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2010 NEWS


11 - Short Telomeres and Stem Cell Exhaustion Model Duchenne Muscular Dystrophy in mdx/mTR Mice

11 - Duchenne muscular dystrophy: Survival by cardio-respiratory interventions

11 - New compound brings hope of muscular dystrophy remedy

4 - Eicosapentaenoic acid decreases TNF-α and protects dystrophic muscles of mdx mice from degeneration


30 -  (Hum. Mol. Genet., Nov 2010) Interleukin-10 reduces the pathology of mdx muscular dystrophy by deactivating M1 macrophages and modulating macrophage phenotype

S. Armando Villalta, Chiara Rinaldi, Bo Deng, Grace Liu, Brian Fedor, and James G. Tidball  - USA

M1 macrophages play a major role in worsening muscle injury in the mdx mouse model of Duchenne muscular dystrophy. However, mdx muscle also contains M2c macrophages that can promote tissue repair, indicating that factors regulating the balance between M1 and M2c phenotypes could influence the severity of the disease. Because IL-10 modulates macrophage activation in vitro and its expression is elevated in mdx muscles, we tested whether IL-10 influenced macrophage phenotype in mdx muscle and whether changes in IL-10 expression affected the pathology of muscular dystrophy. Ablation of IL-10 expression in mdx mice increased muscle damage in vivo and reduced mouse strength. Treating mdx muscle macrophages with IL-10 reduced activation of the M1 phenotype, assessed by iNOS expression, and macrophages from IL-10 null mutant mice were more cytolytic than macrophages isolated from wild-type mice. Our data also showed that muscle cells in mdx muscle expressed the IL-10 receptor, suggesting that IL-10 could have direct effects on muscle cells. We assayed whether ablation of IL-10 in mdx mice affected satellite cell numbers, using Pax7 expression as an index, but found no effect. However, IL-10 mutation significantly increased myogenin expression in vivo during the acute and the regenerative phase of mdx pathology. Together, the results show that IL-10 plays a significant regulatory role in muscular dystrophy that may be caused by reducing M1 macrophage activation and cytotoxicity, increasing M2c macrophage activation and modulating muscle differentiation.

27 - Prevention of muscle fibrosis and myonecrosis in mdx mice by suramin, a TGF-β1 blocker

27 - Chronic Systemic Therapy With Low-dose Morpholino Oligomers Ameliorates the Pathology and Normalizes Locomotor Behavior in mdx Mice

20 - Proteasome Inhibition Improves the Muscle of Laminin {alpha}2 Chain Deficient Mice

14 - Prevention of Muscle Aging by Myofiber-Associated Satellite Cell Transplantation


5 - Antiinflammatory activity of Eugenia punicifolia extract on muscular lesion of mdx dystrophic mice

3 - The risks of therapeutic misconception and n =1 “trials” in rare diseases such as Duchenne dystrophy

3 - Sustained Alpha-Sarcoglycan Gene Expression after Gene Transfer in Limb-Girdle Muscular Dystrophy, Type 2D

1 - Increased catalase expression improves muscle function in mdx mice



23 -  (Hypertension, 2010) Angiotensin II Contributes to Skeletal Muscle Fibrosis, Reduced Locomotor Activity and Autonomic Dysfunction in delta-Sarcoglycan Deficient Mice with Muscular Dystrophy

Rasna Sabharwal, Robert M Weiss, Univ Iowa, Iowa City, IA; Kathy Zimmerman, Vet Affairs
Med Ctr, Iowa City, IA; Mark W Chapleau; Univ Iowa, Iowa City, IA

Sarcoglycan mutations cause muscular dystrophy and dilated cardiomyopathy. We recently reported that sarcoglycan delta deficient (Sgcd / ) mice exhibit reduced locomotor activity and autonomic dysfunction at a young age (11-12wks) prior to development of left ventricular (LV) dysfunction (FASEB J, 2008). We hypothesized that angiotensin II (Ang II) contributes to
skeletal muscle and autonomic dysfunction in Sgcd / mice at this young age. Control and Sgcd / mice were treated with Ang II type 1 receptor (AT1R) blocker losartan (10mg/kg/day, drinking water) for 8 wks beginning at 3 wks of age. Blood pressure (BP), heart rate (HR), activity (telemetry), baroreflex sensitivity (BRS, sequence technique), cardiac vagal and sympathetic tone (HR responses to atropine, propranolol), LV function (echocardiography), and AT1R expression (immunofluorescence) and fibrosis (Masson stain) in skeletal muscle were measured. Comparisons were made between treated and untreated mice. Losartan restored
locomotor activity, decreased HR and reversed autonomic dysfunction in Sgcd / mice while not affecting these measures in control mice (Table). The improved functions were associated with decreases in AT1R expression and fibrosis in Sgcd / skeletal muscle, and occurred independently of changes in LV function.

Summary: Treatment of young Sgcd / mice with losartan reduced AT1R expression and fibrosis in skeletal muscle and normalized locomotor activity and autonomic regulation. We conclude that Ang II contributes to the skeletal muscle and autonomic dysfunction, perhaps via abnormal sensory or cytokine signaling from dystrophic skeletal muscle to brain.

18 - Sildenafil reverses cardiac dysfunction in the mdx mouse model of Duchenne muscular dystrophy

16 - Pivotal Data Presented at the World Muscle Society Congress Suggest Ataluren Slows the Loss of Walking Ability in Patients with Nonsense Mutation Duchenne/Becker Muscular Dystrophy

12 - BMP antagonists enhance myogenic differentiation and ameliorate the dystrophic phenotype in a DMD mouse model

12 - Histological assessment of SJL/J mice treated with the antioxidants Coenzyme Q10 and Resveratrol

9 - Effects of long-term treatment and combination therapeutics for neuromuscular disorders

7 -  Dystrophin Immunity in Duchenne's Muscular Dystrophy

2 - Neuroscience 2010 - 40th Annual Meeting - November 13-17, San Diego.

1) Dystrophinopathy in mdx mice is alleviated by doxycycline, a tetracycline derivative

Anatomia, Biologia Celular, Fisiologia, Biofisica, Univ. Estadual de Campinas UNICAMP, Campinas, Brazil

Muscle degeneration and fibrosis are associated with inflammation in dystrophic muscles of Duchenne muscular dystrophy and in the mdx mice. We evaluated the effect of doxycycline, a tetracycline derivative with anti-inflammatory and anti-apoptotic effects, on muscle degeneration in mdx mice. Mdx mice (n=20; 20 days of age) received doxycycline (n=10; 6mg/ml on drinking water) for 5 weeks, after which the diaphragm (DIA) and biceps brachii (BB) muscles were removed. Control mdx mice (n=10) received water only. Serum creatine kinase levels, an indicative of myonecrosis, were significantly decreased by doxycycline (control-mdx: 1292±223 U/l, doxycycline-mdx: 973±243 U/l; p<0.05, Student's t-test). Histological analysis showed that doxycycline decreased the inflammatory area in both muscles. Doxycycline protected against myonecrosis (3% of Evans blue dye-positive fibers in control-DIA vs 1.2% in doxycycline-DIA; in control-BB, 3.8% of Evans blue positive-fibers vs 1.0% in doxycycline-BB) and promoted a significant increase in peripheral cell nuclei fibers in both DIA and BB. Functional analyses (grip strength) showed that doxycycline did not change muscle force over time. These results indicate that doxycycline may be a possible useful therapeutic alternative to ameliorate muscular dystrophy caused by dystrophin deficiency.

2) Eicosapentaenoic acid decreases TNF and protects dystrophin-deficient muscle fibers of mdx from degeneration

Anatomia, Biologia Celular, Fisiologia, Biofisica, Univ. Estadual de Campinas -UNICAMP, Campinas, Brazil

The lack of dystrophin in dystrophin-deficient fibers of mdx mice and in Duchenne muscular dystrophy leads to sarcolemmal breakdown and progressive muscle degeneration. The increased production of inflammatory cytokines, such as tumor necrosis factor (TNF)-α, seems to contribute to myonecrosis. We examined whether eicosapentaenoic acid (EPA), a -3 polyunsaturated fatty acid with anti-inflammatory properties, could protect different dystrophic muscles from degeneration. Mdx mice (14 days old) received EPA (n=12; 300 mg/kg body weight; daily oral gavage, diluted in mineral oil) for 16 days, after which the sternomastoid (STN), diaphragm (DIA), tibialis anterior (TA) and biceps brachii (BB) muscles were removed. Control mdx mice (n=12) received mineral oil. EPA significantly decreased serum creatine kinase levels (control-mdx: 1208±376 U/L; EPA-mdx: 838±347 U/L; p<0.05, Student's t-test). In all muscles studied, EPA protected against myonecrosis and from changes in sarcolemma permeability leading to a significant increase in peripheral cell nucleated fibres and a concomitant decrease in Evans blue dye-positive fibres (60% of decrease in BB, 70% in DIA and STN and 80% of decrease in TA). EPA reduced the inflammatory area in the DIA (13.2±7.6% of inflammatory area in control-mdx vs 3.4±2.4% of inflammatory area in EPA-mdx; p<0.05, Student's t-test). Grip strength test showed that EPA did not change the increase in muscle strength with time observed in mdx at this age. Immunoblotting indicated that EPA significantly reduced the levels of TNF-α in all muscles studied. These results indicate that EPA has a protective action against dystrophic muscle degeneration, possibly by reducing the levels of TNF-α, supporting further investigations of EPA as a potential therapeutic agent to dystrophinopaties.

3) The effect of a soluble activin receptor type IIB on whole body tension in mdx mice is improved by co-administration of an NF-kappaB inhibitor

1Dept Physiol, AT Still Univ., KIRKSVILLE, MO; 2Acceleron Pharma, Cambridge, MA

The effect of in vivo administration of RAP-031, a soluble activin receptor type IIB (ActRIIB) comprised of a form of the ActRIIB extracellular domain linked to a murine Fc, with and without co-treatment with an NF-κB inhibitor on whole body tension (WBT) was examined using the mdx mouse model for Duchenne muscular dystrophy. Treatment of mdx mice with RAP-031 for 90 days produced a 41 % increase in body mass and a 42.5 % increase in forward pulling tension (FPT) exerted by the limb musculature. Consistent with RAP-031 increasing muscle strength proportionally to body weight, whole body tension (WBT), or FPT normalized for body weight, was unchanged in the RAP-031 treated group compared to vehicle treated. Similar increases in muscle mass and FPT were seen when RAP-031 was used in conjunction with the NF-κB inhibitor ursodeoxycholic acid (UDCA). In contrast to treatment with RAP-031 alone, however, WBT was increased by treatment with RAP-031 + UDCA, supporting the idea that the combination treatment produces muscle that is stronger in proportion to body weight in mdx mice, and that co-treatment with an NF-κB inhibitor may potentiate the beneficial effects of RAP-031 in increasing muscle strength. These results indicate that treatment of dystrophic mice with RAP-031 produces large increases in muscle mass that are associated with corresponding increases in the forward pulling tension exerted by the limb musculature.

4) Granulocyte colony stimulating factor (G-CSF) reduces loss of inputs to alpha-motoneurons during the course of muscular distrophy and after axotomy in mdx mice

1Anat., Univ. of Campinas, Campinas, Brazil; 2Dept Anat, State Univ. Campinas - UNICAMP, Campinas, Brazil

G-CSF is a key hemopoietic factor that has also been demonstrated to have immunoregulatory properties, stimulating anti-inflammatory pathways. Such immune response regulation may positively influence synaptic stability after lesion and during the course of neuromuscular diseases, such as muscular distrophies. Thus, the aim of this study was to investigate the synapse preservation and glial reactivity in the microenvironment surrounding spinal motoneurons in a Duchenne muscular dystrophy animal model (namely the MDX mice) after treatment with G-CSF. In this way, six weeks old male MDX mice were treated with subcucaneous injections of 200 µg/kg/day of G-CSF 7 days before and 7 days after the left sciatic nerve transection. The axotomy was performed after the cycles of muscular degeneration/regeneration, previously described in such model of muscular dystrophy. C57BL/10 mice were used as controls. Seven days after surgery, the animals were sacrificied and their lumbar spinal cords processed for immunohistochemistry (antisera against MHC I - major histocompatibility complex class I, synaptophysin, GFAP - glial fibrillary acidic protein and Iba1 - ionized calcium binding adaptor protein were used) and transmission electron microscopy (TEM). Overall, G-CSF treatment was able to induce upregulation of MHC-I in both strains after axotomy, although MDX mice displayed significantly lower levels. Regarding GFAP expression, G-CSF treatment resulted in a stronger astrogliosis in MDX mice. G-CSF administration resulted in no significant alteration of the microglial reaction, as seen by the anti-Iba1 labeling. Interestingly, G-CSF treatment preserved a significant percentage of pre-synaptic inputs as seen by synaptophysin immunohistochemistry (Placebo - MDX, 4.89×103±76.00 C57BL/10, 6.76×103±108.00, p<0.001; G-CSF MDX, 9.20×103±37.00; C57BL/10, 1.05×104±104.00, p<0.01 - ipsilateral side to the lesion; integrated density of pixels/100µm2). TEM analysis revealed a ~15% greater synaptic covering in both strains after axotomy and G-CSF treatment. Also, the number of synaptic terminals/100µm in apposition to the motor neuron membrane increased after treatment (Axotomized - placebo - MDX, 33.50 ± 0.83; C57BL/10, 39.30 ± 0.49, p<0.001. Axotomized - G-CSF - MDX, 40.26 ± 1.03; C57BL/10, 48.13 ± 1.75, p>0.05). Altogether, the present results indicate that G-CSF treatment is able to reduce the retrograde effects related to the course of the muscular distrophy in MDX mice and following peripheral axotomy. This is possibly associated to the preservation of inputs to spinal motoneurons and to the regulation of the astroglial reaction and MHC I expression.


25 - Past, present and future of myoblast transplantation in the treatment of Duchenne muscular dystrophy

18 - Evaluation of Cyclosporine A modified in skeletal muscle of GRMD dogs - portuguese abstract about Debio-025 in dogs

Conclusion - until now, analysing the above parameters the treated and control groups was not possible to observe clinical improvement in treated group.

18 - Nicotinic acetylcholine receptor activation reduces skeletal muscle inflammation of mdx mice

18 - Bowman Birk inhibitor attenuates dystrophic pathology in mdx mice

17 - (Am J Physiol Regulatory Integrative Comp Physiol, Sep 2010) Leupeptin based inhibitors do not improve the mdx phenotype

Joshua T. Selsby, Klara Pendrak, Monica Zadel, Zuozhen Tian, Jennifer Pham, Ted Carver, Pedro Acosta, Elisabeth R. Barton and H. Lee Sweeney - USA

Calpain activation has been implicated in the disease pathology of Duchenne muscular dystrophy (DMD). Inhibition of calpain has been proposed as a promising therapeutic target, which could lessen the protein degradation, and prevent progressive fibrosis. At the same time, there are conflicting reports as to whether elevation of calpastatin, an endogenous calpain inhibitor, alters pathology. We compared the effects of pharmacological calpain inhibition in the mdx mouse using leupeptin and a proprietary compound (C101) that linked the inhibitory portion of leupeptin to carnitine (to increase uptake into muscle). Administration of C101 for four weeks did not improve muscle histology, function, or serum creatine kinase (CK) levels in mdx mice. Mdx mice injected daily with leupeptin (36 mg/kg) for 6 months also failed to show improved muscle function, histology or CK levels. Biochemical analysis revealed that leupeptin administration caused an increase in m-calpain autolysis and proteasome activity, yet calpastatin levels were similar between treated and untreated mdx mice. These data demonstrate that pharmacological inhibition of calpain is not a promising intervention for the treatment of DMD due to the ability of skeletal muscle to counter calpain inhibitors by increasing multiple degradative pathways.

11 - Effects of irradiating adult mdx mice before full-length dystrophin cDNA transfer on host anti-dystrophin immunity

11 - An Omega-3 Fatty Acid-Enriched Diet Prevents Skeletal Muscle Lesions in a Hamster Model of Dystrophy

11 - Steroid therapy is associated with decreased numbers of dendritic cells and fibroblasts, and increased numbers of satellite cells, in the dystrophic skeletal muscle

7 - Selected abstracts that will be presented in World Muscle Society Meeting 2010 - Kumamoto, Japan, October 12-16.

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wms2010_8      wms2010_9      wms2010_10    wms2010_11    wms2010_12    wms2010_13    wms2010_14  

wms2010_15    wms2010_16    wms2010_17    wms2010_18    wms2010_19    wms2010_20    wms2010_21  

wms2010_22    wms2010_23    wms2010_24    wms2010_25    wms2010_26    wms2010_27

1 - (Journal of Clinical Neuromuscular Disease; 12 (1):1-21, September 2010) Clinical Use of Immunosuppressants in Duchenne Muscular Dystrophy - Review.

Tommaso Iannitti, Stefania Capone, David Feder and Beniamino Palmieri

Duchenne muscular dystrophy (DMD) is a degenerative disease primarily affecting voluntary muscles with secondary consequences on heart and breathing muscles. DMD is an X-linked recessive disease that results in the loss of dystrophin, a key muscle protein. Inflammation can play different roles in DMD; it can be a secondary response to muscle degeneration, a primary cause of degeneration, or can contribute to the disease progression. Several immunosuppressants have been used with the aim to reduce the inflammation associated with DMD. Most recently,myoblast transplantation has shown the possibility to restore the dystrophin lack in the DMD patient’s muscle fibers and this evidence has emphasized the importance of the use of immunosuppressants and the necessity of studying them and their secondary effects. The aim of this review is to analyze the main immunosuppressants drugs starting from the mdx mice experiments and concluding with the most recent human clinical studies.

1 - Not waiting to live, not living to wait....

1 - NIMS to start stem cell therapy treatment


27 - Treatment of Duchenne muscular dystrophy with ciclosporin A: a randomised, double-blind, placebo-controlled multicentre trial

Duchenne muscular dystrophy: an important negative trial

21 - 170th ENMC International Workshop: Bone protection for corticosteroid treated Duchenne muscular dystrophy. 27–29 November 2009, Naarden, The Netherlands

21 - (Muscle and Nerve, 2010) Activin IIB receptor blockade attenuates dystrophic pathology in a mouse model of duchenne muscular dystrophy

Kevin J. Morine; Lawrence T. Bish; Joshua T. Selsby; Jeffery A. Gazzara, Klara Pendrak; Meg M. Sleeper; Elisabeth R. Barton; Se-Jin Lee; H. Lee Sweeney - USA

Modulation of transforming growth factor-beta (TGF-beta) signaling to promote muscle growth holds tremendous promise for the muscular dystrophies and other disorders involving the loss of functional muscle mass. Previous studies have focused on the TGF-beta family member myostatin and demonstrated that inhibition of myostatin leads to muscle growth in normal and dystrophic mice. We describe a unique method of systemic inhibition of activin IIB receptor signaling via adeno-associated virus (AAV)-mediated gene transfer of a soluble form of the extracellular domain of the activin IIB receptor to the liver. Treatment of mdx mice with activin IIB receptor blockade led to increased skeletal muscle mass, increased force production in the extensor digitorum longus (EDL), and reduced serum creatine kinase. No effect on heart mass or function was observed. Our results indicate that activin IIB receptor blockade represents a novel and effective therapeutic strategy for the muscular dystrophies.

21 - Inhibition of the IKK/NF-kappaB pathway by AAV gene transfer improves muscle regeneration in older mdx mice

21 - Continuous monitoring and quantification of multiple parameters of daily physical activity in ambulatory Duchenne muscular dystrophy patients

14 - Blocking the Myostatin Signal With a Dominant Negative Receptor Improves the Success of Human Myoblast Transplantation in Dystrophic Mice

13 - (Human Molecular Genetics, 2010) Flt-1 haploinsufficiency ameliorates muscular dystrophy phenotype by developmentally increased vasculature in mdx mice

Mayank Verma, Yoko Asakura, Hiroyuki Hirai, Shuichi Watanabe, Christopher Tastad, Guo-Hua Fong, Masatsugu Ema, Jarrod A. Call, Dawn A. Lowe and Atsushi Asakura - USA

Duchenne Muscular Dystrophy (DMD) is an X-linked recessive genetic disease caused by mutations in the gene coding for the protein dystrophin. Recent work demonstrates that dystrophin is also found in the vasculature and its absence results in vascular deficiency and abnormal blood flow. This induces a state of ischemia further aggravating the muscular dystrophy pathogenesis. For an effective form of therapy of DMD, both the muscle and the vasculature need to be addressed. To reveal the developmental relationship betweenmuscular dystrophy  and vasculature, mdx mice, an animal model for DMD, were crossed with Flt-1 gene knockout mice to create a model with increased vasculature. Flt-1 is a decoy receptor for vascular endothelial growth factor (VEGF), and therefore both homozygous (Flt-1-/-) and heterozygous (Flt-1+/-) Flt-1 gene knockout mice display increased endothelial cell proliferation and vascular density during embryogenesis. Here, we show that Flt-1+/- and mdx:Flt-1+/- adult mice also display a developmentally increased vascular density in skeletal muscle compared to the wild-type and mdx mice, respectively. The mdx:Flt-1+/- mice show improved muscle histology compared to the mdx mice with decreased fibrosis, calcification and membrane permeability. Functionally, the mdx:Flt-1+/- mice have an increase in muscle blood flow and force production, compared to the mdx mice. Consequently, the mdx:utrophin-/-:Flt-1+/- mice display improved muscle histology and significantly higher survival rates compared to the mdx:utrophin-/- mice which show more severe muscle phenotypes than the mdx mice. These data suggest that increasing the vasculature in DMD may ameliorate the histological and functional phenotypes associated with this disease.

7 - Laminin-111: A Potential Therapeutic Agent for Duchenne Muscular Dystrophy


20 - (Am J Phys Med Rehabil 2010;89:620–624) Duchenne Muscular Dystrophy - The Effect of Glucocorticoids on Ventilator Use and Ambulation

Bach JR, Martinez D, Saulat B - USA

Objective: To describe the effect of glucocorticoid treatment on age at wheelchair dependence and at dependence on part-time       (<23 hrs/day) and continuous noninvasive mechanical ventilation. Design: In this retrospective study, patients with Duchenne muscular dystrophy who received glucocorticoid therapy were compared with those who did not for ages at wheelchair dependence and when beginning part-time (nocturnal) and continuous noninvasive intermittent positive pressure ventilation (NIV). Respiratory symptoms, end-tidal carbon dioxide, oximetry, and vital capacity were noted every 4–12 mos, and NIV was initiated for symptomatic nocturnal hypoventilation. The daily NIV use increased until some required it continuously to survive.
Results: The 117 untreated patients became wheelchair-dependent at 9.7 + 1.3 yrs of age. Four then died from cardiac failure, and five were older than 19 yrs without using NIV. The remaining 108 began nocturnal NIV at 19.2 + 3.7 yrs of age. Ninety of the 108 became continuously NIV-dependent at 21.9 + 4.5 yrs of age, and the 17 treated with glucocorticoid therapy became wheelchair-dependent significantly later at 10.8 + 1.3 yrs of age (P = 0.02). Three died from cardiac failure, and three were older than 19 yrs without using NIV. The remaining 11 began nocturnal NIV at 22.9 + 5.3 yrs of age (P = 0.05). Eight of the 11became continuously NIV-dependent at age 28.9 + 7.3 yrs (P = 0.005). Conclusions: Intermittent glucocorticoid therapy delays wheelchair dependence and may delay ventilator dependence for patients with Duchenne muscular dystrophy.

20 - Angiotensin-converting-enzyme inhibitors versus steroids as first-line drug treatment in Duchenne muscular dystrophy - Letter to the editor

17 - Targeting Fibrosis in Duchenne Muscular Dystrophy

17 - Evaluation of Potential Synergistic Action of a Combined Treatment with Alpha-Methyl-Prednisonole and Taurine on the mdx Mouse Model of Duchenne Muscular Dystrophy

16 - (Neuromuscular Disorders, 2010) Debio-025 is more effective than prednisone in reducing muscular pathology in mdx mice

Erin R. Wissing, Douglas P. Millay, Grégoire Vuagniaux, Jeffery D. Molkentin - USA

Muscular dystrophy results in the progressive wasting and necrosis of skeletal muscle. Glucocorticoids such as prednisone have emerged as a front-line treatment for many forms of this disease. Recently, Debio-025, a cyclophilin inhibitor that desensitizes the mitochondrial permeability pore and subsequent cellular necrosis, was shown to improve pathology in three different mouse models of muscular dystrophy. However it is not known if Debio-025 can work in conjunction with prednisone, or how it compares against prednisone in mitigating disease in dystrophic mouse models. Here we show that Debio-025 reduced the variations in myofiber cross-sectional areas, decreased fibrosis, and decreased infiltration of activated macrophages more efficiently than prednisone. However the use of prednisone and Debio-025 together had no additional effect on these histopathological indexes. Orally administered Debio-025 also reduced creatine kinase blood levels and improved grip strength in mdx mice after 6 weeks of
treatment, and the combination of Debio-025 with prednisone increased muscle function slightly better than prednisone alone. Thus, our results suggest that Debio-025 is as, effective as or slightly better than, prednisone in mitigating muscular dystrophy in the mdx mouse model of disease.

4 - The respiratory management of patients with duchenne muscular dystrophy: A DMD care considerations working group specialty article

4 - Change in Natural History of Duchenne Muscular Dystrophy With Long-term Corticosteroid Treatment: Implications for Management


26 - (Annual Meeting Endocrinology Society - ENDO-2010) Growth Hormone Improves Growth in Duchenne Muscular Dystrophy Boys with Steroid-Induced Growth Failure.

MM Rutter, J Collins, JG Woo, SR Rose, H Sawnani, LH Cripe, KJ Kinnett, K Hor, BL Wong. Cincinnati Children's Hosp Med Ctr, Cincinnati, OH.

Background: Duchenne Muscular Dystrophy (DMD) is a progressive degenerative muscle disorder affecting 1 in 3500 boys. In the absence of a cure, daily high-dose glucocorticoids (GCs) are the mainstay of treatment, slowing disease progression and prolonging ambulation and survival. GCs cause growth failure, weight gain, absent puberty and osteoporosis, which negatively impact quality of life in DMD. Growth hormone (GH) offers potential benefit in DMD: it may help counter GC-induced growth failure, and could have positive effects on body fat, muscle strength and function. However, data regarding efficacy and safety of GH in DMD is lacking.
Objective: To evaluate efficacy and safety of GH in DMD boys with GC-induced growth failure during the first year of treatment.
Methods: We report a case-series of 29 DMD boys on daily GCs, treated in the Cincinnati Neuromuscular Comprehensive Care Center. The boys were treated with growth hormone for severe growth failure. Outcomes included growth velocity, height SD, weight, BMI, neuromuscular and cardiopulmonary function, and side effects.
Results: 29 prepubertal boys (mean age
SD 12.2 2.9y) were treated with GH for 4-32m (mean 12m). They had received daily GCs for 5.5 2.2y. Peak stimulated GH levels were 6.9 3.6 ng/ml. Height z-score (mean SEM) was -3.1 0.2 and height velocity was 1.1 0.3 cm/y before GH. During the first year on GH, height velocity improved to 5.6 0.7 cm/y (p<0.0001). Baseline decline in height z-score before GH (p<0.001) was followed by stabilization at -3.0 0.2 (p=0.2) on GH. There was a trend toward reduction in rate of weight gain at 1 year (2.8 0.7 to 0.6 1.1 kg/y, p=0.3), with decreased weight z-scores from -0.6 0.3 to -1.2 0.4 (p<0.0004). BMI z-score improved from 1.3 0.2 to 0.8 0.2 (p<0.0001). There were no detrimental effects on neuromuscular or cardiopulmonary function attributable to GH. GH was well tolerated, with 3/29 experiencing side effects by 1y. One boy developed worsening insulin resistance / impaired glucose tolerance. Two boys had progression of scoliosis.
Conclusions: GH treatment of DMD boys with GC-induced growth failure improved growth during the first year. Rate of weight gain slowed for some, with improvement in BMI. GH was relatively safe, with no detrimental effects on neuromuscular and cardiopulmonary function. Further study is needed before conclusions can be drawn regarding longer-term safety and efficacy.

 26 - (Annual Meeting Endocrinology Society - ENDO-2010)  Effect of Intravenous Bisphosphonate Therapy among Boys with Duchenne Muscular Dystrophy and Osteoporosis: Clinical Outcomes.

AM Sbrocchi, F Rauch, V Konji, M Tomiak, P Jacob, LM Ward. Children's Hosp of Eastern Ontario, Ottawa, Canada; Shriners Hosp for Children, Montreal, Canada.

Introduction: Boys with Duchenne Muscular Dystrophy (DMD) may develop symptomatic vertebral fractures. Bisphosphonates have been used to treat the spine fragility; however, detailed analyses of the response to therapy are lacking. The objective of this study was to assess the efficacy and safety of IV bisphosphonate treatment in boys with spinal osteoporosis due to DMD.
Methods: This was a one-year, retrospective observational study of 7 boys (age 8.5-14.3 years) with DMD who had received either IV pamidronate (9 mg/kg/year) or zoledronate (0.1 mg/kg/year) to treat painful vertebral fractures. The primary outcome was change in vertebral morphometry at 12 months post-bisphosphonate initiation. Secondary outcomes included back pain status, changes in lumbar spine volumetric bone mineral density (vBMD), and adverse events.
Results: A description of the cohort is presented in the Table. All but one had received glucocorticoid therapy prior to treatment initiation, and all but one was non-ambulatory. There were 27 fracture events noted in the 7 patients at baseline; 15/27 were in the T4-T9 region. Grade 2 (moderate) and Grade 3 (severe) vertebral fractures reconstituted at 12 months, and this was associated with either improvement or complete resolution of back pain. The median spine vBMD also increased. First-dose side effects were present in 4 patients and included fever (N=2), nausea (N=2), myalgias (N=3) and hypocalcemia (N=2).
Conclusion: In boys with spinal osteoporosis and DMD, IV bisphosponate therapy administered over 12 months was associated with improved vertebral morphometry and density; similarly, there was amelioration in back pain.
The therapy was generally well-tolerated.

Table. Clinical Parameters Pre- and 12 Months Post-Treatment

Clinical Characteristics

Results 12 Months Post Bisphosphonate Initiation (N=7)



12 Months Post




Height Z-score

-1.7 (-4.2, -0.5)

-2.0 (-3.5, -0.1)

Weight Z-score

0.4 ( -2.4, 1.8)

-1.7 (-1.9, 1.9)

Vertebral Morphometry



Genant Grade for VF Events, N (%)



Grade 0.5 = 15-19.9% loss in VH

5 (18%)

11 (41%)

Grade 1 = 20-25% loss in VH

4 (15%)

7 (26%)

Grade 2 = 25.1-40% loss in VH

14 (52%)

9 (33%)

Grade 3 = >40% loss in VH

4 (15%)

0 (0%)

Lumbar Spine Volumetric BMD (Z-score)

-1.0 (-3.0, 0.9)

-0.1 (-2.6, 1.4)

Values reported are median (min, max) unless otherwise specified. VH=Vertebral height, BMD= Bone mineral density.

26 - Functional and molecular effects of arginine butyrate and prednisone on muscle and heart in the mdx mouse model of Duchenne Muscular Dystrophy

20 -  (J Am Soc Echocardiogr 2010) Exercise-Induced Left Ventricular Systolic Dysfunction in Women Heterozygous for Dystrophinopathy

RobertM.Weiss,Richard E. Kerber,MD, Jane K. Jones, CarrieM. Stephan, Christina J. Trout, Paul D. Lindower, Kimberly S. Staffey, Kevin P. Campbell, Katherine D. Mathews - USA

Background:Mutations in the X-linked gene encoding dystrophin cause skeletal and cardiac muscle diseases in men. Female ‘‘carriers’’ also can develop overt disease. The purpose of this study was to ascertain the prevalence of cardiac contractile abnormalities in dystrophinopathy carriers. Methods: Twenty-four dystrophinopathy heterozygotes and 24 normal women each underwent standard exercise stress echocardiography. Results: Heterozygotes demonstrated mildly lower left ventricular ejection fractions (LVEFs) at rest compared with controls (0.56 6 0.10 vs 0.62 6 0.07, P = .02). After exercise, the mean LVEF fell to 0.53 6 0.14 in heterozygotes but rose to 0.7360.07 in controls (P < .001). Twenty-one of 24 dystrophinopathy heterozygotes demonstrated >1% of the following: abnormal resting LVEF, abnormal LVEF response to exercise, or exercise-induced wall motion abnormality. Conclusions: Women heterozygous for dystrophinopathy demonstrate significant left ventricular systolic dysfunction, which is unmasked by exercise. This finding has mechanistic implications for both inherited and acquired cardiac disease states.

20 - (Heart Rhythm, 2010) Electrocardiographic Abnormalities and Arrhythmias are Strongly Associated with the Development of Cardiomyopathy in Muscular Dystrophy

Anjan M. Shah, John L. Jefferies, Joseph W. Rossano, Jamie A. Decker, Bryan C. Cannon, Jeffrey J. Kim - USA

Objective: Assess the utility of electrocardiography in patients with muscular dystrophy. Background: Dilated cardiomyopathy is a well-recognized sequela of muscular dystrophy (MD). Early identification of cardiac involvement with timely therapy can favorably impact outcome. We hypothesize that electrocardiography can be a useful adjunct in the identification of DCM in MD. Methods: A retrospective review of patients with MD was performed. ECGs and Holters were analyzed to assess for association between abnormalities and the development of DCM. Results: One-hundred-fifty patients were identified. Forty-three percent of patients
(64/150) developed DCM. ECG abnormalities were found in 65% of patients and correlated well with the presence of DCM with 60/64 (94%) with DCM having an abnormal ECG vs. 38/86 (44%) without DCM (p<0.001). Only 4/52 (8%) of patients with normal ECGs had DCM. The presence of ECG abnormalities was highly sensitive (95.8%) but not specific (40.1%) to the presence of DCM. ECG abnormalities often preceded the development of DCM by a significant period of time (3.7+/-2.6 years). Arrhythmias were common with 17/150 (11%) of the cohort being affected. Those with DCM were significantly more likely to have an arrhythmia with 16/64 (25%) of that group being affected (p < 0.01). The presence of VT was a poor prognostic indicator with 6/11 patients dying within 0.68+/-0.41 years. Conclusions: ECG abnormalities are strongly associated with DCM in patients with MD
and frequently precede cardiac dysfunction by several years. Arrhythmias are common and periodic ECG and Holter evaluations are warranted as they may predict early cardiac involvement.

20 - Electrotransfer of the Full Length Dog Dystrophin in Mouse and Dystrophic Dog Muscles

12 - (Human Molecular Genetics, 2010) Site Directed Gene Repair of the Dystrophin Gene Mediated by PNA-ssODNs

Refik Kayali, Frederic Bury, McIver Ballard and Carmen Bertoni - USA

Permanent correction of gene defects is an appealing approach to the treatment of genetic disorders. The use of single-stranded oligodeoxynucleotides (ssODNs) has been demonstrated to induce single point mutations in the dystrophin gene and to restore dystrophin expression in skeletal muscle of models of Duchenne muscular dystrophy (DMD). Here we show that ssODNs made of peptide nucleic acids (PNA-ssODNs) can achieve gene repair frequencies more than 10 fold higher than those obtained using an older generation of targeting oligonucleotides. Correction was demonstrated in muscles cells isolated from mdx5cv mice and was stably inherited over time. Direct intramuscular injection of PNA-ssODNs targeting the mdx5cv mutation resulted in a significant increase in dystrophin positive fibers as compared to muscles that received the ssODNs designed to correct the dystrophin gene but made of unmodified bases. Correction was demonstrated at both the mRNA and the DNA levels using quantitative PCR and was confirmed by direct sequencing of amplification products. Analysis at the protein level demonstrated expression of full-length dystrophin in vitro as well as in vivo.

These results demonstrate that oligonucleotides promoting strand invasion in the DNA double helix can significantly enhance gene repair frequencies of the dystrophin gene. The use of PNA-ssODNs has important implications in terms of both efficacy and duration of the repair process in muscles and may have a role in advancing the treatment of DMD.

12 - Myocardial fibrosis is unaltered by long-term administration of L-arginine in dystrophin deficient mdx mice: A histomorphometric analysis

12 - Improvement of the mdx mouse dystrophic phenotype by systemic in utero AAV8 delivery of a minidystrophin gene

12 - Urban Mobility in Sao Paulo - Brazilian TV program with participation of Leonardo Feder

3 - (J. Cell Sci., Jun 2010; 123: 2008 - 2013) Sarcolemmal nNOS anchoring reveals a qualitative difference between dystrophin and utrophin

Dejia Li, Akshay Bareja, Luke Judge, Yongping Yue, Yi Lai, Rebecca Fairclough, Kay E. Davies, Jeffrey S. Chamberlain and Dongsheng Duan - USA

Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by dystrophin deficiency. In normal muscle, dystrophin helps maintain sarcolemmal stability. Dystrophin also recruits neuronal nitric oxide synthase (nNOS) to the sarcolemma. Failure to anchor nNOS to the membrane leads to functional ischemia and aggravates muscle disease in DMD. Over the past two decades, a great variety of therapeutic modalities have been explored to treat DMD. A particularly attractive approach is to increase utrophin expression. Utrophin shares considerable sequence, structural and functional similarity with dystrophin. Here, we test the hypothesis that utrophin also brings nNOS to the sarcolemma. Full-length utrophin cDNA was expressed in dystrophin-deficient mdx mice by gutted adenovirus or via transgenic overexpression. Subcellular nNOS localization was determined by immunofluorescence staining, in situ nNOS activity staining and microsomal preparation western blot. Despite supra-physiological utrophin expression, we did not detect nNOS at the sarcolemma. Furthermore, transgenic utrophin overexpression failed to protect mdx muscle from exercise-associated injury. Our results suggest that full-length utrophin cannot anchor nNOS to the sarcolemma. This finding might have important implications for the development of utrophin-based DMD therapies.

3 -  (J Child Neurol, Jun 2010) Read-Through Strategies for Suppression of Nonsense Mutations in Duchenne/Becker Muscular Dystrophy: Aminoglycosides and Ataluren (PTC124)

Richard S. Finkel - USA

Nucleotide changes within an exon can alter the trinucleotide normally encoding a particular amino acid, such that a new "stop" signal is transcribed into the mRNA open reading frame. This causes the ribosome to prematurely terminate its reading of the mRNA, leading to nonsense-mediated decay of the transcript and lack of production of a normal full-length protein. Such premature termination codon mutations occur in an estimated 10% to 15% of many genetically based disorders, including Duchenne/Becker muscular dys trophy. Therapeutic strategies have been developed to induce ribosomal read-through of nonsense mutations in mRNA and allow production of a full-length functional protein. Small-molecule drugs (aminoglycosides and ataluren [PTC124]) have been developed and are in clinical testing in patients with nonsense mutations within the dystrophin gene. Use of nonsense mutation suppression in Duchenne/Becker muscular dystrophy may offer the prospect of targeting the specific mutation causing the disease and correcting the fundamental pathophysiology.

3 -  (J Child Neurol, Jun 2010) The Potential of Exon Skipping for Treatment for Duchenne Muscular Dystrophy

Terence Partridge - USA

Duchenne muscular dystrophy is mainly caused by mutations that disrupt the generation of a translatable mRNA transcript. Most such mutations occur in parts of the gene that are not essential for its function and thus might be eliminated from the transcript to permit translation of a partially functional protein that would convert the disease to a milder clinical form. Two such antisense oligonucleotides of different backbone chemistries have been successful when tested on the mdx mouse, targeting exon 23, containing the nonsense mutation. Subsequently, the morpholino, the more effective of these, has been tested on the dystrophic dog, where it is necessary to skip 2 exons, again with beneficial results. Currently, results of 2 human trials targeting exon 51 have also yielded promising preliminary results.

3 - Widespread Muscle Expression of an AAV9 Human Mini-dystrophin Vector After Intravenous Injection in Neonatal Dystrophin-deficient Dogs

3- Arginine metabolism by macrophages promotes cardiac and muscle fibrosis in mdx muscular dystrophy


25 - (ANN NEUROL 2010;67:771–780) Gentamicin-Induced Readthrough of Stop Codons in Duchenne Muscular Dystrophy

Vinod Malik, Louise R. Rodino-Klapac, Laurence Viollet, Cheryl Wall, Wendy King, Roula Al-Dahhak, Sarah Lewis,
Christopher J. Shilling, Janaiah Kota, Carmen Serrano-Munuera, John Hayes, John D. Mahan, Katherine J. Campbell, Brenda Banwell, Majed Dasouki, Victoria Watts, Kumaraswamy Sivakumar, Ricardo Bien-Willner, Kevin M. Flanigan, Zarife Sahenk, Richard J. Barohn, Christopher M. Walker, Jerry R. Mendell - USA

Objective: The objective of this study was to establish the feasibility of long-term gentamicin dosing to achieve stop codon readthrough and produce full-length dystrophin. Mutation suppression of stop codons, successfully achieved in the mdx mouse using gentamicin, represents an important evolving treatment strategy in Duchenne muscular dystrophy (DMD).
Methods: Two DMD cohorts received 14-day gentamicin (7.5mg/kg/day): Cohort 1 (n 10) stop codon patients and Cohort 2 (n 8) frameshift controls. Two additional stop codon DMD cohorts were gentamicin treated (7.5mg/kg) for 6 months: Cohort 3 (n 12) dosed weekly and Cohort 4 (n 4) dosed twice weekly. Pre- and post-treatment biopsies were assessed for dystrophin levels, as were clinical outcomes.Results: In the 14-day study, serum creatine kinase (CK) dropped by 50%, which was not seen in frameshift DMD controls. After 6 months of gentamicin, dystrophin levels significantly increased (p 0.027); the highest levels reached 13 to 15% of normal (1 in Cohort 3, and 2 in Cohort 4), accompanied by reduced serum CK favoring drug-induced readthrough of stop codons. This was supported by stabilization of strength and a slight increase in forced vital capacity. Pretreatment stable transcripts predicted an increase of dystrophin after gentamicin. Readthrough efficiency was not affected by the stop codon or its surrounding fourth nucleotide. In 1 subject, antigen-specific interferon- enzyme-linked immunospot assay detected an immunogenic dystrophin epitope. Interpretation: The results support efforts to achieve drug-induced mutation suppression of stop codons. The immunogenic epitope resulting from readthrough emphasizes the importance of monitoring T-cell immunity during clinical studies that suppress stop codons. Similar principles apply to other molecular strategies, including exon skipping and gene therapy.

25 - (Muscle Nerve 41: 746–750, 2010) Inefficient Dystrophin Expression after Cord Blood Transplantation in Duchenne Muscular Dystrophy


ABSTRACT: We report a boy who received two allogeneic stem cell transplantations from umbilical cord donors to treat chronic granulomatous disease (CGD). The CGD was cured after the second transplantation, but 2.5 years later he was diagnosed with Duchenne muscular dystrophy (DMD). Examinations of his DNA, muscle tissue, and myoblast cultures derived from muscle tissue were performed to determine whether any donor dystrophin was being expressed. The boy was found to have a large-scale deletion on the X chromosome that spanned the loci for CYBB and DMD. The absence of dystrophin led to muscle histology characteristic of DMD. Analysis of myofibers demonstrated no definite donor cell engraftment. This case suggests that umbilical cord–derived hematopoietic stem cell transplantation will not be efficacious in the therapy of DMD without additional interventions that induce engraftment of donor cells in skeletal muscle.



Duchenne muscular dystrophy (DMD) is one of the most commonly recognized dystrophinopathies. There are no approved therapeutic options available for this disease but recent discoveries have led to hope that effective therapies might be forthcoming. With funding from patient advocacy groups, private investors, and governmental bodies such as the Food and Drug Administration Office of Orphan Product Development (FDA/OOPD), gene modification and other molecular therapies are being actively investigated. However, since DMD patients are few in number and disease manifestations vary considerably in early and late stages of disease, obtaining the data needed for full evaluation of putative therapies may prove challenging. Should ambulation remain the focus of Phase 2/3 studies or should consideration be given to the primary causes of late-stage morbidity and mortality, e.g., cardiac and respiratory dysfunction related to reduced or absent dystrophin production? It seems reasonable to argue that clinical trials planned for DMD should consider the entire population.

22 - (Pediatr Pulmonol. 2010; 45:552-559) Ventilatory parameters and maximal respiratory pressure changes with age in Duchenne muscular dystrophy patients

Jerome Gayraud, Michele Ramonatxo, François Rivier, MD, Véronique Humberclaude, Basil Petrof,  Stefan Matecki

The aim of this longitudinal study was to precise, in children with Duchenne muscular dystrophy, the respective functional interest of ventilatory parameters (Vital capacity, total lung capacity and forced expiratory volume in one second [FEV1]) in comparison to maximal inspiratory pressure (Pimax) during growth. In ten boys the mean age of 9.1 ± 1 years) to mean age of 16 ± 1.4 years followed over a period of 7 years, we found that: (1) ventilatory parameters expressed in percentage of predicted value, after a normal ascending phase, start to decrease between 11 and 12 years, (2) Pimax presented only a decreasing phase since the beginning of the study and thus was already at 67% of predicted value at 12 years while ventilatory parameters was still normal, (3) after 12 years the mean slopes of decrease per year of vital capacity and FEV1 were higher (10.7 and 10.4%) than that of Pimax (6.9%), (4) at 15 years mean values of vital capacity and FEV1 (53.3 and 49.5% of predicted values) was simlar to that of Pimax (48.3%). In conclusion, if at early stages of the disease, Pimax is a more reliable index of respiratory impaiment than ventilatory parameters, the follow-up of ventilatory parameters, when they start to decrease, is a better indicator of disease progression and, at advanced stages they provided same information about the functional impact of disease.

15 -  (Am J Pathol 2010, 177) Matrix Metalloproteinase Inhibitor Batimastat Alleviates Pathology and Improves Skeletal Muscle Function in Dystrophin-Deficient mdx Mice

Akhilesh Kumar, Shephali Bhatnagar AND Ashok Kumar - USA

Duchenne muscular dystrophy (DMD) , caused by mutations in the dystrophin gene, involves severe muscle degeneration, inflammation, fibrosis, and early death in afflicted boys. Matrix metalloproteinases (MMPs) are extracellular proteases that cause tissue degradation in several disease states. In this study, we tested the hypothesis that the expression levels of various MMPs are abnormally increased and that their inhibition will ameliorate muscle pathogenesis in animal models of DMD. Our results show that the transcript levels of several MMPs are significantly up-regulated, whereas tissue inhibitors of MMPs are down-regulated, in dystrophic muscle of mdx mice. Chronic administration of batimastat (BB-94) , a broad spectrum peptide inhibitor of MMPs, reduced necrosis, infiltration of macrophages, centronucleated fibers, and the expression of embryonic myosin heavy chain in skeletal muscle of mdx mice. Batimastat also reduced the expression of several inflammatory molecules and augmented the levels of sarcolemmal protein -dystroglycan and neuronal nitric oxide in mdx mice. In addition, muscle force production in isometric contraction was increased in batimastat-treated mdx mice compared with those treated with vehicle alone. Furthermore, inhibition of MMPs using batimastat reduced the activation of mitogen-activated protein kinases and activator protein-1 inmyofibers ofmdxmice. Our study provides the novel evidence that the expression of MMPs is atypically increased in DMD, that their inhibition ameliorates pathogenesis, and that batimastat could prove to be a significant candidate for DMD therapy.

15 - The Dietary Supplement Protandim® Decreases Plasma Osteopontin and Improves Markers of Oxidative Stress in Muscular Dystrophy Mdx Mice


25 -  50 Researchs that will be presented in Annual Meeting of American Society of Gene & Cell Therapy - May 17-20, Washington, USA

25 - (Abstracts of the UK Neuromuscular Translational Research Conference Oxford, UK 25-26 March 2010)

Results of a systemic antisense study in Duchenne muscular dystrophy

Current progress with the systemic administration trial of AVI-4658, a novel Phosphorodiamidate Morpholino Oligomer
(PMO) skipping dystrophin exon 51 in Duchenne muscular dystrophy (DMD)

Multiexon skipping in Duchenne muscular dystrophy

Lentivirus-mediated stem cell therapy for Duchenne muscular dystrophy

Induction of dystrophin in Duchenne muscular dystrophy patients by antisense oligonucleotide AVI-4658 restores the dystrophin-associated glycoprotein complex

Evaluation of the truncated products of exon and multiple exon skipping in DMD therapy

Translation related clinical trials in duchenne muscular dystrophy (DMD) in the UK

Exploring emotional impact in a proof-of-principle single-blind, controlled, two-doses escalation intramuscular study of a morpholino splice-switching oligonucleotide (AVI-4658) trial to induce dystrophin restoration in children with Duchenne muscular dystrophy

A Novel Ankle foot orthoses/footwear combination to aid walking in Duchenne muscular dystrophy

Parental stress levels in parents of children with muscular dystrophy

UK NorthStar Neuromuscular Clinical Network (NSCN): National audit results in Duchenne muscular dystrophy (DMD) corticosteroid practice, vitamin D status and bone health

An audit of bone density and vertebral fractures during steroid treatment in Duchenne muscular dystrophy

Blocking calcium influx with streptomycin worsens myocardial pathology in the mdx mouse model of muscular dystrophy

Utrophin luciferase knock-in mouse model for in vivo assessment of drug efficacy in preclinical trials for utrophin upregulation

Rescu of severely affected dystrophin/utrophin deficient mice by morpholino-oligomer mediated exon skipping

Chronic long term administration of phosphorodiamidate morpholino oligomer profoundly ameliorates activity, muscle strength and phenotype in dystrophic mdx mice

Preventing dystroglycan phosphorylation as a route to therapy in DMD

22 - (British Journal of Pharmacology, 2010) Co-administration of ibuprofen and nitric oxide is an effective experimental therapy for muscular dystrophy, with immediate applicability to humans

Clara Sciorati, Roberta Buono, Emanuele Azzoni, Silvana Casati, Pierangela Ciuffreda, Grazia D'Angelo, Dario Cattaneo, Silvia Brunelli, Emilio Clementi - Italy

Background and purpose: Current therapies for muscular dystrophy are based on corticosteroids. Significant side effects associated with these therapies have prompted several studies aimed at identifying possible alternative strategies. As inflammation and defects of nitric oxide (NO) generation are key pathogenic events in muscular dystrophies, we have studied the effects of combining the NO donor isosorbide dinitrate (ISDN) and the non-steroidal anti-inflammatory drug ibuprofen.

Experimental approach: α-Sarcoglycan null mice were treated for up to 8 months with ISDN (30 mg/kg) plus ibuprofen (50 mg/kg) administered daily in the diet. Effects of ISDN and ibuprofen alone were assessed in parallel. Drug effects on animal motility and muscle function, muscle damage, inflammatory infiltrates and cytokine levels, as well as muscle regeneration including assessment of endogenous stem cell pool, were measured at selected time points.

Key results: Combination of ibuprofen and ISDN stimulated regeneration capacity, of myogenic precursor cells, reduced muscle necrotic damage and inflammation. Muscle function in terms of free voluntary movement and resistance to exercise was maintained throughout the time window analysed. The effects of ISDN and ibuprofen administered separately were transient and significantly lower than those induced by their combination.

Conclusions and implications: Co-administration of NO and ibuprofen provided synergistic beneficial effects in a mouse model of muscular dystrophy, leading to an effective therapy. Our results open the possibility of immediate clinical testing of a combination of ISDN and ibuprofen in dystrophic patients, as both components are approved for use in humans, with a good safety profile.

17 - Meganucleases can restore the reading frame of a mutated dystrophin

17 - Summary of Ataluren Phase 2b Clinical Trial Results Presented at the American Academy of Neurology Meeting on April 16, 2010

17 - Effect of creatine monohydrate in improving cellular energetics and muscle strength in ambulatory Duchenne muscular dystrophy patients: a randomized, placebo-controlled (31)P MRS study

10 - (FASEB Meeting, 2010) Parallel increasing of {alpha}7 and β1 integrin prevents muscular dystrophy in mdx mice

Jianming Liu1,2 and Stephen J. Kaufman2

1 Cell & Tissue Biology, University of California San Francisco, San Francisco, CA
2 Cell & Developmental Biology, University of Illinois, Urbana, IL

Duchenne muscular dystrophy is a devastating muscle disease that is ultimately lethal. Without the dystrophin complex that normally connects myofibers actin cytoskeleton to the laminin in extracellular matrix, membrane integrity of muscle fibers is greatly compromised. One potential therapeutic treatment for muscular dystrophy is to augment the transmembrane linkages by increasing other laminin receptors such as integrins. We previously showed that transgenic expression of {alpha}7 chain of the integrin in skeletal muscle effectively alleviates the dystrophic pathologies and extends the lifespan of mdx/utrn–/– mice. However, expression of {alpha}7 chain alone in mdx mice provided little improvement of skeletal muscle health. We now found that in transgenic mice expressing high levels of {alpha}7 chain, relatively little {alpha}7 was targeted to the muscle membrane and thus mostly remained within myofibers, likely due to limiting amount of the endogenous integrin β1 protein. We then demonstrated that overexpress β1D integrin results in {alpha}7 chain increase in wild type mice and that commensurate increase of β1 chains in {alpha}7 transgenic mice promotes more functional heterodimers targeting to the sarcolemma. Likewise, increasing the amount of β1D integrin in {alpha}7-mdx transgenic mice also promotes localization of the {alpha}7β1 integrin to the sarcolemma and protects against muscle damages caused by the muscular dystrophy. Our results suggest that parallel increases of {alpha} and β integrin subunits are essential to achieve maximal beneficial effects of integrin based therapies for muscular dystrophy

10 - (FASEB Meeting, 2010) Regenerative medicine based on muscle stem cells

Johnny Huard and Henry J. Mankin

Dept. of Orthopaedic Surgery and Molecular Genetics & Biochemistry, University of Pittsburgh, School of Medicine, Pittsburgh, PA

Members of the Stem Cell Research Center (SCRC) have isolated various populations of myogenic cells from the postnatal skeletal muscle of normal mice by means of the cells’ adhesion characteristics, proliferation behavior, and myogenic and stem cell marker expression profiles. Most of these cell populations have displayed characteristics similar to those of skeletal muscle satellite cells; however, we also have identified a unique population of muscle-derived stem cells (MDSCs). The MDSCs exhibit long-term proliferation and high self-renewal abilities, increased resistance to stress, and multipotency with the ability to differentiate, in vitro and in vivo, into a variety of tissue types including: muscular (skeletal and cardiac), neural, endothelial, osteogenic, and chondrogenic lineages. In contrast to other myogenic cell types, MDSCs are very efficient at engrafting and regenerating a variety of musculoskeletal tissues due to their increased ability to survive post-implantation because of their high expression of anti-oxidants. MDSCs activate host cells and are influenced by environmental cues released within injured tissues, which have been shown to impact the MDSCs regenerative capacity in various tissues. Some of the results regarding the crosstalk between the donor cells and host cells/tissues will be presented. Potential strategies are being explored to increase angiogenesis and prevent scar tissue formation within injured tissues as a means to further improve the regenerative potential of these cells. Finally recent results will be presented that suggest that the blood vessel walls harbor several cell types, including myo-endothelial cells and pericytes that are likely the place of origin of the murine MDSCs discussed above. These human blood vessels derived cells are being tested for their regenerative potential in various tissues. The results outlined above open new avenues by which researchers could use muscle stem cell–based gene therapy and tissue engineering to improve tissue regeneration.

10 - (FASEB Meeting, 2010) Loss of nNOS Signaling Exacerbates Dystrophic Pathology, but not Skeletal Muscle Fatigue in the mdx Mouse Model of Duchenne Muscular Dystrophy

Justin Percival, Sarah Reed, Ken Bible and Stanley Froehner

University of Washington, Seattle, WA

Debilitating fatigue is common in muscle wasting diseases such as Duchenne Muscular Dystrophy (DMD). NO produced by nNOSµ and nNOSβ is a critical regulator of skeletal muscle fatigue. Levels of NOSµ are depleted in the skeletal muscles of DMD patients and may contribute to their fatigue. Increased nitric oxide (NO) levels in the mdx mouse (a model for DMD) significantly reduced dystrophic pathology; however, it is unclear whether the anti-dystrophic effects of exogenous NO are due to activation of downstream targets of nNOSµ or nNOSβ or both. To address nNOS function in mdx muscle, mdx mice were engineered to lack residual nNOSµ and all nNOSβ nNOS-deficient mdx muscles were smaller in size and showed evidence of increased inflammation. Tibialis anterior (TA) muscles from nNOS-deficient mdx mice exhibited reduced maximal tetanic and specific force capacity. Unexpectedly, nNOS-deficient mdx muscle exhibited increased susceptibility to injury by eccentric contractions. Also surprisingly, absence of both nNOS isozymes from mdx muscle did not affect muscle fatigue. In conclusion, the absence of fatigue in nNOS-deficient mdx muscle suggests the existence of mechanisms that compensate for absent nNOS signaling. This novel compensatory mechanism may contribute to the mild phenotype of the mdx mouse compared to its DMD homologue. These data support the hypothesis that nNOS, particularly nNOSβ, modulates clinically relevant features of dystrophin-deficient muscle.

10 - (FASEB Meeting, 2010) Pharmacological suppression of nonsense mutations to treat genetic diseases

David Michael Bedwell1, Ming Du1, Jessica Buckley1, Dan Wang2, Kim Marie Keeling1, Ellen Welch3 and Stuart W Peltz3

1 Dept of Microbiology
2 Dept of Genetics, University of Alabama at Birmingham, Birmingham, AL
3 PTC Therapeutics, Inc, South Plainfield, NJ

We are exploring the use of pharmacological agents to suppress nonsense mutations that cause genetic diseases. This approach could theoretically restore the synthesis of full length, functional proteins and relieve disease phenotypes. Much of our work focuses on cystic fibrosis (CF), a life-threatening genetic disease that compromises function of the lungs and pancreas. CF is caused by mutations in the CFTR gene, which encodes the CFTR protein, a cAMP-activated chloride channel. In cultured cells, we have shown that readthrough agents such as gentamicin and ataluren (formerly PTC124®) suppress CFTR nonsense mutations, resulting in a partial restoration of CFTR protein and function. Using a transgenic mouse model expressing the human CFTR gene with the G542X nonsense mutation found in CF patients, we have shown that these compounds can restore human CFTR expression and function in vivo. Recent data from a phase II clinical study support these conclusions, and show that ataluren can reduce the electrophysiological defects associated with CFTR nonsense mutations in CF patients. This therapeutic approach is also currently being applied to Duchenne Muscular Dystrophy and the lysosomal storage disease Hurler syndrome (MPS I-H). Together, these results suggest that this approach could benefit a genotypic subset of patients with a wide range of genetic diseases caused by nonsense mutations

10 - (FASEB Meeting, 2010) Postnatal PGC-1{alpha} gene transfer attenuates acute injury in mdx mice

Joshua T Selsby and Delphine Gardan-Salmon

Animal Science, Iowa State University, Ames, IA

A promising strategy to treat Duchenne muscular dystrophy aims to replace the aberrant dystrophin protein with utrophin, a dystrophin related-protein. Induction of PGC-1{alpha} increases utrophin and may also support damaged mitochondria. Previously, constitutive PGC-1{alpha} over-expression reduced acute injury in dystrophic skeletal muscle. As dystrophin deficiency results in developmental abnormalities potentially improved by constitutive PGC-1{alpha} over-expression, it is imperative to determine the extent to which post-natal PGC-1{alpha} over-expression prevents acute injury in dystrophic muscle. Neonatal mdx mice were injected in the right limb with 1x1011 gc of AAV 2/6 causing over-expression of PGC-1{alpha} while the left was injected with null virus. At 6 weeks of age, mice performed downhill running (–17°, 10m/min for one hour) to cause an acute eccentric injury, and were sacrificed 72 hours later. Following injury, PGC-1{alpha} over-expression in the soleus caused a 50% reduction in necrotic area and Evan’s blue dye penetration (p<0.05), and tended to reduce central nucleation when compared to contralateral control muscle. These data indicate that PGC-1{alpha} gene transfer following in utero development protects dystrophic muscle from acute injury.

10 -  (FASEB Meeting, 2010) The Effects of Exercise on Ventricular Wall Thickness and Fibrosis in Wild-Type and Dystrophin-Deficient Mice

Jeffrey H Plochocki and Jeffrey M Costas

Arizona College of Osteopathic Medicine, Midwestern University, Glendale, AZ

Dystrophin is a cytoplasmic protein that connects muscle cells to the extracellular matrix. When absent, muscle degeneration and fibrosis occurs that resembles the histopathological presentation of Duchenne muscular dystrophy. In mice that lack dystrophin (Mdx mice), there is muscle degeneration and regeneration in the first few months of life, with fibrosis increasing after that. In this study, we evaluated the relationship between exercise and lateral ventricular wall thickness (LVT) in wild-type and Mdx mice aged 11 weeks. Degree of cardiac fibrosis was assessed using a trichrome stain. Sedentary Mdx mice exhibited significantly larger LVT than wild-type mice (P < 0.05). Exercise treated wild-type mice had larger LVT than sedentary wild-type mice on average, but this difference was not statistically significant. The LVT of sedentary Mdx mice was 28% larger than that of exercise treated Mdx mice (P < 0.05). Exercised treated Mdx mice were the only group that exhibited any noticeable fibrosis in the lateral aspect of the heart. Our findings suggest that exercise within the normal physiological range can accelerate degeneration and fibrosis of the lateral ventricular wall in Mdx mice.

10 - (FASEB J, Apr 2010) MAP kinase phosphatase-1 deficiency impairs skeletal muscle regeneration and exacerbates muscular dystrophy

Hao Shi, Emmanuel Boadu, Fatih Mercan, Annie M. Le, Rachel J. Roth Flach, Lei Zhang, Kristina J. Tyner, Bradley B. Olwin, and Anton M. Bennett - USA

In skeletal muscle, the mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) is a critical negative regulator of the MAPKs. Since the MAPKs have been reported to be both positive and negative for myogenesis, the physiological role of MKP-1 in skeletal muscle repair and regeneration has remained unclear. Here, we show that MKP-1 plays an essential role in adult regenerative myogenesis. In a cardiotoxin-induced muscle injury model, lack of MKP-1 impaired muscle regeneration. In mdx mice, MKP-1 deficiency reduced body weight, muscle mass, and muscle fiber cross-sectional area. In addition, MKP-1-deficient muscles exhibit exacerbated myopathy accompanied by increased inflammation. Lack of MKP-1 compromised myoblast proliferation and induced precocious differentiation, phenotypes that were rescued by pharmacological inhibition of p38{alpha}/{beta} MAPK. MKP-1 coordinates both myoblast proliferation and differentiation. Mechanistically, MyoD bound to the MKP-1 promoter and activated MKP-1 expression in proliferating myoblasts. Later, during myogenesis, MyoD uncoupled from the MKP-1 promoter leading to the down-regulation of MKP-1 and facilitation of promyogenic p38{alpha}/{beta} MAPK signaling. Hence, MKP-1 plays a critical role in muscle stem cells and in the immune response to coordinate muscle repair and regeneration

10 - (Journal of Neuroimmunology, 2010) Differential integrin expression by T lymphocytes: Potential role in DMD muscle damage

Fernanda Pinto-Mariz, Luciana Rodrigues Carvalho, Wallace de Mello, Alexandra de Queiroz Campos Araújo, Márcia Gonçalves Ribeiro, Maria do Carmo Soares Alves Cunha, Thomas Voit, Gillian Butler-Browne, Suse Dayse Silva-Barbosa, Wilson Savino - Brazil and France

The expression and function of integrin-type extracellular matrix receptors, VLA-4 and VLA-5, and laminin receptor VLA-6 on the surface of CD3+CD4+ and CD3+CD8+ defined T cell populations was evaluated in the blood of Duchenne muscular dystrophy (DMD) patients and healthy individuals. Both the number of CD4+ and CD8+ T cell subsets expressing VLA-4 or VLA-5 and the fibronectin-driven T cell migration was significantly higher in DMD patients. These data indicate that interactions of VLA-4 and/or VLA-5 with fibronectin may drive T lymphocytes to specific niches within muscle, contributing to tissue damage and fibrosis in DMD patients.

2- (Am. J. Pathol., Apr 2010) Antibody-Directed Myostatin Inhibition Improves Diaphragm Pathology in Young but not Adult Dystrophic mdx Mice

Kate T. Murphy, James G. Ryall, Sarah M. Snell, Lawrence Nair, René Koopman, Philip A. Krasney, Chikwendu Ibebunjo, Kathryn S. Holden, Paula M. Loria, Christopher T. Salatto, and Gordon S. Lynch - Australia

Duchenne muscular dystrophy (DMD) is characterized by progressive skeletal muscle wasting and weakness, leading to premature death from respiratory and/or cardiac failure. A clinically relevant question is whether myostatin inhibition can improve function of the diaphragm, which exhibits a severe and progressive pathology comparable with that in DMD. We hypothesized that antibody-directed myostatin inhibition would improve the pathophysiology of diaphragm muscle strips from young mdx mice (when the pathology is mild) and adult mdx mice (when the pathology is quite marked). Five weeks treatment with a mouse chimera of anti-human myostatin antibody (PF-354, 10 mg/kg/week) increased muscle mass (P < 0.05) and increased diaphragm median fiber cross-sectional area (CSA, P < 0.05) in young C57BL/10 and mdx mice, compared with saline-treated controls. PF-354 had no effect on specific force (sPo, maximum force normalized to muscle CSA) of diaphragm muscle strips from young C57BL/10 mice, but increased sPo by 84% (P < 0.05) in young mdx mice. In contrast, 8 weeks of PF-354 treatment did not improve muscle mass, median fiber CSA, collagen infiltration, or sPo of diaphragm muscle strips from adult mdx mice. PF-354 antibody-directed myostatin inhibition completely restored the functional capacity of diaphragm strips to control levels when treatment was initiated early, but not in the later stages of disease progression, suggesting that such therapies may only have a limited window of efficacy for DMD and related conditions.

1 - (J. Biochem., Apr 2010; 147: 463 - 470) Transdermal delivery of a readthrough-inducing drug: a new approach of gentamicin administration for the treatment of nonsense mutation-mediated disorders

Masataka Shiozuka, Akira Wagatsuma, Tadafumi Kawamoto, Hiroyuki Sasaki, Kenichi Shimada, Yoshikazu Takahashi, Yoshiaki Nonomura, and Ryoichi Matsuda - Japan

To induce the readthrough of premature termination codons, aminoglycoside antibiotics such as gentamicin have attracted interest as potential therapeutic agents for diseases caused by nonsense mutations. The transdermal delivery of gentamicin is considered unfeasible because of its low permeability through the dermis. However, if the skin permeability of gentamicin could be improved, it would allow topical application without the need for systemic delivery. In this report, we demonstrated that the skin permeability of gentamicin increased with the use of a thioglycolate-based depilatory agent. After transdermal administration, the readthrough activity in skeletal muscle, as determined using a lacZ/luc reporter system, was found to be equivalent to systemic administration when measured in transgenic mice. Transdermally applied gentamicin was detected by liquid chromatography-tandem mass spectrometry in the muscles and sera of mice only after depilatory agent-treatment. In addition, expansion of the intercellular gaps in the basal and prickle-cell layers was observed by electron microscopy only in the depilatory agent-treated mice. Depilatory agent-treatment may be useful for the topical delivery of readthough-inducing drugs for the rescue of nonsense mutation-mediated genetic disorders. This finding may also be applicable for the transdermal delivery of other pharmacologically active molecules.


30 - (Bioorganic & Medicinal Chemistry,2010) Repairing faulty genes by aminoglycosides: Development of new derivatives of geneticin (G418) with enhanced suppression of diseases-causing nonsense mutations

Igor Nudelamn, Dana Glikin, Boris Smolkin, Mariana Hainrichson, Valery Belakhov and Timor Baasov - Israel

New pseudo-di- and pseudo-trisaccharide derivatives of the aminoglycoside drug G418 were designed, synthesized and their ability to readthrough nonsense mutations was examined in both in vitro and ex vivo systems, along with the toxicity tests. Two novel lead structures, NB74 and NB84, exhibiting significantly reduced cell toxicity and superior readthrough efficiency than those of gentamicin, were discovered. The superiority of new leads was demonstrated in six different nonsense DNA-constructs underling the genetic diseases cystic fibrosis, Duchenne muscular dystrophy, Usher syndrome and Hurler syndrome.

27 - The development of antisense oligonucleotide therapies for Duchenne muscular dystrophy: Report on a TREAT-NMD workshop hosted by the European Medicines Agency (EMA), on September 25th 2009

19 - (Am. J. Pathol. 2010,176(4):1863-1877)Therapeutic Potential of Proteasome Inhibition in Duchenne and Becker Muscular Dystrophies
Elisabetta Gazzerro, Stefania Assereto, Andrea Bonetto, Federica Sotgia, Sonia Scarfì, Angela Pistorio, Gloria Bonuccelli, Michele Cilli, Claudio Bruno, Federico Zara, Michael P. Lisanti, and Carlo Minetti - Italy

Duchenne muscular dystrophy (DMD) and its milder allelic variant, Becker muscular dystrophy (BMD), result from mutations of the dystrophin gene and lead to progressive muscle deterioration. Enhanced activation of proteasomal degradation underlies critical steps in the pathogenesis of the DMD/BMD dystrophic process. Previously, we demonstrated that treatment with the proteasome inhibitor MG-132 rescues the cell membrane localization of dystrophin and the dystrophin glycoprotein complex in mdx mice, a natural genetic mouse model of DMD. The current work aims to thoroughly define the therapeutic potential in dystrophinopathies of Velcade, a drug that selectively blocks the ubiquitin-proteasome pathway. Velcade is particularly intriguing since it has been approved for the treatment of multiple myeloma. Therefore, its side effects in humans have been explored. Velcade effects were analyzed through two independent methodological approaches. First, we administered the drug systemically in mdx mice over a 2-week period. In this system, Velcade restores the membrane expression of dystrophin and dystrophin glycoprotein complex members and improves the dystrophic phenotype. In a second approach, we treated with the compound explants from muscle biopsies of DMD or BMD patients. We show that the inhibition of the proteasome pathway up-regulates dystrophin, {alpha}-sarcoglycan, and {beta}-dystroglycan protein levels in explants from BMD patients, whereas it increases the proteins of the dystrophin glycoprotein complex in DMD cases.

17 - (J.Clin.Invest., March,2010) Chronic administration of membrane sealant prevents severe cardiac injury and ventricular dilatation in dystrophic dogs

DeWayne Townsend, Immanuel Turner, Soichiro Yasuda, Joshua Martindale, Jennifer Davis, Michael Shillingford, Joe N. Kornegay and Joseph M. Metzger - USA

Duchenne muscular dystrophy (DMD) is a fatal disease of striated muscle deterioration caused by lack of the cytoskeletal protein dystrophin. Dystrophin deficiency causes muscle membrane instability, skeletal muscle wasting, cardiomyopathy, and heart failure. Advances in palliative respiratory care have increased the incidence of heart disease in DMD patients, for which there is no cure or effective therapy. Here we have shown that chronic infusion of membrane-sealing poloxamer to severely affected dystrophic dogs reduced myocardial fibrosis, blocked increased serum cardiac troponin I (cTnI) and brain type natriuretic peptide (BNP), and fully prevented left-ventricular remodeling. Mechanistically, we observed a markedly greater primary defect of reduced cell compliance in dystrophic canine myocytes than in the mildly affected mdx mouse myocytes, and this was associated with a lack of utrophin upregulation in the dystrophic canine cardiac myocytes. Interestingly, after chronic poloxamer treatment, the poor compliance of isolated canine myocytes remained evident, but this could be restored to normal upon direct application of poloxamer. Collectively, these findings indicate that dystrophin and utrophin are critical to membrane stability–dependent cardiac myocyte mechanical compliance and that poloxamer confers a highly effective membrane-stabilizing chemical surrogate in dystrophin/utrophin deficiency. We propose that membrane sealant therapy is a potential treatment modality for DMD heart disease and possibly other disorders with membrane defect etiologies.

13 - (Cardiovasc Res, Mar 2010) The histone deacetylase inhibitor suberoylanilide hydroxamic acid reduces cardiac arrhythmias in dystrophic mice

Claudia Colussi, Roberta Berni, Jessica Rosati, Stefania Straino, Serena Vitale, Francesco Spallotta, Silvana Baruffi, Leonardo Bocchi, Francesca Delucchi, Stefano Rossi, Monia Savi, Dante Rotili, Federico Quaini, Emilio Macchi, Donatella Stilli, Ezio Musso, Antonello Mai, Carlo Gaetano, and Maurizio C. Capogrossi - Italy

Aims The effect of histone deacetylase inhibitors on dystrophic heart function is not established. To investigate this aspect, dystrophic mdx mice and wild-type (WT) animals were treated 90 days either with suberoylanilide hydroxamic acid (SAHA, 5 mg/kg/day) or with an equivalent amount of vehicle.

Methods and results The following parameters were evaluated: (i) number of ventricular arrhythmias in resting and stress conditions (restraint test) or after aconitine administration; (ii) cardiac excitability, conduction velocity, and refractoriness; (iii) expression and distribution of connexins (Cxs) and Nav1.5 sodium channel. Ventricular arrhythmias were negligible in all resting animals. During restraint, however, an increase in the number of arrhythmias was detected in vehicle-treated mdx mice (mdx-V) when compared with SAHA-treated mdx (mdx-SAHA) mice or normal control (WT-V). Interestingly, aconitine, a sodium channel pharmacologic opener, induced ventricular arrhythmias in 83% of WT-V mice, 11% of mdx-V, and in 57% of mdx-SAHA. Epicardial multiple lead recording revealed a prolongation of the QRS complex in mdx-V mice in comparison to WT-V and WT-SAHA mice, paralleled by a significant reduction in impulse propagation velocity. These alterations were efficiently counteracted by SAHA. Molecular analyses revealed that in mdx mice, SAHA determined Cx remodelling of Cx40, Cx37 and Cx32, whereas expression levels of Cx43 and Cx45 were unaltered. Remarkably, Cx43 lateralization observed in mdx control animals was reversed by SAHA treatment which also re-induced Nav1.5 expression.

Conclusion SAHA attenuates arrhythmias in mdx mice by a mechanism in which Cx remodelling and sodium channel re-expression could play an important role.

10 - (Journal of Pineal Research 2010, 48(3): 282-289) Melatonin treatment normalizes plasma pro-inflammatory cytokines and nitrosative/oxidative stress in patients suffering from Duchenne muscular dystrophy

Mariam Chahbouni, Germaine Escames, Carmen Venegas, Belén Sevilla, José Antonio García, Luis C. López, Antonio Muñoz-Hoyos, Antonio Molina-Carballo, Darío Acuña-Castroviejo - Spain

Duchenne muscular dystrophy (DMD), a lethal disorder characterized by dystrophin absence, courses with chronic inflammation, sarcolemmal damage, and skeletal muscle degeneration. Among the multiple pathogenic mechanisms proposed for DMD, oxidative stress and inflammation are directly involved in the dystrophic process. Unfortunately, there is no current treatment for DMD, and the inflammatory process is an important target for therapies. Based on the antioxidant and anti-inflammatory properties of melatonin, we investigated whether melatonin treatment may reduce the dystrophic process. Ten DMD patients aged 12.8 ± 0.98 yr, were treated with melatonin (60 mg at 21:00 hr plus 10 mg at 09:00 hr), and plasma levels of lipid peroxidation (LPO), nitrites (NOx), interleukin (IL)-1β, IL-2, IL-6, tumor necrosis factor-α, interferon-γ, and plasma markers of muscle injury, were determined at 3, 6 and 9 months of treatment. Healthy age- and sex-matched subjects were used as controls. The results show a significant increase in LPO, NOx, and cytokine levels in plasma of DMD patients compared with controls. Melatonin administration reduced these values to control levels at 3 months of treatment, decreasing further 9 months later. In parallel, melatonin also reduced plasma levels of creatine kinase (CK; 50%), lactate dehydrogenase (28%), aspartate aminotransferase (28%), alanine aminotransferase (20%), and myoglobin (13%). These findings strongly support the conclusion that melatonin administration significantly reduced the hyperoxidative and inflammatory process in DMD patients, reducing the muscle degenerative process.

6 - (Journal of Child Neurology, 2010) Use of Corticosteroids in a Population-Based Cohort of Boys With Duchenne and Becker Muscular Dystrophy

Dennis J. Matthews, Katherine A. James, Lisa A. Miller,  Shree Pandya, Kimberly A. Campbell, Emma Ciafaloni, Katherine D. Mathews, Timothy M. Miller, Christopher Cunniff, MD, F. John Meaney, Charlotte M. Druschel, Paul A. Romitti, P, and Deborah J. Fox - USA.

The use of corticosteroids for treatment of Duchenne and Becker  muscular dystrophy in clinical practice from 1991 through 2005 was reviewed in a large population-based cohort (MD STARnet) of boys in 4 regional sites and 6 clinics of the United States. Corticosteroid use increased from 20% (11 of 56 individuals) in 1991 to 44% (93 of 218 individuals) in 2005. Average use varied by site and ranged from 15% to 49%. The median age of corticosteroid initiation was 6.9 years (range, 3.7-17.4 years). Dosage and growth information was available for 102 participants and showed a median dose as 0.729 mg/kg for prednisone and 0.831 mg/kg for deflazacort. T. The most common reasons that corticosteroids were discontinued included weight gain, behavioral side effects, and loss of ambulation, resulting in full-time wheelchair use. Substantial variations in clinical practice were identified among study sites.

3 - PTC Therapeutics and Genzyme Corporation Announce Preliminary Results from the Phase 2b Clinical Trial of Ataluren for Nonsense Mutation Duchenne/Becker Muscular Dystrophy


20 - (American Academy of Neurology Meeting, April 2010) Preliminary Results with AVI-4658 of Dystrophin Expression, Safety and Pharmacokinetics from the First Systemic Administration Study in Boys with Duchene Muscular Dystrophy (DMD), with a Phosphorodiamidate Morpholino Oligomer (PMO) to Skip Exon 51

Francesco Muntoni, London, United Kingdom, Kate Bushby, Newcastle upon Tyne, United Kingdom, Cirak Sebahattin, London, United Kingdom, Michela Guglieri, Newcastle upon Tyne, United Kingdom, Shirley Leow, Stephen B. Shrewsbury, Bothell, WA

OBJECTIVE: We have previously identified a PMO to skip exon 51 in DMD patients, restore the reading frame and enable dystrophin expression. The current study, on repeated IV treatment to select an effective, tolerated dose, should complete in February 2010. BACKGROUND: DMD is the commonest inherited muscular dystrophy, affecting 1 in every 3,500 boys. DMD boys present with leg weakness by age 5, wheelchair confinement by age 10-12; respiratory insufficiency requiring mechanical ventilation by late teens, and cardiomyopathy, placing a huge burden on patient and parents/caregivers. Out of frame deletions abolishing the production of the muscle protein dystrophin are the commonest mutations. DESIGN/METHODS: Open label, dose escalation study in ambulant DMD boys aged 5-15 years with relevant deletions, of 12 weekly administrations of AVI-4658; muscle biopsy to assess dystrophin expression at baseline and 14 weeks. Clinical parameters are followed for 26 weeks, consisting of safety (adverse events, physical examinations, laboratory tests), skeletal muscle, pulmonary and cardiac function, and pharmacokinetics at 1st, 6th and 12th doses. A DSMB guides dose escalation decisions (across the doses 0.5, 1.0, 2.0, 4.0, 10.0 and 20.0 mg/kg). RESULTS: Cohorts 1, 2, 3 and 4 completed 12 weeks of dosing (October 2009). Cohort 1 has completed follow up to 26 weeks. Cohorts 5 and 6 are proceeding with dosing. No drug related SAEs or severe drug related AEs have been reported so far. To date, maximum cumulative PMO dose approaches 3,000mg. Preliminary analysis of the exon skipping, RNA and dystrophin protein expression will be presented and safety data updated. CONCLUSIONS/RELEVANCE: The first PMO for DMD has been well tolerated at all doses to date. Preliminary data is expected to show dystrophin expression at one or more doses and will allow dose selection for confirmatory clinical studies and extended (compassionate) use Supported by: Medical Research Council, UK and AVI Biopharma.

20 -   (American Academy of Neurology Meeting, April 2010) Preclinical Safety of AVI-4658, a Phosphorodiamidate Morpholino Oligomer (PMO) Being Developed To Skip Exon 51 in Duchenne Muscular Dystrophy

Peter Sazani, Stephen Shrewsbury, Bothell, WA

OBJECTIVE: The current studies were designed to assess the safety of AVI-4658 (and PMO in general). BACKGROUND: AVI-4658 is a PMO that skips dystrophin exon 51, restores the reading frame and enable dystrophin expression in selected DMD boys, proven by our recent single IM dose study in the UK. To enable clinical trials in the US, three 12-week GLP studies in animals were performed. Published data suggests the older phosphorothioate antisense oligonucleotides have dose limiting toxicities. DESIGN/METHODS: (1) mdx mice were dosed IV with 0, 12, 120 or 960mg/kg (the maximum feasible dose (MFD)), or subcutaneously at 960mg/kg; wild type C57 mice at 0 and 960 mg/kg (i.e., 7 groups) with AVI-4658. (2) A second identical study with AVI-4225, the PMO to skip exon 23 of in the dystrophic mouse and restore dystrophin, was also performed. (3) Cynomolgus monkeys were dosed IV with 0, 5, 40 or 320mg/kg (MFD) and 320mg/kg subcutaneously. A 28 day recovery period was included in all studies. RESULTS: In mice, both AVI-4658 and AVI-4225 were well tolerated at doses including 960 mg/kg/injection, with no adverse effects. Findings were generally limited to the kidney, and were generally reversible, as shown in the 28 day recovery groups. No evidence of kidney function change was detected. In cynomolgus monkeys, AVI-4658 was also well tolerated at all doses including 320 mg/kg/injection, with no adverse effects. Findings were similar to those seen in the mouse studies. CONCLUSIONS/RELEVANCE: AVI-4658, the first PMO for DMD, was extremely well tolerated at all doses in dystrophic mice, normal mice and primates. In addition, AVI-4225, which restores dystrophin in mdx mice, also led to no adverse effects. Based on this preclinical package, and encouraging safety and dystrophin expression results from a concurrent UK clinical study, US clinical studies are anticipated.

20 -  (American Academy of Neurology Meeting, April 2010) A Phase I/IIa Systemic Study on Antisense Oligonucleotide Compound PRO051 in Patients with Duchenne Muscular Dystrophy

Nathalie M. Goemans, Leuven, Belgium, Mar Tulinius, Gothenburg, Sweden, Gunnar Buyse, Leuven, Belgium, Sjef J. de Kimpe, Judith C. van Deutekom, Leiden, The Netherlands

OBJECTIVE: In this phase I/IIa open label study we evaluated the systemic delivery of the antisense oligonucleotide compound PRO051 in Duchenne Muscular Dystrophy (DMD) patients. BACKGROUND: DMD patients suffer from progressive muscle degeneration due to mutations in the DMD gene and the resulting absence of dystrophin at the muscle fiber membrane. PRO051, an antisense oligonucleotide compound, induces specific exon 51 skipping during pre-mRNA splicing, and can induce novel dystrophin expression in a subpopulation of DMD patients (Van Deutekom N Eng J Med 2007;357(26):2719-2722). DESIGN/METHODS: Twelve DMD patients received 5 weekly subcutaneous injections. Four dosing cohorts were applied (0.5 mg/kg, 2 mg/kg, 4 mg/kg, 6 mg/kg), three patients each. Muscle biopsies were taken at baseline for patients in cohort 1, and at two and seven weeks after the last administration for all patients. Adverse events were recorded and safety assessments (laboratory analysis and ECG) were performed at regular intervals. RESULTS: PRO051 induced specific exon 51 skipping in cohorts 2, 3 and 4, and novel dystrophin expression in a dose related manner in all cohorts. The treatment was well tolerated, none of the patients discontinued. A review of the safety data revealed no clinically significant changes in laboratory values and ECG. Antibodies against dystrophin were not detected. CONCLUSIONS/RELEVANCE: This is the first study showing successful systemic administration of an antisense oligonucleotide compound in DMD patients. An extension study is ongoing to collect at least 6 months safety data in all 12 patients at 6 mg/kg. A phase 3 study is in preparation. Supported by: Prosensa Therapeutics BV, the Netherlands.

20 -  (American Academy of Neurology Meeting, April 2010) Gentamicin Treatment of Duchenne Muscular Dystrophy Reinforces the Potential for Mutation Suppression Therapy

Vinod Malik, Louise R. Rodino-Klapac, Laurence Viollet, OH, Cheryl Wall, Wendy King, Roula Al-Dahhak, Sarah Lewis, Christopher J. Shilling, Janaiah Kota, Columbus, OH, John Hayes, Forest Grove, OR, John D. Mahan, Katherine J. Campbell, Columbus, OH, Brenda Banwell, Toronto, ON, Canada, Majed Dasouki, Victoria Watts, Kansas City, KS , Kumaraswamy Sivakumar, Ricardo Bien-Willner, Scottsdale, AZ, Kevin M. Flanigan, Zarife Sahenk, Columbus, OH, Richard J. Barohn, Kansas City, KS, Christopher M. Walker, Jerry R. Mendell, Columbus, OH

OBJECTIVE: Establish if the biopotency of gentamicin demonstrated in the mdx mouse can be confirmed in a clinical setting and if so, how could it be administered considering that readthrough would be an ongoing requirement. Address the percent increase in dystrophin expression required to provide clinically meaningful outcomes, and the potential immunogenicity of newly expressed dystrophin epitopes following treatment. BACKGROUND: Mutation suppression, also referred to as readthrough of stop codons, to restore the dystrophin gene is undergoing clinical trials in Duchenne muscular dystrophy (DMD). DESIGN/METHODS: Duchenne muscular dystrophy (DMD) subjects included: 1) Cohort 1 (n = 10) stop codon patients and Cohort 2 (n=8) frameshift controls receiving 14-days of gentamicin (7.5 mg/day). 2) Cohort 3 (n =12) and Cohort 4 (n=4) received an unprecedented six month delivery of weekly or twice-weekly gentamicin permitting an accumulating dystrophin pool to reach potential therapeutic levels. Readthrough was assessed in pre-and post-treatment biopsies and by clinical outcomes. RESULTS: In the 14-day biopotency study serum CK dropped by 50%, not seen in frameshift DMD controls. After 6-months of gentamicin, dystrophin levels significantly increased (p = 0.027) reaching levels 13% to 15% of normal accompanied by a drop in serum CK, stabilization of strength by manual muscle testing and a slight increase in forced vital capacity without adverse events. Stable transcripts that escaped nonsense mediated decay predicted the greatest increase of dystrophin following gentamicin. The efficiency of readthrough was not affected by either the stop codon or the fourth nucleotide surrounding the stop. Novel immunogenic epitopes were found in post-treatment biopsies by antigen specific IFN-g ELISpots. CONCLUSIONS/RELEVANCE: The results support on-going efforts to achieve drug-induced mutation suppression of stop codons. Immunogenic epitopes resulting from readthrough emphasize the importance of monitoring T cell immunity during clinical gene manipulation trials including mutation suppression, exon skipping and gene therapy. Supported by: NIH, NINDS, MDA, Jesse's Journey, The University of Kansas Medical Center GCRC

20 - (American Academy of Neurology Meeting, April 2010) Transient Expression of a Therapeutic Dystrophin Transgene in Duchenne Muscular Dystrophy Revealed by T Cell Mediated Immunity

Jerry Mendell, Katherine Campbell, Louise Rodino-Klapac, Zarife Sahenk, Christopher Shilling, Sarah Lewis, Columbus, OH, Dawn Bowles, Steven Gray, Chengwen Li, Chapel Hill, NC, Gloria Galloway, New Albany, OH, Vinod Malik, Brian Coley, Reed Clark, Columbus, OH, Juan Li, Xiao Xiao , Jade Samulski, Scott McPhee, R. Samulski, Chapel Hill, NC, Christopher Walker, Columbus, OH

OBJECTIVE: Describe the immune response following gene therapy for Duchenne muscular dystrophy (DMD) using adeno-associated virus (AAV) to transfer the mini-dystrophin gene. BACKGROUND: Gene therapy for DMD is a potentially promising means of gene replacement to restore dystrophin expression. This study reports the first clinical trial of viral mediated gene transfer in DMD. DESIGN/METHODS: Six DMD subjects were enrolled in a double-blind gene transfer study to the biceps muscle. Four were receiving glucocorticoid therapy at the time of gene transfer. The mini-dystrophin transgene used in this study encoded the actin binding domain, 5 rod spectrin repeats (R1, R2, R22, R23, and R24), 3 hinge domains (H1, H3 and H4), and the cysteine-rich domain. Expression was under control of the human cytomegalovirus immediate early promoter. Vector genomes were packaged in a hybrid AAV 2 capsid with 6 amino acid substitutions designed to minimize recognition by serum neutralizing antibodies. RESULTS: Four of six subjects with frame-shifting deletions in the DMD gene had detectable T cell responses to mini-dystrophin. Most of the targeted epitopes were non-self, located in sequences unique to the therapeutic dystrophin protein. However in at least one instance the dystrophin-specific T cells were present at low frequency before vector treatment and expanded rapidly after mini-dystrophin expression. The target of this unexpected memory T cell response was a self-epitope expressed from the defective dystrophin gene in revertant muscle fibers. CONCLUSIONS/RELEVANCE: This study illustrates the potential for a host response to foreign transgene products that are caused by large deletions or frame-shifting mutations. Recall of auto-reactive T cells has important clinical significance beyond gene therapy for DMD. Similar cell-mediated immune responses could be elicited by any strategy that increases expression of functional dystrophin in subjects with DMD and should be considered in the design and monitoring of experimental therapies for this disease.
Supported by: The MDA and Jesse's Journey.

20 -  (American Academy of Neurology Meeting, April 2010)  Initial Efficacy and Safety Evaluation in Cynomolgus Monkeys of AVI-5038, a Peptide Conjugated Phosphorodiamidate Morpholino Oligomer (PPMO) Being Developed To Skip Exon 50 in Duchenne Muscular Dystrophy

Peter Sazani, Stephen Shrewsbury, Bothell, WA

OBJECTIVE: To evaluate the efficacy of AVI-5038 at inducing skipping of dystrophin exon 50, as determined by RT-PCR, and also perform an initial toxicology assessment. BACKGROUND: AVI-5038 is a PPMO that induces dystrophin exon 50, and is designed to restore the reading frame and enable dystrophin expression in DMD patients. DESIGN/METHODS: Cynomolgus monkeys were dosed IV with 0, 3, or 9 mg/kg with AVI-5038, once weekly for 4 weeks. Following a 21 day recovery period, animals were sacrificed and a toxicological evaluation was performed. Selected muscle tissues were also evaluated by RT-PCR for evidence of skipping of dystrophin exon 50. RESULTS: AVI-5038 was well tolerated, with no adverse effects detected at doses up to 9 mg/kg. Toxicological findings were generally limited to the kidney, and included basophilic granules and instances of tubular degeneration / regeneration that was dose dependant. No clear evidence of kidney function change was detected, as shown by clinical chemistry and urinalysis evaluations. Significant levels of exon skipping were detected by RT-PCR in all major muscle groups evaluated, including diaphragm, heart, and quadriceps, at 9 mg/kg. CONCLUSIONS/RELEVANCE: AVI-5038, the first PPMO for DMD, was extremely well tolerated at all tested doses in primates. In addition, the safety data indicate that higher doses could be used to produce greater efficacy. Exon skipping was induced by the PPMO in healthy primate's muscles following systemic administration.

20 -  (American Academy of Neurology Meeting, April 2010) Outcome Measures Validation Study for Mesoangioblasts Transplantation in Children Affected by Duchenne Muscular Dystrophy

Serena Bonfiglio, Alberto Lerario, Andrea Tettamanti, Sarah Marktel, Sara Napolitano, Stefano Previtali, Marina Scarlato, Maria Grazia Natali Sora, Nereo Bresolin, Giancarlo Comi, Roberto Gatti, Fabio Ciceri, Giulio Cossu, Yvan Torrente, Milano, Italy

OBJECTIVE: The aim of this study is to establish a reliable tool of reproducible assessment of muscle strength in children affected by Duchenne muscular dystrophy (DMD) which will be selected for mesoangioblasts transplantation. BACKGROUND: We have developed a potential treatment for DMD based on infusion of cells (mesoangioblasts) from a healthy donor capable. The results of the current functional study will hopefully establish reliable qualitative and quantitative tool to assess results of a future cell therapy clinical trial with mesoangioblasts. DESIGN/METHODS: This is a single centre, prospective, non-randomised, study of validation of outcome measures on 30 ambulant patients aged 5 to 12 years old affected by DMD including a cohort of 15 healthy aged matched males. We perform two days evaluation each three month for one year. During each assessment the following outcome measures are applied to DMD subjects: North Star Scale and 6 minute walking test during the first day; quantitative assessment using the Kin Com 125 machine during the second day. The controls subjects will perform quantitative assessment twice in a year. Twice during this evaluation year patients perform spyrometry, cardiac assessment and lower limb MRI. RESULTS: We divided the patients into 3 subgroups of age (5-7 years, 8-9 years, 10-12 years). The results of this preliminary part of the study show specific correlation between functional and quantitative tests in stronger children. Kin Com measurements correlate appropriately with functional tests for 10-12 years old DMD boys, while show a major variability in muscle strength for 8-9 years old DMD boys. The comparison with healthy subjects showed a difference of muscle strength that increases with age. CONCLUSIONS/RELEVANCE: This preliminary study demonstrates that our assessment may represent a useful tool to monitor the progress of DMD in ambulant children to determine the pre-transplantation story of the children who will be later treated with mesoangioblasts.

20 -  (American Academy of Neurology Meeting, April 2010) The 6-Minute Walk Test in Duchenne Muscular Dystrophy: Longitudinal Observations

Craig McDonald, Erik Henricson, Sacramento, CA, Richard Abresch, Davis, CA, Jay Han, Alina Nicorici, Erica Goude, Sacramento, CA, Gary Elfring, Allen Reha, Samit Hirawat, Langdon Miller, S. Plainfield, NJ

OBJECTIVE: To develop the six-minute walk test (6MWT) as a clinically-valid outcome measure for ambulatory boys with Duchenne muscular dystrophy (DMD). BACKGROUND: We evaluated longitudinal changes in the ambulation of boys with (DMD) and age-matched controls, ages 4-13 years, using a 6MWT modified for use in DMD therapeutic trials. DESIGN/METHODS: We tested 18 boys with confirmed DMD and 22 healthy boys, ages 4-13 years using a our previously reported 6MWT methodology. Boys were tested at baseline and after
1 year. RESULTS: The median [range] test-retest interval was 58 [35-84] weeks in boys with DMD and 66 [51-114] weeks in healthy boys. At baseline, 13/18 (72%) boys with DMD were using corticosteroids. The groups were similar in age, height, and weight. Mean six-minute walk distance (6MWD) declined (p=0.054) in boys with DMD (mean [SD] change = -54 [100] m) but increased in healthy boys (mean [SD] change = 16 [54] m). Mean stride length decreased in boys with DMD (mean [SD] change = -0.03 [0.18] m) but increased in healthy boys (mean [SD] change = 0.03 [0.18] m). Cadence decreased in boys with DMD (mean [SD] change = -9 [15] strides/min) but was stable in healthy boys (mean [SD] change = 0.02 [9] strides/min). In boys with DMD, change in 6MWD correlated strongly with change in stride length (r=0.94, p<0.0001) and change in cadence (0.71, p=0.0045). In healthy boys, change in 6MWD correlated strongly with change in stride length (r=0.73, p=0.0001) but not at all with change in cadence (r=0.002). CONCLUSIONS/RELEVANCE: This modified 6MWT shows clinically important changes over an 1-year period. These changes are age-dependent, consistent with the known natural history of DMD. Improvement or stabilization of 6MWD during the course of a long-term therapeutic trial would represent a clinically meaningful benefit for boys with DMD. Supported by: PTC Therapeutics, Inc.

20 -  (American Academy of Neurology Meeting, April 2010) Risk Factors for Fractures in the Muscular Dystrophy Surveillance, Tracking and Research Network Cohort

Lisa A. Miller, Kathy James, Denver, CO, Katherine Mathews, Iowa City, IA, Shree Pandya, Rochester, NY, Susan Apkon, Seattle, WA, Chris Cunniff, Tucson, AZ

OBJECTIVE: To determine the occurrence of fractures and assess risk factors for fractures among the Muscular Dystrophy Surveillance, Tracking and Research Network (MD STARnet) cohort, a large population-based cohort of individuals with well-characterized dystrophinopathy. BACKGROUND: Fracture occurrence is a significant problem in Duchenne muscular dystrophy, with reported rates between 21% and 44%. DESIGN/METHODS: All males with definite or probable dystrophinopathy identified through MD STARnet who were greater than 3 years of age and had a birth year from 1982 to 2006 were included. Data were abstracted annually from medical records for all individuals with a potential dystrophinopathy in the surveillance regions of Arizona, Colorado, Iowa, Georgia and Western New York. Cox proportional hazard modeling, a complex multivariate survival analysis, was used to assess risk factors for fractures. For each patient, wheelchair use (part-time or full-time); bisphosphonate, steroid, and calcium/vitamin D use prior to fracture; and the duration of each were determined at each month of fracture occurrence. RESULTS: There were 187 first fractures among 578 individuals (32.4 percent). The most common site was the femur (31.4 percent), followed by the tibia/fibula (16.0 percent). The fracture risk for individuals who were in a wheel chair full-time was 3.2 times higher than for individuals who were ambulating, but for every month of full-time wheel chair use, risk decreased by 2.0 percent. For each additional month of steroid use beyond 6 months, fracture risk increased by 1.2 percent. Calcium/vitamin D and bisphosphonate use prior to fracture did not significantly affect risk. CONCLUSIONS/RELEVANCE: Our results confirm the high rate of fractures in individuals with dystrophinopathy and their relation to steroid use. In this population, the risk of fracture is higher during early wheel chair use and decreases over time. Fracture risk in these individuals is likely complex and the result of multiple factors. Supported by: Centers for Disease Control and Prevention Cooperative Agreement (5U01DD000191).

20 -  (American Academy of Neurology Meeting, April 2010) Disparities in the Diagnosis of Duchenne and Becker Muscular Dystrophy: Data from the MDSTARnet, 1999-2007

Christopher Cunniff, Jennifer Andrews, Tucson, AZ, Emma Ciafaloni, Rochester, NY, Deborah Fox, Troy, NY, Caleb Holtzer, Zhenqiang Lu, Tucson, AZ, Lisa Miller, Denver, CO, John Meaney, Tucson, AZ

OBJECTIVE: To characterize the association of sociodemographic factors with delays at specific steps in the diagnosis of Duchenne and Becker Muscular Dystrophy (DBMD). BACKGROUND: Prior studies of DBMD observed diagnostic delays ranging from 1.9 to 4.3 years, with diagnosis occurring around age 5 years. No studies report on sociodemographic disparities in the diagnostic process. DESIGN/METHODS: We analyzed medical records for 593 boys in the MDSTARnet database and assessed mean age differences by sociodemographic group at first sign or symptom, initial medical evaluation, and first creatine kinase (CK) test using generalized linear regression models and T tests. We assessed the uptake of diagnostic testing and mutation analysis using the Cox Proportional Hazard Model. RESULTS: Family history information was available for 549 boys. Of these, 174 (29.3%) had a known family history of DBMD prior to diagnosis. Boys with a family history were diagnosed at 33 months and experienced each step to diagnosis at a younger age than boys without a family history (p<.001). Boys without a family history were diagnosed on average at 66 months. Within this group, age at evaluation was younger for boys with more educated mothers (p<.001). Whites underwent CK and DNA testing earlier than Blacks and Hispanics (p<.005). Mean ages for boys grouped by socioeconomic status or place of residence were not different at any time point. The rate of point mutation analysis following a negative deletion/duplication test was greater among boys with more educated mothers (p<.001). CONCLUSIONS/RELEVANCE: In the MDSTARnet population, children without a family history are diagnosed at age 5 years, 6 months, despite longstanding symptoms. Differences in race/ethnicity and mother education are associated with longer delays at multiple steps in the diagnostic process. The reasons for such delay are not known but may result from lack of awareness of symptoms or decreased access to diagnostic services. Supported by: A cooperative agreement from the Centers for Disease Control and Prevention through the Association of American Medical Colleges, grant number U36/CCU319276, AAMC ID number MM-1064-09/09. Publication and report contents are solely the responsibility of the authors and do not necessarily represent the official views of the AAMC or the CDC. This study was also funded by the Centers for Disease Control and Prevention under the Cooperative Agreement for Surveillance and Epidemiologic Research of Duchenne and Becker Muscular Dystrophy DD000187, DD000189, DD000190, DD000191.

20 -  (American Academy of Neurology Meeting, April 2010) Respiratory Care Trends for Duchenne and Becker Muscular Dystrophies (DBMD): Data from the MD STARnet, 2001-2007

Daniel A. Mandel, Atlanta, GA, Daniel W. Sheehan, Buffalo, NY, Shree Pandya, Rochester, NY, Christina P. Westfield, Deborah J. Fox, Troy, NY, Sarah K. Nabukera, Katherine Mathews, Iowa City, IA, Christopher Cunniff, Tucson, AZ, Carolyn M. Constantin, Atlanta, GA, David J. Birnkrant, Cleveland, OH

OBJECTIVE: To profile forced vital capacity (FVC) monitoring for males who have DBMD and to evaluate non-invasive positive pressure ventilation (NPPV) and mechanical insufflator/exsufflator (MI/E) use among males with very low FVC measurements. BACKGROUND: Semiannual FVC measurements are recommended for patients age > 12 who have Duchenne muscular dystrophy. FVC measurements of < 1 L are associated with complications of hypoventilation and early mortality. NPPV and MI/E are used to support patients with poor pulmonary function. DESIGN/METHODS: MD STARnet is a population-based surveillance system that identifies all patients who have DBMD in defined geographic areas. MD STARnet records were analyzed for 132, 177, and 199 males age > 12 in 2001, 2004, and 2007, respectively. Inclusion criteria for FVC < 1L were: > 5 recorded FVC measurements; > 2 consecutive FVC measurements < 1 L; and no subsequent FVC measurements > 1L. Sample sizes for FVC < 1 were 11, 22, and 33 in 2001, 2004, and 2007, respectively. Logistic regression models were used to investigate NPPV and MI/E use over time, clustering on patient. RESULTS: The percentage of males who have DBMD age > 12 years with a recorded FVC measurement ranged from 60-65% in the three study years. The rates of NPPV use significantly increased among males with FVC < 1L (Wald X2 = 11.5, p = .003) over the study period. NPPV rates ranged from a low of 27% in 2001 to a high of 85% in 2007. Rates of MI/E use followed a less clear pattern: 18% in 2001; 9% in 2004, 58% in 2007. CONCLUSIONS/RELEVANCE: Data show consistent FVC monitoring for males who have DBMD and increasing use of NPPV for males who have very low FVC measurements between 2001 and 2007. MI/E was relatively underused compared with NPPV. Supported by: CDC cooperative agreements DD000187, DD000189, DD000190, and DD000191.

20 -  (American Academy of Neurology Meeting, April 2010) Neuropsychological Profile of Adult Patients with Duchenne Muscular Dystrophy

Natalia Sierra, Lilia Mesa, Alberto Dubrovsky, Pablo Sojo, Teresa Torralva, María Roca, Fernando Chloaca, Laura Pirra, Facundo Manes, Buenos Aires, Argentina

OBJECTIVE: To analyze the cognitive profile of adult patients with normal IQ diagnosed with Duchenne Muscular Dystrophy (DMD). BACKGROUND: Research on muscular dystrophies has focused extensively on the impact of peripheral neural structures affected by the degenerative nature of the disease. However, investigating the cognitive impairment in these patients may contribute to the understanding of the pathophysiological changes occurring on the central nervous system (CNS) and its relationship with peripheral structures. DESIGN/METHODS: Ten patients with diagnosis of DMD aged between 17 and 28 years were assessed with a general comprehensive cognitive battery as well as a specific executive battery. All patients had within normal IQ scores. RESULTS: Tasks associated with a motor component such as the Complex Rey Figure (z= -2,02) and TMT-A (z= -2,31) and TMT-B (z=-2,47) were impaired. Performance deficits were also found on tasks of verbal inhibitory control (z=-3,02) with normal scores on reading speed (z=-0,69) and color naming (z=-0,81) on the Stroop task. Decreased scores were observed for tasks of theory of mind (z=-1,19) and decision-making (Iowa Gambling Task), although performance on the latter did not correlate significantly with theory of mind or verbal inhibitory control scores (both p > .05). CONCLUSIONS/RELEVANCE: These results are in accordance with previous studied (e.g. Hinton et al, 2007) and suggest that normal IQ patients with DMD present theory of mind and verbal inhibitory control deficits. Our results also revealed decision-making impairments, although apparently not associated with theory of mind and executive deficits.

20 -  (American Academy of Neurology Meeting, April 2010) Pulmonary Function Characteristics of Boys with Duchenne and Becker Muscular Dystrophy by Age Groups and Steroid Use: One-Year Data from the CINRG Longitudinal Study Project

R. Ted Abresch, Craig M. McDonald, Davis, CA, Jay J. Han, Sacramento, CA, Robert Leshner, Washington, DC, Diana Escolar, McLean, VA, Avital Cnaan, Eric Hoffman, Adrienne Arrieta, Tina Duong, Fenming Hu, Washington, DC, Julaine Florence, Saint Louis, MO, CINRG Investigators, Washington, DC

OBJECTIVE: To describe the pulmonary function characteristics of Duchenne Muscular Dystrophy (DMD) over a one-year time period. BACKGROUND: Lack of well-characterized pulmonary function data inhibits the development of therapeutic clinical trials in DMD. DESIGN/METHODS: Pulmonary function tests (PFTs) were performed in subjects with confirmed DMD at ages <7, 7-12, 13-18 and > 18 in 20 centers from the Cooperative International Neuromuscular Research Group at baseline and 12 months. Fifteen percent of subjects were steroid na
ve. Pulmonary function measures included absolute and % predicted forced vital capacity (FVC and %FVC), forced expiratory volume in 1 second a (FEV1, %FEV1), peak expiratory flow rate (PEFR, %PEFR), maximum inspiratory pressure (MIP, %MIP), and maximum expiratory pressure (MEP, %MEP). Significance was accepted at p < 0.05. RESULTS: There was a significant one-year increase in FVC (0.15l 0.1 [sd]), FEV1 (0.15l 0.2) and PEFR (0.49 l/s 0.5) in the <7 year age groups (n=10). There was a significant one-year increase in FVC and FEV1 (0.11l 0.2 and 0.13l 0.2, respectively; n=85), as well as MIP and MEP (3.7 cmH20 12.5 and 5.7 cmH20 10.9, respectively; n = 102) in the 7-12 year age groups. There was a significant one-year decline in %FVC and %PEFR (-5.8% 5.7 and 5.0% 10.3, respectively) in the 13-18 year age groups (n=57). At age 19 (n=23) there was a significant one-year decline in FVC (-0.14l 0.2), %FVC (-3.0% 3.4), FEV1 (-0.14l 0.2), %FEV1 (-3.6% 3.9), %PEFR (-3.5% 6.5) and %MIP (-2.7% 2.8). No other PFT measures exhibited significant differences over a one-year period. CONCLUSIONS/RELEVANCE: Pulmonary function testing reflects changes associated with growth at age groups <7 and 7-12. DMD subjects exhibit significant one-year PFT declines in the 13-18 and >18 year age groups. Supported by: National Institute of Disability and Rehabilitation Research Grant H133B980008-03; National Institutes of Health Grant 1U54HD053177-01A1; Department of Defense Grant 0616USAMRAA.

20 -  (American Academy of Neurology Meeting, April 2010) Functional Motor Performance Characteristics of Boys with Duchenne Muscular Dystrophy by Age Groups and Steroid Use: One-Year Data from the CINRG Longitudinal Study Project

Craig McDonald, Erik Henricson, Sacramento, CA, Richard T. Abresch, Davis, CA, Jay J. Han, Sacramento, CA, Robert Leshner, Washington, DC, Diana Escolar, McLean, VA, Eric Hoffman, Avital Cnaan, Addrienne Arrietta, Fenming Hu, Angela Zimmerman, Tina Duong, Washington, DC, Julaine Florence, Saint Louis, MO, CINRG Investigators, Washington, DC

OBJECTIVE: To describe the changes in timed function testing (TFT) in Duchenne muscular dystrophy (DMD) over one-year and the predictive value of TFTs for determining loss of ambulation. BACKGROUND: In DMD, TFT measures are used as clinical endpoints for therapeutic clinical trials and may predict loss of ambulation over one-year. DESIGN/METHODS: TFTs were performed in 255 subjects with confirmed DMD at ages <7, 7-12, and >13 in 20 centers from the Cooperative International Neuromuscular Research Group at baseline and 12 months. Fifteen percent of subjects were steroid na
ve. TFTs (in seconds) included run/walk 10 meters, climb 4 steps, and standing from lying. Significance was accepted at p < 0.05. RESULTS: There was a mean decrease in all TFTs over one-year for the <7 year group (n=56): run/walk 10 meters (-0.46 1.7[sd]), climb 4 steps (-1.83 4.4) and standing from lying (-0.65 2.9). There was a significant one-year increase in all TFTs in 7-12 year olds (n=65): run/walk 10 meters (+1.38 1.8), climb 4 steps (+2.47 4.9) and standing from lying (+3.42 6.1). Those >13 also increased time to run/walk 10 meters (+3.21 5.2, n=10) and climb 4 steps (+1.58 1.3, n=7). Only one subject >13 was able to perform standing from lying. Loss of ambulation over 12 months was compared for three groups: baseline run/walk 10 meters < 6 seconds, 6-12 seconds, and >12 seconds. Survival analysis for the milestone of loss of ambulation showed all three groups to be significantly different using a Log-rank test (p<0.0001). CONCLUSIONS/RELEVANCE: In DMD, TFTs show relative improvement with time in younger subjects <7 years. For those 7 and older TFTs show disease-related progression in both steroid-users and steroid-nave subjects. Timed to run/walk 10 meters is predictive of loss of ambulation over the following 12 months. Supported by: National Institute of Disability and Rehabilitation Research Grant H133B980008-03; National Institutes of Health Grant 1U54HD053177-01A1; Department of Defense Grant 0616USAMRAA

20 -  (American Academy of Neurology Meeting, April 2010) A Cooperative International Neuromuscular Research Group (CINRG) Study of the Relationship between Impairment, Activity Limitation, Participation and Quality of Life in Persons with Confirmed Dystrophinopathies: One Year Follow-Up of Skeletal Muscle Strength and Timed Motor Performance

Erik Henricson, Craig McDonald, Sacramento, CA, Richard Abresch, Davis, CA, Jay Han, Sacramento, CA, Robert Leshner, Eric Hoffman, Washington, DC, Diana Escolar, McLean, VA, Avital Cnaan, Fengming Hu, Angela Zimmerman, Tina Duong, Washington, DC, Julaine Florence, Saint Louis, MO, Adrienne Arrieta, CINRG Investigators, Washington, DC

OBJECTIVE: To evaluate 12-month change in skeletal muscle strength and timed motor function tests in individuals with Duchenne muscular dystrophy (DMD) aged 2-28 years. BACKGROUND: Lack of adequate natural history data in steroid-treated DMD and lack of well-characterized outcome measures across age ranges limits the ability to assess therapeutic effectiveness in clinical trials in DMD. DESIGN/METHODS: We enrolled males with confirmed DMD from 20 participating centers from 10 countries of the Cooperative International Neuromuscular Research Group (CINRG). Ambulatory and transitioning participants underwent strength assessment (modified MRC manual muscle test (MMT), quantitative muscle tests (QMT) of grip, elbow flexion/extension, knee flexion/extension) and timed function tests ((TFT) stand from supine, run/walk 10m, climb 4 stairs) at baseline, and months 3, 6, 9, 12. RESULTS: 255/347 males with DMD aged 2 to 28 years of age underwent strength and functional testing, and 15% were glucocorticoid-na
ve. Significant changes over 12 months included: Children <7 years decreased time to climb 4 steps by 1.83(SD=4.4)s (p<0.0001 N=56) and increased quantitative grip strength by 2.96(SD=2.5)lbs (p<0.0001 N=37) and knee flexors by 1.18(SD=2.6)lbs (p<0.04 N=37). Children aged 7-12 years increased time to run/walk 10m by 1.38(SD=1.8)s (p<0.0001 N=65), time to climb 4 stairs by 2.47(SD=4.9)s (p<0.0001 N=65) and time to stand from supine by 3.42(SD=6.1)s (p<0.0001 N=65). Children aged 13-18 years decreased quantitative elbow extensor strength by 1.23(SD=1.1)lbs (p<0.001 N=61) and elbow flexor strength by 0.98(SD=1.3)lbs (p<0.01 N=61) and decreased manual muscle test score by 0.32(SD=0.4) points (p<0.01 N=61). Adults aged >18 years who were testable decreased quantitative grip strength by 0.6(SD=0.9)lbs (p<0.02 N=31). CONCLUSIONS/RELEVANCE: Few measures of strength and function in steroid-treated boys with DMD show significant disease-related changes over one-year. Relative stability over this time suggests that clinical trials must demonstrate improvement rather than stabilization using these measures. Supported by: National Institute of Disability and Rehabilitation Research Grant H133B980008-03 National Institutes of Health Grant 1U54HD053177-01A1 Department of Defense Grant 0616USAMRAA.

20 -  (American Academy of Neurology Meeting, April 2010) Multiple Sclerosis in a Patient with Duchenne Muscular Dystrophy

Adrienne A. Salomon, Negar Sodeifi, Babak Movassaghi, David Libell, Morgantown, WV

OBJECTIVE: To describe a rare association of multiple sclerosis (MS) in a patient with Duchenne Muscular Dystrophy (DMD). BACKGROUND: MS is an autoimmune disorder that most commonly begins between ages 20 and 40, but can be seen at any age. The average age at diagnosis is 31 years in males. DMD is an X-linked disorder which affects the DYS gene. It is the most common muscular dystrophy and carries an average life expectancy ranging from early teens to mid30s. No reported association between these two conditions was found. DESIGN/METHODS: Case report involving a 23-year-old man with known DMD who presented with unilateral left eye blurry vision. The patient admitted to having suffered a similar episode of visual changes approximately one year prior. Examination revealed left eye visual acuity of 20/100 with abnormal enhancement of the left optic nerve on MRI consistent with optic neuritis. MRI of the brain demonstrated multiple periventricular and juxtacortical lesions with post-contrast enhancement. Lumbar puncture was significant for increased oligoclonal bands with elevated IgG index (1.63) and myelin basic protein (3.5
g/L). While the patient's diagnosis of DMD had previously been established by neurological examination, family history, elevated creatine kinase levels, and DNA analysis, the patient's new findings were consistent with a diagnosis of MS based on McDonald criteria. RESULTS: The patient received high-dose intravenous solumedrol for three days. The patient improved minimally during his hospital course and was discharged on a steroid taper. Follow-up examination confirmed resolution of his blurry vision with no further symptoms at six months. CONCLUSIONS/RELEVANCE: We report the rare association between DMD and MS. The patient's clinical course suggests that DMD did not impact the clinical course or treatment of his MS. The fact that many DMD patients do not survive long enough to manifest symptoms of MS may partly explain the rare association of these two disorders.

20 -  (American Academy of Neurology Meeting, April 2010) Expression of Heat Shock Proteins in Skeletal Muscle from Idiopathic Inflammatory Myopathy and Duchenne Muscular Dystrophy Patients

Jan L. De Bleecker, Kim K. Creus, Ghent, Belgium, Jean-Jacques Martin, Antwerp, Belgium, Joachim Weis, Aachen, Germany, Boel De Paepe, Ghent, Belgium

OBJECTIVE: To investigate heat shock protein families 70 (HSP70) and HSP90 in idiopathic inflammatory myopathies (IIM) and Duchenne Muscular dystrophy (DMD). BACKGROUND: HSP70 and HSP90 chaperones assure proper protein folding and activity. Furthermore, HSP90 enhances the cytotoxic activity of inflammatory cells. In view of current approaches exploring anti-HSP90 therapy in inflammatory diseases, more in-depth knowledge of the individual pros and cons of chaperones could be relevant. DESIGN/METHODS: The expression of HSP70 and HSP90 was investigated in muscle biopsies from controls, and from IIM and DMD patients using immunofluorescence, in situ hybridization and Western blotting. RESULTS: Inflammatory cells in IIM and DMD expressed low levels of HSP70, HSP90 expression was increased in macrophages and cytotoxic T-cells in proximity of invaded nonnecrotic myofibers of PM/IBM. HSP90alpha mRNA was localized in endomysial infiltrates of PM/IBM along with faint HSP90beta expression. Part of the invaded nonnecrotic myofibers showed sarcolemmal staining for HSP70 and HSP90 proteins. The sarcoplasm of most small fibers, and some normal appearing myofibers were strongly HSP70 positive. Double staining showed important overlap between HSP70 and HSP90alpha in small NCAM+ fibers, and rare co-localization with HSP90beta. Western blotting detected HSP70 and HSP90 proteins in all muscle tissues, but protein levels were increased in all (HSP70) or part of (HSP90) IIM/DMD patients. In normal controls that had received glucocorticoids prior to biopsy, nuclear over cytoplasmic protein ratios were increased for HSP70, and decreased for HSP90. CONCLUSIONS/RELEVANCE: Our data appoint pathological and physiological roles for HSP70 and HSP90, ascribing these factors both adverse and beneficiary potential. On the one hand, HSP90 was associated with the active invasion targeting the nonnecrotic myofibers in PM/IBM. On the other hand, a strong expression of HSP70 occured in myofibers at different regeneration-stages, with important but no absolute overlap with HSP90 in the small regenerating muscle fibers, indicating both general and member-specific involvement. Supported by: L'Association Fran
aise contre les Myopathies (AFM, France) L'Association Belge contre les Maladies neuro-Musculaires (ABMM, Belgium) The Muscular Dystrophy Association (MDA, USA).

20 -  (American Academy of Neurology Meeting, April 2010) Adult Murine Derived Mesoangioblasts Successfully Recovered Dysferlin Expression in a Murine Model of Dysferlinopathy

Jordi Díaz-Manera, Barcelona, Spain, Thierry Touvier, Rossana Tonlorenzi, Laura Perani, Arianna Dellavalle, Graziella Messina, Patrizia Pessina, Milano, Italy, Eduard Gallardo, Isabel Illa, Barcelona, Spain, Yvan Torrente, Giulio Cossu, Milano, Italy

OBJECTIVE: Our aim was to treat a mouse model of dysferlinopathy with transplantation of adult derived murine mesoangioblasts (mMABs). BACKGROUND: Mutations in dysferlin gene produce a muscular dystrophy characterized by adult onset and progressive weakness leading to a severe phenotype. The A/J mouse is a good model to study cell therapy as it completely lacks dysferlin and develops a slowly progressive muscular dystrophy. Mesangioblasts (MABs) are vessel associated progenitors that has been successfully used in preclinical models for cell therapy for muscular dystrophy including the a-sarcoglycan null mice and the Golden Retriever dogs affected by Duchenne's disease. DESIGN/METHODS: Murine mesoangioblasts were obtained from skeletal muscle of 8 days old wild type mice (C57 strain), and labeled with a lentiviral vector expressing nuclear LacZ. To avoid an immunological response, A/J-SCID mice were generated by crossing A/J and SCID strains. Firstly, we performed a single intramuscular injection of 5x105 mMABs both in cardiotoxin treated and untreated muscles from 5 months old mice. Then we proceed with a single injection of 5x105 mMABs in the right femoral artery. We analyzed the expression of dysferlin 3 weeks after the injection using immunofluorescence, quantitative RT-PCR and Western-Blot. RESULTS: The first dystrophic features in AJ-SCID mice appeared at 4-5 months of age, without significant differences in the progression and distribution of them compared with control A/J mice. Three weeks after transplantation, multiple areas of injected muscles expressed dysferlin which was absent in non-injected contralateral muscles. Presence of the protein in the membrane of Lac-Z + fibers was also demonstrated in intra-arterially injected animals. The expression of dysferlin was significantly higher in muscles treated with cardiotoxin. CONCLUSIONS/RELEVANCE: Treatment with wild type mMABs successfully recovered the expression of dysferlin in A/J-SCID mice. This fact suggests that adult derived mesoangioblasts may be a promising candidate for future cell-therapy protocols in dysferlinopathy patients. Supported by: European Federation of Neurology grant for young neurologist.

 20 -  (American Academy of Neurology Meeting, April 2010) rAAV5 Mediated Delivery of Dysferlin as a Therapeutic Strategy for LGMD2B and Miyoshi Myopathy

Louise R. Rodino-Klapac, Kimberly M. Shontz, Chrystal Montgomery, Vinod Malik, Nancy Davis, Paul M. L. Janssen, K. Reed Clark, Columbus, OH, Robert H. Brown, Worcester, MA, Jerry R. Mendell, Columbus, OH

OBJECTIVE: To develop an adeno-associated virus (AAV) mediated therapeutic transgene to treat dysferlinopathies including limb-girdle muscular dystrophy (LGMD) type 2B and Miyoshi myopathy (MM). No therapeutic treatments are currently available for these disorders. BACKGROUND: The size of the DYSF cDNA (6.5 kb) negates packaging into traditional AAV serotypes (capacity < 4.7 kb) known to express well in muscle (i.e. rAAV1, 2, 6, 8). Potential advantages of a full cDNA versus a truncated transgene include: maintaining structural-functional protein domains, evading protein misfolding, and avoiding novel epitopes that could be immunogenic. This work describes in vivo delivery of AAV5.DYSF to limb muscle and diaphragm of dysferlin deficient (Dysf-/-) mice by both intramuscular and vascular (femoral artery) approaches. DESIGN/METHODS: A cassette containing the DYSF cDNA driven by the muscle specific MHCK7 promoter was packaged into an AAV2/5 vector. Physiological characterization of three dysferlin deficient mouse strains (AJ, SJL, and 129-Dysftm1Kcam/J ) revealed functional deficits in the diaphragm but not skeletal muscle. Efficacy of rAAV5.MHCK7.DYSF gene replacement was tested following intramuscular and intravascular delivery to skeletal muscle and diaphragm of 4-6 week old Dysf -/- mice. Functional improvement was measured by tetanic force and resistance to fatigue in the diaphragm at 2 months. RESULTS: Robust dysferlin gene expression was achieved in a dose-dependent manner by both intramuscular and vascular approaches. Western blot analysis confirmed the immunostaining results demonstrating a 237kDa band in treated samples that was absent in controls. Band intensity correlated with dose. Gene transfer improved both force generation and resistance to fatigue in the functionally impaired diaphragm in Dysf-/- mice. CONCLUSIONS/RELEVANCE: These results provide proof of principle that a full-length dysferlin cDNA can be delivered efficiently to muscle using AAV5 leading to physiological improvement. Future studies in a larger animal model using a vascular approach targeting multiple muscles will guide clinical trial design for LGMD2B and MM patients.
Supported by: Day Foundation.

19 - (Biochimica et Biophysica Acta (BBA) - Biomembranes, February 2010) Morpholinos and Their Peptide Conjugates: Therapeutic Promise and Challenge for Duchenne Muscular Dystrophy

Hong M. Moulton and Jon D. Moulton - USA

Exon-skipping efficacy of phosphodiamidate morpholino oligomers (PMOs) or the conjugates of PMOs with cell-penetrating peptides (PPMOs) have been tested in various animal models of Duchenne muscular dystrophy (DMD), including mdx mice, utrophin-dystrophin double-knockout mice and CXMD dogs as well as in DMD patients in clinical trials. The studies have shown that PMOs can diffuse into leaky muscle cells, modify splicing of DMD transcripts, induce expression of partially-functional dystrophin and improve function of some skeletal muscles. PMOs are non-toxic, with a report of mdx mice tolerating a 3 g/Kg dose and no drug-related safety issue in human has been reported. However, because of their poor cell uptake and rapid renal clearance, large and frequently repeated doses of PMOs are likely required for functional benefit in some skeletal muscles of DMD patients. In addition, PMOs do not enter cardiomyocytes sufficiently to relieve heart pathology, the efficacy of delivery to various muscles varies greatly and delivery across the tissue of each skeletal muscle tissue is patchy. PPMOs enter cells at far lower doses, enter cardiomyocytes in useful quantities, and deliver more evenly to myocytes both when different muscles are compared and when assessed at the level of single muscle tissue sections. Compared to PMOs, far lower doses of PPMOs can restore dystrophin sufficiently to reduce disease pathology, increase skeletal and cardiac muscle functions and prolong survival of animals. The biggest challenge for PPMO is determining safe and effective doses. The toxicity of PPMOs will require caution when moving into the clinic. The first PPMO-based DMD drug is currently in pre-clinical development for DMD patients who can benefit from skipping exon 50.

19 - Perioral skin biopsy to study skeletal muscle protein expression

19 - Surgical management of severe scoliosis with high risk pulmonary dysfunction in Duchenne muscular dystrophy: patient function, quality of life and satisfaction.

19 - Cells of extraembryonic mesodermal origin confer human dystrophin in the Mdx model of duchenne muscular dystrophy

19 - Systemic Myostatin Inhibition via Liver-Targeted Gene Transfer in Normal and Dystrophic Mice

13 - Functional improvement of patients with progressive muscular dystrophy by bone marrow and umbilical cord blood mesenchymal stem cell transplantations

4 - (Molecular Therapy 2010;18(2):386–393) Complete Genetic Correction of iPS Cells From Duchenne Muscular Dystrophy

Yasuhiro Kazuki, Masaharu Hiratsuka, Masato Takiguchi, Mitsuhiko Osaki, Naoyo Kajitani, Hidetoshi Hoshiya, Kei Hiramatsu, Toko Yoshino, Kanako Kazuki, Chie Ishihara, Shoko Takehara, Katsumi Higaki, Masato Nakagawa, Kazutoshi Takahashi, Shinya Yamanaka and Mitsuo Oshimura - Japan

Human artificial chromosome (HAC) has several advantages as a gene therapy vector, including stable episomal maintenance that avoids insertional mutations and the ability to carry large gene inserts including the regulatory elements. Induced pluripotent stem (iPS) cells have great potential for gene therapy, as such cells can be generated from the individual's own tissues, and when reintroduced can contribute to the specialized function of any tissue. As a proof of concept, we show herein the complete correction of a genetic deficiency in iPS cells derived from Duchenne muscular dystrophy (DMD) model (mdx) mice and a human DMD patient using a HAC with a complete genomic dystrophin sequence (DYS-HAC). Deletion or mutation of dystrophin in iPS cells was corrected by transferring the DYS-HAC via microcell-mediated chromosome transfer (MMCT). DMD patient- and mdx-specific iPS cells with the DYS-HAC gave rise to differentiation of three germ layers in the teratoma, and human dystrophin expression was detected in muscle-like tissues. Furthermore, chimeric mice from mdx-iPS (DYS-HAC) cells were produced and DYS-HAC was detected in all tissues examined, with tissue-specific expression of dystrophin. Therefore, the combination of patient-specific iPS cells and HAC-containing defective genes represents a powerful tool for gene and cell therapies.

4 - Bringing down the barriers in translational medicine for inherited neuromuscular diseases.

4 - Evaluation of Skeletal and Cardiac Muscle Function after Chronic Administration of Thymosin beta-4 in the Dystrophin Deficient Mouse


12 - Cytokinetics Announces Positive Data From Phase I Clinical Trial of CK-2017357: Statistically Significant Increases in Muscle Force Production Demonstrated During Treatment With Novel Skeletal Muscle Activator

12 - (International Journal of Cardiology, 2009) Relationship of natriuretic peptide and transthoracic echocardiographic findings in 135 subjects with muscular dystrophy

Miyuki Kawakubo, Nobusada Funabashi, Maiko Takahashi, Makoto Sueishi, Yasufumi Motoyoshi, Takashi Mikata, Idai Uchida, Takehiro Asakawa, Riyo Takahashi, Midori Takamatsu, Yukie Matsuoka, Masaaki Minegishi, Hideki Naga, Rei Yajima, Akihisa Kataoka, Kwangho Lee, Issei Komuro - Japan

Purpose: To examine the usefulness of plasma atrial (ANP) and brain natriuretic peptide (BNP) levels and transthoracic echocardiogram (TTE) findings in predicting left ventricular (LV) dysfunction in muscular dystrophies (MD).

Materials and methods: 135 MD subjects (83 Duchenne MD (DMD), all males, mean age 22 ± 7 years, 20 Becker MD (BMD), all males, 45 ± 16 years, 21 limb-girdle MD (LGMD), 14 males, 52 ± 13 years, and 11 facioscapulohumeral MD (FSHD), all males, 58 ± 13 years) underwent TTE and measurement of BNP and ANP.

Results: In DMD, TTE revealed asynergy of the LV posterior-wall (72%), interventricular septum (IVS) (29%) and LV apex-wall (24%); in BMD subjects: TTE revealed asynergy of LV posterior-wall (50%) and IVS (25%); in LGMD and FSHD subjects: TTE revealed asynergy of LV posterior-wall (33 and 27%, respectively). LV end-diastolic (LVDdI) and end-systolic diameter index (LVDsI) were significantly larger in DMD than FSHD, and LV ejection fraction (LVEF) was significantly lower in DMD than LGMD and FSHD. In DMD, when LVEF was > = 35%, BNP and ANP levels remained low, but when LVEF < 35%, BNP and ANP levels showed steep increase. Relationship between LVEF and BNP or ANP levels was curvilinear (R2 = 0.69 and 0.55, respectively, p < 0.001) using following equation: BNP = 673491×EF− 2.897, ANP = 5716.6 × EF− 1.422, respectively. When LVDdI > = 40 mm/m2 or LVDsI > = 35 mm/m2, LVEF < = 35%, plasma BNP and ANP levels increased steeply and the relationship between LVDdI, LVDsI, or LVEF and plasma BNP or ANP levels was curvilinear.

Conclusion: In DMD, LVEF was significantly lower, asynergy of LV posterior-wall was more frequent and BNP and ANP levels were dramatically higher when LVDdI > = 40 mm/m2, LVDsI > = 35 mm/m2 or LVEF < 35%.


9 - (PNAS, 2010) Leaky RyR2 trigger ventricular arrhythmias in Duchenne muscular dystrophy

Jérémy Fauconnier; Jérôme Thireau; Steven Reiken; Cécile Cassana; Sylvain Richarda; Stefan Matecki; Andrew R. Marks; Alain Lacampagne - France

Patients with Duchenne muscular dystrophy (DMD) have a progressive dilated cardiomyopathy associated with fatal cardiac arrhythmias. Electrical and functional abnormalities have been attributed to cardiac fibrosis; however, electrical abnormalities may occur in the absence of overt cardiac histopathology. Here we show that structural and functional remodeling of the cardiac sarcoplasmic reticulum (SR) Ca2+ release channel/ryanodine receptor (RyR2) occurs in the mdx mouse model of DMD. RyR2 from mdx hearts were S-nitrosylated and depleted of calstabin2 (FKBP12.6), resulting in “leaky” RyR2 channels and a diastolic SR Ca2+ leak. Inhibiting the depletion of calstabin2 from the RyR2 complex with the Ca2+ channel stabilizer S107 (“rycal”) inhibited the SR Ca2+ leak, inhibited aberrant depolarization in isolated cardiomyocytes, and prevented arrhythmias in vivo. This suggests that diastolic SR Ca2+ leak via RyR2 due to S-nitrosylation of the channel and calstabin2 depletion from the channel complex likely triggers cardiac arrhythmias. Normalization of the RyR2-mediated diastolic SR Ca2+ leak prevents fatal sudden cardiac arrhythmias in DMD.