G.O. 7 Pharmacological chaperones as an alternate treatment for Pompe disease

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T.O. 5 Lack of myostatin results in satellite independent muscle fibre hypertrophy and mitochondrial depletion H. Amthor 1,2,*, M. Schuelke 3, A. Otto 2, K. Patel 2, T. Partridge 4, T. Voit 1, P. Zammit 5 1 University Hospital of Essen, Department of Pediatrics, Essen, Germany; 2 Royal Veterinary College, London, UK; 3 Charite´ University Hospital, Department of Neuropediatrics, Berlin, Germany; 4 Medical Research Council, Hammersmith Hospital, Imperial College, London, UK; 5 Kings College, Randall Division of Cell and Molecular Biophysics, Imperial College, London, UK At the 9th WMS congress in Gothenburg, we reported that despite having a larger muscle mass, myostatin knockout mouse were not stronger compared to wildtypes. In fact they exhibited a significantly decreased specific force when maximum tetanic force generation was expressed as a function of muscle size. Here, we show that muscle from mstn / mouse enlarged predominantly through an increase in individual fibre size. Interestingly, individual muscle fibres from mstn / mouse, although being bigger, contained a similar number of myonuclei compared to wildtype fibres, indicating no increased recruitment of satellite cells during hypertrophic growth. We determined the number of satellite cells attached to single myofibres and found no increase in satellite cells per myofibre from mstn / mouse. We investigated the ability of satellite cells to proliferate by performing single myofibre explant cultures from mstn / and wildtype muscles. Satellite cells from mstn / mouse fibres did not proliferate faster than from wildtype mouse. In a complimentary experiment, myofibres from wildtype mouse were treated with recombinant myostatin protein, which had no effect on satellite cell proliferation. Thus, excessive muscle growth in lack of myostatin did not result from increased satellite cell activity and nuclear density of myostatin depleted fibres was decreased. In extensor digitorum muscle from mstn / mouse, we revealed a fibre type conversion towards fast glycolytic fibres and loss of oxidative fibres. However, staining for the activity of mitochondrial enzymes revealed a decrease in SDH activity that could not be accounted for by sole fibre type conversion. We investigated the ratio of the number of mtDNA (MT-CO1) copies per single copy nuclear gene (Ndufv1) using quantitative real-time PCR and found an about 50% decrease in extensor digitorum longus and soleus muscle from mstn / muscle that was unrelated to fibre type composition. Hence, the reduction of the mtDNA/myonucleus ratio in muscle from mstn / mouse truly reflects a mitochondrial depletion. The effect of the decreased mtDNA/ myonucleus ratio is likely to be enhanced due to the decreased nuclear to cytoplasmic ratio in muscle from mstn / mouse resulting in a greatly reduced number of mitochondria per cytoplasm. We discuss that myostatin limits muscle fibre size and stimulates proliferation of mitochondria and that such effect optimises aerobic metabolism and force output. doi:10.1016/j.nmd.2006.05.252

T.O. 6 Systemic delta-sarcoglycan gene transfer into cardiomyopathic BIO14.6 hamsters by AAV C. Vitiello 1,2, A. Auricchio 1, S. Faraso 1,2, N. Sorrentino 1,2, D. Di Napoli 3, S. Castaldo 3, E. Nusco 1, S. Aurino 1,2, V. Saccone 1,2, G. Piluso 2, V. Nigro 1,2,* 1 Telethon Institute of Genetic and Medicine, Naples, Italy; 2 Dipartimento di Patologia Generale, Seconda Universita` degli Studi di Napoli, Naples, Italy; 3 Centro di Biofarmacologia, Ospedale A. Cardarelli, Naples, Italy

The delta-sarcoglycan (delta-SG) deficient Syrian hamster strain BIO14.6 is one the most used animal model for human primitive cardiomyopathy and muscular dystrophy. The main goal of our project is a rescue of cardiac and muscular functionality of BIO14.6 hamsters using adeno-associated viral (AAV) vectors injection. We observed that therapy could be much more successful in younger animals (16 days old BIO14.6 hamsters), in which we injected 2 · 1012 GC of the AAV2/8 vector expressing the human delta-SG. After four months from the first intraperitoneal injection, we performed a second intravenous injection. We used 3 · 1012 GC in a different serotype (AAV2/1). One month later, we observed a very high expression of delta-SG in the muscular and cardiac tissues and, moreover, an improvement in their muscular functionality under exercise. These results are promising because all the injected animals are viable and to date do not show overt cardiomyopathy. Recently, we used double-stranded (ds) AAV vector for more rapid and efficient AAV-mediated transgene expression. We injected 1 · 1012 GC in younger (10 day-old) BIO14.6 hamsters and the treatment is under study. doi:10.1016/j.nmd.2006.05.253

NEW DEVELOPMENTS ACROSS THE NEUROMUSCULAR FIELD: ORAL PRESENTATIONS G.O. 7 Pharmacological chaperones as an alternate treatment for Pompe disease B.A. Wustman *, R. Khanna, A.C. Powe Jr., R. Dhulipala, E. Benjamin, C.W. Pine, W. Liang, K. Mascioli, H.-H. Chang, M. Toth, P. Huertas, K. Valenzano Amicus Therapeutics, Cranbury, USA Pompe disease (GSD-II) is caused by a deficiency of acid alphaglucosidase (GAA) activity. Juvenile- and adult-onset are the most common forms of the disease, characterized by skeletal muscle (limb girdle) weakness and respiratory failure. The underlying cause of muscle wasting is still unclear since glycogen accumulation does not correlate with muscle weakness. Interestingly, mistrafficking of membrane stabilizing proteins, such as alpha-dystroglycan, sarcoglycans and dystrophin, and their presence in inclusion bodies also containing various UPR-related heat shock proteins have been observed in muscle biopsies. These results suggest that global protein mistrafficking and accumulation may contribute to muscle wasting and disease progression. To test this hypothesis, we characterized various patient fibroblasts for GAA accumulation and global protein mistrafficking. Our results show that these cells indeed have lysosomal proliferation, massive accumulation of mutant GAA in LAMP-1 positive compartments, and formation of perinuclear inclusion bodies that stain positive for GAA and negative for LAMP-1. The number of inclusion bodies and density of lysosomal-like structures increased significantly as the cells reached confluence. XBP-1 splicing assays demonstrate that patient cells are less responsive to the ER stress inducer tunicamycin than wild-type cells, suggesting that disease fibroblasts may have adapted to a state of chronic stress. To determine whether the GAA enzyme activity and protein trafficking can be rescued, fibroblasts were treated with various concentrations of a pharmacological chaperone. Our results show that chaperone treatment yields 4–8-fold increases in GAA activity in several infantile- and adult-onset cell lines. To test for in vivo effectiveness, wild-type mice were treated with increasing doses of chaperone to assess drug accessibility to disease relevant tissues, including skeletal muscles, heart, brain and liver. Our results show an increase in GAA activity in all tissues

Abstracts / Neuromuscular Disorders 16 (2006) 644–726 examined. These results suggest that pharmacological chaperone strategy may be a therapeutic approach for treatment of Pompe disease. doi:10.1016/j.nmd.2006.05.254

G.O. 8 Gene therapy trials in the ovine model of McArdle’s disease J. McC Howell 1,2,*, K.R. Walker 3, J. Nalbantoglou 4, N. Laing 3, G. Karpati 4 1 Department of Veterinary Biology and Biomedical Science, Murdoch University, Perth, WA, Australia; 2 Center for Neuromuscular and Neurological Disorders, University of Western Australia, Perth, WA, Australia; 3 Center for Medical Research, University of Western Australia, Perth, WA, Australia; 4 Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada The ovine model of McArdle’s disease exhibits many of the clinical effects and morphological changes seen in human patients and sheep have a similar muscle mass to humans. Thus the ovine model provides a unique and authentic test bed for therapeutic strategies aimed at treating human McArdle’s disease. Gene therapy trials have been undertaken to deliver myophosphorylase cDNA or LacZ cDNA to the muscle of affected and carrier sheep using AdV5, AAV2 and plasmids. The plasmids were delivered with and without electroporation or sonoporation. All vector construct combinations resulted in transduction. Of the viral vectors more transduced fibres were seen and lasted for a longer time following the use of AdV rather than the AAV vectors and plasmids with electroporation resulted in greater transduction than the use of plasmid alone or plasmid with sonoporation. The phosphorylase produced was active and was confined to the area of the injection sites. The muscle isoform was expressed and the brain and liver isoforms were re-expressed at these sites. The majority of human McArdle’s patients requiring treatment will be adults. A vector capable of transducing mature muscle, an alternate delivery method such as systemic delivery and the overcoming of immune issues will be required for gene therapy to be practical in patients. doi:10.1016/j.nmd.2006.05.255

G.O. 9 Dynamin 2 mutations are associated with dominant intermediate Charcot-Marie-Tooth disease and dominant centronuclear myopathy K.G. Claeys 1,2,*, S. Zu¨chner 3,4, M. Kennerson 5,6, K. Verhoeven 1, C. Ceuterick 7, J.J. Martin 2,7, J. Berciano 8, J.M. Vance 3, G. Nicholson 5,6, V. Timmerman 1, P. De Jonghe 1,2 1 Department of Molecular Genetics, Flanders Interuniversity Institute for Biotechnology, University of Antwerp, Antwerp, Belgium; 2 Department of Neurology, University Hospital Antwerp, Antwerp, Belgium; 3 Center for Human Genetics, Duke University Medical Center, Durham, North Carolina, USA; 4 Department of Neuropathology, University Hospital, RWTH Aachen, Aachen, Germany; 5 Northcott Neuroscience Laboratory, ANZAC Research Institute, New South Wales, Australia; 6 Molecular Medicine Laboratory, Concord Hospital, Concord, New South Wales, Australia; 7 Department of Neuropathology, Institute Born Bunge, University of Antwerp, Antwerp, Belgium; 8 Service of Neurology, University Hospital ‘‘Marque´s de Valdecilla’’, Santander, Spain Dynamin 2 (DNM2) is one of these interesting genes in which distinct mutations can result in different phenotypes. Zu¨chner et al. (2005) reported mutations in the pleckstrin homology domain of DNM2 as

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the genetic cause of dominant intermediate Charcot-Marie-Tooth disease type B (DI-CMTB) in three unrelated families originating from Belgium (K558del), Australia (K558E), and North America (D551_E553del; K550fs). We recently observed an additional DNM2 mutation (G358R) in a Spanish family. Mutations in DNM2 have also been shown to cause autosomal dominant centronuclear myopathy (CNM; Bitoun et al., 2005). Interestingly, the mutations in CNM cluster in the middle domain of the DNM2 protein, outside the pleckstrin homology domain, suggesting that the location of the mutation is crucial for the underlying pathomechanism. Although DI-CMTB and CNM are both neuromuscular disorders, their phenotypes are clearly distinct. In contrast to classical CMT1 and CMT2, families with DICMT have a broad range of nerve conduction velocities from normal (>49 m/s for the motor median nerve) to 25 m/s. In the current study, we examined the clinical and electrophysiological phenotypes in the patients of the DI-CMTB pedigrees. Interestingly, in the Australian and Belgian families, which carry two different mutations affecting the same amino acid (Lys558), CMT cosegregated with neutropenia, which has not previously been associated with CMT neuropathies. We also observed a DNM2 mutation (R465W) in two apparently unrelated CNM families. Patients in our CNM pedigrees initially presented with a distal myopathy that in one proband mimicked CMT because the patient also had pes cavus. However, electromyography showed clear myogenic changes in the distal muscles, and a muscle biopsy showed the typical abnormalities observed in CNM. In one of the families, patients later on also developed severe proximal weakness in both lower and upper limbs. In one patient restricted opening of the mouth was a striking feature. We describe in more detail the clinical, electrophysiological, and neuropathological findings in our DI-CMTB and CNM families. doi:10.1016/j.nmd.2006.05.256

G.O. 10 Dynamin 2 mutations and impairment of EGF-induced MAPK activation M. Bitoun 1,*, J.A. Bevilacqua 1, B. Prudhon 1, S. Maugenre 1, F. Lubieniecki 2, S. Monges 2, A.L. Taratuto 2, M. Fardeau 1, B. Eymard 1, E. Uro-Coste 3, N.B. Romero 1, P. Guicheney 1 1 Unite´ INSERM 582, Institute of Myology, CHU Pitie´-Salpeˆtrie`re, Paris, France; 2 Hospital Nacional de Pediatria J.P. Garrahan, Buenos Aires, Argentina; 3 Service d’Anatomie Pathologique, CHU Rangueil, Toulouse, France Centronuclear myopathy (CNM) is a rare congenital myopathy characterized by delayed motor milestones, facial and muscular weakness often associated with ptosis and ophthalmoplegia. Considerable variability in the phenotypic manifestations has been observed ranging from benign classical forms, with slowly progressive myopathy during adolescence or later, to more severe neonatal presentation. The characteristic muscle histopathology comprises a triad constituted by centrally located nuclei, type 1 fibre predominance and hypotrophy, and a typical arrangement of sarcoplasmic strands radiating like spokes of a wheel from the centre to the periphery of the fibres. Recently, we identified four missense mutations in dynamin 2 gene (DNM2, 19p13.2) in patients affected by autosomal dominant form of CNM. DNM2 is a large GTPase involved in endocytosis and membrane trafficking, centrosome cohesion and Mitogen-Activated Protein Kinase (MAPK) activation. These mutations were restricted to the middle domain of the DNM2, known as responsible for the centrosome targeting of the protein and we showed a reduced labelling of the transfected mutants in the centrosome. Here, we report two additional DNM2 mutations in the Pleckstrin Homology (PH) domain which mediates interaction with phosphoinositides in patients with a more severe phenotype. Mutations of this domain have been recently identified in the dominant