Resumos que serão apresentados no 10o Congresso Anual da Sociedade Mundial de Músculo que será realizado no Brasil de 28 de setembro a 1 de outubro de 2005 em Foz do Iguaçu.


A new original compound, which inhibits calpain 1-2 and reactive oxygen species, reduces the dystrophic progression of mdx mice

B. Pignol, R. Burdi, M. Didonna, J.F. Rolland, P. Ferro, P. Confalonieri, R. Cooper, G. Butler-Browne, A. De Luca – France and Italy


Calpain activation and overproduction of deleterious reactive oxygen species (ROS) are often associated with cell death in neuromuscular disorders. We have previously demonstrated that the association of calpain inhibitors with antioxidants act in a synergistic manner to protect the cell

against death (7th WMS, G.P.3-6). The aim of this study was to demonstrate the potential beneficial effect of a compound which possesses a dual activity (antioxidant and anti-calpain1-2) both in vitro on the model of Human skeletal muscle (HSM) cells death induced by influx of calcium

and in vivo on mdx mice. In vitro: BN82270 inhibits calpain activity, lipid peroxidation and totally protects HSM cells against death by maitotoxininduced calcium influx, with higher efficiency compared to methylprednisolone, a drug clinically used in DMD patients. In vivo: dystrophic mdx

mice were treated with the drug at 30 mg/kg/day s.c for 3–6 weeks (a dose which acutely reduces muscle calpain activity). In diaphragm muscle, BN 82270 protects healthy fibers (peripherally located nuclei) and decreases the percentage of diseased fibers with centrally located nuclei (degenerated/regenerated at least once) or necrosis. Functional measurements were performed after chronic exercise on a treadmill, which markedly worsen the dystrophic progression. BN82270 fully counteracted the exercise-induced fore limb strength observed in mdx mice. Ex vivo, the treatmentsignificantly reduced (46+14%) the elevated creatine kinase (CK) plasma level used as a diagnostic sign of muscular dystrophy. Also, ELISA analysis showed the ability of BN 82270 to markedly reduce the pro-fibrotic cytokine Transforming Growth Factor-b1 (TGFb-1), in both mdx

diaphragm and limb muscles. Conclusions. The beneficial effect of BN82270 on many dystrophic signs of mdx mice reinforces the role of calpain/ROS in the pathology and indicates that this approach may lead to drug candidates for Duchenne Muscular Dystrophy treatment in the future.



Tamoxifen increases muscular strength of the mdx dystrophic mice

D. Feder, A. Cavalsan, R.F. Vasquez, R.C. Lin, S.B. Zyngier, F.J. Velloso, L.L. Fogaca, H.C. Silva, M. Vainzof – São Paulo - Brazil


The mdx mice is a well-known model of Xp21 dystrophin-deficient muscular dystrophy. Although a good genetic and biochemical model, the mdx shows no muscle weakness, but under physical exercise, a loss of muscular strength can be detected. Here, we have tested the possible therapeutic beneficial effect of Tamoxifen in the degenerative process of the dystrophic muscle, analyzing muscle strength of tamoxifen treated mdx mice, under intensive physical exercise. Twenty-two mice aged 4 weeks were divided into three groups: control (n=8) treated with 0.5 mL saline ip, Tamoxifen 5 mg/kg body weight ip (n=8) daily and Tamoxifen 10 mg/kg ip

(n=6) daily. The exercise protocol was done in a wheel revolving at 18 cm/s, for 10 min, twice a day, 5 days/week, up to 12 weeks. Whole-body strength was measured weekly using a force transducer coupled to a computer. The mice treated either with 5 mg/kg (6.26+1.44 dynes/g body

weight) or 10 mg/kg (6.46+2.52) showed a significant increase of muscular strength (P<0.05) compared to the control group (3.66+0.77) starting in the 5th week, and maintaining significance up to the end of the experiment. Histological and histochemical analysis of the complex of the gastrocnemius muscle are under analysis, but preliminary results suggest a less degree of degeneration in the Tamoxifen treated groups. Tamoxifen is an anti-tumoral drug and act on TGF-beta. Its possible therapeutic effect in the degenerative process of the dystrophic muscle could ameliorate the clinical course of dystrophic patients. FAPESP-CEPID, CNPq, PRONEX, NEPAS-FMABC.



Effects of NF-kB blockade on muscle gene expression in mdx mice

S. Messina, M. Aguennouz, N. Macellani, M.C. Monici, D. Arcelli, P. Seminara, S. Volinia, F. Squadrito, G. Vita - Italy


Nuclear Factor Kappa-B (NF-kB) is a major transcription factor modulating the cellular immune, inflammatory and proliferative responses. Previous studies provided evidences of a pathogenic role played by activation of NF-kB in mdx mice and Duchenne muscular dystrophy (DMD). We have recently demonstrated that treatment with IRFI 042, a vitamin E analogue inhibiting NF-kB, ameliorates muscle function, decreases serum CK level and muscle necrosis and enhances regeneration in mdx mice. The aim of the study was to identify the genes differentially expressed in skeletal muscle of IRFI 042-treated mdx mice, searching for a molecular hallmark responsible for the treatment positive effect. Microarray experiments were performed on leg muscles using a GeneChips microarray slide with a whole mouse genome of 44K genes (Agilent Technologies,

Italy). Selective chemokine upregulation was further studied by RT-PCRand immunoblot. Results in vehicle-treated animals provided evidence for coordinated activity of numerous components of a chronic inflammatory response, including cytochine and chemochine signaling, leukocyte adhesion and complement system activation. Moreover, genes of extracellular matrix and those involved in regeneration process were upregulated. After treatment with IRFI 042 we noticed a decreased expression of chronic inflammatory response components and an enhanced expression of genes involved in repair processes. These data better clarify the mechanisms underlying the NF-kB blockade-induced benefit in mdx mice and suggest new treatment avenues in DMD.



Efficiency of splicing inversely correlates with clinical severity in dystrophinopathy due to deep intronic splice site activation mutations

T. Tuohy, C. Anderson, K. Hart, M.T. Howard, K.M. Flanigan, - Utah –USA


Purpose: Both Becker and Duchenne Muscular Dystrophy (BMD and DMD) can be caused by deep intronic mutations which create splice sites resulting in the inclusion of pseudoexons in the DMD mRNA. Such mutations account for at least half of the 5–7% of mutations not detected in

lymphocyte-derived DNA samples by modern mutation detection methods. We detected two such mutations in patients with dystrophinopathy. One patient, with BMD, carries an intron 11 mutation (c.1331+17770C>G) which creates a splice donor site, resulting in the incorporation of a 79 nucleotide (nt) out-of-frame insertion. A second patient, with DMD, carries an intron 45 mutation (c.6614+3313G>T), resulting in a 139 nt out-offrame insertion. We hypothesized that the milder phenotype may result from less efficient alternate splicing. Methods: In order to investigate the

relationship of splicing efficiency to phenotype, quantitative RT-PCR was performed using forward amplification primers which span the junction between the wild type exon (11 or 45) and the pseudoexon (1 k or 45a) and compared to amplification with primers spanning the wild-type (11–12 or 45–46) junction. Each reaction product was normalized against the EEF gene, and compared to results found in normal control muscle tissue. Results: In the DMD sample, the alternatively-spliced transcript was present at the level of 41% compared to a normal control, and no wild-type transcript was detected. In the BMD sample, the alternatively spliced transcript was present at the level of 40%, but the wild type transcript was detected at a level of 13%. These results suggest that clinical phenotype is dependent upon splicing efficiency in the setting of cryptic splice site mutations.



Effective correction of the mdx dystrophin gene mutation in the mouse model of Duchenne muscular dystrophy

M. Todaro, S.H.A. Wong, M. Kita, A.F. Quigley, K.N. Lowes, R. Marotta, M.J. Cook, A.J. Kornberg, R.M.I. Kapsa – Melbourne - Australia


Targeted correction of mutations in the genome holds great promise for the repair/treatment of disease causing mutations either on their own applied directly to the affected tissue, or in combination with other techniques such as stem cell transplantation. Various DNA or RNA/DNA

based Corrective Nucleic Acid (CNA) molecules such as chimeraplasts single stranded oligonucleotides triplex forming oligonucleotides and SFHR have been used to change specific mutant loci. Significant variation in the success of these techniques from laboratory to laboratory can be attributed to factors such as differences between application protocols, loci and/or target cell/tissue type. However, a number of recent publications have highlighted the potential of PCR-based locus detection methods to generate artifactually over-estimated positive evidence of mutation correction. This potential for artifact universally affects all of the mutation correction methods in use today, and usually arises from the presence of the corrective nucleic acid after extraction of DNA from the treated cells. It is important to establish however that in all instances, such artifact may be eradicated from results by relatively simple but effective controls/steps to

provide accurate information regarding the effectiveness of protocols in promoting mutated locus correction. This study reports the steps that prevent artefractual contamination of genuine correction data and highlights the use of gene correction in vivo in dystrophic mdx mouse muscle



Green tea polyphenols improve histology and mechanical properties of skeletal muscle in the mdx5Cv dystrophic mouse

O.M. Dorchies, S. Wagner, T.M. Buetler, O. Vuadens, P. Kucera, U.T. Ruegg – Switzerland


In Duchenne Muscular Dystrophy (DMD), the cytoskeletal protein dystrophin is absent leading to numerous cellular dysfunctions that culminate in muscle cell necrosis. Subsequently, an inflammatory response develops in the necrotic muscle tissue, resulting in increased oxidative

stress, responsible for further tissue damage. In the mdx dystrophic mouse, both inflammation and oxidative stress have been identified as aggravating factors for the course of the disease. Indeed, we have shown previously that Green Tea Extract (GTE), a mixture of powerful antioxidant polyphenols could reduce muscle necrosis in mdx mice. Here, we investigated the ability

of 2 doses of GTE and of its major constituent, (K)-epigallocatechin gallate (EGCG), in improving mdx5Cv muscle function and histology. Threeweek- old mdx5Cv mice were fed for either 1 or 5 weeks with a control chow or a chow containing GTE or EGCG. Normal C57Bl/6J mice were used as control. Muscle histology showed that GTE and EGCG given for 1 week reduced muscle necrosis. After a 5-week treatment, the muscle histology from treated and untreated mice was similar, although plasma total antioxidant potential was increased. Electrically evoked mechanical

properties of the triceps surae were recorded. Phasic and tetanic forces of treated mice were increased to values developed by normal mice. Moreover, muscles from treated mice displayed improved resistance to fatigue (+30 to +50% of untreated). These results suggest that administration of GTE or EGCG to mdx5Cv mice (i) caused a delay in muscle necrosis, and (ii)

stimulated muscle adaptation towards a slower or more resistant phenotype.



Green tea polyphenols promote mouse skeletal muscle cell maturation in vitro

O.M. Dorchies, C. Hirn, P. Mittaud, J. Reutenauer, G. Schiffmann, O. Vuadens, U.T. Ruegg - Swirzerland


Our work aims at discovering novel palliative treatments for Duchenne muscular dystrophy (DMD). Recent findings indicate that green tea extract (GTE) and its major polyphenol ( ) epigallocatechin

gallate (EGCG) are serious candidates for this purpose. Using the mdx5Cv dystrophic mouse as a model for DMD, we have shown previously that GTE and EGCG protect skeletal muscle from necrosis and enhance muscle force and resistance to fatigue. Moreover, we have demonstrated that these substances protect cultured myotubes from oxidative stress induced cell death. Here, we report that GTE and EGCG also display unexpected pro myogenic properties. Primary cultures of skeletal muscle cells were established from both normal and dystrophic mice and treated with GTE and EGCG for 1–7 days. As judged by in situ staining of myosin heavy chains (MyHC), we found

that GTE and EGCG concentration-dependently stimulated the rate of formation of myotubes within the first 2–4 days of application. The amount of myotubes reached similar level with both agents compared to control thereafter. Western-blot analysis was performed on myotube cultures treated for 7 days. GTE and EGCG promoted the expression of several muscle-specific proteins, such as dystrophin (in control cultures), sarcomeric alpha actinin, and MyHC, while myogenin was unchanged. By contrast, the expression of desmin was down-regulated and redistributed to Z discs. Our results suggest that green tea polyphenols display pro myogenic properties by acting directly on skeletal muscle cells. These findings suggest a beneficial action for muscle regeneration and strengthening in dystrophic condition.



Evaluation of the therapeutic effect of insulin-like growth factor 1 on muscular dystrophy by gene expression analysis

S. Noguchi, M. Fujita, T. Sasaoka, I. Nishino - Japan


Several therapeutic studies, such as gene therapies, cell implantations and pharmaceutical therapies, have been attempted toward muscular dystrophies. Recently two growth factor-related molecules were highlighted by showing the effects on improvement of dystrophic phenotype of

mdx mice. One was insulin-like growth factor-1 (IGF-1), and another was myostatin. In this study, we used sarcoglycanopathic mice (sgcbK/K mouse at 3–5 weeks) and tried to evaluate the effect of IGF-1 administration on their dystrophic phenotypes by pathological observation and comprehensive gene expression analysis. We administered recombinant IGF-1 by two ways: by subcutaneous injection for a week and by osmotic pump implanted subcutaneously for 4 weeks. In pathological examination, by both administrations, the gastrocnemius muscles showed remarkable

reduction in necrotic fibers and increase in centrally nucleated fibers. In gene expression analysis using oligonucleotide array with 22,000 gene probes, 544 genes were changed in expression after these treatments (P < 0.05). Inflammatory response-related genes and translation-related genes

were principally upregulated and muscle contraction-related genes were downregulated. By ANOVA test, expression pattern of the genes with varied expression is similar to that in older sgcbK/Kmice with later stage of dystrophic phenotype. Furthermore, by immunostaining, not Akt but MAPK activation was observed in treated muscles. These results suggest that IGF-1 contributes to the improvement of dystrophic phenotype of sgcbK/Kmouse by enhancement of regeneration process via activation of MAPK pathway.



Are CD34C stem cells from umbilical cord able to produce muscle dystrophin in vitro?

T. Jazedje, E. Zucconi, V.A. Nunes, N.M.S. Vieira, L. Marti, A.F. Ribeiro, T.R. Gollop, E. Ferreira, M. Vainzof, M. Zatz – São Paulo - Brazil


Muscular dystrophies include an heterogeneous group of genetically determined progressive disorders of the muscle which present with a great clinical variability ranging from very severe childhood forms to mild ones with adult onset. The most common and severe is X-linked Duchenne

Muscular Dystrophy (DMD), caused by mutations in the dystrophin gene and total absence of dystrophin protein in muscle. Becker Muscular Dystrophy (DMB), also caused by mutations in the dystrophin gene, is milder than DMD because patients have some functional dystrophin in their

muscles. Many different gene therapy approaches are currently under investigation trying to correct the defective gene. The possibility to replace the abnormal muscle by a normal one through stem cells transplantation represents a new alternative with the advantage to be useful for different

forms of muscular dystrophy independently of the primary gene defect. The objective of the present research was to analyze the potential of umbilical cord blood CD34C stem cells to differentiate in muscle cells and express dystrophin, in vitro. The results suggest that CD34C stem cells fuse to

muscle cells in culture and express dystrophin, although the expression seems to be relatively low. Besides, we observed a spontaneous differentiation of some CD34C stem cells in dystrophin producers muscle cells, suggesting that this type of progenitor cell has the potential to be

committed to muscle. However, studies with animal models will be necessary to evaluate the real potential of these cells for the treatment ofmuscular dystrophies. Supported by: FAPESP-CEPID, CNPq



Analysis of umbilical cord blood stem cell transplantation and dystrophin expression in golden retriever muscular dystrophy dogs

E. Zucconi, V.A. Nunes, T. Jazedje, N.M.S. Vieira, M.D.F. Carvalho, C.E. Ambrosio, M. Vainzof, M.A. Miglino, M. Zatz – São Paulo - Brazil


Duchenne muscular dystrophy (DMD) is an X-linked inherited disorder, caused by mutations in the dystrophin gene that leads to muscle fiber necrosis and premature death. Although great efforts, no effective therapy is available for this disease; nonetheless, allogenic cellular therapies have been proposed as a new strategy to treat DMD. To evaluate whether umbilical cord blood stem cells could contribute to skeletal muscle repair at clinically significant levels, we are investigating GRMD (Golden Retriever Muscular Dystrophy) dogs, that present a severe dystrophin deficiency and a comparable form of the human disease. Hematopoietic cells were collected from fresh cord blood of newborn dogs and mononuclear fraction was separated by gradient of density (dZ1.077). A

subpopulation of stem cells that present the myogenic marker CD34 was isolated by immunomagnetic sorting and characterized by flow cytometry. Healthy DLA-identical cells were used as grafts (via local or systemic) in 2–10-months-old immunosuppressed dogs. To verify the dystrophin expression, immunofluorescence staining and western blotting were performed in pre and posttransplantation muscle biopsies collected at 2 and 6 months after transplantation. Muscle frozen sections taken after stem cell transplantation and labeled with anti-dystrophin antibody showed a small number of dystrophin-positive fibers with a different pattern of distribution and labeling when compared with samples from the same dog before transplantation. Analysis of protein expression in muscle biopsies by western blotting indicated the presence of a faint band of dystrophin. Further analyses are currently underway to verify if this protein is due to revertant fibers or if it has an exogenous origin. Supported by FAPESP,CEPID, CNPq.



Skeletal muscle remodelling by murine adipose and skin-derived mesenchymal stem cells

K. Lowes, M. Kita, A. Quigley, M. Cook, A. Kornberg, R. Kapsa – Melbourne - Australia


Mesenchymal stem cells (MSCs) are multipotential cells that have shown promise as a potential source of cells for cell replacement strategies. We have isolated Sca-1C, CD45-, CD34-, CD31- and c-kit- putative MSCs from murine adipose tissue and skin, termed mAdSs and mDmSs,

respectively, by culture in a highly defined, serum-free growth medium. To investigate their muscle remodelling potential, mAdSs and mDmSs isolated from ROSA(GT)26 mice were engrafted into gastrocnemius muscles of mdx mice, a model of Duchenne Muscular Dystrophy. Muscles were analysed 5 weeks post-transplantation for expression of b-galactosidase and dystrophin. b-galactosidaseC/dystrophinC myofibres were observed in mice transplanted with both mAdSs and mDmSs although there was marked variation in success of engraftment. These data show that

mDmSs and mAdSs have the capacity to remodel non-self tissue. The b- galactosidaseC/dystrophinC fibres were located away from the engraftment site in regions of myofibre ablation suggesting that dispersion of donor cells into the muscle parenchyma as well as appropriate regenerative environmental cues are necessary for successful engraftment.

We are currently investigating means of optimising the remodelling capacity of mAdSs and mDmSs including various donor and recipient muscle pre-conditioning regimens. Additionally, cells expressing b-galactosidase were observed in the injection tract of all mice suggesting

that many mAdSs and mDmSs survived the engraftment procedure and remained viable for at least 5 weeks post transplantation. Long-term survival of the engrafted cells suggests that mAdSs and mDmSs may also be ideal vehicles for gene therapy applications in muscle.



Neurogenic and myogenic remodelling capacity of muscle derived stem cells

A.F. Quigleya, M. Kita, K. Lowes, P. Smith, K. Sweerts, A.J. Kornberg, M.J. Cook, R.M.I. Kapsa – Melbourne - Australia


Neuromuscular disorders typically involve the loss of neuronal and muscle tissues. Cell replacement strategies potentially provide an effective treatment for cell loss in neuromuscular disorders; however, issues with immune rejection of non-host cells limit therapeutic application. We are developing autologous cell replacement strategies for neuromuscular and neurodegenerative disorders such as Duchenne Muscular Dystrophy. We investigated the possibility that muscle contains a population of cells capable of remodeling both skeletal and neuronal tissues. A population of predominantly non-adherent myospheres was isolated from the skeletal muscle of C57Bl6-GT(Rosa)26 mice and expanded in defined culture conditions to maintain a Sca-1C, CD34-, CD45- expression profile. RTPCR analysis of myospheres showed absence of myogenic markers such as GDF8, Pax3 and Pax7 and expression of stem cell markers such as nestin

and prominin. Myospheres were transplanted into the left lateral ventricle, or the hippocampus, of C56BL6 neonate or adult mice respectively. Brains were removed 3 weeks post transplantation and analysed for the presence of beta-gal positive cells. A number of donor derived beta-galC cells were observed in the lateral ventricle or the hippocampus in treated mice. Myospheres also displayed muscle-remodeling capacity when injected into  the gastrocnemius of irradiated mice. Treated muscles showed the presence of beta-galC/dysC immature muscle fibres. These experiments raise two possibilities that myospheres are a heterogeneous cell population of which

subpopulations independently remodel neural and muscle tissues, or that these cells are multipotent and have the potential to remodel both tissues.



Expression profile shifts in clonal populations of muscle derived stem cells

R.M.I. Kapsa, A.F. Quigley, M. Kita, K.N. Lowes, M.J. Cook, A.J. Kornberg – Melbourne - Australia


The myodegenerative disorder Duchenne Muscular Dystrophy (DMD) affects 1 in every 3500 live-born males, and is caused by the functional absence of a dystrophin due to frame-shift or nonsense mutations in the 2.5 MB DMD gene located at Xp21.1. Restoration of functional dystrophin in dystrophic mdx mouse muscle (nonsense mutation) has been achieved to varying levels of success by cell transplantation, viral and non viral gene therapies. Our strategy for dystrophin restoration in dystrophic muscle involves Targeted Corrective Gene Conversion (TCGC) in mature, nondividing

muscle fibres, combined with ex-vivo TCGC of mutations in cell types that display optimal myoremodelling capacity, followed by transplantation of corrected cells into the dystrophic muscle (see Todaro et al., this conference). This forms the basis of an autologous cell transplantation protocol that may be developed in the mdx mouse model for hereditary neuromuscular disorders. One of the issues compromising autologous transplantation in muscular dystrophy is a depleted pool of myogenic precursor cells in the affected individual’s dystrophic muscle. We investigated clonal populations of myogenic stem cells to determine if and how single muscle-derived stem cells can give rise to populations of muscle precursors capable of reconstituting muscle to different extents. Myoblasts were derived from C57BL10 mice and isolated using the preplate (pp) method. PP6 cells were extensively passaged before a single cell was isolated and expanded to a clonal population in myogenic media. This clonal population was fractionated based on adherence, and grown under specific media conditions. Transcript analyses showed that expression of myogenic markers such as myf-5, myf-6, pax-7 and myo-D in all populations was similar to the parental culture; however, there was marked variation in the expression of the stem cell marker Sca-1 (0-30%). Similarly, all clonal cell populations were negative for CD45, CD34, CD31 and c-kit by FACS analysis. This study demonstrates that single clones isolated from a population of MDSCs are capable of reconstituting heterogeneous MDSC populations with an associated difference in their respective muscle remodeling potential.



Design of U7 snRNA for skipping of exon 51 in DMD: a promising tool for future clinical trials

A. Goyenvalle, A. Vulin, F. Leturcq, J.C. Kaplan, O. Danos, L. Garcia – Paris - France


Background: Most cases of Duchenne muscular dystrophy (DMD) are caused by dystrophin gene mutations that disrupt the mRNA reading frame. In some cases, forced exclusion (skipping) of a single exon can restore the reading frame, giving rise to a shorter, but still functional dystrophin protein (so called quasi-dystrophin). One potential treatment of the disorder has utilized antisense oligoribonucleotide (AO) to induce removal of diseaseassociated exons during pre-mRNA processing. Indeed, this approach has been successfully used in DMD cells in vitro with antisense sequences against splice junctions of exon 51. Skipping of this exon would theoretically restore a functional quasi-dystrophin in a significant subset of DMD patients with D45-50, D47-50, D48-50, D49-50, D50 and D52 genotypes. However, since the AO are not self-renewed, they cannot

achieve long-term correction. To overcome this limitation, we have introduced antisense sequences into small nuclear RNAs (snRNA) and vectorized them in AAV and lentiviral vectors. Method: We have designed AAV and lentiviral vectors harboring chimeric U7 snRNA carrying

antisense sequences against exon 51 of the human dystrophin gene (U7- ex51). Lentiviral vectors expressing this U7-ex51 were tested on human myoblasts, whereas AAV vectors were injected in the transgenic hDMD mice (in collaboration with J. Van Deutekom, Leiden, NL). Results: We

confirmed the skipping of the exon 51 in vitro in human myoblasts after transduction with the lentiviral vector encoding U7-ex51 by RT-PCR. We also detected the skipping of the exon 51 after intramuscular injection of an AAV-U7ex51 vector in the transgenic hDMD mouse. We have also tested the efficacy of these vectors to restore dystrophin expression in myoblasts from patients with D48-50 and D49-50 deletions. Conclusion: In this study, we provide evidence that efficient skipping of exon 51 can be achieved in human cells and also in vivo after intramuscular injection in a transgenic hDMD mice through U7snRNA shuttle. These results offer very promising tools for clinical treatment of DMD.



Dystrophin rescue by exon-skipping in the canine model of DMD

A. Vulin, A. Goyenvalle, S. Blot, O. Danos, L. Garcia  - France


Background: Among well described animal models of Duchenne Muscular Dystrophy, the GRMD dog represents the best model of DMD patients in terms of size and in the pathological expression of the disease. GRMD is caused by a 30 splice-site point mutation in intron 6, which induces the skipping of exon 7 and thus results in a frame shift that prematurely aborts dystrophin synthesis. By forced exclusion (skipping) of two exons (6 and 8) in the case of GRMD, it is possible to restore an open reading frame. Antisense oligonucleotides allow specifically to target and inhibit individual genes for the treatment of the disorder. However, since the AO are not self-renewed, they cannot achieve long-term correction. To overcome this limitation, we have introduced antisense sequences into small nuclear RNAs (snRNA) and vectorized them in AAV vectors. Method: To determine efficient antisense oligonucleotides (AO), we tested in particular exon splicing enhancer (ESE) motifs, which target internal exon sequences, crucial to pre-mRNA splicing. We designed different 20Omethyl oligoribonucleotides targeting either ESE or donor or acceptor splice sites of exons 6 and 8 in GRMD. These AONs were tested first in vitro. Then we have designed AAV vector harbouring chimeric U7 snRNA carrying efficient antisense sequences against exons 6 and 8 of the dog dystrophin gene and AAV were injected in GRMD. Results: Among different antisense oligonucleotides tested in GRMD, the combination of two AONs targeting, respectively, ESE from exon 6 and 8 induced D5-9 inframe skipping. In this study, we show the efficacy of these ESE sequences for exon skipping in culture myotubes. We also detected this correct exons skipping after intramuscular injections of the AAV-U7-ESE6/8 vector in GRMD and observed a restoration of dystrophin expression. In order to spread the benefit to the entire limb, we are currently evaluating systemic delivery of the vector. Conclusion: In this study, we provide evidence that efficient skipping of two exons can be achieved in GRMD cells and also in vivo after intramuscular injections through U7snRNA shuttle. Theoretically over 75% of Duchenne patients could benefit from skipping of a single exon and multiexon skipping would significantly extend this percentage to most DMD mutations.



AAV-mediated delivery of myostatin propeptide increases muscle mass in mouse and improves dystrophic phenotype

A. Vulin, L. Arandel, C. Fleury, O. Danos, L. Garcia - France


Background: Myostatin is known to negatively regulate skeletal muscle mass. It is a member of the transforming growth factor-b superfamily, which is predominantly expressed in skeletal muscle. Like other TGF-b, myostatin contains a signal sequence, a N-terminal propeptide domain, and a disulfidelinked dimmer of C-terminal fragments. After proteolytic cleavage, myostatin binds non-covalently to its propeptide, forming an inactive complex that inhibits the binding to the activin type II receptors. Overexpression of myostatin propeptide in transgenic mice leads to an increase of muscle mass. Method: To show the effect of the propeptide overexpression in adult mice, we designed an AAV vector encoding the myostatin propeptide (AAV-Promyo). Normal and dystrophic mice received the vector AAV-Promyo by either intramuscular or intra-arterial injections.

After different time points, mice were sacrificed and muscles were weighed and analysed. Results: High levels of propeptide myostatin mRNA were detected in each injected muscle.Weobserved the increase of muscle mass in all injected muscles (16–30%) and at all time points between 15 and 75 days. All muscles treated were significantly larger and histological analyses of all sections show no obvious muscle damage. Nevertheless, the mean fiber diameter was larger in injected muscle with the AAV-propeptide and we did not detect significant increase of myofibers number in most muscles. Conclusion: The increase of muscle mass in adult mice is attributable to fiber hypertrophy rather than hyperplasia. Interestingly, the effect of myostatin propeptide is local and limited to the injected muscle. The injected propeptide does not diffuse and remains probably associated at the basal lamina in producing cells. In dystrophic animals, the injections of AAVPromyo improved the phenotype at histological level by decreasing the fibrosis and cellular infiltration. Among several inhibitors of myostatin, the overexpression of propeptide seems to be an effective agent for the regulation of the myostatin activity.



Creatine biosynthesis mitigates the phenotype of dystrophin-deficiency in mdx mice

B.S. Tseng, W.C. McClure, R.E. Rabon, D. Isbrandt, H. Ogawa – USA , Germany and Japan


Duchenne Muscular Dystrophy (DMD) is the most common, most severe and lethal progressive pediatric muscular dystrophy. A mouse (mdx) model of the human DMD disease lacks the same dystrophin protein, but these mice are not severely crippled like DMD boys. Identifying specific

secondary adaptations in the mdx mouse may provide important clues for understanding the more benign dystrophin-deficient phenotype. After extensive microarray screens of multiple mdx studies, we find guanidineacetate methyltransferase (GAMT), the key enzyme for creatine synthesis,

highly upregulated compared to wild type. This same gene GAMT is found to be downregulated in three independent human DMD expression profiling studies. Using biochemical, molecular, cellular and imaging techniques, preliminary findings include: (1) unlike wild-type, GAMT is upregulated at the mRNA and protein level in whole mdx muscle and mdx single fibers, and (2) total creatine, GAMT’s end-product is found to be near normal in mdx muscle. These studies will illuminate a metabolic aspect by which the mdx dystrophin-deficient mouse could resist overwhelming muscle degeneration. We predict de novo creatine synthesis occurs in muscles of mdx mice and serves to maintain near-normal creatine levels, which enables better ATP turnover in the face of dystrophin-deficient leaky membranes. This will be the first detailed analysis of de novo creatine biosynthesis in the mdx mouse. Long-term objective is to delineate molecular underpinnings of the benign phenotype of dystrophin-deficient mdx mice and with new insights develop novel translational treatments for boys with DMD.



The mitotic clock in skeletal muscle disease: immortalization of human myoblasts and consequences for cellular therapy

V. Mouly, A. Bigot, R.N. Cooper, D. Furling, V. Jacquemin, K. Mamchaoui, E. Negroni, W. Wright, G.S. Butler-Browne – Paris - France


Satellite cells, which are the physiological post-natal progenitors of skeletal muscle, are responsible for growth and repair of muscle fibres. The potential of skeletal muscle to regenerate will depend on the number of satellite cells available and their capacity to proliferate and differentiate. We have determined that the number of satellite cells decreases with age in

humans, and that the proliferation of human satellite cells is limited by cellular senescence. Their proliferative capacity decreases during growth, but stays relatively constant during aging in healthy adults. This is correlated with measurements of telomere length, which decreases with

proliferative age and can be used as a marker of the mitotic clock. In degenerative diseases such as muscular dystrophies, the proliferative capacity of satellite cells and their telomere length are dramatically decreased as compared to age-matched controls. An ex-vivo model has

been developed used to optimize the conditions of cell therapy in human skeletal muscle, and telomere length measurements provide a reliable marker to evaluate the proliferative potential of human satellite cells for clinical trials. Techniques have also been developed to obtain immortalized human myoblasts from diseased tissue which provide a valuable model to develop new therapeutic strategies.




Improved muscle healing through enhanced regeneration and reduced fibrosis in myostatin null mice

M.S. Salerno, S. McCroskery, M. Thomas, L. Platt, A. Hennebry, T. Nishimura, M. Sharma, R. Kambadur - Japan


Numerous stimulatory growth factors that can influence muscle regeneration are known. Recently, it has been demonstrated that neutralization of muscle growth inhibitory factors, such as Myostatin

(Mstn), also lead to increased muscle regeneration in mdx mice which are known to have cycles of degeneration. However, the precise mechanism by which Mstn regulates muscle regeneration has not yet been fully determined. To investigate the role of Mstn in adult skeletal muscle regeneration, wild type and myostatin-null mice (MstnK/K) were injured with Notexin. Forty eight hours after injury, there was accelerated migration and enhanced accretion of myogenic cells (MyoD positive) and

macrophages (Mac 1-positive cells) at the site of regeneration in MstnK/K muscle as compared to wild type muscle. Inflammatory cell numbers decreased more rapidly in the MstnK/K muscle, indicating that the whole process of inflammatory cell response is accelerated in MstnK/K mice.

Consistent with this result, addition of recombinant Mstn reduced the activation of satellite cells (SC) and chemotactic movements of both myoblasts and macrophages ex vivo. Examination of regenerated muscle (28 days after injury) also revealed that in MstnK/K mice there was increased expression of decorin mRNA, reduced fibrosis and improved healing as compared to wild type mice. Based on these results, we propose that Mstn negatively regulates muscle regeneration not only by controlling SC activation but also by regulating the migration of myoblasts and macrophages to the site of injury. Thus, antagonists of Mstn appear to be potentially useful pharmacological agents for the treatment of disorders of overt degeneration and regeneration.



Stem cells in the treatment of muscular dystrophy

J. Huard – Pittsburgh - USA


Members of my laboratory have isolated various populations of myogenic cells from the postnatal skeletal muscle of normal mice on the basis of the cells¡‹ adhesion characteristics, proliferation behavior, and myogenic and stem cell marker expression profiles. Although most of these cell populations have displayed characteristics similar to those of satellite cells, we also have identified a unique population of muscle-derived stem cells (MDSCs). MDSCs exhibit long-term proliferation and high selfrenewal rates and can differentiate toward various lineages, including muscle (skeletal and cardiac), neural, endothelial, osteogenic, and chondrogenic lineages, both in vitro and in vivo. The transplantation of MDSCs, in contrast to that of other myogenic cells, has improved the

efficiency of dystrophic muscle regeneration and the delivery of dystrophin to the dystrophic muscle of mdx mice. The ability of MDSCs to proliferate in vivo for an extended period of time, combined with their capacity to exhibit self-renewal, multipotent differentiation, and immune-privileged

behavior, reveals at least a partial basis for the improvements observed after MDSC transplantation. Recent studies performed by members of my laboratory have shown that transplantation of female MDSCs (F-MDSCs) rather than male MDSCs (M-MDSCs) significantly improve skeletal muscle regeneration, despite the similar myogenic and stem cell marker

expression by both cell types. This talk will summarize results showing that differences in self-renewal capacity, aging, and stress tolerance may partially explain the increased muscle regeneration efficiency exhibited by F-MDSCs. My presentation will also address the influence of environmental cues with dystrophic or injured skeletal muscle on the differentiation of MDSCs into fibrotic cells. I will discuss potential strategies by which to prevent scar tissue formation within injured muscle by blocking TGF-b1 activity. Finally, I will present results showing that MDSCs can create grafts of dystrophin-positive myocytes in the hearts of mdx mice after intracardiac implantation. Although the vast majority of the injected cells differentiated toward a skeletal muscle phenotype, some of the injected MDSCs acquired a cardiac phenotype through fusion with host

cardiomyocytes within the injected myocardium. The results outlined in my presentation suggest new avenues by which researchers could use muscle stem cell-based gene therapy and tissue engineering for the treatment of muscular dystrophy.



Gene and cell therapy strategies for Duchenne muscular dystrophy

J.S. Chamberlain, P. Gregorevic, M. Reyes, M. Blankinship, S. Li, E. Kimura, L. Judge, J. Allen - Seattle, USA


Our laboratory is exploring methods for transferring dystrophin expression cassettes to muscle for potential use in gene or cell therapy for DMD. We are also examining the origin and potential myogenicity of mononuclear, non-satellite cells in muscle tissue. Recombinant adenoassociated

viral vectors pseudotyped with the serotype 6 capsid (rAAV6) efficiently transduce striated muscles. These vectors have a cloning capacity of about 5 kb, necessitating the use of highly truncated ‘microdystrophin’ cDNA clones and compact, muscle-specific promoters for delivery. Intravenous injection of rAAV6 vectors into mice at doses greater than 10E12 results in efficient transduction of all striated muscles when the muscle-specific CK6 promoter is used. Lower doses can be used with stronger promoters. Preliminary studies indicate that rAAV6 is also highly

efficient at transducing canine muscles, and that the systemic delivery method is applicable in that species. Transfer of micro-dystrophins tomuscles of mdx mice, a model for DMD, shows that the dystrophin–glycoprotein complex (DGC) is restored to the sarcolemmal membrane with the exception of nNOS. Interestingly, expression of larger dystrophin mini-proteins, such as DH2-R19, also fails to restore expression of nNOS, despite an apparent complete functional rescue of the muscles. We have also developed a series of lentiviral vectors that efficiently transduce and

stably integrate into a variety of different types of stems cells. Using FACS analysis, we show that these vectors efficiently transduce all mononuclear cells isolated from mdx mouse muscles. Extended culturing of muscle mononuclear cells has led to the isolation of lines of multipotent adult

progenitor cells (MAPC), previously isolated only from bone marrow. MAPC can form cell types from all three germinal layers, including muscle. Bone marrow transplantation from a GFP-expressing donor into lethally irradiated mdx mice results in numerous GFP-positive, CD45-negative mononuclear cells in muscle, from which GFP-positive MAPC can be derived. Over time, the number of GFP-positive MAPC increases in mdx muscles, but not in wild-type muscles. Finally, we show that these mdx muscle MAPC are efficiently transduced with lentiviral vectors expressing

mini-dystrophin leading to the formation of dystrophin-positive myotubes.



The intravenous administration of naked DNA for muscular dystrophy

J.A. Wolff, G. Zhang, J. Hagstrom, J. Hegge, C. Thiooudellet, S. Blot, T. Huss, S. Braun – USA and France


Duchenne/Becker muscular dystrophy is an attractive candidate for gene therapy because its molecular and cellular pathogenesis is wellunderstood but better methods of gene transfer are required. We have recently made an important improvement on our intravascular approach for

delivering naked plasmid DNA (pDNA) to limb muscles. Extending our previous intra-arterial approach, we have found that a simple intravenous injection of naked pDNA into a distal, peripheral vein of a limb confined by a proximally placed blood pressure cuff enables high levels of foreign gene expression throughout all the limb muscles in mammals. Plasmid DNA delivery to myofibers is facilitated by its rapid injection in sufficient volume to enable extravasation of the nucleic acid solution into muscle tissue. The procedure works well in both small and large animals, including monkeys, indicating that the procedure should be useful in humans as well. The only

toxicity from the procedure is some minimal and transient muscle necrosis. Repetitive administrations are possible because the procedure only requires access to a peripheral vein and naked plasmid DNA is not toxic and does not induce neutralizing antibodies that inhibit expression. Data in the mdx mouse and GRMD dog models for Duchenne muscular dystrophy indicate that the procedure can be used to express mouse and dog dystrophin protein, respectively, at levels that should have a therapeutic effect. Long-term expression was achieved as well. The eventual aim of the gene therapy is to preserve hand function to improve quality of life.



Exon skipping and the dystrophin gene: molecular by-pass surgery

S.D. Wilton, S. Fletcher - Perth, Australia


Antisense oligonucleotide (AO) induced exon skipping has generated great interest as a possible treatment to reduce the severity of Duchennemuscular dystrophy. These AOs may be regarded as informational drugs that can be rationally designed to anneal to motifs involved in the splicing of the dystrophin gene transcript. AOs are able to bind to the single-stranded pre-mRNA to prevent normal spliceosome assembly and induce specific exon skipping. Removal of an exon carrying a nonsense mutation or removing one of more exons flanking frame-shifting genomic deletions is anticipated to result in an internally truncated, but still functional dystrophin protein. Becker muscular dystrophy patients with mild symptoms clearly demonstrate that substantial portions of the

dystrophin protein, especially within the rod domain, can be deleted with minimal consequences. Preparations are underway to start clinical trials to demonstrate safety and proof of principle that AOs can by-pass genomic deletions in the dystrophin gene. Initial exons chosen for skipping will address the more common deletions, with intramuscular injections of AOs planned to demonstrate that these compounds can restore some dystrophin expression. Systemic administration will be

essential for this molecular by-pass surgery to be efficacious and consideration is being given to commencing the clinical trials with systemic delivery of AOs. Each new AO will be regarded as a new drug and must undergo extensive safety and toxicology studies. Some AO chemistries have already been used in human trials as anticancer or antiviral treatments, which may provide helpful safety, dosage and delivery information.




Effects of granulocyte colony-stimulating factor on the regeneration of injured skeletal muscle in mice

K. Goto, A. Kojima, S. Morioka, T. Naito, T. Akema, T. Sugiura, Y. Ohira, T. Yoshioka -  Japan


It has been considered that muscle satellite cells play an important role in growth and regeneration of injured skeletal muscles. As the content of satellite cells in skeletal muscle tissues is small, a part of satellite cells in the regenerative phase may be differentiated stem cells mobilized from other tissues, such as bone marrow. Administration of granulocyte colonystimulating factor (G-CSF), which is one of the cytokines, induces mobilization of bone marrow stem cells (BMSCs) from bone marrow. Therefore, administration of G-CSF could facilitate the regeneration of

injured skeletal muscles. The purpose of the present study was to investigate the effects of G-CSF administration on the regeneration of injured mammalian skeletal muscles in vivo. Male C57BL/6J mice (10 weeks old) were divided into no-administration control and G-CSF-administration

groups. To activate a necrosis-regeneration cycle, 0.1 mL of cardiotoxin (CTX, 10 mM) was injected into the left tibialis anterior muscle of both groups. G-CSF (10 mg/kg/day) was injected i.p. for the G-CSF-administration group 24 and 48 h before intramuscular injection of CTX. G-CSF was also administrated i.p. 9 times every other day after CTX injection. Results clearly demonstrated that the administration of G-CSF facilitated the regeneration of skeletal muscles.



Improved proliferation of DMD myoblasts by silencing the cyclindependent kinase inhibitor p21 - a new therapeutic approach

A. Kliche, A.V. Moers, S. Endesfelder, H. Lochmuller, A. Speer – Germany


Correction of the primary protein defect is a main goal of experimental therapeutics in DMD. But one could device also alternative approaches to diminish the progression of the dystrophic process. In this context we were interested to improve the proliferation of primary myoblasts of DMDpatients by a reduction of cyclin-dependent kinase p21, which we found elevated in DMD patients. After transient transfection of myoblasts with antisense oligonucleotides (ASO) or short interfering RNAs (siRNA)in cell culture, proliferation was analysed using BrdU assay comparing specific transfected cells with untransfected cells and cells transfected with scrambled ASO and luciferase siRNA, respectively. Four out of five dystrophin deficient cell culture samples revealed an increase in proliferation between 7 and 18% compared to untransfected cells and between 8 and 36% compared to cells transfected with scrambled ASO. Transfection with siRNA was performed for selected samples to prove the opinion that siRNA is more effective in gene silencing than ASO. If using untransfected cells as reference, the increase of proliferation was higher for siRNA compared to ASO (20–47 versus 7–18%) but this must be carefully interpreted with respect to non-specific effects on gene expression by siRNA. A viral vector for inducible p21 siRNA expression was constructed and used to infect human myoblasts succesfully as shown by GFP marker gene expression.



TGF-b induced failure of muscle regeneration and myopathy in fibrillin-1 deficient mice

R.D. Cohn, J.Pardo, B.L. Loeys, T.M. Holm, D.P. Judge, H.C. Dietz – Baltimore - USA


A large subset of patients with Marfan syndrome, an autosomal dominant connective tissue disorder caused by mutations in the fibrillin-1 gene, exhibit significant muscle hypoplasia and loss of fat stores. Recent evidence suggests that fibrillin-1 deficiency does not simply manifest loss of structural integrity of the tissues, but rather involves excessive activation and signaling superfamily of cytokines. Transforming growth factor b of the TGF-beta (TGF-beta) has been shown to be a potent inhibitor of terminal differentiation of cultured myoblasts, however the functional impact of TGF-beta signaling on myogenesis in vivo remains obscure. We find for the first time that excessive TGF-beta bioactivity in vivo leads to significant myopathic alterations in mice. Mice homozygous for a missense mutation (C1039G) in fibrillin-1 exhibit significant muscle hypoplasia and muscle hypotrophy. Heterozygote C1039G mice demonstrate myopathic alterations at 3 months of age with striking variation in fiber size, central ucleation, endomysial fibrosis and increased number of split fibers suggestive of abnormal muscle regeneration. Similar myopathic changes can be observed in skeletal muscle samples of patients with Marfan syndrome. Analysis of skeletal muscle repair after cardiotoxin injection reveals impaired muscle regeneration in fibrillin-1 deficient mice secondary to perturbed satellite cell proliferation. Intraperitoneal administration ofTGF-beta neutralizing antibody rescues the myopathic phenotype ofheterozygous C1039G mice. Moreover, TGF-beta antagonism restoressatellite cell function and thus, leads to normal muscle repair in heterozygous C1039G mice challenged with cardiotoxin. These data demonstrate the first and compelling evidence for a primary role of TGFbeta dysregulated signaling in the pathogenesis of myopathic states caused by impaired muscle repair and thus provides both rationale and motivation to explore the therapeutic utility of TGF-beta antagonism in vivo.



Utrophin expression and localization in the canine Golden Retriever muscular dystrophy model

L.L.Q. Fogaca, T.L. Gouveia, P. Kossugue, C.E. Ambrosio, M.A. Miglino, M. Zatz, T.M. Nguyen, G. Morris, M. Vainzof – São Paulo – Brazil


Golden Retriever Muscular Dystrophy (GRMD) is one of the canine models for human Duchenne muscular dystrophy, caused by deficiency of dystrophin in the muscle. Utrophin is an autosomal homologue of dystrophin. In normal mature muscle utrophin localize to neuromuscular junctions, while in fetal and dystrophic muscle, utrophin can also be detected in the sarcolemma of muscular fibres. The relation between this modified pattern of expression and the dystrophic process is still unknown. Only a few studies have been done in the dystrophic canine model, mainly using human anti-utrophin antibodies. Here, we analysed utrophin in the GRMD model using the canine-specific antibody Mancho-3. Different muscles from animal in diverse stages of the disease are under analysis. Anti-utrophin Mancho-3 antibody labeled strongly the neuromuscular junctions in normal adult canine muscle. In quadriceps muscles of 1-year old affected dog, utrophin labelled the

sarcolemma similarly to the pattern observed in human DMD. On western blot, a large amount of utrophin, of the expected molecular weight was detected in the dystrophic muscle. Utrophin was also detected through western blot analysis in cultured myotubes from normal and affected dogs aged 3 days and 1 year. In the canine model for muscular dystrophy, these preliminary data suggest a similar pattern of expression of utrophin as observed in the human form. Complementary

analyses will evaluate the effect of age and degree of degeneration and regeneration in the mechanism of utrophin expression in the dystrophic muscle. FAPESP-CEPID, PRONEX, CNPq.



Genetic analysis in C. elegans suggest that muscle necrosis in dystrophin-deficient animals is caused by an improper attachment of actin filaments to the Z-discs

K. Gieseler, E. Martina, L. Granger, C. Venoux , M.C. Mariol, L. Segalat – Lyon - France


The molecular mechanisms underlying muscle necrosis in dystrophinopathies remain elusive. Our group addresses this question by using the genetically amenable animal model Caenorhabditis elegans. C. elegans sarcomeres have the same overall composition and architecture as those of vertebrates. The part of the sarcomere called the Z disk (also called dense body in C. elegans) act both as points of attachment for actin filaments and as force transmission devices. The Z disks are deeply embedded in the basal membrane by a set of specialized proteins, whereas, the upper part of the disk is composed of proteins required for binding actin. To decipher the cause of muscle necrosis in dystrophin-deficient C. elegans, we reasoned that mutations which cause a similar necrosis would probably point to genes of interest. Using a non- bias approach, we identified C. elegans mutants which mimick the phenotype of C. elegans dystrophin mutants. This allowed us to identify two genes (actinin and dyc-1/CAPON), which encode proteins linked to the Z disk. Actinin is a known actin-binding protein that participates to the attachment of the actin filaments to the Z disk. The function of Dyc-1 is still unclear, but it is localized at the edge of  the Z-disk, where actin filaments are anchored. This result points to the Z disk as a critical zone in the natural history of dystrophin-dependent muscle degeneration.



The role of the inhibitor of neurite outgrowth Nogo in regenerating skeletal muscle

D. Heuss, T. Eberle, T. Leuschner, M. Haslbeck, B. Neundorfer - Germany


Many tissues of human body have the ability to regenerate after injury, such as skin, bone and skeletal muscle. The role of potential negative regulators of regeneration of skeletal muscle fibers still remains obscure. Nogo, a member of the myelin-associated proteins, is known to be a potent

inhibitor of neurite outgrowth. It is usually expressed by oligodendrocytes and bind to the Nogo-66 receptor (NgR). For Nogo also a peripheral role could be demonstrated showing marked expression in cultured endothelial and smooth muscle cells. The aim of our study was to determine the role of Nogo/NgR for regeneration of skeletal muscle in inflammatory myopathies. Diagnostic muscle biopsy specimens were obtained form patients with dermatomyositis (DM, nZ6) and polymyositis (PM, nZ7). Skeletal muscle biopsies of normal persons served as controls (nZ8). Immunhistochemical staining was performed with antibodies against Nogo and NgR. Additional staining was done with NCAM, a marker for regenerating skeletal muscle fibers. Furthermore, immunofluorescense was drawn up to specify our results and for colocalization. Regenerating fibers show a colocalization for Nogo and NgR in polymyositis. In dermatomyositis, especially perifascicular NCAM-positive atrophic muscle fibers coexpress Nogo and NgR. In conclusion, our results show that the myelin-associated inhibitor of neurite outgrowth Nogo and its receptor NgR are upregulated in regenerating skeletal muscle fibers. Nogo could have a paracrine and

possible autocrine function as negative regulator of regeneration and collateral innervation of skeletal muscle fibers. This pathway seems to play a role maybe for maintenance of tissue homeostasis. Blocking its function as negative regulator of innervation/regeneration could be considered as a therapeutical target in myopathies.



Systemic delivery of delta-sarcoglycan into BIO14.6 hamsters by AAV vectors

C. Vitiello, A. Auricchio, M. Allocca, S. Faraso, D. Di Napoli, S. Castaldo, S. Aurino, V. Saccone, G. Piluso, V. Nigro - Italia


A major obstacle limiting gene therapy for diseases of the heart and skeletal muscles is an inability to deliver genes to all muscle fibers of an adult organism. The BIO 14.6 Syrian hamster has been one of the most used animal model for inherited cardiomyopathy and muscular dystrophy. The

molecular defect is a deletion at the delta-sarcoglycan locus; recently it became a target for gene delivery to muscle by recombinant adenoassociated virus (AAV) vectors. Our plan was to evaluate the usefulness of a single intra-jugular injection using AAV vectors of pseudotype 2/1. We

performed systemic injections of fýˆ1012 copies of an AAV vector expressing beta-galactosidase into both control and BIO 14.6 hamsters, in the presence or absence of the Vascular Endothelial Growth Factor (VEGF). All animals were male and 6–7-weeks old. Both normal and cardiomyopathic hamsters survived after the systemic injection of AAV vectors, expressing beta-galactosidase. They did not show any immune reaction. Interestingly, we observed a good efficiency of infection, especially at cardiac level, where up to 80% of the muscle expressed the

galactosidase. At this number of virus particles, VEGF does not show an influence on the efficiency of infection. We then systemically injected both control and BIO14.6 hamsters using the AAV vector 2/1 containing the human cDNA for delta-sarcoglycan. Forty-five days after the injection, all

hamsters still have good health. Expression data of delta-sarcoglycan and associated proteins will be evaluated.



LARGE restores glycosylation of a-dystroglycan in congenital muscular dystrophies with glycosylation defects

R. Barresi, D.E. Michele, M. Kanagawa, H. Schachter, I. Nishino, K.P. Campbell – Canada and Japan


Fukuyama congenital muscular dystrophy (FCMD), muscle–eye–brain disease (MEB), and Walker–Warburg syndrome (WWS) are congenital muscular dystrophies with associated similar developmental brain defects. Genes involved in these diseases encode for putative or known

glycosyltransferases: mutations in fukutin are responsible for FCMD, POMGnT1 for MEB, and POMT1 for a percentage of WWS. The proteins mutated in these disorders may function either directly or indirectly in the transfer of sugars to a-dystroglycan (a-DG). In skeletal muscle,

dystroglycan bridges the cytoskeleton and extracellular matrix and forms a link that protects the fiber from contraction-induced damage. The receptor activity of a-DG is modulated by the presence of sugar chains, and abnormally glycosylated a-DG loses the binding activity for its

extracellular ligands. We investigated changes in the processing and function of a-DG resulting from genetic manipulation of LARGE, the putative glycosyltransferase mutated in Largemyd mice and MDCn patients. We show that overexpression of LARGE induces the synthesis

of a-DG species enriched in glycans with high affinity for extracellular ligands. In Largemyd mice, LARGE expression ameliorates the dystrophic phenotype. Importantly, LARGE circumvents the posttranslational processing defect of a-DG in cells from patients with genetically distinct types of

congenital muscular dystrophy. Glycan-enriched a-DG produced by LARGE gene transfer in patients’ cells displays restored receptor functions and acquires the ability to coordinate the organization of laminin on the cell surface. Our findings indicate that modulation of LARGE expression or activity is a viable therapeutic strategy for glycosyltransferase-deficient

congenital muscular dystrophies.



Preclinical study of autologous transplantation of myoblasts in the pharyngeal muscles of dogs: preliminary study to correct dysphagia in patients with oculo-pharyngeal muscular dystrophy

S. Perie, J. Lacau St Guily, S. Blot, B. Bouazza, V. Mouly, G.S. Butler-Browne - France


Oculo-pharyngeal muscular dystrophy is characterized by the selective affection of upper esophageal sphincter (UES) and pharyngeal muscles resulting in dysphagia. The most common treatment is a UES myotomy. However, this procedure does not prevent the progressive degradation of the pharyngeal muscles. To evaluate the tolerance and feasibility of autologous myoblast transplantation in the pharyngeal muscles, isolated from unaffected muscles, a preclinical study has been carried out in dogs. Biopsy of quadriceps 1 month prior to autotransplantation was performed and myoblasts were amplified in culture and labelled with a fluorescent dye (PKH26). The surgery consists of both an UES myotomy and injection of myoblasts above myotomy, in the pharyngeal muscles. One month later, a biopsy of the pharyngeal muscles was performed. A videofluoroscopy of swallowing was performed before and after autotransplantation. The injections of myoblasts were successful without any adverse event, and no modification of swallowing was observed by videofluoroscopy. Labelled myoblasts were detected in the biopsies after 1 month as shown by PKH 26 detection. No significant modification of the overall architecture of the muscle fibres was observed. These results validated the procedure and a phase I clinical trial is presently being performed. Five patients have received autologous myoblast transplantations.



Cardiac involvement of the model mice for nuclear envelopathy

R. Ozawa, Y. Hayashi, S. Noguchi, I. Nishino – Tokio - Japan


Emery–Dreifuss muscular dystrophy is an inherited muscular disorder characterized by the triad of progressive weakness in humero-peroneal muscles, early contractures and cardiomyopathy with conduction block. We produced emerin KO mice as a model of X-linked Emery–dreifuss

muscular dystrophy by insertion of a neomycin resistance gene into exon 6 of the coding gene. In addition, we produced and analyzed three kinds of mutant mice; emerin KO, lamin A/C KO and emerin and lamin A/C double KO. Emeirn KO mice display normal growth rate and have no muscle

weakness, but they show prolonged PR interval by electrocardiography. Double KO mice displayed marked growth delay and muscle weakness. Electrocardiography showed significant elongation of PR intervals. Mean PR interval were 34.48G3.25 ms in wild-type, 33.80G3.5 ms in emerin KO mice, 34.61–3.88 ms in laminA/C KO mice and 45.13G7.21 ms in double KO mice. Connexin 40 is a gap junction protein, which is distributed at the His-Purkinje fibers and atrium. The cardiac muscle of emerin KO mice showed normal distribution of connexin 40. In contrast, lamin A/C KO mice showed decreased connexin 40 staining in the Purkinje fibers. Double KO mice showed lacking of connexin 40 at AV node. This result suggests that connexin 40 has an important role in conduction block in lamin A/C KO and double KO mice.



AAV gene transfer in limb girdle muscular dystrophies type 2A and 2D

I. Richarda, M. Bartoli, F. Fougerousse, S. Martin, C. Roudaut, J.


Limb-girdle muscular dystrophies (LGMD2) are a group of genetically distinct disorders characterized by symmetrical and selective atrophy of the proximal limb muscles, elevated serum creatine kinase and a necroticregeneration pattern on muscular biopsies. The cause of LGMD2A is deficiency in calpain 3, a calcium-dependent cysteine protease whose exact function is still elusive. The cause of LGMD2D is deficiency in a-sarcoglycan, a transmembrane protein part of a complex associated with dystrophin. We evaluated the potential of adeno-associated virus (AAV) for gene therapy of these two diseases in corresponding animal models. rAAV vectors carrying the calpain 3 or a-sarcoglycan cDNA under the influence of muscle-specific promoters were constructed. After intra-muscular or intra-arterial delivery, we observed efficient transgene

expression in muscle tissue from the vector at the RNA and protein level without evident toxicity. In addition, the protein products seem to be correctly targeted, at the sarcomere for calpain 3 and the sarcolemma for a-sarcoglycan. More importantly, transfer produced therapeutic efficacy at the physiological levels, including full rescue of the contractile force deficits. Our results demonstrate feasibility for clinical application of AAV-mediated gene transfer in human LGMD2 patients. In this perspective, we are setting up a large-scale and cost-effective rAAV production process in insect cells using the baculovirus expression system. We also have implemented a study of the natural

history of LGMD2A to be able to define precisely the best clinical target.



The mitotic clock in skeletal muscle disease: immortalization of human myoblasts and consequences for cellular therapy

V. Mouly, A. Bigot, R.N. Cooper, D. Furling, V. Jacquemin, K. Mamchaoui, E. Negroni, W. Wright, G.S. Butler-Browne – Paris - France


Satellite cells, which are the physiological post-natal progenitors of skeletal muscle, are responsible for growth and repair of muscle fibres. The potential of skeletal muscle to regenerate will depend on the number of satellite cells available and their capacity to proliferate and differentiate. We have determined that the number of satellite cells decreases with age in

humans, and that the proliferation of human satellite cells is limited by cellular senescence. Their proliferative capacity decreases during growth, but stays relatively constant during aging in healthy adults. This is correlated with measurements of telomere length, which decreases with

proliferative age and can be used as a marker of the mitotic clock. In degenerative diseases such as muscular dystrophies, the proliferative capacity of satellite cells and their telomere length are dramatically decreased as compared to age-matched controls. An ex-vivo model has been developed used to optimize the conditions of cell therapy in human skeletal muscle, and telomere length measurements provide a reliable marker to evaluate the proliferative potential of human satellite cells for clinical trials. Techniques have also been developed to obtain immortalized

human myoblasts from diseased tissue which provide a valuable model to develop new therapeutic strategies.