 (AAV8) vector in DMD muscular cells B. Maniangou, B. Cadot, M. Dorna, I. Holtzman, M. Pontoriero, A. Jollet, K. Mamchaoui, S. Lorain, T. Voit, F. Pietri-Rouxel, S. Benkhelifa-Ziyyat Myology Center of Research, UM76 UPMC – U974 Inserm – FRE3617 CNRS, Institut de Myologie, Paris, France Adeno Associated virus serotype 8 (AAV8) is of particular interest as a vector for pre-clinical and clinical trial for Duchenne Muscular Dystrophy (DMD). In several cell lines, this vector has been shown to enter cells through clathrin-mediated endocytosis followed by a trafficking through the microtubule network in various endosomal compartments toward the nucleus. To efficiently transduce cells, AAV must undergo multiple levels of regulation in these cellular compartments. In DMD, dystrophin deficiency results in disturbed balance of cellular events i.e., fiber