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Wms25 2013.Pdf Abstracts / Neuromuscular Disorders 23 (2013) 738–852 841 patients, such as prednisone, will be screened using the newly established When compared to the mdx muscles, histopatological features of both efficacy study platform. Sgcb-null and age-matched mdx mice were similar at all examined ages except that in Sgcb-null mice the extent of connective tissue was generally http://dx.doi:10.1016/j.nmd.2013.06.697 greater. This was particularly evident in the quadriceps muscle where the endomysial connective tissue was prominent and the extent of the various collagens was significantly greater in the Sgcb-null mice at all ages com- pared to mdx. Furthermore, differently than in the Scgb-null mouse, where P.20.8 the amount all of three collagen isoforms increased steadily, in the mdx AAV genome loss from dystrophic mouse muscles during AAV-U7snRNA- they remained stable. mediated exon skipping therapy The Sgcb-null mouse represents a useful model for evaluating the path- M. Le Hir 1, A. Goyenvalle 2, C. Peccate 1, G. Pre´cigout 2, K.E. Davies 3, 1 2 1 ogenetic mechanisms of muscle fibrosis and for development of anti-fibro- T. Voit , L. Garcia ,S.Lorain 1 Association Institut de Myologie Hospital Pitie Salpetriere, Um76 UPMC tic treatments. – UMR 7215 CNRS – U974 Inserm – Institut de Myologie, Paris, France; 2 Universite´ de Versailles Saint-Quentin-en-Yvelines, UFR des http://dx.doi:10.1016/j.nmd.2013.06.699 sciences de la sante´, Montigny-le-Bretonneux, France; 3 University of Oxford, Department of Physiology, Anatomy and Genetics, Oxford, United Kingdom P.20.10 In the context of future AAV-based clinical trials for Duchenne myop- Human adipose mesenchymal stem-cells injections in golden retriever mus- athy, AAV genome fate in dystrophic muscles is of importance considering cular dystrophy (GRMD) dogs: a four-year follow-up the viral capsid immunogenicity that prohibits recurring treatments. We M. Zatz, N. Vieira, M. Secco, E. Zucconi, M. Valadares, C.R. Bueno, M. followed AAV genome copy numbers after AAV-U7 delivery in the Vainzof, J. Gomes, V. Landini, T. Andrade mdx dystrophic mouse. We showed that AAV genomes encoding non- University of S. Paulo, Human Genome Research Center, Sa˜o Paulo, therapeutic U7 were lost from mdx muscles within three weeks after Brazil intra-muscular injection. In contrast, AAV genomes encoding U7ex23 restoring expression of a slightly shortened dystrophin were maintained The possibility to treat Duchenne dystrophy (DMD) through cell ther- endorsing that the arrest of the dystrophic process is crucial for maintain- apy has been widely investigated but before starting clinical trials in ing viral genomes in transduced fibers. Indeed, muscles treated with low humans many questions still need to be addressed in pre-clinical studies. doses of AAV-U7ex23, resulting in sub-optimal exon-skipping, displayed In addition to clarifying the role of the exogenous cells in dystrophic mus- much lower titers of viral genomes, showing that sub-optimal dystrophin cle, one important concern is whether there is an increased risk of tumors restoration does not prevent AAV genome loss. We also followed thera- or any other long-term side effects following stem-cells injections. Since peutic viral genomes in severe dystrophic dKO mice over time after sys- murine models have a short life span, the best animal model to address temic treatment with scAAV9-U7ex23. Dystrophin restoration decreased these questions is the Golden Retriever Muscular Dystrophy (GRMD) significantly between 3 and 12 months in various skeletal muscles, which dog, which reproduces the full spectrum of human DMD. They present was correlated with important viral genome loss, except in the heart. Alto- clinical signs within the first weeks of life and most of them do not survive gether these data show that the success of future AAV-U7 therapy for beyond age two. However, due to their phenotype variability each dog Duchenne patients would require optimal doses of AAV-U7 to induce must be compared separately before and after any experiment. We have substantial levels of dystrophin to stabilize the treated fibers and maintain injected human adipose-derived mesenchymal stromal cells (hAMSCs), the long lasting effect of the treatment. from two different donors, in three GRMD dogs, without immunosup- pression. They were injected in the cephalic vein with 5 Â 107 cells/kg http://dx.doi:10.1016/j.nmd.2013.06.698 within their first year of life: weekly in the first month of injections and then monthly. Dolar and Yuan were born in September of 2008 and Rum in July of 2011. In Dolar and Yuan, who received cells from donor 1, we observed that hAMSCs injected systemically were able to reach and P.20.9 express human dystrophin in the host GRMD dystrophic muscle up to Muscle fibrosis in the sgcb-null mouse model versus the mdx model 6 months after transplantation. When cells from donor 2 were injected S. Gibertini, S. Zanotti, M. Curcio, P. Savadori, M. Mora in Rum, no human dystrophin was found in the host muscle. However, Fondazione IRCCS Istituto Neurologico C. Besta, Neuromuscular Diseases the three dogs showed clinical improvement following hAMSCS injections and Neuroimmunology, Milano, Italy with an apparent stabilization afterwards, suggesting a beneficial effect The Sgcb-null mouse model, with beta-sarcoglycan knocked-down, from factors released by the injected cells. Most importantly, after almost develops severe muscular dystrophy with early fibrosis like the human four years of follow-up for Dolar and Yuan there was no tumor or other limb girdle muscular dystrophy type 2E. The mdx mouse, lacking dystro- side effect suggesting that hAMSCS transplantation is a safe procedure, phin, is the most used model for Duchenne muscular dystrophy (DMD). which may have important applications for future therapy in muscular Unlike DMD, the mdx mouse has mild clinical features and shows little dystrophy patients. endomysial fibrosis in limb muscles. We have characterized the progres- http://dx.doi:10.1016/j.nmd.2013.06.700 sion of muscle fibrosis, at molecular and histopathological level, in the Sgcb-null mouse and compared results to findings in the mdx. We evalu- ated expression of collagen I, III and VI, and of decorin and TGFbeta1 by immunohistochemistry or immunoblotting, and transcript levels by Real- P.20.11 Time PCR, in the quadriceps and diaphragm muscles, at 2, 4, 8, 12, 26 and Dystrophin rescue needed to recover a correct location of nNOS and the 52 weeks in the sgcb-null mouse, and at 12, 26 and 52 weeks in the mdx. return to a normalized RyR1 status in treated GRMD dogs We found severe histopathological features from 4 weeks on, and concom- C. Gentil 1, C. le Guiner 2, Y. Cherel 3, M. Montus 4, P. Moulier 2, itant deposition of extracellular matrix components in the Sgcb-null L. Servais 5, T. Voit 1,F.Pietri-Rouxel 1 mouse, with collagen I, III and VI significantly increased since early ages 1 UM76-UPMC/ U974-Inserm/UMR7215-CNRS – Institut de Myologie, both in the quadriceps and diaphragm. Paris, France; 2 Inserm UMR 1089/Atlantic Gene Therapies, Universite´ de 842 Abstracts / Neuromuscular Disorders 23 (2013) 738–852 Nantes IRT1 8, Nantes, France; 3 INRA UMR 703/Oniris Atlantic Gene tological pattern was assessed by histomorphological analysis of the Therapies, Nantes, France; 4 Genethon, Evry, France; 5 Institut de Myol- immunological detection of dystrophin expression, sclerosis (immunopex- ogie, Department of Therapeutic Trials and Databases, Paris, France oxydase against Collagen I), regeneration (developmental Myosin Heavy Chain), calcium accumulation, anisocytosis, macrophagic infiltration, The most promising gene therapy approaches to treat Duchenne Mus- and lymphocytic infiltration. The dystrophin expression was characterized cular Dystrophy (DMD) is currently the use of the exon skipping strategy by a diffuse pattern with single or small groups of positive fibers in most as shown by clinical trials underway. However, truncated dystrophins fascicles. A significant decrease of sclerosis in muscles exhibiting more resulting from exon skipping may be more or less efficacious depending than 60% of dystrophin positive fibers and an important decrease of regen- on the effect of the truncation on the protein function. In particular, the eration (in muscles containing up to 30% of dystrophin), was observed. neuronal nitric oxide synthase (nNOS) binding site to the dystrophin These results show the efficiency of the rAAV8-U7 strategy to restrict has been defined on the repeats 16 and 17 (R16/17). Considering that the natural degradation of the dystrophic muscle. R16/17 are encoded by exons 42–45 of dystrophin mRNA, an alteration Supported by the Association Francßaise contre les Myopathies and in this zone could impact on the integrity of the nNOS binding site pre- Advanced Diagnostics for New Therapeutic Approaches, a program ded- venting the proper binding of nNOS to dystrophin. icated to personalized medicine, coordinated by Institut Me´rieux and sup- Several studies have provided convincing evidence that mis-localiza- ported by OSEO. tion of nNOS and cytosolic NO impair skeletal muscle contractibility via nitrosylative modification of the sarcoplasmic reticulum calcium- http://dx.doi:10.1016/j.nmd.2013.06.702 release-channel RyR1. These data point towards nitrosylation-induced leakiness of the RyR1 as a molecular mechanism underlying muscle weak- ness and highlight that the dystrophin rescue has to take into account its functional properties such as nNOS binding. P.20.13 Considering these facts, we have evaluated the level of dystrophin res- Gene therapy of Duchenne Muscular Dystrophy using rAAV vectors: Exon toration needed to recover, qualitatively and quantitatively, a correct loca- skipping and microdystrophin approaches in GRMD dogs tion of nNOS. For this study, we have collected biopsies from GRMD C. Le Guiner 1, M. Montus 2, L. Servais 3, Y.
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