Seleção dos resumos apresentados no 3o Encontro "Novos Rumos em Biologia e Doença do Músculo Esquelético (Third Meeting New Directions in Biology and Disease of Skeletal Muscle), New Orleans, April-2008

14. Real-time interstitial oxygen content measurements reveals novel mechanism for degeneration of skeletal muscles in dystrophin-deficient mdx mice.

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

21. Quantification of muscle pathology and inflammation during the early disease time course of mdx mice

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)

Duchenne muscular dystrophy is a genetic disorder characterized by severe muscle wasting and early death in affected boys. The primary cause of this disease is mutations in the dystrophin gene resulting in the loss of the dystrophin protein from muscle fibers. In the absence of dystrophin, muscles undergo massive degeneration and inflammation. Inflammation contributes substantially to dystrophic muscle pathology; however, the time course of this process has not been clearly characterized before and during acute disease onset. In mdx mice age 21 days, 3.5% (P0.05) of the tibialis anterior (TA) muscle cross sectional surface was degenerating; by age 28 days, 30% (P0.05) of the muscle surface was regenerating. Immune cell infiltration peaked at age 28 days and covered 3-4% (P0.05) of the TA muscle cross sectional surface. Flow cytometry revealed skeletal muscle from mdx mice age 35 days contained a higher proportion of CD11b+ F4/80+ macrophages compared to w ild type, but macrophages did not appear to express more chemokine receptor (CCR) 2 on their surface. Although there was no increase in CCR2, expression of monocyte chemoattractant protein-1 (MCP-1) was elevated in mdx TA muscles at age 21 days. Inflammation is a significant feature of dystrophic muscle pathology that appears to be regulated by the interaction of cytokines and immune cells.

27.In-vivo monitoring of disease progression in mdx mice

Vohra, Ravneet S (Dept of Physical Therapy, Univ. of Florida, Gainesville, FL, 32610); Mathur, Sunita (Dept of Physical Therapy, Univ. of Florida, Gainesville, FL, 32610); Germain, Sean (Dept of Physical Therapy, Univ. of Florida, Gainesville, FL, 32610); Vandenborne, Krista (Dept of Physical Therapy, Univ. of Florida, Gainesville, FL, 32610); Bryant, Nathan D (Dept of Physiology and Functional Genomics, Univ. of Florida, Gainesville, FL, 32610); Walter, Glenn A (Dept of Physiology and Functional Genomics, Univ. of Florida, Gainesville, FL, 32610)

Purpose: To develop MRI as a valid measure of disease progression in mdx mice. Methods: mdx mice (5-48 wks old) underwent MRI (4.7T) of their lower hindlimbs. Muscle volumes of the posterior and anterior compartment muscles were computed from high resolution, 3D T1-weighted, transaxial MR images (21.5x42.9x375 μm3) at 5, 9, 13 and 17 wks of age. At 17wks, hindlimb muscles were excised and wet weights determined. Muscle damage was quantified in the gastroc (GAS) and tibialis anterior (TA) from T2-weighted images. Magnetization transfer (MT) imaging was used to examine fibrosis in both young and old mdx mice and compared to histological sections. Results: An increase in muscle volume was seen using MRI from 5-17wks of age (total volume=0.17 ± 0.02 cm3 (mean±SEM) at 5 wks, 0.28 ± 0.01 at 17 wks; p=0.006). There was a significant correlation between muscle volume and wet weights (r=0.70-0.85). Muscle damage was highest at 8 wks of age in the GAS (16.4±6.1%) and 7 weeks of the age in the TA (13.8±6.3%), decreasing between 9 to 11 weeks (5.8±0.4% and 3.1±0.7%, respectively). There was an age-dependent reduction in MT of young vs. old mdx mice (GAS: 0.79±0.02 vs 0.76±0.03; TA: 0.81±0.02 vs. 0.78±0.004), which corresponded to greater fibrosis. Conclusion: MRI is a non-invasive tool which can be used to track disease progression in mdx mice, including changes in muscle volume, % damage and fibrosis. MRI may be applied in studies examining therapies for muscular dystrophy.

37. Functional skeletal muscle regeneration from differentiating embryonic stem cells

Perlingeiro, Rita; Darabi, Radbod; Gehlbach, Kimberly; Bachoo, Robert; Kamath, Shwetha; Osawa, Mitsujiro; Kamm, Kristine; Kyba, Michael (Univ. of Texas Southwestern Medical Center, Dallas, TX 75390-9133)

Little progress has been made towards the use of embryonic stem cells (ES) to study and isolate skeletal muscle progenitors. This is in part due to the rarity of skeletal muscle precursors within the EB, as well as the lack of reliable identification and isolation criteria. We have developed an ES cell line in which expression of Pax3, the master regulator of the embryonic myogenic program, can be induced by doxycycline (dox). Our results show that expression of Pax3 during EB differentiation enhances paraxial mesoderm, and cells with myogenic potential within this population. However, transplantation of Pax3-induced cells lead to teratoma formation, indicating the presence of residual undifferentiated cells. By sorting for PDGFalphaR, a paraxial mesoderm marker, and absence of Flk-1, a lateral plate mesoderm marker, we show that early muscle precursors with significant potential for muscle regeneration can be isolated from differentiating ES cell cultures. Purifi ed cells demonstrated significant potential for muscle regeneration as observed upon transplantation into immuno-deficient mice. Intramuscular and systemic transplantation of these cells into dystrophic mice results in extensive engraftment of adult myofibers with enhanced contractile function. These data demonstrate the therapeutic potential of ES cells in muscular dystrophy.

42. Regulation of MMP-2 by CTGF and its relationship with fibrosis

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.

43. Transplantation of uncultured mononuclear cells from skeletal muscle retain long term regenerative capacity

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.

45. Regulation of TGF-beta dependent signaling by decorin and LRP-1 in skeletal muscle cells

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.

48. Muscle-derived stem cells regenerate skeletal muscle and peripheral nerve, but undergo microenvironment-induced transformation

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.


54. Novel Enzyme Immunoassays for the Detection of Slow and Fast Isoforms of Skeletal Troponin I: Clinical Characterization of Skeletal Muscle Injury Using Troponin I Isoforms

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.

62. Blocking TGF-β receptors I and II affect C2C12 myoblast fusion, in a Smad-independent pathway

Rebeca, Droguett (Pontificia Universidad Catolica de Chile) Enrique, Brandan (Pontificia Universidad Catolica de Chile)

TGF-beta, strong inhibitor of myogenesis, signals through RIbeta and RIIbeta. However, during myogenesis, these receptors increase on the cell surface, therefore we evaluated their relevance during this process. Inhibiting RIbeta kinase activity with SB431542, myogenin induction increases, but expression of late differentiation markers, such as creatine kinase and myosin are decreased. Using SB431542 or a dominant negative form of RIIbeta, myoblast fusion is strongly affected, and integrin beta1 and N-cadherin, critical molecules in myotube formation decrease. Overexpression of Smad7 (strong inhibitor of Smad proteins pathway) did not affect myoblast fusion, suggesting that receptors signaling is Smad-independent. Among non-canonical pathways we evaluated Akt and p38 pathways, but these were unaffected. Finally, TGF-beta1, 2 and 3 seems to be not the ligand that activated these receptors during myogenesis, because blocking antibody of these factors did not affect myoblasts fusion. These results suggest that cell signaling through the TGF-beta receptors, and activation of a Smad-independent pathway, are essential for myoblast fusion.

63. Involvement of Ozz-E3, a muscle-specific ubiquitin ligase, in muscle regeneration

Zanoteli, Edmar (St. Jude Children's Research Hospital, Memphis, TN 38105) Campos, Yvan (St. Jude Children's Research Hospital, Memphis, TN 38105) Vergani, Naja (St. Jude Children's Research Hospital, Memphis, TN 38105) Qiu, Xiaohui (St. Jude Children's Research Hospital, Memphis, TN 38105) Oberoi, Kawleen (St. Jude Children's Research Hospital, Memphis, TN 38105) Hu, Huimin (St. Jude Children's Research Hospital, Memphis, TN 38105) Harris, A. John (University of Otago, Dunedin, New Zealand) d'Azzo, Alessandra (St. Jude Children's Research Hospital, Memphis, TN 38105)

Ozz, an E3 ligase specific for striated muscle, is a member of the Suppressor of Cytokine Signaling (SOCS) family of proteins. Ozz expression during myogenesis coincides with myofiber formation and growth. The induction of necrosis, by injecting cardiotoxin (ctx) in skeletal muscle of mice, showed a reduction of Ozz protein expression up to 3 days after lesion. Ozz levels normalized around day 4; increased progressively up to day 10 and went back to normal at 21 days. Analysis of Ozz mRNA revealed a progressive up-regulation of the Ozz up to day 4 after lesion, followed by normalization. Immunohistochemical study showed an increased Ozz expression throughout the cytosol, but especially in regions juxtaposed to the nuclei, 3 days after lesion. At 5-7 days post injury, the expression of Ozz in the cytosol became more diffuse. A number of positive nuclei for Ozz expression were also seen in the developing myotubes. The overall morphology of the Ozz–/– skeletal mu scle did not seem to differ from that of wild-type muscle after ctx treatment. However, at the ultrastructural level there were few developing fibers in Ozz–/– muscle with distinct sarcomeric alterations. The expression pattern of Ozz during muscle regeneration and the alterations seen in Ozz–/– fibers support the notion that Ozz-E3 plays a role in myofiber maturation and growth during regeneration.

64. Skeletal muscle cells express and respond to connective tissue growth factor (CTGF/CCN2), a profibrotic cytokine

Vial, Cecilia (FONDAP-Biomedicina, Facultad de Ciencias Biológicas, P. Universidad Católica de Chile. Santiago, Chile.) Lidia, Zuñiga (FONDAP-Biomedicina, Facultad de Ciencias Biológicas, P. Universidad Católica de Chile. Santiago, Chile.) Enrique, Brandan (FONDAP-Biomedicina, Facultad de Ciencias Biológicas, P. Universidad Católica de Chile. Santiago, Chile.)

Muscular dystrophies are characterized by a progressive wasting of the musculature ending up in an extensive fibrosis with an increased extracellular matrix (ECM) deposition. Connective tissue growth factor (CTGF) is involved in several fibrosis, however, its role in skeletal muscle is unknown. Here we show that myoblasts and myotubes synthesize CTGF in response to transforming growth factor-beta and lysophosphatidic acid. In myoblasts and myotubes CTGF induced several ECM molecules such as fibronectin, collagen type I and III. It had an inhibitory effect on muscle differentiation evaluated by the nuclear translocation of the muscle regulatory factor myogenin, and of myosin expression. CTGF treatment of myoblasts induced their dedifferentiation, downregulating MyoD and desmin, two markers of committed myoblasts, together with a strong reorganization of actin filaments, but not tubulin filaments. Finally we show that CTGF effect in myoblasts depends on ERK1/2 signa ling pathway. These results provide novel evidence for the underlying mechanisms and participation of skeletal muscle cells in the synthesis and role of CTGF inducing fibrosis, inhibiting myogenesis and dedifferentiating myoblasts.

65. Regulatory mechanisms of TGF-beta signaling attenuation during myogenesis.

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

TGF-beta (TGFb) is a strong inhibitor of myogenesis. It binds to transducing receptors (TGFbR I and II) on the cell surface, activating a Smad-dependent pathway. To understand how successful skeletal muscle formation occurs in vivo, in the presence of TGFb, we have studied three mechanisms of TGFb signaling attenuation. i) A decrease of Smad protein levels together with a decrease of TGFbeta dependent Smad2 phosphorylation and Smad4 nuclear translocation. ii) At extracellular milieu, the proteoglycans decorin, biglycan and betaglycan all diminished the TGFb availability decreasing its biological activity and, iii) TGFb dependent signaling is regulated by electrical activity in rat primary myotubes; the inhibition of electrical activity increases TGFbR I levels, and the promotion of electrical activity in myotube cultures causes TGFbR I to decrease diminishing signaling. Altogether, these findings support three novel regulatory mechanisms acting on TGFb signaling c ascade in myogenesis.

68. IGF-I E-peptides are active in vitro and in vivo

Barton, Elisabeth R (University of Pennsylvania, Philadelphia, PA 19104)

Insulin-like growth factor I (IGF-I) is a strong therapeutic candidate for muscle disease because it aids in the repair and maintenance of tissue health. The igf1 gene not only produces IGF-I, but also generates multiple E-peptide extensions through alternative splicing. The E-peptides might modulate the actions of IGF-I, or they might have independent activity. C2C12 cells were exposed to synthetic E-peptides to determine if the possessed inherent bioactivity. Both EA and EB peptides caused a significant increase in cell number at physiologically relevant doses. Proliferation was enhanced in the presence of serum, suggesting that co-factors may modulate the effects of the E-peptides. Viral administration of IGF-IA, IGF-IB, and IGFStop (a construct that lacks any E peptide) into muscles of C57 mice was utilized to determine mechanisms responsible for E-peptide activity. Microarray analysis revealed responses that were driven by increased IGF-I regardless of the p resence of E-peptide, such as cell survival. In contrast, distinct expression patterns were observed for each IGF isoform, which included matrix remodeling genes. These studies provide evidence that the igf1 gene produces multiple biologically active proteins that may work in concert to enhance muscle repair.

71. Examination Data at Enrollment for 510 Subjects in the United Dystrophinopathy Project

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.

74. Cardiac Ankyrin Repeat Protein is a biological marker of atrophy in dystrophic skeletal muscles

Danièle, Nathalie F (Généthon CNRS FRE 3018, Evry, France) Laure, Lydie (Généthon CNRS FRE 3018, Evry, France) Suel-Petat, Laurence (Généthon CNRS FRE 3018, Evry, France) Bartoli, Marc (Généthon CNRS FRE 3018, Evry, France) Richard, Isabelle (Généthon CNRS FRE 3018, Evry, France)

Muscular dystrophies (MD) are a group of genetic diseases characterized by progressive muscle degeneration and weakness. Muscle atrophy is a very common clinical feature of these pathologies. In an attempt to identify potential therapeutic targets for the correction of muscle wasting in muscular dystrophies, the expression of several pivotal proteins involved in protein metabolism was investigated in 4 MD animal models. Amongst all the proteins considered, the expression of CARP, a regulator of transcription factors, appears to be the only one systematically rising. CARP forced over-expression in muscle fibres fails to induce an atrophic phenotype, indicating that CARP per se cannot initiate the phenomenon. CARP is also persistently up-regulated in a condition leading to atrophy (definitive sciatic nerve denervation), whereas MAFbx and MURF1, two E3 ubiquitin ligases known to be involved in muscle wasting, are only transiently over-expressed. These results suggest that CARP might be of particular importance in the late phase of atrophy. Finally, we conclude that CARP is a major marker of muscular pathologies and therefore propose its down-regulation as a potential therapeutic target.

82. Inhibition of smad3 phosphorylation by halofuginone prevents muscle fibrosis and improves muscle performance in muscle dystrophies

Halevy, Orna (The Hebrew University of Jerusalem, Rehovot, Israel); Turgeman, Tidhar (Institute of Animal Sciences, the Volcani Center, Bet Dagan, Israel); Hagai, Yosei (The Hebrew University of Jerusalem, Rehovot, Israel); Huebner, Kyla (University of Manitoba, Winnipeg, Manitoba, Canada); Jassal, Davinder S (St. Boniface General Hospital, Winnipeg, Manitoba, Canada); Anderson, Judy E (University of Manitoba, Winnipeg, Manitoba, Canada); Pines, Mark (Institute of Animal Sciences, the Volcani Center, Bet Dagan, Israel)

Fibrosis is a characteristic feature of the muscle in patients with DMD and CMD, leading to cardiorespiratory failure and delayed motor development respectively. In the present study, we evaluated the efficacy of Halofuginone (Halo), an anti-fibrotic compound, in reducing muscle fibrosis in mdx and Dy2J/Dy2J mice, animal models of DMD and CMD, respectively. Halo prevented the age-dependent increase in collagen synthesis in the diaphragm of young mdx mice and the exacerbation of fibrosis and myopathy in mdx mice treated with cyclosporine A. A similar effect was observed in the gastrocnemius and tibial cranialis of Dy2J/Dy2J mice. The mitigation of diaphragm fibrosis in mdx mice was associated with a decrease in the number of central myonuclei, suggesting a reduction or a delay in the progression of muscle damage and regeneration. Moreover, enhanced motor coordination and balance, and a major improvement in cardiac muscle function were observed. Phosphorylation of S mad2/3, a key signaling molecule downstream to TGF-beta and myostatin, was inhibited by Halo in vivo and in cultures of C2 cell line and of primary mouse myoblasts derived from the mdx and Dy2J/Dy2J mice. This inhibition was partially due to Smad2/3 association with phosphorylated Akt. Since the inhibition of TGF-beta-dependent fibrosis by Halo results in a major improvement in muscle performance, Halo has strong potential as a novel anti-fibrotic drug for muscular dystrophies.

83. Hematopoietic cell transplantation provides an immune tolerant platform for myoblast transplantation in dystrophic dogs

Parker, Maura H (Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA); Kuhr, Christian (Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA); Tapscott, Stephen J (Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, WA); Storb, Rainer (Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA)

Duchenne Muscular Dystrophy (DMD) is the most common and severe form of muscular dystrophy in humans. The goal of myogenic stem cell transplant therapy for DMD is to increase dystrophin expression in existing muscle fibers and provide a source of stem cells for future muscle generation. Although syngeneic myogenic stem cell transplants have been successful in mice, allogeneic transplants of myogenic stem cells were ineffective in several human trials. To determine whether allogeneic muscle progenitor cells can be successfully transplanted in an immune tolerant recipient, we induced immune tolerance in two DMD affected (xmd) dogs through hematopoietic cell transplantation (HCT). Injection of freshly isolated muscle-derived cells from the HCT donor into either fully or partially chimeric xmd recipients restored dystrophin expression up to 6.72% of wild-type levels, reduced the number of centrally located nuclei, and improved muscle structure. Dystrophin expression w as maintained for at least 24 weeks. Taken together, these data indicate that immune tolerance to donor myoblasts provides an important platform from which to further improve myoblast transplantation, with the goal of restoring dystrophin expression to patients with DMD.

84. Antagonism of myostatin ameliorates the dystrophic phenotype in mdx mice

Senna Salerno, Monica; Nicholas, Gina; Berry, Carole ; Thomas, Mark; Siriett, Victoria; Hennebry, Alex; Sharma, Mridula; Kambadur, Ravi (AgResearch Ltd/ Orico Ltd)

In dystrophic conditions there is constant activation of satellite cells due to repeated cycles of muscle degeneration and subsequent regeneration. Recently we showed that myostatin is a powerful inhibitor of satellite cell activation and myoblast proliferation and differentiation. Here we have investigated the therapeutic potential of novel recombinant myostatin antagonists in ameliorating dystrophic muscle loss in the mdx mouse model. When tested for their ability to modulated myoblast growth in vitro, the recombinant antagonists (Mstn-ant1, 2 and 3) showed the capacity to significantly increase myoblast proliferation. Furthermore, in competition assays, they were able to overcome the anti-proliferative effect of exogenous myostatin in a dose dependent manner. Three trials of varying duration were performed to assess the ability of the recombinat proteins to antagonize myostatin in vivo: an 8 week trial with Mstn-ant1 and 2; a 4 week trial with Mstn-ant3; and a 10 day trail with Mstn-ant1. A variety of markers were used to determine the efficacy of the antagonists in ameliorating the dystrophic phenotype. High levels of serum creatine kinase (CK) is considered a marker for muscle fiber damage. Serum CK levels were reduced in the antagonist treated mice in all trials when compared to saline controls. A morphological improvement, with an increase in the regenerating area and concomitant reduction in the necrotic area, was also observed for the muscles of Mstn-Ant1, 2 and 3 treated mice when compared to controls, with an accompanying increase in both Pax7 and MyoD protein expression. However, while individual hind limb muscles from mice treated with Mstn-ant1 and 2 weighed significantly less than muscles from mice treated with saline in the 8 week trial, no weight reduction was observe in the muscles from mice in the 4 week or the 10 day trial. This suggests that a minimum treatment period is required for the decrease in muscle weig ht due to inflammation and cell infiltration. The results presented here thus suggest that peptide antagonists of myostatin are a viable option for ameliorating the dystrophic phenotype in mdx mice.

85. Antagonism of myostatin as a treatment for muscle wasting conditions

Senna Salerno, Monica; Siriett, Victoria; Nicholas, Gina; Berry; McFarlane, Craig; Plummer, Erin; Sharma, Mridula; Kambadur, Ravi (AgReseach Ltd/ Orico Ltd)

Muscle satellite cells are a distinct lineage of myogenic progenitors responsible for postnatal muscle growth. When required, satellite cells are activated to proliferate and differentiate into myoblasts that contribute to the formation of new and existing myofibers. Myostatin is a transforming growth factor-beta superfamily member that functions as a potent inhibitor of muscle growth. Myostatin has been shown to inhibit satellite cell activation in mice, which could have significant consequences during muscle wasting and regeneration conditions such as sarcopenia, cachexia, wound healing and muscular dystrophy. We have previously demonstrated that myostatin has an active role in the regulation of myogenesis. More recently, we have conducted studies to test the therapeutic value of a myostatin antagonist during a variety of muscle wasting conditions. Antagonism of myostatin following notexin injury in aged mice led to satellite cell activation, increased Pax7 and My oD protein levels, and greater myoblast and macrophage cell migration resulting in enhanced muscle regeneration. Aged mice treated with the antagonist also displayed a 12% increase in grip strength as compared to the control group. We have also investigated the therapeutic potential of myostatin antagonists in ameliorating dystrophic muscle loss in the mdx mouse model. Treatment of mdx mice with a myostatin antagonist led to decreased CK serum levels, increased MyoD and Pax7 expression, and a morphologically improved dystrophic muscle, due to a reduction in necrotic areas and a concomitant increase in regenerating areas. Using the same antagonists in a Dexamethasone induced cachexia model, we have shown a significant increase in both Pax7 and MyoD expression, as well as a tendency for increased muscle mass in the antagonist treated mice. We therefore propose that the antagonism of myostatin has significant therapeutic potential in the alleviation of sarcopenia, the amelio ration of cachexia symptoms and the dystrophic phenotype, and in muscle regeneration following injury.

86. Readthrough-inducing ointment: the new approach for the treatment of genetic disorders caused by nonsense mutations

Shioizuka, Masataka (Department of Life Sciences, The University of Tokyo, Tokyo 153-8902, Japan) Sasaki, Hiroyuki (Institute of DNA Medicine, The Jikei University School of Medicine, Tokyo 105-8461, Japan) Kawamoto, Tadafumi (Radioisotope Research Institute, Tsurumi University, Yokohama 230-8501, Japan) Wagatsuma, Akira (Department of Life Sciences, The University of Tokyo, Tokyo 153-8902, Japan) Nonomura, Yoshiaki (Microbial Chemistry Research Foundation, Tokyo 141-0021, Japan) Matsuda, Ryoichi (Department of Life Sciences, The University of Tokyo, Tokyo 153-8902, Japan)

The ability of aminoglycoside antibiotics, such as gentamicin (GM), to promote readthrough of premature termination codons (PTC) has attracted interest in these drugs as potential therapeutic agents in nonsense mutations. In order to measure readthrough efficiency with quantitative accuracy, we have established transgenic mouse strain containing dual-reporter gene with PTC. The objective of this study was to evaluate the transdermal drug delivery (TDD) potential for treatment of genetic diseases caused by nonsense mutations. Here, we show that skin permeability can be increased through the use of chemical enhancer. It was found that the readthrough activity by transdermal administration of GM resulted similar to those by subcutaneous injection. In vivo permeation was verified by LC-MS/MS and the reduction in the intercellular adhesiveness was observed in prickle cell layer and basal layer by electron microscopic analysis. These readthrough therapy on TDD may provide significant value in preventing genetic diseases associated with nonsense mutations. This work was supported by a Research Grant for Nervous & Mental Disorders and by a Grant for Research in Brain Science from the Ministry of Health, Labor and Welfare, Japan.

87. Systemic AAV-9 delivery in normal dog leads to high-level persistent transduction in whole body skeletal muscle

Yue, Yongping (Univ. of Missouri, Columbia, MO 65212) Ghosh, Arka (Univ. of Missouri, Columbia, MO 65212) Long, Chun (Univ. of Missouri, Columbia, MO 65212) Bostick, Brian (Univ. of Missouri, Columbia, MO 65212) Smith, Bruce F (Auburn University, Auburn, AL 36849) Kornegay, Joe N (Univ. of North Carolina-Chapel Hill, NC 27599) Duan, Dongsheng (Univ. of Missouri, Columbia, MO 65212)

Adeno-associated virus (AAV) is a promising vector for muscle disease gene therapy. The dog model represents an ideal intermediate system prior to human trials. Previous attempts to deliver AAV directly to canine muscle have largely failed to achieve efficient transduction because of a strong immune response. In this study, we evaluated systemic AAV-9 gene delivery in newborn dogs by local and systemic delivery. Transgene expression was examined at different time points after AAV infection by biopsy or whole body necropsy. In contrast to the previous reports of low expression and strong immune reaction in dog muscle, we observed efficient transduction at 4 weeks following intramuscular gene delivery. Importantly, systemic gene delivery resulted in impressive whole body skeletal muscles transduction for up to 6 months. In more than 20 different muscle groups (including head, neck, chest, abdominal, thoracic and pelvic limbs), we observed 80% transduction in the majority of muscles throughout the entire muscle length. Taken together, our results provide the first evidence that systemic AAV delivery can reach multiple muscles in a large animal and that body size is not a barrier to intravascular AAV gene transfer. Our results raise the hope of whole body correction for many muscle diseases such as Duchenne muscular dystrophy.

88. Functional resolution of fibrosis in mdx mouse dystrophic heart and skeletal muscle by halofuginone

Anderson, Judy E (University of Manitoba, Winnipeg, MB, R3T 2N2, Canada) Huebner, Kyla D (University of Manitoba, Winnipeg, MB, R3E 0W3, Canada) Jassal, Davinder S (University of Manitoba, Winnipeg, MB, R3E 0W3, Canada) Halevy, Orna (Hebrew University of Jerusalem, Rehovot, Israel) Pines, Mark (Volcani Center, Bet-Dagan, Israel)

The effect of halofuginone (Halo) on established fibrosis in older mdx dystrophic muscle was investigated. Mice (8-9-months) treated with Halo (or saline) for 5, 10 or 12 weeks were assessed weekly for grip strength and voluntary running. Echocardiography was performed at 0, 5, and 10 weeks. Respiratory function and exercise-induced muscle damage were tested. Heart, quadriceps, diaphragm and tibialis anterior muscles were collected to study fibrosis, collagen I and III expression, collagen content using a novel collagenase-digestion method, and cell proliferation. Hepatocyte growth factor (HGF) and alpha-smooth muscle actin (SMA) proteins were assayed in quadriceps. Halo decreased expression of collagens I and III, muscle collagen 1α(I) and SMA content. Muscle-cell proliferation increased at 5 weeks, and HGF increased by 10 weeks treatment. Halo markedly improved function, including cardiac function, reduced muscle damage, improved respiratory function, and impro ved recovery from exercise. Halo reduced the impact of dystrophy by partly resolving pre-existing fibrosis and reducing collagen synthesis in cardiac and skeletal muscles. Marked improvements in vital-organ functions implicate Halo as a strong candidate drug to reduce morbidity and mortality in DMD.

89. Cardiac specific expression of the ΔH2-R19 mini-dystrophin gene only partially restored heart function in aged mdx mice

Brian, Bostick (Univ. of Missouri, Columbia, MO 65212) Yue, Yongping (Univ. of Missouri, Columbia, MO 65212) Long, Chun (Univ. of Missouri, Columbia, MO 65212) Marschalk, Nate (Univ. of Missouri, Columbia, MO 65212) Chen, Jing (Univ. of Missouri, Columbia, MO 65212) Duan, Dongsheng (Univ. of Missouri, Columbia, MO 65212)

Heart failure is a leading cause of death in Duchenne muscular dystrophy (DMD). Effective treatments for DMD cardiomyopathy are lacking. A 6 kb ΔH2-R19 mini-dystrophin gene has been shown to completely rescue skeletal muscle pathology and restore skeletal muscle force. The potential of this minigene for heart rescue is unknown. To address this issue, we developed a series of transgenic mouse lines expressing the ΔH2-R19 minigene in the heart. We evaluated heart function after these mice were backcrossed to the congenic mdx background. Transgenic ΔH2-R19 minigene expression significantly strengthened the sarcolemma integrity and reduced Evans blue dye uptake in the heart. Surprisingly, ECG defects were only partially normalized. Closed-chest Millar catheter assay revealed a normal systolic function. Stroke volume and cardiac output were improved but did not reach those of the normal mice. Taking together, our results provide critical insight towards the understanding of the dystrophin structure-function relationship in the heart. It also calls for developing novel therapeutic strategies to treat DMD cardiomyopathy.

90. Systemically-administered biglycan upregulates utrophin, counters dystrophic pathology and improves muscle function in mdx mice: a novel pharmacological approach for DMD therapy

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.

91. Non-invasive monitoring of skeletal muscle therapy protocols by 1H-NMR imaging

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.

92. Imatinib mesilate (Gleevec®) ameliorates the dystrophic phenotype in exercised mdx mice

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.

93. Treatment with a soluble activin receptor type IIb results in increased muscle mass in marmosets.

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.

94. A novel myostatin inhibitor attenuates muscle loss and improves muscle function in response to hypoxia

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.

95. The six minute walk test as a clinical trial outcome measure in Duchenne muscular dystrophy (DMD): Reliability and correlation with disease progression and clinical timed function testing

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.

97. TAT-Utrophin crosses cell barriers to combat dystrophin deficiency

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.

98. Prednisolone and Inactivity Effects on Recovery Following Eccentric Injury in mdx Mice

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.

101. Osteopontin Is A Modulator Of Early Necrotic And Late Fibrotic Processes In Mdx Muscle

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.

102. The Relationship Between Regional Body Composition and Quantitative Strength in Facioscapulohumeral Muscular Dystrophy (FSHD)

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.

103. Assessment of Regional Body Composition with Dual Energy X-Ray Absorptiometry in Duchenne Muscular Dystrophy: Correlation of Lean Body Mass and Strength.

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.

104. Stable Genome Alteration of the Dystrophin Gene for DMD Due to Frame-Shift Mutations Using Oligonucleotide-Mediated Exon Skipping.

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.

105. Leucine increases muscle stress output of MDX mice

Voelker, Kevin A (Virginia Tech, Blacksburg, VA 24061) Meaney, Mary Pat (Virginia Tech, Blacksburg, VA 24061) Sabat, Agnes E (Virginia Tech, Blacksburg, VA 24061) Escobar, Jeffrey (Virginia Tech, Blacksburg, VA 24061) Grange, Robert W (Virginia Tech, Blacksburg, VA 24061)

Duchenne muscular dystrophy (DMD) is a lethal, progressive disease, characterized by muscle wasting and weakness. The mechanisms responsible for the onset and progression for the disease are not presently known. Thus, therapeutics to counter loss of muscle function would be advantageous. The branched chain amino acid leucine enhances muscle growth through increased phosphorylation of the translation factors 4E-BP1 and S6K1. We tested the hypothesis that MDX mice provided drinking water with leucine (114mM, MDXL) from age 3-7 weeks would increase extensor digitorum longus (EDL) muscle mass and stress output compared to MDX mice given normal drinking water (MDX). After 4 weeks of treatment, mean body mass (MDXL, 27.0 g; MDX, 25.5 g) and EDL muscle length, mass and cross-sectional area were not different between MDXL and MDX mice. Nevertheless, the MDXL vs the MDX stress–frequency response was increased (p < 0.05). Max stress output increased 20% in MDXL comp ared to MDX (30.5 vs 25.4 g/mm2, respectively, p<0.05). MDXL muscles demonstrated no changes in stiffness, fatigue, or fatigue recovery compared to MDX. A 4-week treatment with leucine enhanced stress output in mdx skeletal muscle, but not due to increased muscle mass. The mechanism(s) responsible is(are) at present undetermined.

106. LMP-420: A Transcriptional Inhibitor of TNF with Therapeutic Potential for Muscle Diseases

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.

107. Reduced muscle necrosis and long-term benefits in dystrophic mdx mice after cV1q (blockade of TNF) treatment

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.

110. Moving to human trials- the TREAT-NMD approach

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.

112. Optimization of therapeutic antisense-mediated exon skipping for Duchenne muscular dystrophy

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.

113. Preclinical Drug Studies In Mdx Mice: A Challenge To Speed Up Pharmacotherapy Of Duchenne Muscular Dystrophy

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

114. Myostatin blockade in a murine knockout model of limb girdle muscular dystrophy type 2G (LGMD2G)

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).

116. Pre-clinical and clinical development of 2OMePS antisense oligonucleotides for Duchenne Muscular Dystrophy

J.C.T. van Deutekom1, S.J. de Kimpe1, P.F. Ekhart1, J.T.H.M. van den Akker1, J.M.A. Sitsen1, J.A.M. Janson1, C. de Winter2, A. Aartsma-Rus2, G.J.B. van Ommen2, J.J.M. Verschuuren3, N.M. Goemans4, and G.J. Platenburg1. 1Prosensa Therapeutics B.V., Leiden, The Netherlands 2Leiden University Medical Center, Department of Human Genetics, Leiden, the Netherlands 3Leiden University Medical Center, Department of Neurology, Leiden, the Netherlands 4University of Leuven, Department of Pediatric Neurology, Leuven, Belgium

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.

117. Muscle function recovery in dystrophic dog after exon skipping gene therapy

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