Baby, Santhosh M (Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA, 19104, USA) Bogdanovich, Sasha (Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA, 19104, USA) Lahiri, Sukhhamay (Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA, 19104, USA) Wilson, David F (Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA, 19104, USA) Khurana, Tejvir S (Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA, 19104, USA)

Although the primary cause of dystrophy is the lack of dystrophin, the mechanisms by which dystrophin-deficiency causes progressive muscle damage and respiratory-cardiac failure remains obscure. Several theories exist to explain degeneration of dystrophin-deficient muscles including; disorder of the microcirculation and vascular impairment, ischemia, impaired mitochondrial function and oxidative stress. We have determined interstitial oxygen pressure by a non-invasive, real-time oxygen-dependent quenching of phosphorescence method. Surprisingly, at rest the oxygen pressure in the interstitial space of mdx muscle is substantially higher than normal muscle. Further, mdx mice did not show as much hypoxic- and exercise-dependent oxygen depletion as occurs in normal mice, suggesting decreased oxygen uptake and/ or vascular function in mdx muscle. We hypothesize that later stages of the dystrophic phenotype is characterized by the loss of vascular barrier integrity/ function accompanied by increased oxidative stress due to decreased oxygen utilization in mdx muscle

Evans, Nicholas P (Department of Human Nutrition, Foods and Exercise, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0430, USA) Guri, Amir J (Department of Human Nutrition, Foods and Exercise, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0430, USA) Bassaganya-Riera, Josep (Department of Human Nutrition, Foods and Exercise, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0430, USA) Grange, Robert W (Department of Human Nutrition, Foods and Exercise, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0430, USA)

Droppelmann, Cristian A (Ponticia Universidad Católica de Chile, Alameda Libertador Bernardo O´Higgins 340, Santiago, Chile.) Mezzano, Valeria (Ponticia Universidad Católica de Chile, Alameda Libertador Bernardo O´Higgins 340, Santiago, Chile.) Brandan, Enrique (Ponticia Universidad Católica de Chile, Alameda Libertador Bernardo O´Higgins 340, Santiago, Chile.)

In order to understand the fibrotic process observed in muscular pathologies like Duchenne Muscular Dystrophy (DMD) we analyzed the participation of Connective Tissue Growth Factor (CTGF), a profibrotic factor overexpressed in fibrotic wounds which induce connective tissue synthesis, and metalloproteinase type 2 (MMP-2 or gelatinase A), an important extracellular matrix remodeling enzyme. We analyzed the expression of MMP-2 in 3T3 fibroblasts in response to rCTGF using relative quantitative PCR and zymography analyses. We observed that CTGF regulates the MMP-2 expression and gelatinase activity in 3T3 fibroblasts in conditioned medium. We evaluated through western blot the presence of fibronectin (FN), a marker of fibrosis, in response to rCTGF in presence of metalloproteinase inhibitor GM-6001 and in a stable kock-down cell line for MMP-2. In the GM-6001 treated and knock-down cells an increase in the amount of FN relative to control was viewed. Moreover, in thes e cells we observed a decrease in amount of FN in response to CTGF. This behavior is analogous to the observed in fibroblasts of mdx dystrophic mice. These results suggest that regulation of the expression and activity of MMP-2 can play an important role in the fibrosis genesis.

Wallace, Gregory Q (The University of Chicago, Chicago, Illinois, 60637 USA); Kenik, Jordan S (The University of Chicago, Chicago, Illinois, 60637 USA); Lapidos, Karen A (The University of Chicago, Chicago, Illinois, 60637 USA); McNally, Elizabeth M (The University of Chicago, Chicago, Illinois, 60637 USA)

Satellite cells are muscle repair cells that are activated after damage or disease to regenerate muscle fibers. Thus, isolating and transplanting these cells is an attractive strategy for treating muscle diseases, but have been limited by their decreased regenerative capacity when exposed to culture conditions. Additional challenges for stem cell-based therapies include maintaining long-term survival of transplanted cells and avoiding immune responses. We have isolated adult muscle mononuclear cells (AMMCs) from normal, strain-matched adult mice and injected them directly into muscles of immunocompetent, delta-sarcoglycan null mice. AMMCs were 35 times more efficient at restoring sarcoglycan than primary myoblasts that had been expanded in culture. AMMC-derived muscle fibers expressed sarcoglycan protein throughout their entire length consistent with enhanced migratory ability. Moreover, recipient fibers resisted muscle degeneration and exercise-induced damage. Donor-derived fibers increased with time after transplantation, suggesting that AMMCs survive for at least six months to augment the satellite cell pool. Interestingly, AMMCs regenerated large clusters of sarcoglycan-positive muscle fibers in aged dystrophic muscle.

Cabello-Verrugio, Claudio (FONDAP-Biomedicina, Facultad de Ciencias Biológicas, P. Universidad Católica de Chile. Santiago, Chile.); Brandan, Enrique (FONDAP-Biomedicina, Facultad de Ciencias Biológicas, P. Universidad Católica de Chile. Santiago, Chile.)

Skeletal muscle differentiation is strongly inhibited by TGF-beta. Decorin is one of the proteoglycans can bind TGF-beta and regulate its activity. This soluble proteoglycan is endocytosed in skeletal muscle cells by low-density lipoprotein receptor-related protein (LRP-1). Interestingly, decorin is essential for TGF-beta dependent inhibition of myogenesis. The aim of this work is to know how decorin favors TGF-beta activity. Studies using myoblasts lacking decorin, in which the levels of LRP-1 were diminished by means of siRNA, suggest that decorin favors TGF beta dependent signaling through a mechanism that involves to LRP-1. At the intracellular level, the TGF-beta Smad dependent signaling pathway was not altered by the absence of decorin and/or LRP-1, which suggests the participation of a non canonical signaling pathway. Using pharmacologic inhibitors we established that PI3K is needed to regulate TGF-beta signaling by decorin through LRP-1. At the extracellular level an increase of the interaction between decorin and LRP-1 occurs when TGF-beta is present, which suggests the formation of a complex among them. These results show a novel regulatory mechanism of TGF-beta signaling in skeletal muscle cells.

Lavasani, Mitra (Stem Cell Research Center, Children's Hospital of Pittsburgh, PA 15213) Pollett, Jonathan B (Department of Bioengineering, Univ. of Pittsburgh, PA 15261) Usas, Arvydas (Department of Orthopaedic Surgery, Univ. of Pittsburgh, Pittsburgh, PA 15213) Pollett, Aaron F (Department of Molecular Genetics and Biochemistry, Univ. of Pittsburgh, Pittsburgh, PA 15261) Huard, Johnny (Univ. of Pittsburgh Cancer Institute, Pittsburgh, PA 15232)

Here, we describe a population of stem cells isolated from skeletal muscle of female newborn mice using a preplate technique that can differentiate into muscle, neuronal, and glial cells in vitro and in vivo. These cells were able to form myosin heavy chain positive myotubes in vitro, and regenerate muscle fibers 17 weeks post-implantation in mdx mice. These cells had a high predilection towards expressing both neuronal and glial cell markers and were able to generate neurospheres in neurogenic media. When implanted into sciatic nerve defects the cells enhanced the rate of nerve regeneration and restored function. However, several weeks after regenerating the sciatic nerve, neoplastic growths formed. The resulting tumors were malignant Triton tumors that expressed myogenic, neurogenic, and glial markers. While the stem cells used in this study were not oncogenic in nature (no tumors were observed when implanted subcutaneously or injected intravenously for more tha n 1 year), the neoplasias were composed almost entirely of donor cells. Furthermore, cells isolated from the tumors generated were serially transplantable. We posit that the progenitor cells used were transformed in a time- and microenvironment-dependent manner, when they received concomitant neurogenic and myogenic signals. Interestingly, this transformation could be abrogated by further differentiation of the cells toward the neurogenic lineage prior to implantation.

50. The use of follistatin to improve the transplantation of muscle-derived stem cells

Zhu, Jinhong (Department of Bioengineering, Univ. of Pittsburgh) Li, Yong (Department of Pathology, Univ. of Pittsburgh) Gharaibeh, Burhan (Stem Cell Research Center, Department of Orthopaedic Surgery, Univ. of Pittsburgh) Huard, Johnny (Stem Cell Research Center, Department of Orthopaedic Surgery, Univ. of Pittsburgh)

Myoblast transplantation has been proposed as a potential treatment for Duchenne muscular dystrophy (DMD), but has been hindered by numerous limitations (eg. poor survival). We have observed that the injection of muscle-derived stem cells (MDSCs) displays a greater muscle regeneration capacity than myoblasts, but the success of stem cell transplantation remains limited. Myostatin (MSTN) is a key inhibitor of muscle growth and may represent a potential target to improve the success of cell transplantation. We recently observed that MDSCs isolated from MSTN-/- muscle displayed better regeneration in host muscle than wide-type (WT) MDSCs, suggesting that blocking of MSTN signaling pathway enhances the efficiency of cell transplantation. Follistatin (FLST) is a potent antagonist of MSTN and may represent an approach to improve cell transplantation. We found that FLST significantly stimulated myogenic differentiation of myoblasts, as well as expressions of myoD, Myf5, and myogenin in myoblasts. FLST overexpressing (OE) transgenic mice showed significantly increased muscle regeneration and decreased fibrosis after injury, in contrast to WT mice. Moreover, we have been able to isolate MDSCs from FLST/OE mice, and compare their regeneration index with WT-MDSC after injected into the skeletal muscles of mdx/scid mice. Dystrophin immunostaining revealed that FLST/OE-MDSCs regenerated a larger number of dystrophin-positive muscle fibers than WT-MDSCs. Our results suggest that a combination of stem cell therapy with gene therapy (eg. FLST) may provide an effective treatment for DMD.

Moussazadeh, Mitra - (Pathway Diagnostics, Malibu, CA 90265) Robins, Terry S (Pathway Diagnostics, Malibu, CA 90265)

The diagnosis of skeletal muscle injury has been a challenge since the common serum markers for this condition namely, myoglobin, lactate dehydrogenase, and creatine kinase (CK), lack specificity to skeletal tissue. Therefore, the need for skeletal-specific biomarkers is clear. In recent years, skeletal troponin I (sTnI) has gained wide attention as a serum biomarker of skeletal muscle damage. sTnI is a myofilament regulatory protein and exists in two isoforms, ssTnI and fsTnI, associated with the slow (type I) and the fast (type II) twitch muscle fibers, respectively. Depending on the origin of muscle injury, one or both of these isoforms may be released into the blood stream. We have developed enzyme immunoassays which specifically react with the fast and the slow isoforms of sTnI. These ELISAs are very sensitive with LLOQ of <1ng/mL and do not cross-react with cardiac troponin I. Using these assays, we have analyzed serum specimen from hospitalized sepsis, rhabdomyolysis, muscular dystrophy, and ventilator patients, as well as a set of serum specimen from random volunteers, for the presence of fsTnI and ssTnI isoforms. Results demonstrate that on average, sTnI isoforms are present at very low (<4ng/ml) to undetectable levels in normal healthy subjects; whereas, they are widely detectable in varying ratios, in sepsis, muscular dystrophy, and ventilator patients ranging between 5 – 100 ng/mL, and in statin-induced rhabdomyolysis, ranging from 50 to 2200 ng/mL. We have observed disease-specific ratios of ssTnI and fsTnI in serum with no direct correlation to CK. We believe that circulating sTnI isoforms may serve as important indices of skeletal muscle damage and may aide in determining the origin and severity of muscle injury, as well as specific diseases or clinical conditions associated with the injury.

Soltanzadeh, Payam (University of Utah, Salt Lake City, UT) Pestronk, Alan (Washington University Medical Center, Saint Louis, MO) Mathews, Katherine (University of Iowa, Iowa City, IA) Finkel, Richard (Children’s Hospital of Philadelphia, Philadelphia, PA) Mendell, Jerry R (Ohio State University Medical Center, Columbus, OH) Wong, Brenda (Cincinnati Children’s Hospital Medical Center, Cincinnati, OH) Weiss, Robert B (University of Utah, Salt Lake City, UT) Flanigan, Kevin M (University of Utah, Salt Lake City, UT)

Introduction: Mutations in the DMD gene, encoding dystrophin, are responsible for the dystrophinopathies. These include Duchenne, Becker, and Intermediate Muscular Dystrophies (DMD, BMD, and IMD); X-linked dilated cardiomyopathy; and manifesting carrier states. The United Dystrophinopathy Project (UDP) is a large multicenter consortium which has established a genotype/phenotype database and patient registry, using recent advances in molecular diagnostic methods to determine genotypes. Here we provide an overview of the phenotypic features of patients at the time of enrollment into UDP. Methods: Dystrophinopathy patients are enrolled through muscular dystrophy clinics at each of the 7 participating centers, and clinical subtype is determined by the expert clinician at each center. Historical data is recorded, and most patients undergo a yearly standardized physical examination. Genotyping is performed by a central laboratory. Results: Currently, 849 patients are enrolled, of whom 510 (including 383 DMD and 101 BMD) have undergone the standardized examination. We present baseline data regarding manual muscle scores, timed functional tests, functional grades, and pulmonary function tests in this large cohort. Conclusion: Data from the UDP will help to establish the natural history of the dystrophinopathies in an era of modern clinical practice, and will provide cohorts of characterized patients for clinical trials.

Amenta, Alison R (Department of Neuroscience, Brown University, Providence, RI 02912) McKechnie, Beth A (Department of Neuroscience, Brown University, Providence, RI 02912) Bogdanovich, Sasha (Department of Physiology and Pennsylvania Muscle Institute, University of Pennsylvania School of Medicine, Philadelphia, PA, 19104)Abedi, Mehrdad (Department of Hematology/Oncology, Roger Williams Medical Center, Providence, RI 02908) Khurana, Tejvir S (Department of Physiology and Pennsylvania Muscle Institute, University of Pennsylvania School of Medicine, Philadelphia, PA, 19104) Fallon, Justin R (Department of Neuroscience, Brown University, Providence, RI 02912)

An attractive approach for DMD therapy is the pharmacological upregulation of utrophin, a dystrophin homolog that is prominent in developing muscle. The extracellular matrix protein biglycan is normally present at high levels in immature muscle and regulates the expression of signaling and structural proteins at the sarcolemma including alpha- and gamma- sarcoglycans and the dystrobrevin-syntrophin-nNOS complex. Here we tested whether biglycan treatment can ameliorate pathology and improve function in the muscles of mdx mice, the canonical animal model for DMD. A single systemic injection of recombinant human biglycan protein (rhBGN) improved the health of mdx muscle as indicated by reduced myofiber death and mononuclear cell infiltration up to three weeks later. rhBGN treatment upregulated utrophin expression as judged by immunohistochemistry and western blotting. Studies in mdx:utrophin-/- double mutant mice indicated that the ability of rhBGN to counter mu scle pathology is utrophin-dependent. Repeated rhBGN at 3wk intervals prolongs utrophin upregulation and ameliorates muscle pathology for at least three months. Finally, rhBGN treatment improved muscle function as measured by reduced susceptibility to eccentric contraction-induced muscle damage. We propose that rhBGN could be a therapeutic for DMD.

Baligand, Céline (NMR laboratory, AIM and CEA, Pitié-Salpêtrière Hospital, Paris,France); Vauchez, Karine; Vilquin, Jean-Thomas; Fiszman, Marc (INSERM U582, AIM, Pitié-Salpêtrière Hospital, Paris,France); Carlier, Pierre G (NMR laboratory, AIM and CEA, Pitié-Salpêtrière Hospital, Paris,France)

High resolution 1H-NMR imaging, combined with NMR contrast agent cell labeling is an emerging methodology for non-invasive monitoring of cell therapy in vivo. Because of the high sensitivity of iron oxide particulates, most studies have been performed using SPIOs loading protocols. However, potential recapture by macrophages after cell death might be a serious issue and would lead to an overestimation of cell survival. More recently, lanthanide chelates have also been shown to detect grafted cells in vivo. The choice of the appropriate contrast agent is still under consideration. We investigated SPIO and Gd-DTPA sensitivity and ability to reflect the presence of grafted cells. Taking advantage of the rapid rejection process of xenogenic transplantation, we grafted Gd-DTPA labeled, SPIO labeled and unlabeled human myoblasts into a leg muscle of immunocompetent mice. We compared the time-courses of signal detection and cells disappearance. Label outcome was monitored by NMR imaging and human cells rejection was assessed in parallel by immunohistochemistry, in a 3-month longitudinal study. As expected, human cells were eliminated from host tissue within 1 week. Quantitative NMR evaluation showed persistence of the Gd-DTPA label in the grafted area for 2 weeks and a total disappearance on day 21. Most importantly, SPIO label was still visible after 3 months, confirming its poor relevance for therapeutic cell monitoring in vivo.

Bizario, João C S; Cerri, Daniel G; Rodrigues, Lilian C; Callejon, Daniel R; Caldas, Juliana R; Fukuhara, Paula S (Faculdade de Ciências Farmacêuticas de Ribeirao Preto - USP, Ribeirao Preto, 14049-900, SP, Brazil); Castro, Fabiola A (Faculdade de Ciências Farmacêuticas de Ribeirao Preto - USP, Ribeirao Preto, 14049-900, SP, Brazil); Costa, Maria Cristina R (Universidade de Ribeirão Preto - UNAERP, Ribeirao Preto, 14096-900, SP, Brazil)

Duchenne Muscular Dystrophy (DMD) is a neuromuscular disorder caused by mutations in the dystrophin gene. It is characterized by progressive skeletal muscle degeneration that leads to weakness and early death by respiratory and cardiac breakdown. There is no specific treatment to DMD. Preclinical tests to find new drugs that can stop or retard DMD progression are usually performed in exercised mdx mouse. One important feature in DMD is the massive muscle infiltration by immune cells and the replacement by fibrous or fatty tissue. Immunomodulators have recently emerged for DMD trials. Imatinib mesilate is a specific inhibitor of tyrosine kinases, such as Bcr-Abl, PDGFR-beta and c-Kit receptors. It also inhibits the profibrogenic activity of TGF-beta. The present study aimed to evaluate imatinib mesilate in mdx mice submitted to treadmill exercise. Four-week old mice were analyzed in the beginning and in the end of a physical activity program during six weeks conside ring histopathological evaluation of gastrocnemius and diaphragm muscles, serum creatine kinase dosage and whole body strength increment. Comparative analyses showed that 0,125 mg/mouse/day resulted in amelioration of the muscular conditions, increased force increment (p<0.0001, unpaired t test, n=15 and 21 for the untreated and treated group, respectively) and decreased CK levels (p=0.0022, n=7). Histological analyses of the gastrocnemius showed abrupt decreasing of the area occupied by injured myofibers infiltrated by Blue Evans dye (p=0.0009, n=3), while diaphragm showed no significant difference. Serum levels of cytokines were evaluated suggesting immunomodulation activity of the drug. Taken together, these data suggest that Gleevec® can ameliorate the dystrophic phenotype in mdx mice, and could be used as potential drug to future clinical tests.

Lachey, Jennifer L (Acceleron Pharma, 149 Sidney Street, Cambridge, MA) Wachtman, Lynn M (New England Research Primate Research Center, Harvard School of Medicine Southborough, MA) Mansfield, Keith G (New England Research Primate Research Center, Harvard School of Medicine Southborough, MA) Pearsall, R. Scott (Acceleron Pharma, 149 Sidney Street, Cambridge, MA) Lane, Joan H (Acceleron Pharma, 149 Sidney Street, Cambridge, MA) Seehra, Jasbir S (Acceleron Pharma, 149 Sidney Street, Cambridge, MA)

Myostatin, or GDF-8, is a TGF-beta family member that negatively regulates muscle mass. Myostatin overexpression causes muscle wasting while its inhibition leads to increased muscle. Mice, cows, dogs and humans with genetic removal of myostatin have increased muscle mass thereby demonstrating its function is evolutionarily conserved. Activin receptor type IIb (ActRllb) is the high affinity receptor for myostatin and other related GDFs. Soluble ActRIIb increases muscle mass in myostatin null mice, suggesting other negative regulators of muscle exist. To study the effects of ActRIIb inhibition in a species closer to humans, common marmosets were treated with a soluble ActRIIb-Fc fusion protein (ACE-031). Weekly administration of ACE-031 at 3 mg/kg resulted in significantly increased lean tissue mass (Echo MRI) at 2 and 4 weeks. At 4 weeks, the treated group had gained 4.3 times more lean tissue (vehicle: +3.45 ± 7.7 g, ACE-031: +14.83 ± 8.5 g) as compared with control animals. These findings show that inhibition of the negative regulators of skeletal muscle with ACE-031 increases total lean tissue in a non-human primate model, suggesting utility in the treatment of muscle disorders in humans.

Pistilli, Emidio E (University of Pennsylvania and Pennsylvania Muscle Institute, Philadelphia, PA) Bognovich, Sasha (University of Pennsylvania and Pennsylvania Muscle Institute, Philadelphia, PA) Mosqueira, Matias (University of Pennsylvania and Pennsylvania Muscle Institute, Philadelphia, PA) Lachey, Jen (Acceleron Pharma, Cambridge, MA) Seehra, J. (Acceleron Pharma, Cambridge, MA) Khurana, Tejvir S (University of Pennsylvania and Pennsylvania Muscle Institute, Philadelphia, PA)

Reductions in respiratory function contribute significantly to the morbidity and mortality of neuromuscular diseases; respiratory failure being the most common cause of death in DMD. In addition to changes in respiratory function, skeletal muscle function is compromised in DMD, possibly via hypoxia-mediated mechanisms. We have previously demonstrated that hypoxia is a stimulus for the rapid onset of muscle atrophy (Willmann et al., ICNMD 2006). Therefore, we hypothesized that myostatin blockade would attenuate loss of muscle mass & function induced in a hypoxic environment. We treated male C57BL/10 mice with either a soluble ActR-IIB receptor (RAP-031, Acceleron Inc.) or PBS for a 2-week period followed by exposure to hypoxia. Bodyweight of RAP-031 treated mice was 9% greater than control mice following hypoxia. Weights of the EDL, soleus, gastrocnemius, quadriceps, and tibialis anterior muscles were a>20% greater in RAP-031 treated mice following hypoxia, with a concomitant improvement of muscle function in the EDL. These results suggest that myostatin blockade via a soluble ActR-IIB receptor can attenuate body- and muscle weight loses in response to hypoxia.

McDonald, Craig M (University of California, Davis) Henricson, Erik K (University of California, Davis) Abresch, Richard T (University of California, Davis) Nicorici, Alina R (University of California, Davis)

Background: The six-minute walk test (6MWT) is a commonly used measure of cardiorespiratory endurance. Researchers have begun to use it as a strength-related outcome measure in clinical trials in neuromuscular disease. We evaluated the 6MWTs differentiation between boys with DMD and healthy controls, test-retest variability in DMD boys and correlation of the test with timed functional measures. Methods: We enrolled boys 5-12 years old with DMD (n=15) and without (n=20). Boys with DMD were tested 7 days apart using a modified American Thoracic Society 6MWT and standard clinical timed function testing. Healthy controls underwent testing for one time point only. Results: Distance traveled differed between boys with DMD and healthy controls across all ages. In boys aged 5-6, those with DMD averaged 367 +/- 74M vs. the healthy controls 574 +/- 35M (p<.01). In boys aged 7-9, those with DMD averaged 354 +/- 31M vs. the healthy controls 622 +/- 50M (p<.001). In boys aged 10-12, those with DMD averaged 265 +/- 146M vs. the healthy controls 646 +/- 49M (p<.001). As age increased in the DMD group, % predicted scores decreased from 82% at age 5 to 56% at age 12. Test-retest correlation was high (r=.92). 6MWT correlated well in DMD with time to walk 10M (r=.80), time to walk 25M (r=.80), time to climb 4 standard stairs (r=.77). Conclusion: A modified 6MWT in ambulatory boys with DMD is reproducible, differentiates DMD and controls at all ages, and correlates with disease severity.

Sonnemann, Kevin J (Dept. of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455) Heun, Hanke (Dept. of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455) Baltgalvis, Kristen (Program in Physical Therapy, University of Minnesota, Minneapolis, MN 55455) Lowe, Dawn (Program in Physical Therapy, University of Minnesota, Minneapolis, MN 55455) Ervasti, James M (Dept. of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455)

Upregulation of the dystrophin homolog utrophin by gene delivery or pharmacological means is thought to provide one possible mechanism to restore membrane integrity and combat the phenotypic effects of dystrophin deficiency. However, despite significant effort, no therapeutic interventions are currently available. Here we identify a novel protein-delivery therapy and show that repeated intraperitoneal injections of cell-penetrating recombinant TAT-utrophin protein into the dystrophin-deficient mdx mouse elevated utrophin levels in all tissues examined, partially restored muscle membrane integrity, reduced the prevalence of muscle fibrosis and degeneration, and improved physiological performance in a dose-dependent manner. These results triple the known size capacity for effective TAT-mediated cell transduction and establish the feasibility of TAT-utrophin as a novel protein-based therapy for the treatment of skeletal muscle and cardiac diseases caused by loss o f dystrophin.

Baltgalvis, Kristen A (Univ. of Minnesota, Minneapolis, MN 55455) Lowe, Dawn A (Univ. of Minnesota, Minneapolis, MN 55455)

Current treatment for DMD is chronic administration of the glucocorticoid prednisone. Prednisone can improve muscle function, but the mechanism is unknown. The purpose of this study was to determine if prednisone treatment in dystrophic mice aids in muscle recovery following a physiological injury. Male mdx mice received a prednisolone pellet (n=16; 1.5 mg) or placebo pellet (n=8) at 4-wks of age; half of the prednisolone mice were restricted in their physical activity. After 6-wks of treatment, the left anterior crual muscles underwent 150 eccentric contractions in vivo. Isometric torque was measured 2 min after the injury protocol and functional recovery was assessed 2 wks later. All groups underwent about a 75% reduction in isometric torque following the injury protocol indicating that prednisolone does not protect muscle from being injured. Two wks later, recovery of in vivo isometric torque, in vitro EDL specific force, and muscle masses were slightly better in the Prednisolone than the Placebo mice. The Prednisolone mice that were restricted in their physical activity recovered the best in terms of EDL specific force (24% higher than Placebo and 12% higher than Prednisolone). These data suggest that while prednisolone may not prevent force loss following eccentric injury, it may help in the recovery process, particularly if physical activity is minimized.

Spencer, Melissa J (David Geffen School of Medicine at UCLA, Los Angeles, CA 90095) Lopez, Sylvia A (David Geffen School of Medicine at UCLA, Los Angeles, CA 90095) Kramerova, Irina (David Geffen School of Medicine at UCLA, Los Angeles, CA 90095) Kudryashova, Elena (David Geffen School of Medicine at UCLA, Los Angeles, CA 90095) Liu, Scot (David Geffen School of Medicine at UCLA, Los Angeles, CA 90095) Hoffman, Eric P (Children's National Medical Center, Washington, DC.) Montecino-Rodriguez, Encarnacion (David Geffen School of Medicine at UCLA, Los Angeles, CA 90095) Miceli, M. Carrie (David Geffen School of Medicine at UCLA, Los Angeles, CA 90095)

Leukocytes invade dystrophin-deficient muscles and presumably exacerbate necrosis and promote fibrosis. To understand the nature of the T cell infiltrate in mdx muscles, we isolated leukocytes and analyzed the T cell receptor (TCR) Vbeta usage. T cells bearing the Vbeta8.1/8.2 TCR predominated, which were CD4 / CD8 negative and expressed NK markers such as DX5, suggesting they might be NKT cells. The Vbeta8.1/8.2 cells from mdx muscle expressed Valpha14 but did not bind CD1d, indicating that they are not conventional NKT cells. Treatment of mdx mice with an antibody that inactivates NK and NKT cells was not effective in reducing disease markers. Cell sorting of Vbeta8.1/8.2 cells showed that they express Osteopontin (Opn), a secreted phosphoprotein that can bind to integrins. Ablation of Opn in the mdx mouse (by crossing with the Opn knock out) showed that some disease markers were improved at 4 wks of age, such as serum CK and strength by wire test. These impro vements were not maintained at 6 mo except that the Opn null/mdx had a significant reduction in diaphragm fibrosis. These studies suggest that Opn may be a viable therapeutic target for treating early necrotic and late fibrotic processes in muscular dystrophy.

Skalsky, Andrew J (University of California Davis, Sacramento, CA 95817); Han, Jay J (University of California Davis, Sacramento, CA 95817); Abresch, Richard T (University of California Davis, Sacramento, CA 95817); Shin, Chris S (University of California Davis, Sacramento, CA 95817); McDonald, Craig M (University of California Davis, Sacramento, CA 95817)

Objectives: Assess in Facioscapulohumeral muscular dystrophy (FSHD) and able-bodied controls (1) the regional body composition using a three compartment model of lean tissue mass (LTM), fat tissue mass (FTM), and bone mineral content (BMC) and (2) the correlation of regional LTM and the corresponding regional strength. Design: Cross-sectional, criterion standard, case-control study evaluating eighteen FSHD subjects and eighteen anthropometrically matched controls. A DEXA scanner was used to obtain regional and whole body composition measurements of LTM, FTM, and BMC. A dynamometer determined peak isometric strength of flexion and extension for both the elbow and knee. Results: FSHD showed a significantly increased regional and whole body fat tissue mass (FTM) (p<0.001 to 0.017), decreased regional and whole body lean tissue mass (LTM) (p<0.001 to 0.010) except for the forearm, and decreased peak isometric strength for all regions measured (p<0.001 to 0.020). There was also a significant correlation between the quantitative strength and LTM for both FSHD and controls (r=0.791 to 0.906; p<0.001). Conclusions: FSHD subjects have higher regional and whole body FTM and lower LTM, and the regional LTM correlates well with strength in both groups.

Skalsky, Andrew J (University of California Davis, Sacramento, CA 95817); Han, Jay J (University of California Davis, Sacramento, CA 95817); Abresch, Richard T (University of California Davis, Sacramento, CA 95817); Shin, Chris S (University of California Davis, Sacramento, CA 95817); McDonald, Craig M (University of California Davis, Sacramento, CA 95817)

Objectives: Assess (1) the regional body composition especially regional percentage lean mass with dual energy x-ray absorptiometry (DEXA) in Duchenne muscular dystrophy (DMD) and able-bodied controls and (2) the correlation of regional lean mass and the corresponding regional strength. Design: Cross-sectional, criterion standard, case-control study evaluating 23 DMD subjects and 23 controls. A DEXA scanner was used to obtain regional and whole body composition measurements of lean body mass. A dynamometer determined peak isometric strength of flexion and extension for both the elbow and knee. Results: DMD showed a significantly decreased regional lean body mass percentage (p<0.001) and increased regional fat mass percentage (p=0.015 to 0.064). There was also a significant correlation between the elbow and knee quantitative strength and regional lean body mass for controls (r=0.832 to 0.947; p<0.001); however, DMD only showed a significant correlation between kne e quantitative strength and regional lean body mass (r=514; p=0.012). In addition, the knee strength per regional lean body mass trended down with increased age in DMD (r=-0.180) but trended up in the controls (r= 0.359). Conclusions: DMD subjects have lower regional lean body mass and increased regional fat body mass, and the regional lean body mass correlates with strength.

Bertoni, Carmen (Univ. of California, Los Angeles, CA 90095); Rando, Thomas (Stanford University, Palo Alto, CA)

Somatic gene therapy represents the best option in the treatment of Duchenne muscular dystrophy (DMD). We have explored gene repair of the dystrophin gene in animal models of DMD using oligonucleotides. This type of approach presents several advantages over other non viral mediated technologies. Gene correction is permanent, genes that have undergone repair remain under the control of their own regulatory mechanisms and problems of long term toxicity resulting from continuous administration of the therapeutic agent are reduced.

The designing of oligonucleotides capable to direct the repair process specifically on the genomic sequence has the potential to significantly increase the repair process. We have tested the ability of oligonucleotides containing CpG modifications to activate MBD4, a major protein component of the base excision repair mechanism. The initial studies were performed in vitro and were followed by studies of dystrophin gene repair in vivo using the mdx model for DMD. As a target for the single base substitution we have chosen the splice boundary of exon 23 of the mouse dystrophin gene in order to induced exon skipping to bypass the nonsense mutation and induce expression of internally deleted but functional dystrophin proteins. Dystrophin protein expression was followed over prolonged period of time in treated muscles to asses for stability of repair. This data were complemented by studies at the messenger and genomic level.

Costa, Maria Cristina R (AADM/UNAERP, Universidade de Riberao Preto, SP 14096900, Brazil); Araujo, Daniela D (AADM/UNAERP, Universidade de Riberao Preto, SP 14096900, Brazil); Cianciolo, George J (Duke University Medical Center, Durham, NC 27710, USA); Rodrigues, Lilian C (AADM/UNAERP, Universidade de Riberao Preto, SP 14096900, Brazil); Pimenta, Aline C (AADM/UNAERP, Universidade de Riberao Preto, SP 14096900, Brazil); Toschi, Ana Carolina C (AADM/UNAERP, Universidade de Riberao Preto, SP 14096900, Brazil); Bizario, Joao Carlos S (AADM/UNAERP, Universidade de Riberao Preto, SP 14096900, Brazil)

The mdx mouse, a Duchenne Muscular Dystrophy model, shows susceptibility to muscle injury and high levels of serum creatine kinase (CK); a phenotype intensified by exercise. Muscular degeneration is exacerbated by a chronic inflammatory process in which tumor necrosis factor alpha (TNF) has a central role. TNF blockade, with either specific antibodies or soluble receptors, reduces necrosis in mdx mice. LMP-420, a purine nucleoside analog, is an orally-active inhibitor of TNF transcription that could be more easily administered than TNF antagonists requiring injection. The effect of 25 mg/kg/day of LMP-420 in 4 week-old mdx mice submitted to a treadmill running program for 5 weeks was evaluated using the following parameters: quantitative histopathological analyses of gastrocnemius and diaphragm and CK levels. LMP-420 treated mice showed significantly-decreased CK levels and amelioration of muscular degeneration, fewer inflammatory infiltrates, decreased degenerati on/regeneration ratios and Evans blue dye infiltrated myofibers. Our data suggest that the inhibition of TNF production by a small molecule transcriptional inhibitor is beneficial for retarding dystrophinopathy and may lead to an option for future clinical trials for DMD.

Radley, Hannah G (Univ. of Western Australia, Perth, WA 6009) Grounds, Miranda D (Univ. of Western Australia, Perth, WA 6009)

Tumour Necrosis Factor (TNF) is a potent inflammatory cytokine that appears to exacerbate damage of dystrophic myofibres in-vivo. The mono-clonal murine specific antibody cV1q (Centocor USA) that blockades TNF demonstrated significant anti-inflammatory effects in dystrophic mdx mice (an animal model for DMD). cV1q administration significantly protected dystrophic myofibres against necrosis in both young and in adult mdx mice subjected to 48 hours voluntary exercise. Long-term studies (up to 90 days) in voluntarily exercised mdx mice showed beneficial effects of cV1q treatment with reduced histological evidence of myofibre damage and a striking decrease in serum creatine kinase levels. A convincing measure of improved muscle function in cV1q treated mdx mice was that they ran significantly more than control mdx mice, further demonstrating protective effects of anti-TNF therapy on dystrophic muscle function. In the absence of exercise long-term studies with cV1q di d not reduce dystropathology in mdx mice. These long-term studies with cV1q in exercised (and unexercised) mdx mice (1) emphasise the importance of exercise for drug testing in this dystrophic mouse model and (2) confirm the benefits of anti-TNF drugs on dystrophic muscle. These data support an important role for inflammation in exacerbation of muscular dystrophy and suggest new drug interventions (much already in wide clinical use) to reduce the clinical severity of DMD.

Bushby, Katie M (University of Newcastle upon Tyne, UK) Straub, Volker W (University of Newcastle upon Tyne, UK)

TREAT-NMD is a European Union funded Network of Excellence aimed at accelerating trials of cutting edge therapies in rare inherited neuromuscular diseases. As such is seeks to harmonise practice in this area and integrate efforts so that there is greater collaboration and progress can be enhanced. The network addresses the pathway from the identification of promising molecules and how these are identified in a consistent way to the delivery of therapies to the patients. A crucial aspect of this is the "trial readiness" of the target population. The initial phases of the network have focussed on DMD and SMA, disseminating and developing standards of care and diagnosis and working on registries and trial networks. The approach is now broadening to other neuromuscular diseases. The network does not seek to be exclusive, and is working collaboratively with many groups worldwide. Funding is from the European Union Framework 6 programme.

Aartsma-Rus, Annemieke (Leiden University Medical Center, Leiden, the Netherlands) Heemskerk, Hans A (Leiden University Medical Center, Leiden, the Netherlands) de Winter, Christa L (Leiden University Medical Center, Leiden, the Netherlands) de Kimpe, Sjef (Prosensa BV, Leiden, the Netherlands) den Dunnen, Johan T (Leiden University Medical Center, Leiden, the Netherlands) 't Hoen, Peter-Bram A (Leiden University Medical Center, Leiden, the Netherlands) van Deutekom, Judith C (Prosensa BV, Leiden, the Netherlands) van Ommen, Gert-Jan B (Leiden University Medical Center, Leiden, the Netherlands)

Antisense-mediated exon skipping is one of the most promising therapies for Duchenne muscular dystrophy. It employs antisense oligoribonucleotides (AONs) to induce exon skipping during pre-mRNA splicing of DMD transcripts, restoring the reading frame and generating partially functional Becker-like dystrophins. Proof of concept was obtained in cultured muscle cells from 15 patients. Recently, exon 51 skipping and local dystrophin restoration was shown in 4 patients treated with a single intramuscular AON injection (van Deutekom et al., this meeting). Currently, we are optimizing systemic delivery of AONs for subsequent clinical trials. In parallel, we are assessing the specificity and bio-distribution of different AON chemistries after local and systemic treatment. Notably, our hDMD model contains a complete copy of the human DMD gene stably integrated into the mouse genome and allows testing of human specific AONs in vivo. First results indicate that morpholino an d 2’-O-methyl phosphorothioate AONs are equally efficient for most human target sequences, but that morpholinos may be less sequence specific. Finally, we are furthering our work on multiexon skipping (AJHG 2004, 74:83-92) to broaden the mutational scope of this approach.

De Luca, Annanaria (Unit Pharmacology; Dept. Pharmacobiology; University of Bari, 70125 Bari - Italy)

Prompt-to-use drugs targeting disease-related pathways can lead to a safer than steroids pharmacotherapy of Duchenne Muscular Dystrophy. Moreover, drugs may help to avoid rejection and enhance efficiency of gene/cellular therapies in the future. Here comparative data of a large in vivo preclinical screening on the model of exercised mdx mice are presented. Outcome of drug treatment is evaluated on a large array of in vivo and ex vivo disease-related parameters by means of a multidisciplinary approach. Immunosuppressive/anti-citokine drugs (cyclosporine A, etanercept) contrast exercise-induced weakness, degeneration-related impairment of muscle chloride channels, high levels of creatine kinase and of pro-fibrotic TGF-beta1 as well as muscle degeneration. Similar results are observed after a partial restoration of dystrophin by a chronic gentamicin treatment, suggesting that the inflammation-dependent degeneration can be highly sensitive to dystrophin presence, and supporting the possible synergism of therapies able to control inflammation and to enhance dystrophin level. By contrast, the alteration of calcium homeostasis is unrelated to inflammatory cascade and is more strongly affected by the absence of dystrophin. This alteration requires more direct drug action on the pathways related to abnormal calcium entry, as obtained with either the phosphodiesterase inhibitor pentoxifylline or taurine

Markert, Chad D (Wake Forest University Health Sciences, Dept. of Neurology, Winston-Salem, NC 27157) Machingal, Masood (Wake Forest Institute for Regenerative Medicine, Winston-Salem, NC 27157) Grange, Robert W (Virginia Polytechnic Institute and State University, Dept. of Human Nutrition, Foods and Exercise, Blacksburg, VA 24061) Childers, Martin K (Wake Forest University Health Sciences, Dept. of Neurology, Winston-Salem, NC 27157)

LGMD2G has been described in humans as a muscular pathology occurring due to absence of the sarcomeric protein, TCAP. Until now, research in LGMD2G has been limited by the lack of an available animal model. We propose to perform the first comprehensive assessment of function in TCAP-null mice, and to further characterize the phenotype by describing muscle function following tissue damage. In keeping with a clinical interest in treatment of muscle wasting, we will investigate whether myostatin blockade ameliorates dystrophic muscle pathology observed in TCAP (-/-) mice by crossing TCAP (-/-) mice with myostatin (-/-) mice. We anticipate that the resulting TCAP/myostatin (-/-) double knockout will improve muscle function observed in TCAP (-/-) mice, and establish a foundation for use of myostatin-blocking agents in patients. Here, we will describe prerequisite experiments: 1) confirmation of knockout (KO) status, and 2) preliminary rotor-rod performance data, w hich indicates a tendency towards a functional deficit in TCAP-null mice. KO was confirmed using standard western blotting techniques with both a custom N-terminus-specific TCAP antibody, and a commercially available TCAP antibody. Performance on the rotor-rod was assessed longitudinally and was poorer in TCAP KO mice vs controls (n=5-34 mice/group) at 2 months (62± 36 vs 77±20 sec, mean±SD), 3 months (65±33 vs 72±34 sec), and 4 months (56±27 vs 71±14 sec).

Duchenne muscular dystrophy (DMD) patients suffer from a progressive, severe muscle-wasting disease due to frame-disrupting mutations in the DMD gene and a complete loss of functional dystrophin. Antisense oligonucleotide compounds (AONs) have recently shown therapeutic promise for DMD patients. By inducing specific exon skipping during mRNA splicing AONs have been successful in repairing the open reading frame in cultured muscle cells from patients, as well as in the mdx mouse model. This resulted in novel expression of internally deleted, partially functional dystrophin proteins as found in the less severe Becker muscular dystrophy patients (BMD). We recently completed a successful proof-of-concept study, demonstrating safety, tolerability, and dystrophin restoring effect of a single, intramuscular dose of antisense oligonucleotide PRO051. Based on positive results with 2OMePS antisense compounds in non-clinical pharmacodynamic, pharmacokinetic and toxicity studies in mice, rats and monkeys, we are now preparing for phase I/II studies. An ascending-dose, open label study will be done in 5-16 year old DMD patients. Eligible patients will receive PRO051 via systemic injections. Several safety parameters and muscle function and strength will be assessed before, during and after treatment. Muscle biopsies will be taken to determine specific exon skipping and dystrophin protein expression.

Adeline Vulin, Inès Barthélémy, Stéphane Blot and Luis Garcia (UPMC-Inserm UMR S 787, 105 Bd de l'Hopital, 75005 Paris, France, Laboratoire de Neurobiologie ENVA, 7 Avenue du Général de Gaulle, Maisons Alfort, France)

Duchenne Muscular Dystrophy (DMD) is an X-linked recessive disorder due to mutations in the gene that encodes dystrophin. Most of these mutations consist in large genomic deletions, although their extent is not directly correlated with the severity of the phenotype. Out-of-frame deletions lead to abortion of translation, dystrophin deficiency and severe DMD phenotypes, while internal deletions that produce in frame mRNAs leading to shorter proteins are responsible for a milder myopathy known as Becker Muscular Dystrophy (BMD) 1. About 80% of the out-of-frame mutations could be theoretically rescued after restoring the translational frame by using exon skipping strategies 2. Here we used gene transfer in a large animal model of DMD, the Golden Retriever Muscular Dystrophy (GRMD) dog 3, to achieve the precise skipping of multiple exons spaced over 125,000 bp of the dystrophin pre-mRNA and the re-expression of a functional protein. This led to sustained correction of the dystrophic phenotype in extended muscle areas and muscle strength recovery. Exon skipping was obtained with U7snRNAs (U7smOPT) 4 carrying antisense sequences designed to mask determinants of exon 6 and 8 definition. These U7smOPT were introduced into skeletal muscle fibres by using Adeno Associated Viral (AAV2/1) vectors. After two months, levels of dystrophin were almost normal in transduced fibres. Histological examination revealed that the dystrophin glycoprotein complex was restored and that spontaneous muscle damages were stopped. Muscle architecture was fully corrected and fibres displayed the hallmarks of mature and functional units. Muscle force as well as NMR imaging indices reflecting muscle structural and functional integrity were improved. Our study documents for the first time the recovery of dystrophin at the scale of entire limbs in a large animal and thus represents a critical milestone for the development of clinical trials in human patients