Abstracts that will be presented in World Muscle Society Meeting 2014

Experimental Studies in DMD

Novel inhibitor of hematopoietic prostaglandin D synthase improves the muscle disorder in an experimental model of Duchenne muscular dystrophy

K. Tanaka, K. Aritake, M. Tayama, E. Sasaki, T. Utsugi, T. Sasaoka, Y. Urade

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 mdxmice but hardly in wild-type mice. The urinary tetranor-PGDM concentration was significantly higher in mdxmice 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.

Effects of S48168/Arm210, a new rycal® compound, on pathology related signs of exercised dystrophic mdx mouse

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 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.

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

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 indystrophic mice towards wild type controls. Although apocynin and diapocynin had beneficial effects in dystrophic mice, diapocynin was superior in improving locomotion.

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, C. Le Guiner, M. Montus, Y. Cherel, V. Francois, J.L. Thibaud, C. Wary, B. Matot, T. Larche, L. Guigand, M. Dutilleul, H. Goubin, J.Y. Deschamps, C. Domenger, M. Allais, J. Le Duff, M. Devaux, N. Jaulin, M. Guilbaud, O. Adjali, V. Latournerie, et al.

We have previously demonstrated that a recombinant adeno-associated 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.

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

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 centronucleation, disorganized cytoskeleton, presence of fibrosis. We have recently described a loss of virion genomes from both dogs and mice models of DMD treated with therapeutic molecules vectorized in AAV. Indeed, the pathophysiological state of DMD muscle should impact on virions fate and subsequently affect crucial steps for AAV effectiveness as viral uncoating, viral genome maintenance and consequently, the transduction efficiency of AAV. Our project aims to characterize cellular uptake and intracellular transport of AAV8 in DMD muscular cells, with the goal of optimizing AAV vector use to get the best transduction efficiency with the lowest AAV dose. Our first data showed that AAV8-GFP was less efficient to transduce DMD and control primary muscular cells compared to HeLa cells. Moreover, AAV8 traffics through same endosomal compartment in DMD and control myoblasts, but at different rates during early time points of the transduction. These results suggest that in muscle cells, AAV8 uses different entry and trafficking pathways from those previously described in HeLa cells and that dystrophic cellular status could affect subcellular processing of the vector particles. We will specify the relationship between AAV8 vector entry, trafficking, uncoating, and transduction efficiency in vitro in primary myoblasts/myotubes of DMD patients and controls.

Optimization of micro-dystrophin cassettes for AAV-mediated gene therapy

J.S. Chamberlain, J. Ramos, G. Banks, S.D. Hauschka

Gene therapy approaches for DMD using recombinant adeno-associated viral (rAAV) vectors have the potential to halt dystrophic progression and reverse aspects of the muscle pathophysiology. Advantages of rAAV-delivery of micro-dystrophins include long lasting (years) expression of the delivered gene cassette, potential for systemic delivery to all muscles of the body (including the heart) and applicability to all patients regardless of their mutation. However, AAV vectors have a limited cloning capacity of <5 kb and have been associated with cellular immune responses against the delivered gene products. We have been testing numerous design modifications of micro-dystrophin expression cassettes to increase expression, improve functionality of micro-dystrophins and minimize immune recognitions of the delivered protein. Expression and immune modulation are primarily regulated by promoter/enhancer elements, and modification of muscle-restricted enhancer plus promoter elements enables strong expression in muscle but not in immune effector cells. Further enhancement of expression is obtained by suing synthetic, codon-optimized cDNAs to express the encoded micro-dystrophin protein. To maximize dystrophin functionality we have tested novel micro-dystrophins with modified hinge domains and with various combinations of spectrin-like repeat domains, including repeats that help localize nNOS. Our results show that optimized micro-dystrophins can halt all ongoing dystrophy in dystrophic mdx mice and lead to a significant, albeit not full functional reversal of mechanical properties in treated muscles. The use of rAAV vectors expressing optimized vectors using MCK regulatory elements and, codon optimized cDNAs and hinge/repeat domain optimized micro-dystrophins has significant potential for clinical application in patients with DMD.

Preclinical evaluation of tamoxifen and other selective estrogen receptor modulators in mdx5Cv dystrophic mice

O.M. Dorchies, H.M. Ismail, S. Tardy, J. Reutenauer-Patter, E. Dahmane, L.A. Décosterd, D. Picard, U.T. Ruegg, L. Scapozza

We are investigating the effects of selective estrogen receptor modulators (SERMs) in mdx5Cv dystrophicmice (Dys), a model for Duchenne muscular dystrophy (DMD). SERMs display either pro-estrogenic or anti-estrogenic activities in a tissue-dependent manner. Tamoxifen (TAM), the most well characterised SERM, has been used for over 30 years to treat estrogen-sensitive breast cancer in both women and men and has been reported to be also well tolerated in pre-pubertal boys. In 2013, we published that oral treatment of Dys mice from 3 weeks of age for 15 months with TAM (10 mg/kg/day) improved muscle force and the structure of diaphragm and heart. TAM and its metabolites were present in nanomolar concentrations in plasma and muscles, suggesting signalling through high affinity targets, likely the estrogen receptors alpha and beta that were several-fold more abundant in dystrophic muscle than in normal ones. Next, we tested TAM in adult Dys mice in order to investigate its efficacy in the low-intensity chronic stage of the disease, which resembles most closely the DMD condition. TAM at doses as low as 0.1 mg/kg/day improved motor performance of active mice and enhanced the contractile characteristics of the triceps surae. At 3 mg/kg/day, TAM corrected most endpoints close to normal values. We are currently testing other SERMs (all at 3 mg/kg/day): the chlorinated TAM analogues clomiphene and toremifene, the 3-hydroxylated TAM derivative droloxifene, the second generation SERM unrelated to TAM raloxifene (RAL), and the pure anti-estrogen fulvestrant (Faslodex). Overall, the ranked efficacy was as follows: TAM > toremifene > clomiphene  > droloxifene ≈ RAL > Faslodex. Our data as well as our current understanding of estrogenic signalling in dystrophic muscle suggests that TAM and other SERMs with pro-estrogenic activities on muscle might be beneficial for DMD and maybe also for other muscular dystrophies.

The Sodium/Hydrogen Exchanger (NHE-1): A promising novel target for DMD

U. Burki, E. Greally,S. Laval, S. Schäfer, V. Straub

Abnormal intracellular calcium load is one of the most consistent findings in Duchenne muscular dystrophy(DMD) but previous attempts to improve the dystrophic pathology with calcium antagonists have been unsuccessful. However, there is an intriguing possibility of restoring calcium homeostasis indirectly by exploiting the relationship between the Sodium/Hydrogen Exchanger (NHE-1) and the Sodium/Calcium Exchanger (NCX). NHE-1 has been reported to be abnormally overactive in DMD models, leading to an increased influx of sodium which in turn switches the NCX into reserve mode leading to calcium influx. Using selective NHE-1 inhibitors the influx of sodium can be reduced thereby allowing the NCX to revert back to normal mode, where calcium is extruded, thus reducing the cellular calcium load. In collaboration with our industrial partner Peacock Pharma, we have demonstrated the potential of NHE-1 inhibition in the mdx mouse model for DMD by chronically treating animals with a novel NHE-1 inhibitor. The pilot study showed significantly reduced calcium uptake in treated mice (n = 6) using manganese enhanced MRI (MEMRI). Manganese acts as a contrast agent in T1 weighted MRI and is thought to be taken up by cardiac and skeletal muscle by the same receptors as calcium. There was also an increase in functional grip strength, although not statistically significant the results are nevertheless encouraging and indicate the potential of this novel approach in DMD. This is currently being investigated more thoroughly using a greater number of animals (n = 12) treated chronically via drug in chow over 6-months and assessed using MEMRI and grip test. In addition, in vitro calcium-flux assays (e.g. Fluo-4) are used to determine the potency and investigate the mechanism of action of this drug. The objective of our study is to provide the requisite pre-clinical data that would enable these drugs to progress towards clinical trials.