The contractile phenotype of a novel dystrophin-negative mouse strain with enhanced voluntary exercise capability

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Abstracts 2017 / Neuromuscular Disorders 27 (2017) S96–S249

P.309 Success of preclinical drug trials using reliable and reproducible endpoints in mouse models of neuromuscular diseases K. Nagaraju, A. Mullen, A. MacKinnon, K. Uaesoontrachoon, E. Hoffman, S. Srinivassane Agada Biosciences Inc, Halifax, Canada Preclinical efficacy evaluation in mouse models of human diseases is an important component of drug development. It has been reported that phase II clinical trial success rates have fallen significantly in recent years, with a lack of efficacy being the most frequent reason for failure. These failures could be due to 1) the poor predictive power of disease models, 2) questionable targets, 3) lack of rigor in preclinical trial design, 4) poor control for potential bias, or 5) variable reporting standards. The quality and reproducibility of preclinical trials depend on the thoroughness of the preclinical study, including the design, execution, analysis, and reporting of the preclinical data. Here we have evaluated 34 interventions in mouse models of Duchenne muscular dystrophy and limb girdle muscular dystrophy 2B using a well characterized set of functional testing (grip strength, in vitro force contractions, treadmill exhaustion, voluntary wheel running, echocardiography and plethysmography), histological (inflammation, central nucleated fibers, degeneration, regeneration, fibrosis), biochemical (serum CK) and molecular (gene expression and western blot) based on the TREAT-NMD preclinical standard operating procedures. Each of these studies were done using an appropriate sample size, blinding, randomization and statistical analysis. In the 34 interventions using 1187 mice, only 3 interventions showed efficacy, indicating that 91% of interventions failed to show efficacy. Our studies indicate that this failure could be due to questionable therapeutic targets. It is important to perform robust proof of concept studies and target validation studies before advancing to preclinical mouse trials. http://dx.doi.org/10.1016/j.nmd.2017.06.349

PRECLINICAL TREATMENT: PART 2 P.310 A novel drug screening approach to identify new drug candidates for the treatment of Duchenne muscular dystrophy A. Hick 1, I. Prokic 1, F. Bousson 1, C. Fugier 1, B. Gobert 1, M. Hestin 1, E. Riguet 2, T. Cherrier 3, J. Chal 4, O. Pourquie 4, M. Guyot 1, J. Bonnefoy 1 1 Anagenesis Biotechnologies, Illkirch-Graffenstaden, France; 2 SmartDrugs Therapeutics, Fenouillet, France; 3 IGBMC, Illkirch-Graffenstaden, France; 4 Harvard Stem Cell Institute, Boston, USA Duchenne muscular dystrophy (DMD) is the most common type of muscular dystrophy. DMD is a very severe disorder presenting with rapidly progressing muscle wasting and premature death. No current treatment is available and drugs improving the course of the disease are limited and with various side effects. The aim of this work is to develop a drug screening assay to find new drug candidates for DMD. A high throughput screening test was developed using a unique in vitro differentiation protocol recapitulating key signaling events in skeletal muscle development. This protocol allows the production of fully differentiated myotubes associated with satellite-like cells expressing the Pax7 marker, starting from human induced pluripotent stem cells (hiPSCs) carrying two common DMD mutations. Practically, a population of muscle progenitors enriched in Pax7+ satellite-like cells is placed in 384 wellplates in a medium allowing their spontaneous differentiation into myotubes and exposed to the test compounds. The readout selected is the surface of the wells covered by myotubes, where increased myotube area reflects an increased commitment of Pax7+ cells towards myotube formation. This has the potential to translate into a therapeutic benefit in patients as recent data supports the existence of a defect in satellite cell commitment towards muscle fiber generation in DMD boys. Several hits coming out of the first screening campaign (5’000 compounds) were already known in the literature to have an

effect on muscle function in mdx mice and/or DMD patients, thus validating the predictivity of the HTS assay. Furthermore, a selection of hits was tested in vivo in the mdx mouse model with or without combination with the standard of care, and all were found to improve skeletal muscle function and resistance to muscle damage. This approach should lead to the identification of promising drug candidates for DMD patients. http://dx.doi.org/10.1016/j.nmd.2017.06.350

P.311 Detailed natural history of the mdx-DBA model M. van Putten, M. Overzier, K. Putker, B. Kogelman, W. Adamzek, L. van der Weerd, J. Plomp, A. Aartsma-Rus Leiden University Medical Center, Leiden, Netherlands Mdx-BL10 mice are, despite their very mild pathology, the most commonly used model for Duchenne muscular dystrophy. Mdx mice on a DBA2/J genetic background (mdx-DBA) are more severely affected due to their dysfunctional Anxa6 and Ltbp4 genes (respectively involved in regeneration and fibrosis). Detailed knowledge on the natural disease history and standardized outcome measures are critical for preclinical studies, but are lacking for mdx-DBA mice. To address this, we assessed functional performance (fore-limb grip strength and wire and grid hanging tests) in n = 10 mdx-DBA and DBA2/J wildtype mice of both genders, twice monthly for a duration of 34 weeks and compared histopathology with similar sized sedentary groups. Muscle pathology between sedentary and functionally challenged mice did not differ, indicating that the functional test regime was not detrimental. In all functional tests, performance of mdx-DBA was severely impaired compared to wild type and interestingly, mdxDBA females outperformed males. Respiratory function assessed at 7, 14 and 34 weeks of age was also affected in mdx-DBA mice. Creatine kinase levels were elevated in mdx-DBA mice, but lower than those of mdx-BL10 mice. Histopathology of the gastrocnemius, triceps, diaphragm and heart was compared between mdx-DBA, mdx-BL10 and wild type mice and revealed fibrosis, centralized nuclei, altered fiber size distribution and calcifications for both mdx models, but was more severe in mdx-DBA mice. The diaphragm was the most severely affected muscle. Expression of regenerative, immunological, fibrotic and adipose genes was upregulated in dystrophic mice. We are currently assessing the impact of forced treadmill running on pathology in mdx-DBA and mdx-BL10 mice, and the applicability of MRI to non-invasively monitor pathology. Our studies offer a comprehensive natural history data set which will be useful in the design of standardized tests and future pre-clinical studies in mdx-DBA mice. http://dx.doi.org/10.1016/j.nmd.2017.06.351

P.312 The contractile phenotype of a novel dystrophin-negative mouse strain with enhanced voluntary exercise capability C. Wingate 1, G. Pinniger 1, P. Arthur 1, A. Bakker 1, K. Nowak 2 1 University of Western Australia, Perth, Australia; 2 University of Western Australia, Harry Perkins Institute of Medical Research, Perth, Australia Duchenne muscular dystrophy (DMD) is a debilitating X-linked disease caused by mutations in the gene encoding the protein dystrophin. The lack of dystrophin leads to progressive skeletal muscle damage and wasting, which significantly decreases locomotory ability. DMD ultimately results in death from respiratory or cardiac failure, as no cure is currently available. Mdx mice, an animal model of DMD, exhibit significantly lower levels of voluntary running exercise than control mice due to their diseased skeletal muscles. In this study, we bred mdx mice with a mouse strain with a naturally high voluntary exercise capability (‘CC’ mouse). Dystrophin-deficient progeny of this cross (mdx/CC mice) display significant improvements in daily voluntary running distance (P < 0.001) and maximum running velocity (P < 0.01) compared to mdx mice. Therefore, we measured the contractile properties of fast- and slowtwitch-predominant skeletal muscles from mdx and mdx/CC mice to investigate

Abstracts 2017 / Neuromuscular Disorders 27 (2017) S96–S249 the physiological mechanisms responsible for the increased exercise ability of mdx/CC mice. Six-week-old mdx controls (n = 14) and mdx/CC (n = 15) mice were anaesthetised, the extensor digitorum longus (EDL) and soleus hind-limb muscles surgically removed and attached to an in vitro muscle test system. Contractile measurements assessed included fatigability and response to damaging eccentric contractile activity. Fatigability of the EDL and soleus muscles from mdx/CC mice was not significantly different to mdx mice (P > 0.05). However, both soleus and EDL muscles from mdx/CC mice showed a significant increase in resistance to eccentric-contraction induced damage (P < 0.05). This increased resistance may play a major role in the enhanced exercise ability of the mdx/CC mouse strain. Therefore, elucidating the genes responsible for this improved performance of dystrophin-deficient skeletal muscle could lead to novel therapeutic targets for DMD patients. http://dx.doi.org/10.1016/j.nmd.2017.06.352

P.313 Genetic and phenotypic diversity in a panel of seven new DMD mouse models in the founder strains of the Collaborative Cross L. Bogdanik, K. Carpentier, L. Cantor, C. Lutz The Jackson Laboratory, Bar Harbor, USA In Duchenne muscular dystrophy, sarcolemma damage, chronic inflammation, and regeneration of myofibers create a complex cellular environment that inexorably progresses toward muscle tissue loss, fibrosis, and strength reduction. The interplay of these different mechanisms is still incompletely understood: for instance, deciphering which parts of the immune reaction promote regeneration or fibrosis is critical for the refinement of immunomodulatory therapies. Here, we present a new collection of genetically and phenotypically diverse mouse mutant strains with dystrophin mutations, developed to better understand the role of the different mechanisms of the muscle pathology, and provide improved preclinical platforms for the development of therapies. Recently, it has been shown that polymorphisms between the genetic backgrounds of the traditional (C57BL10.mdx) and newer (DBA/2.mdx) DMD mouse models, and the type of inflammation developing in their muscles, account for differences in fibrosis, regeneration, and muscle loss. We expanded the repertoire of DMD mouse models by creating non-sense targeted mutations of dystrophin exon 23 in seven of the eight founder strains of the Collaborative Cross, a panel of inbred strains that capture nearly 90% of the known genetic variations present in laboratory mice: A/J, C57BL/6J, 129S1/ SvImJ, NOD/LtJ, NZO/HlLtJ, PWK/PhJ, and WSB/EiJ. We used zinc-fingernucleases to induce equivalent mutations in the exon carrying the historical mdx mutation, in perfectly inbred, stable and characterized genetic backgrounds. We present the cross-comparison of these seven new strains for their muscle loss, fibrosis, regeneration and immune reactions, to draw correlations between genotype, nature of the inflammation, and balance between repair and destruction of the muscle tissue. In conclusion, this new panel of DMD mouse models may offer precision preclinical models tailored to test specific therapeutic approaches for Duchenne muscular dystrophy. http://dx.doi.org/10.1016/j.nmd.2017.06.353

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Several growth factors have been involved in the process of muscle fibrosis in Duchenne muscular dystrophy (DMD). Nintedanib, a tyrosine kinase inhibitor, is effective in fibrotic disorders such as idiopathic lung fibrosis. We studied whether nintedanib could be effective slowing down fibrosis in DMD. We studied the influence of nintedanib on proliferation, chemotaxis and gene expression of human fibroblasts in vitro. We studied the effect of nintedanib in muscle function and structure in 10 months old mdx treated-mice (n = 7) with 60 mg/kg nintedanib daily for one month compared to age matched mdx mice (n = 5) and C57BL/6N controlmice (n = 5) using digigait, electromyography and histological studies. We used ANOVA and Student T test to study possible differences. Nintedanib significantly decreased human fibroblast (p < 0.001) proliferation,and chemotaxis (p < 0.05) and it reduced collagen I and III and fibronectin expression in vitro. We did not observe differences in muscle function of mdx mice treated and non-treated compared to controls. EMG detected motor unit action potentials of bigger amplitudes and shorter duration in treated mice compared to non-treated mice. Histological studies showed a significant reduction in the fibrotic tissue area in muscle sections of diaphragm (p < 0.001) and quadriceps (p = 0.03) of treated compared to non-treated animals. Real Time PCR and WB studies showed a reduction in the expression of collagen I and III and fibronectin in muscles obtained from the treated mice compared to controls. Nintedanib is an effective antifibrotic drug in a murine model of DMD, reducing muscle fibrotic area and inflammation. This effect could be produced by a reduction of the proliferation ratio of fibroblasts. This promising result suggests a possible role of tyrosine kinase inhibitors in the treatment of muscle dystrophies. http://dx.doi.org/10.1016/j.nmd.2017.06.354

P.315 Short term estradiol administration improves muscle funcion, decreases CK levels and upregulates p75 neurotrophin receptor in mdx mice F. Munell 1, S. Ferrer Aparicio 1, E. Martínez Saez 2, M. Pérez-Garcia 2 1 Vall d’Hebron Research institute, Barcelona, Spain; 2 Vall d’Hebron University Hospital, Barcelona, Spain Despite intense efforts, no cure is currently available for Duchenne muscular dystrophy and glucocorticoids are the only drug effective for slowing disease progression. In skeletyal muscle, estrogens have been proven to stimulate growth and regeneration and reduce inflammation and fibrosis. We have used young mdx mice to demonstrate that short-term estradiol administration caused a notable decrease in CK levels and improved motor function. Aiming to decipher the mechanisms of action of estradiol, we analyzed differentially expressed genes in the muscle of mdx mice treated either with estradiol or placebo for 4 weeks, and identified the low-affinity receptor for neurotrophins p75NTR as one of the most upregulated genes. p75NTR protein levels were also increased. No concomitant increase in TrkA and neurotrophins expression was observed. Treatment of mdx mice with a small molecule p75NTR ligand also demonstrated effectiveness in improving muscle function and decreasing CK levels. Remarkably, high levels of p75NTR protein were found in skeletal muscle of patients with DMD. These results suggest that modulation of this pathway could become a novel therapeutic strategy for DMD. http://dx.doi.org/10.1016/j.nmd.2017.06.355

P.314 Nintedanib as a new therapeutic agent for Duchenne muscular dystrophy: preclinical in vitro and in vivo studies P. Piñol 1, E. Fernández-Simón 1, X. Suárez 1, N. de Luna 1, A. Molins 1, N. de Oliva 2, A. Martínez 2, L. Escudero 3, D. Sánchez 3, X. Navarro 2, I. Illa 1, E. Gallardo 1, J. Díaz-Manera 1 1 Universitat Autònoma de Barcelona, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain; 2 Universitat Autònoma de Barcelona, Bellaterra, Spain; 3 Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla, Spain

P.316 Taurine: an anti-inflammatory and antioxidant with strong potential benefits for Duchenne muscular dystrophy P. Arthur, J. Terrill, M. Grounds University of Western Australia, Perth, Australia Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease where current treatments, such as corticosteroids, are limited in efficacy and