Abstracts from 11th International Congress of World Muscle Society - Bruges, Belgium, October 2006

 

1) A phase I/II clinical trial in Duchenne muscular dystrophy using IM and IV delivered antisense oligonucleotides: The MDEX consortium

V. Arechavala, K. Bushby, G. Dickson, I. Graham, M. Kinali, K. Liu, J. Morgan, F. Muntoni, L. Popplewell, T. Partridge, F. Thorogood, K. Wells, N. Wells, M. Wood and H. Yin – UK  

Duchenne muscular dystrophy (DMD) is the most common childhood form of muscular dystrophy, and out-of-frame deletions and duplications the most common type of mutations. Antisense oligonucleotides (AO) can induce exon skipping and restore the reading frame in animal models and cultured DMD cells. We are involved in a Department of Health (DOH) funded phase I/II clinical trial which first stage it to administer intramuscularly AO in DMD children with deletions which can be rescued by skipping of exon 51. During the first year of this 4 year project we have performed preparatory clinico-pathological studies to establish the grade of preservation of distal leg muscles and the frequency of revertant fibres in non ambulant DMD children, using both MRI and biopsies taken at the time of planned surgery, as we will need to target this age range in the initial IM studies. We have established a biobank of cell cultures from DMD patients with a variety of deletions to validate AO in the human. From a preclinical point of view, we have optimised sequence targets, by testing eight different exon 51 AO, which eventually have allowed us to choose an optimal sequence. We have chosen to use a morpholino backbone AO for the clinical trial. We have also developed a transgenic approach using a luciferase reporter gene to facilitate the study of systemically delivered AO. We expect to start the IM arm of the trial in exon 51 deleted DMD boys by the end of 2006 and to be able to analyse the data by summer of 2007. If this approach will be found to be safe and able to restore dystrophin production, we plan to commence a systemic (I.V.) delivery trial with exon 51 by the end of 2007. We wish to thank Dr. J.Versnel, Study Coordinator, and the Muscular Dystrophy Campaign, The DMD Parent Project UK, the DMD Family Support group and the Department of Health UK for financial support. We thank Prof. Steve Wilton, Perth, Australia for the generous sharing of AOs sequence and advise, and Dr. J. van Deutekom, Leiden, The Netherlands, for collaborative studies aimed at validating the different AO in the hmDMD mouse.

2) Gene expression profiling to monitor therapeutic and adverse effects of antisense-induced exon skipping for Duchenne muscular dystrophy

P.A.C. ’t Hoen, C.G.C. van der Wees, A. Aartsma-Rus, R. Turk, A. Goyenvalle, O. Danos, L. Garcia, G.J.B. van Ommen, J.C.T. van Deutekom and J.T. den Dunnen - Nederland and France

The objective of our study was to assess the utility of genome-wide gene expression profiling for the preclinical evaluation of the efficacy and safety of disease treatment. As a model we used antisense oligonucleotide (AON) induced exon skipping for Duchenne muscular dystrophy (DMD). Muscles from mdx mice, a well-characterized animal model for DMD, were injected with antisense constructs that restore the open reading frame in the Dmd gene. Both synthetic AONs complexed with different carriers to enhance cellular uptake and recombinant adenoassociated virus (rAAV) expressed antisense sequences were evaluated. At different time points after treatment muscle RNA was isolated and gene expression profiles were generated using oligonucleotide microarrays. Polyethylenimine (PEI)-complexed AONs restored the reading frame slightly more effectively than uncomplexed, F127- or Optison-complexed AONs. However, PEI induced the expression of many immune genes, reflecting an aggravation of the inflammation present in untreated mdx mice. Expression profiles in Optison and F127-injected muscles were similar to those of saline-treated muscles, implying that these carriers did not evoke adverse responses. Due to the moderate levels of exon skipping obtained in these experiments, a significant shift towards wild-type expression levels could not be detected. Injection with rAAV vectors resulted in much higher skipping levels, concomitant production of dystrophin and a greatly improved histological appearance of the muscle. When the exon skipping levels increased, the expression of genes previously shown to be elevated in muscular dystrophies, partly or completely returned to wild-type expression levels. Reductions in inflammation and fibrosis were among the most prominent changes observed. Our results show that genome-wide gene expression profiling is a powerful tool for the evaluation of both desired and adverse effects of new pharmacological therapies. It is sensitive and can detect changes that are not yet visible histologically. In addition, its ability to simultaneously monitor a large number of different biological processes may assist in the early detection of potential undesired side effects, thereby reducing overall development time and cost considerabl
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3) Towards safe and efficient full-body delivery of antisense oligonucleotides for the treatment of Duchenne muscular dystrophy

J.A. Heemskerk,  C.L. de Winter, N. Heuvelmans, P. van Kuik, S. de Kimpe, G. Platenbur and J.C.T. van Deutekom - Netherland

Duchenne muscular dystrophy (DMD) is caused by frame-shifting mutations in the DMD gene that lead to dysfunctional truncated dystrophin proteins. By inducing the specific skipping of exons from the pre-mRNA, the reading frame and thus the dystrophin expression can be restored. The resulting proteins carry an internal deletion, but include the essential C-terminal parts, comparable to the partly-functional dystrophins found in the mostly milder Becker muscular dystrophy patients. Exon skipping can be induced with antisense oligonucleotides (AONs) targeting sequences involved in the recognition of exons by the splicing machinery. We have shown that this strategy has therapeutic potential in several patient cell cultures and in the mdx mouse model after intramuscular injections. Our current focus is to find the best AON formulation, dose, regimen, and administration method to deliver AONs safely and efficiently to muscle cells throughout the body. In this study, AON (2′-O methyl PS RNA) uptake in mdx and wild type mice was compared. In addition, different injection sites (intravenous, subcutaneous and intraperitoneal) were evaluated. An AON-specific ELISA showed that AON uptake by muscle fibres was approximately four times higher in mdx mice than in wild type mice, while uptake was equal in other organs. This implies that the damage of the muscle fibre membrane in mdx mice indeed enhances AON uptake. It was further shown that the uptake was highest after IV injection, followed by SC injection and finally IP injection. The levels of AON uptake corresponded to the levels of exon skipping as determined by RT-PCR analysis; the highest skipping levels were observed after IV injection (over 40% in various skeletal muscle groups, 30% in the diaphragm and 5% in the heart). This resulted in significant amounts of dystrophin, even in the heart. Serum creatine kinase levels were decreased when compared to placebo treated littermates, and muscle endurance experiments with a RotaRod measuring device showed a clear improvement.

4) Antisense oligonucleotide design for therapeutic antisense-mediated exon skipping for Duchenne muscular dystrophy

A. Aartsma-Rus, C.L. de Winter, W.E. Kaman, A. A.M. Janson and J.C.T. van Deutekom - Netherlands

Both the severe Duchenne and the milder Becker muscular dystrophies (DMD and BMD, respectively) are caused by mutations in the dystrophin gene. We are pursuing a therapy that aims to restore the open reading frame in DMD transcripts. Using antisense oligoribonucleotides (AONs) the pre-mRNA splicing is modulated such that one or more exons are skipped, thus allowing reading frame restoration and the generation of BMD-like proteins. We have so far designed 123 AONs that target 39 different DMD exons; 81 of these are effective at variable levels. The AONs are complementary to exon-internal sequences and are mainly directed at open structures within the target RNA (as predicted by m-fold). It is thought that the majority of exons contains exonic splicing enhancer sites (ESEs). The splicing factor family of SR proteins binds to these sites and thus facilitates exon inclusion by recruiting other splicing factors such as U1 snRNP to the splice sites. Retrospective analysis of our AON series with ESE predicting software revealed that effective AONs target significantly more putative ESEs than ineffective AONs. As for some exons the skipping levels are low, we hypothesized that for these exons two mutually exclusive ESEs might be present. Indeed using a combination of AONs within a single exon induced the skipping of thus far ’unskippable’ exons and improved exon skipping levels for inefficiently skipped exons. These results indicate that exon-internal AONs are likely to act by steric hindrance of SR protein binding to ESEs and that we can thus use ESE predicting software to fine-tune the design of new AONs.

5) Improvement of muscle mass after injection of AAV vectors expressing either myostatin shRNA or Activin receptor IIb shRNA

J. Dumonceaux, S. Marie and L. Garcia - France

Duchenne muscular dystrophy (DMD) is a severe degenerative disorder of skeletal and cardiac muscle. DMD patients show a progressive muscle weakness which begins in early childhood. Our goal is to oppose this continuous muscle wasting by altering the myostatin, a negative regulator of skeletal muscle mass. This protein binds to its cell-surface receptor (Activin receptor IIb, AcvRIIb) to inhibit both proliferation and differentiation of myoblasts. Indeed, it has been shown that myostatin deficient mice have a double mass phenotype compared to wild type animals. Our hypothesis is that inactivation of myostatin function may rescue muscle wasting in DMD patients as well as in its murine model, the mdx mouse. We investigated RNA interference strategy to inhibit functions of myostatin. We have decided to target both AcvRIIb and myostatin. Mice were first injected intra muscularly using an AAV vector expressing either the AcvRIIb shRNA or myostatin shRNA. Preliminary results show that a 50% down regulation of AcvRIIb mRNA in the TA of mdx mice results in a 15% increase in muscle mass. We also show that myostatin shRNA interfered with 95% of myostatin mRNA expression. In addition, when mice were injected systemically with this AAV expressing myostatin shRNA, we observed, 25 days after injection, that the body weight of the injected group is 15% more important than the non-injected group, suggesting a body-wide delivery of the vector. In conclusion, we show the possibility to manipulate muscle mass in dystrophic mice by interfering with the expression of myostatin activities. We are now investigated the possibility to combine myostatin inhibition and dystrophin rescue in a large animal model of DMD.
 

6) Autologous transplantation of muscle-derived AC133+ stem cells

M. Belicchi, C. Marchesi, R. Benchaouir, G. D’Antona, V. Mouly, G. Buttler-Browne, G. Cossu, N. Bresolin, L. Garcia and Y. Torrente - Italy and France

We recently reported that human circulating AC133+ progenitor cells can be induced to differentiate into skeletal muscles of scid-mdx dystrophic mice. However, in blood specimens of dystrophic patients there are low numbers of identifiable AC133+ stem cells that are difficult to be expanded in vitro. Here, we show that autologous transplantation of muscle-derived AC133+ cells in Duchenne muscles is safety and feasible. No local or systemic side effects were observed in treated DMD patients included in a randomised double blinded phase I study. Surprisingly, two treated patients had an increased ratio of capillary per muscle fibers with a switch from slow to fast myosin+ myofibers and partial recovery of some contractility force as showed by functional studies. We thus extended these clinical observations combining gene and stem cell treatment by the genetic engineering of AC133+ stem cells using exon-skipping approach which rescue the dystrophin expression. In these experiments the intramuscular and intra-arterial transplantation of engineered DMD stem cells caused a significant amelioration of skeletal muscle structure and function when delivered to scid-mdx dystrophic mice. We speculate that transplantation of DMD engineered AC133 positive stem cells could represent a future treatment for Duchenne muscular dystrophy.
 

7) NT-proBNP is not associated with dilated cardiomyopathy in Becker and Duchenne muscular dystrophies

S.M. Schade van Westrum, L. Dekker, E. Endert, R.J. de Haan, A. A.M. Wilde, M. de Visser and A.J. van der Kooi -  Netherlands

N-terminal proBrain natriuretic peptide (NT-proBNP) is a stable protein produced by myocytes of mainly the left cardiac ventricle as a response to stretch. It is increasingly used as predictor for the presence of cardiac failure and as parameter of therapeutic effect. In Becker and Duchenne muscular dystrophies (BMD and DMD) dilated cardiomyopathy (DCM) with cardiac failure is frequently observed among patients and carriers. Although the curvilinear relationship between BNP and systolic dysfunction is known, the relationship between the NT-proBNP level and the presence of DCM in patients and carriers is not. To assess whether NT-Pro BNP can serve as predictor of dilated cardiomyopathy in DMD/BMD patients and carriers, we included 394 individuals (84 DMD (17%), 92 BMD (24%), 147 DMD-carriers (37%), and 81 BMD-carriers (21%)). All were interviewed, neurologically examined and the following tests were done. NT-proBNP in peripheral blood was measured (normal: <0.35 pmol/l). M-mode and 2D echocardiography was used to asses the presence of DCM, defined as an enlarged left ventricle corrected for age and height with a global left ventricle dysfunction or fractional shortening less than 28%. A
χ2 test was used for associations and ROC-analysis for sensitivity and specificity of NT-proBNP for DCM. In DMD patients mean age was 13.6 years (SD 6.8), 62% was wheelchair bound. The mean age of BMD patients was 30 years (SD 13), 73% had some functional weakness of the leg muscles. Of the DMD-carriers (mean age 44 years, SD 11.8) and of BMD-carriers (43.3 years, SD 12.4), 14% and 13%, respectively, had myalgia, cramps or weakness. Overall, clinical symptoms of cardiac failure were present in 39%. The mean NT-proBNP was 0.64 pmol/l (SD 0.64). 62 (17%) out of 365 individuals in whom echocardiography was feasible, had DCM. Nine patients with DCM had a normal NT-proBNP, none were treated for DCM. The association between an elevated NT-proBNP and DCM was not significant (p = 0.12). A ROC-analysis showed no NT-proBNP value where sensitivity and specificity of any significance was reached (area under ROC curve = 0,58). NT-proBNP is not associated with echocardiographic defined dilated cardiomyopathy in DMD/BMD patients and carriers.
 

8) Revising the cardiac phenotype of Duchenne muscular dystrophy

L.W. Markham, A. Barone, K. Kinnett, R. Spicer, B. Wong, D.W. Benson and L. Cripe - USA

Duchenne muscular dystrophy (DMD) is a progressive skeletal and cardiac myopathy with a reported classic electrocardiogram (ECG) consisting of sinus tachycardia along with additional ECG abnormalities. Inappropriate sinus tachycardia has been postulated as precipitating the development of ventricular dysfunction. The objectives of this study were to determine the prevalence of sinus tachycardia and other ECG abnormalities in association with ventricular dysfunction. We hypothesized that the DMD ECG profile would change with age and the degree of ventricular dysfunction. From 1991 to 2006, 105 DMD boys had 503 ECGs and 322 echocardiograms performed. Subjects were divided into five groups based on age of initial cardiac evaluation – A, 0–5 years (n = 17); B, 6–10 years (n = 34); C, 11–15 years (n = 24); D, 16–20 years (n = 19); and E, >20 years (n = 11). Left ventricular (LV) dysfunction was defined as a shortening fraction <28%. By group, mean age in years at evaluation was: A, 4.3; B, 8.5; C, 13.5; D, 18.5; and E, 23.1. Sinus tachycardia (A, 0%; B, 12%; C, 30%; D, 48%; and E, 55%) increased significantly with age (p < 0.01). The percent with LV dysfunction (A, 5%; B, 15%; C, 33%; D, 53%; and E, 64%) also increased significantly with age (p < 0.01). Sinus tachycardia occurred in association with LV dysfunction (p < 0.001). Sinus tachycardia has a sensitivity of 84%, specificity of 95%, predictive value positive of 87% and predictive value negative of 93% for LV dysfunction. Additional ECG abnormalities were frequently noted in later stages of cardiac disease. In DMD, sinus tachycardia increases with age, rarely occurs prior to LV dysfunction, and is temporally related to LV dysfunction. The “classic” ECG findings of DMD as reported in the literature are only seen late in the cardiac disease process. Additional ECG abnormalities become more prevalent with age and correlate with the degree of ventricular dysfunction

9) Impaired platelet adhesion on collagen surfaces and secretion defect in Duchenne muscular dystrophy patients

V. Labarque, N. Goemans, K. Freson, M.F. Hoylaerts and C. Van Geet - Belgium

1Center for Molecular and Vascular Biology, Leuven, Belgium
2University Hospital Gasthuisberg, Leuven, Belgium

Duchenne muscular dystrophy (DMD) patients experience a significantly increased blood loss during spinal surgery without spontaneous bleeding tendency. Previous studies explained this by decreased platelet adhesion to glass beads, due to a deficiency of glycoprotein IV [Forst, Neuromuscul Disord 1998] or by the absence of an isoform of dystrophin, Dp71, in platelets [Austin, J Biol Chem 2002]. Others hypothesized the defect of primary hemostasis in DMD could be due to impaired vessel reactivity [Turturro, Neuromuscul Disord 2005]. However, none of these hypotheses have been confirmed unequivocally. In this study, we further investigated platelet function in DMD patients. We studied 11 patients in whom the diagnosis of DMD was confirmed on muscle biopsy or by mutation analysis. They showed no coagulation abnormalities and had a normal von Willebrand factor antigen and function as well as a normal expression of GP Ib, IIb and IV. Ristocetin- and ADP-induced platelet aggregations were decreased in 36% and 18% of the patients, respectively. During perfusion studies with whole blood, platelet adhesion on calf skin collagen was reduced at 2700/s (P = 0.01) as well as at 1300/s (P = 0,048). Patients with absent Dp71 (n = 2) did not show a reduced platelet adhesion. However, one of our patients showed a decreased ATP-secretion on stimulation with ADP or collagen, three others had an impaired serotonin release. Platelet Function Analysis, shape change, FAK-phosphorylation and spreading experiments on fibrinogen were normal. In this study, we found that DMD patients manifest an impaired platelet adhesion on collagen. However, we could not confirm a role for GP IV or Dp71, while at least in some patients, this platelet dysfunction could be explained by a secretion defect. As the platelet dysfunctions are minor and variable, we conclude that the increased blood loss in DMD patients could be due to the concurrence of reduced platelet function and disturbances in local factors, such as vessel reactivity and endothelial function. Future studies should focus both on these environmental factors as well as on defects in pathways following platelet activation.

10) The therapeutic effect of myostatin-blockade on muscular dystrophic mice and gene expression analysis of the treated muscles

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

Several therapeutic studies, such as gene therapies, cell implantations and pharmaceutical therapies, have been attempted toward muscular dystrophies. Recently, two growth factor-related molecules were highlighted by showing the effects on improvement of dystrophic phenotype of mdx mice. One was insulin-like growth factor-1 (IGF-1), and another was myostatin. Previously, we reported the therapeutic effect of IGF-1 on sarcoglycanopathic mice. In this study, we made recombinant folistatin-related gene (FLRG) protein in cultured cells and measured its blockade activity against myostatin in vitro. Then we administered recombinant FLRG protein into sarcoglycanopathic mice (sgcb−/− mouse at 3–5 weeks) and tried to evaluate its effect on their dystrophic phenotypes by pathological observation on comparison with those of IGF-1 administration. After administration of recombinant FLRG by osmotic pump implanted subcutaneously for 4 weeks, serum creatine kinase level was significantly decreased after treatment. In pathological examination, the gastrocnemius muscles showed remarkable reduction in necrotic fibers and increase in centrally nucleated fibers similarly to IGF-1 administration. Using oligonucleotide arrays with 22,000 gene probes, we also analyzed expression profiles of skeletal muscles of FLRG-administrated sgcb−/− mice. FLRG has a therapeutic effect on muscular dystrophic mice in similar levels as with IGF-1. Microarray analysis is a powerful tool for evaluation of the therapeutic effect.
 

11) The effects of prednisone on exercised-induced muscle damage in mdx mice

J. Collins, B. Wong,  A. McCullough and J. Quinlan - USA

Dystrophin deficiency results in muscle membrane instability and exercise enhances contraction-induced injuries in dystrophin-deficient muscles. Corticosteroids are known to delay progression of muscle weakness in children with Duchenne Muscular Dystrophy, but the mechanism of action is unclear. To evaluate the effects that prednisone had on exercised-induced muscle damage in mdx mice. mdx mice were treated with prednisone or placebo for 4 weeks. They were subjected to either a one time exhaustive run or a 4 week gentle daily run on a rate-controlled treadmill. Intra-peritoneal Evans blue dye (EBD) was injected thirty minutes prior to the exhaustive or final daily run. Histopathological markers of membrane stability (EBD penetrated fibers) and necrosis (CD11b) were examined in mdx rectus femoris muscles. In mice with exhaustive runs, the mean EBD positive area was 6.2 ± 1.6% (SEM) in prednisone-treated mice (n = 6) compared to 12.6 ± 2.5% (SEM) in placebo-treated mice (n = 6, p < 0.05). No significant difference was seen in the prednisone (n = 5, 6.4 ± 1.5%) and placebo (n = 5, 8.8 ± 1.4%, [p < 0.06]) groups with daily exercise. The mean CD11b stained areas were significantly different in prednisone-treated (6.9 ± 0.65%, n = 5) daily run mice compared to placebo (13 ± 2.9%, [n = 5, p < 0.05]). Prednisone stabilized the mdx sarcolemma in strenuous exercise and alleviated muscle cell damage and necrosis.
 

12) Quantitative ultrasound measurements of bone density DMD and SMA patients

A. Berardinelli, K. Gorni, L. Pedrotti, R. Mora, B. Bertani and E. Fazzi - Italy

Osteoporosis is characterized by loss of both bone mass and microarchitectural integrity, resulting in an increased risk of fractures with associated morbidity and mortality. Bone mass in the elderly is related to the rate of involutional bone loss and mainly to the peak bone mass reached in childhood and adolescence. The peak bone mass appears as being the key issue problem in prophylactic strategy of osteoporotic therapy. Reduced bone density is now well recognized in children and adolescents affected by Duchenne Muscular Dystrophy (DMD) even before motor impairment is clinically relevant; only one study has been performed so far about Spinal Muscular Atrophy (SMA) patients as far as we know, surprisingly a normal bone density nevertheless an even more severe motor impairment. The aim of this study was to determine quantitative ultrasound velocity, which is related to bone mass, in patients with DMD and SMA. The work is in progress and more patients will be included by next summer. Each patient is included accordingly with his follow-up time table. The sample so far consists of 14 patients, 12 males and 2 females; 4 non-ambulant; 10 affected by Duchenne (DMD), 2 by Becker Muscular dystrophy (BMD) and 2 by Spinal Muscular Atrophy (SMA). The rage range was from 3 yrs 8 mths to 18 yrs. Quantitative ultrasound velocity was measured by Sunlight Omnisense, a device designed to measure SOS (Speed of Sound) at multiple skeletal sites, by axial transmission method. The purpose of the study is to investigate whether osteopenia/osteoporosis is always associated with neuromuscular pathology and if that is related to motor impairment. In order to critically interpret the sonographic results of the children included in our study, we have drawn a diagram of reference with the data obtained by the same device in the control group. Speed of sound values, indicative of bone density, obtained in children affected by DMD and SMA were compared with those obtained by means of the same device in a control group of 700 Italian healthy children and adolescents aged 6–19 years. Correlation between degree of disability and SOS has been analysed. 5 subjects had normal SOS values, 6 were classified as osteopenic and 3 as osteoporotic. Only 1 child in the “osteopenic” group had a possible pathologic bone fracture in the cuboid some years ago. Six DMD patients are on steroids. Due to the small sample we cannot perform any statistical analysis so far. As described in the literature the most of our DMD patients had reduced SOS, but we also found some DMD in the “Normal” group, apparently with no correlation with steroid therapy, age, or motor impairment. Both of the younger DMD patients (3 years 8 months/3 yrs 11 mths) are in the “not normal” group.
 

13)  Bone health in Duchenne muscular dystrophy

B.L. Wong, M.Y. Shao, M. Hurtubise, A. El-Bohy, M. Knue, P. Morehart and J.E. Heubi - USA

Patients with Duchenne muscular dystrophy (DMD) are at risk for osteoporosis and fractures secondary to muscle weakness, loss of mobility and chronic corticosteroid therapy. To assess the prevalence of reduced bone mineral density (BMD) and fractures in patients with DMD, a cross-sectional study of 45 patients with DMD was conducted between June and August 2005. The primary outcome variable was BMD of distal femur [DF]] (R1 – cancellous bone, R3 – cortical bone, R2 – between R1 and R3) and lumbar spine (LS) as measured by DEXA z scores. The secondary outcome variables were fractures (per history and radiographs of spine and lone bones), DEXA lean body mass adjusted for weight, cumulative corticosteroid dose, calcium intake, serum 25 OH vitamin D levels and functional mobility level. Patients: n = 45 (ages 3.3–26, mean age 12.3 years), 24 on chronic steroids, 22 wheel chair dependent and 23 independent ambulators. For all DMD patients, DEXA z score (±SEM) for LS was −1.05 (±0.20); DF R1: −2.9 (±0.32), R2: −2.56 (±0.43), R3: −2.60 (±0.48) and whole body (WB): −1.78 (±0.25). DEXA z scores for LS were −1.08 (±0.34) vs −1.03 (±0.25); DF R1: −4.36 (±0.45) vs −1.63 (±0.23) [p < 0.0001]; R2: −4.33 (±0.63) vs −1.02 (±0.37) [p < 0.0001]; R3: −4.71 (±0.69) vs −0.76 (±0.38) [p < 0.0001] for non-steroid treated vs. treated subjects, respectively. LS, all 3 DF and WB DEXA z scores did not correlate with 25 OH Vitamin D levels and dietary calcium intake. DF R1, R2 and R3 z scores correlated with weight adjusted lean body mass (p < .0001) and with functional mobility levels (p < .0001). Analysis of covariance for comparing the steroid group and the non-steroid group adjusted for one of the four covariates (BMI, height, calcium intake and functional mobility level) found height and functional mobility level separately had significant effects as a covariate for the 3 DF z scores (p < 0.0001). When all above parameters were placed in the regression model as covariates with steroids, functional mobility level had significant effects for all 3 DF scores (p < 0.005), and height was significant for DF R3 scores (p = 0.04). Calcium intake was 1404.1 (±387.7) mg/day. Vitamin D intake was 498 (±62) IU/day and 25 OH vitamin D level was 26.78 (±1.67) ng/ml. Twenty of 45 (44%) patients had a history of fractures; 9/20 (45%) were on steroids; 5 patients had spine compression fractures (all on steroids). WB DEXA z scores correlated with fracture history (p = 0.0003). BMD is reduced in patients with DMD despite adequate calcium and vitamin D intake. Osteoporosis in the lower extremities is more profound in non-steroid treated DMD patients compared to steroid treated patients and is correlated with worsening functional mobility and decreased muscle mass. Further larger studies are needed to define the risk factors for osteoporosis and fractures in DMD patients to enable effective treatment interventions.
 

14) Functional ability monitoring in Duchenne muscular dystrophy using posture and walking time recording in a home environment

C. Bloetzer, B. Najafi, K. Aminian and P.Y. Jeannet -  Switzerland

Developing new tools to better assess disease progression in neuromuscular disorders is important, particularly in the light of new therapeutic possibilities. In this pilot study, the daily functional ability of Duchenne (DMD) patients and healthy controls was measured using a small and light, wireless ambulatory system (ASUR) attached to the child’s t-shirt. ASUR is composed of three accelerometers and one gyroscope which identify body postures and periods of walking. Seven DMD patients (age 4–10 years) and 2 healthy controls (age 5 and 7 years) were studied in their home environment for one day while parents kept a diary of their activities. A validated motor function measure scale (MFM) was performed in patients 6 years or older (n = 5) before the recordings. There was good correlation of recorded data with parent’s diary. For patients 8 years or younger (n = 5), mean time spend sitting (Si) was 31% ± 6.5; standing (St) 41% ± 6.4; lying (Ly) 3% ± 1.7; walking (Wa) 25% ±  4.5; mean number of walking episodes (WE) was 371 ± 165; and mean number of continuous walking periods for more than 30 s (Wp30) was 54 ± 17; mean MFM score was 84.4/100 (n = 3). In patients older than 8 years (n = 2), Si was 53.5% ± 6; St 28% ± 13; Ly 5.5% ± 8; Wa 13% ± 1; WE 302 ± 143; Wp30 26.5 ± 5; mean MFM score 87/100. In controls, Si was 16% ± 4; St 54% ± 10; Ly 9% ± 8; Wa 21% ± 3; WE 567 ± 83; Wp30 29 ± 1. One patient (age 4 years) monitored before prednisolone treatment and one month later increased Wa from 22 to 30%, We from 650 to 859, Wp30 from 46 to 93. Older and more disabled DMD patients spend more time sitting, less time standing and walking and had less walking episodes than younger patients and controls. Improvement of functional ability after initiation of steroid treatment was quantified in one patient. These promising preliminary results on a small cohort need to be confirmed by a larger study, currently under way.
 

15)  Is there a relationship between hamstring length and function in ambulant boys with Duchenne muscular dystrophy?

C. Nicholson, M. Main, E. Mercuri and F. Muntoni - UK and Italy

Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disorder characterised by weakness, falls, toe walking, joint contractures, difficulty rising from the floor and early loss of independent ambulation. It has been reported that tendo-achilles (TA) tightness interferes with function in ambulant boys with DMD and physiotherapy treatment including stretches and use of night splints, helps to maintain TA length. No studies found have considered the effect of hamstring length on function in ambulant boys with DMD, although it is usual for physiotherapists to teach hamstring stretches as part of daily management. Popliteal angle has been found to be a reliable measure of hamstring length with studies in normal paediatric populations showing increasing tightness up to the age of 9 years. These studies give normal range as being 20–50°. Our aim was to determine whether there was a significant relationship between hamstring length and function and try to establish when hamstring stretches are indicated for boys with DMD. Sixty boys with a confirmed diagnosis of DMD seen regularly at Hammersmith Hospital were included in the study. Measurements of hamstring length, TA and ilio-tibial band (ITB) tightness, timed rise from the floor, Hammersmith motor ability score, and muscle power measured using the Medical Research Council (MRC) scale, were recorded during routine assessments. The results show, of the 60 boys, only 6 had a popliteal angle greater than 50°. Using Spearman’s correlation coefficient there was no significant relationship between hamstring length and functional score (p < 0.503) with a weak correlation between hamstring length and timed rise from the floor (p < 0.006). There was a significant relationship between function and hip extensor power (p = 0.001) and between timed rise from the floor and hip extensor power (p < 0.001). Hamstring length, therefore, was not proven to be a major factor in causing functional difficulties in ambulant boys with DMD. Further, from our results, hamstring tightness does not appear to be a common feature of ambulant boys with DMD and the appropriateness of routine hamstring stretches for these boys, has not been established.
 

16) Predictive factors for the development of scoliosis in Duchenne muscular dystrophy

M. Kinali, R.K. Knight, M. Main, E. Mercuri, S. Messina, A.Y. Manzur and F. Muntoni - UK and Italy

Scoliosis is a complication in up to 90% of boys with Duchenne muscular dystrophy (DMD). Physiotherapy, rehabilitation in lightweight knee–ankle–foot orthoses (KAFOs), and glucocorticosteroids contribute to prolongation of walking and standing and might reduce the risk of scoliosis development. We evaluated possible predictive factors for development and progression of scoliosis in a large DMD population. Case notes of 123 boys with DMD
> 17 years, followed at our centre over a 10 year period were reviewed. Stepwise multiple regression analysis was used to correlate the age of onset and severity of scoliosis with: glucocorticosteroid treatment, ages at loss of ambulation and rehabilitation into KAFOs, length of KAFO usage, lower limb contracture asymmetry and age at loss of standing. 95/123 boys (70%) developed scoliosis with a Cobb angle  30°. Mean age (SD) at scoliosis onset was 12 years 8 months (SD 1 year 7 month). 37/123 boys (30%) received intermittent prednisolone (0.75 mg/kg/day, 10 days/month) for a median 1 year (range 2 month to 9 year), starting between 7 years 8 months and 12 years 5 months (mean 9 year 7 month). 96/123 (78%) were rehabilitated into KAFOs at 10 years 2 months (SD 1 year 7 month). Ages at loss of ambulation in KAFOs were 12 years 2 months and at loss of standing 12 years 10 months (SD 2 year 1 month). Forty-three boys (35%) had scoliosis surgery at 15 years (SD 1 year 2 month; preoperative Cobb angle 62.2° [SD 15.9°]). Of the 80/123 boys managed conservatively, including those who refused surgery, 12/80 had no scoliosis, 16/80 had mild scoliosis (31–120°). Age at onset of scoliosis correlated with loss of independent ambulation (p < 0.005). Combination of age at rehabilitation into KAFOs and duration of glucocorticosteroid treatment correlated with scoliosis severity at 17 years (p < 0.005). Our data demonstrate that age at loss of independent ambulation is an important predictive factor for scoliosis development. Other aspects of intervention such as rehabilitation into KAFOs and glucocorticosteroid treatment may also influence scoliosis outcome.
 

17)  Does deflazacort treatment impact the surgical outcomes for boys with Duchenne muscular dystrophy?

W.D. Biggar, V.A. Harris and B.A. Alman - Canada

The objective of this study was to compare the surgical outcomes for 3 groups of boys with DMD: (a) 41 steroid-naïve boys, (b) 8 boys previously treated with deflazacort, and (c) 9 boys on deflazacort at the time of surgery. This was a retrospective review of 58 boys with DMD between 1990 and 2006 and followed at Bloorview Kids Rehab. For the 41 steroid-naïve boys who had spine stabilization surgery at age 15.1 ± 1.8 years, 39 of 41 required assistance for feeding within 1 year of surgery, 13 had severe weight loss, 9 had severe leg/hip pain lasting longer than 12 weeks, 6 had an infection at the surgical site and 4 of those 6 required a surgical revision. Eight boys had been treated previously with deflazacort: (age deflazacort started 8.6 ± 2.4 years, age deflazacort stopped 12.4 ± 2.9 years, years on deflazacort 3.8 ± 1.4, age at surgery 14.9 ± 2.2 years). None had deflazacort within 1 year of surgery (2.5 ± 1.7 years). Pneumonia developed in 1 boy, a surgical infection in 3 boys, and 1 of these 3 developed Clostridium difficile colitis. None had severe weight loss. Nine boys were on deflazacort (0.2–0.7 mg/kg/day) at the time of surgery: (deflazacort started 11.5 ± 1.6 years, years on deflazacort 3.2 ± 1.3, age of surgery 14.7 ± 2.2 years). One boy had severe post-operative leg/hip pain. None had infections, poor wound healing or severe weight loss. All remained independent for feeding more than 1 year after surgery. In addition,we reviewed the surgical outcomes for another 135 boys ages 4–21 years treated with daily deflazacort, 2 had a typical presentation for acute appendicitis and a normal recovery, 3 had fractured femurs treated uneventfully with internal fixation, and 10 had one or more elective dental extractions. Our findings suggest that surgical outcomes are not adversely affected by deflazacort treatment in boys with DMD. Furthermore, in this small series, when deflazacort is started in older boys and at a low dose or if deflazacort is started in younger boys and then discontinued, the progression of scoliosis seems unaffected.
 

18) CINRG pilot trial of oxatomide in steroid-naive Duchenne muscular dystrophy

G.M. Buyse, D.M. Escolar, N. Goemans, E. Henricson, M. van den Hauwe, A. Jara Vallejos, C. Shao, K.M. Patel, R. McCarter, R. Leshner, J. Florence and J. Mayheww - Belgium and USA

Mast cells have been implicated in the pathophysiology of Duchenne muscular dystrophy (DMD) and its animal models. In addition the mast cell stabilizer oxatomide has shown promise in a pre-clinical screening platform that uses the homologous mdx mouse model of DMD. With CINRG (Cooperative International Neuromuscular Research Group), we have tested the hypothesis that oxatomide specifically targets the pathophysiological cascade of DMD by blocking the mast cell mediator cascade and inhibiting dendritic cell’s release of cytokines, and hence may improve muscle strength or delay progression of muscle weakness in DMD patients. We tested the efficacy and safety of oxatomide in 14 ambulant steroid-naive DMD boys aged 5–10 years, in a pilot open-label two-center clinical trial with a 3 months medication-free lead-in period followed by a 6 months treatment period with oral oxatomide (30 mg/day). Drug efficacy was tested by measuring muscle strength quantitatively (QMT, 10 muscle groups) and manually (MMT, 34 muscle groups), and by timed functional tests. Repetitive measure analysis was applied using linear mixed-effects models. Comparison of linear evolutions of muscle strength and function between lead-in and treatment periods showed no significant differences. There was a strong trend that patients treated for 6 months with oxatomide showed less deterioration in QMT measured muscle strength than in an historical matched control group. There were no serious adverse events and overall oxatomide was well tolerated. Within the limitations of a smaller non-controlled trial, we conclude that 6 months oxatomide treatment does not improve muscle strength or function in a group of 5–10 year old DMD patients. A minor disease-modifying effect of oxatomide in DMD cannot be excluded.
 

19) Lower urinary tract symptoms in patients with Duchenne muscular dystrophy

E. van Wijk, E.J. Messelink and I.J.M. de Groot - Netherlands

Duchenne muscular dystrophy (DMD) is a progressive disease that mainly affects striated muscles. However, involvement of gastrointestinal muscles (smooth muscles) and bladder dysfunction have also been reported. Lower urinary tract symptoms (LUTS) are frequently reported by boys with DMD at the outpatient rehabilitation clinic, though the prevalence of LUTS and the effect of LUTS on social activities in the DMD population are unknown. Therefore, we conducted a questionnaire-based survey on the prevalence and social effects of LUTS in the Dutch DMD population. A postal questionnaire was sent to members of the Dutch DMD patient organisations. The questionnaire was based on International Continence Society standards and contained general questions and specific questions about LUTS. Boys and men with DMD from the age of four were included. DMD was confirmed by DNA or muscle biopsy. Two hundred and one out of 217 respondents were included. 171/201 patients (85%) had one or more LUTS. One hundred and twenty-nine of the 171 (67%) patients with LUTS had 3 or more complaints. Post micturition dribble, straining, feeling of incomplete emptying, slow stream and (urge) incontinence were most frequently mentioned. 44/201 (22%) were too late toilet trained and 5 patients above the age of 12 were incontinent. The effect of LUTS on social activities varied per complaint, though a mean of 42% of patients (range 18–76%) experienced the LUTS as a problem. In 51/201 (25%) patients, quality of life was reduced by LUTS. 47/171 (27%) of patients had never mentioned their complaints to a doctor. The LUTS were not strongly correlated to age, functional status, scoliosis operation and/or constipation. Virtually all boys and men with DMD have LUTS, and often reduces quality of life. LUTS seem to be integrated, though underreported symptoms of DMD, and are not related to progression of disease. Further research is necessary to unravel the pathophysiology of LUTS in DMD and to direct future treatment.
 

20)  Depression and functional evaluation in Duchenne muscular dystrophy according to family functionality

R.E. Escobar, A. Miranda, M. Alfaro, C.K. Rocha, F. Calderon and E. Espejel - Mexico and USA

Depression in Duchenne muscular dystrophy (DMD) could be the result of the progression of natural history of this disorder, but some other factors could also be related such as family functionality.
The aim of this study was to compare depression and functional evaluation in DMD patients with family functionality. A comparative study was conducted. Patients with DMD diagnosed through clinical, enzymatic and electromyographic means were selected, aged between 8 and 14 years. Family functionality was evaluated with a Familial Functionality Profile questionnaire applied in parents and patients and according to the results two groups were formed, patients with functional and dysfunctional families. Depression was evaluated with the Children Depression Scale questionnaire. Functional evaluation were assessed through Brooke, Vignos and Swinyard functional grading scales. Family functionality and depression questionnaires have been validated for Mexican families. Analysis was performed with descriptive statistics, Mann-Whitney U test and chi squared or Fisher exact test. Thirty-five patients were invited but only 21 accepted to participate. There were no significant statistical differences between the groups in depression or functional grading scales (p > 0.05). But there was a slight tendency to increase depression values according to questionnaire results and a decrease in functional grading scales. In this study, we found that there is no differences between depression and functional evaluation in patients with Duchenne muscular dystrophy according to family functionality. It is possible that the 14 patients who refused to participate maybe were too depressed to do it.
 

21) Protease inhibitors as potential treatment strategy for muscular dystrophies

T. Meier -  Switzerland

Protease inhibitors as potential treatment strategy for muscular dystrophies Thomas Meier Santhera Pharmaceuticals, Liestal, Switzerland;
www.santhera.com. Absence of the functional dystrophin protein in Duchenne muscular dystrophy (DMD) leads to elevated levels of intracellular calcium which is considered as a key step in the cellular pathology of the disease. The ubiquitous calcium-dependent cytosolic cysteine protease calpain is activated in dystrophin-deficient muscle, and consequently, calpain inhibition is regarded as a potential treatment option for DMD. Subsequent proteolysis of myofibrillar proteins by the ubiquitin–proteasome pathway further contributes to the loss of muscle tissue. Genetic and pharmacological studies validate both calpain and the proteasome as potential drug targets for DMD. Since currently available calpain and proteasome inhibitors are not suitable for the chronic use in DMD patients we have developed and optimized novel calpain/proteasome inhibitors as a potential treatment option for DMD. These dual-property inhibitors have superior overall pharmacological properties as compared to reference calpain and proteasome inhibitors. We further demonstrate that a selected calpain/proteasome inhibitor accumulates dose-proportionally in muscle tissue where it significantly improves quantitative histological parameters, restores the expression of beta-dystroglycan at the sarcolemma and ameliorates motor performance of mdx mice. Our approach to simultaneously inhibit calpain and the proteasome not only qualifies as promising pharmacological treatment option for DMD but for all forms of muscular dystrophies where impaired calcium homeostasis and elevated proteolytic activity contributes to the manifestation of the disease.
 

22) Antisense-induced exon skipping in Duchenne muscular dystrophy patients

A. A.M. Janson, I.H.B. Ginjaar, A. Aartsma-Rus, G.-J.B. van Ommen, A. van der Kooi, S. de Kimpe, P. Ekhart, G. Platenburg, J.J.G.M. Verschuuren and J.C.T. van Deutekom - Netherlands

Antisense oligonucleotides (AONs) are emerging as small molecule drugs with a high, sequence-specific, corrective potential on RNA level. Especially for Duchenne muscular dystrophy (DMD), AONs have demonstrated to be an efficient and relatively simple and safe alternative to gene therapy approaches based on gene replacement. By inducing specific exon skipping, AONs allow restoration of the open reading frame of mutated DMD transcripts and turn on the synthesis of internally truncated, but largely functional Becker-like dystrophins. We have successfully demonstrated its high efficiency and specificity in cultured muscle cells from a series of DMD patients affected by different mutations, as well as in muscle tissue of the mdx and hDMD mouse models. Over the last 6 months, we have performed a small-scale, single-centre, clinical trial study at the Leiden University Medical Centre. It was based on a single, local, intramuscular injection of an antisense oligo (with a 2′OMePS RNA chemical backbone) that efficiently induces the skipping of exon 51. Six Dutch DMD patients were selected in the age group of 8–16 years, with well-characterized mutations correctable by exon 51 skipping (i.e., deletion of exons 49–50, 50, or 52). They were pre-screened by confirming the mutation on RNA level and by showing the therapeutic effect of the antisense oligo in cultured muscle cells. The oligo was administered in a 1–2 cm3 area in the tibialis anterior muscle in which the progression of muscle damage was determined to be limited by MRI analysis. A small biopsy of the treated area was taken at 4 weeks post-injection. Samples were analyzed for local exon 51 skipping and novel dystrophin production. In addition, patients were carefully screened for any adverse effects.
 

23) Rescue of dystrophin in the GRMD dog by multi-exon skipping using engineered U7 snRNAs

A. Vulin, A. Goyenvalle, I. Barthélémy, F. Leturcq, J.C. Kaplan, J. Chelly, O. Danos, S. Blot and L. Garcia - France

Among well described animal models of Duchenne muscular dystrophy, the GRMD dog is likely the best model of DMD patients in terms of size and pathological expression of the disease. GRMD is caused by a 3′ splice-site point mutation in intron 6, which induces the skipping of exon 7 and thus results in a frame shift that prematurely aborts dystrophin synthesis. By forced exclusion of two exons (6 and 8), it is possible to restore an open-reading frame. Antisense oligonucleotides (AO) allow specifically to target and inhibit individual genes for the treatment of the disorder. However, since the AO are not self-renewed, they cannot achieve long term correction. To overcome this limitation, we have introduced antisense sequences into small nuclear RNAs (snRNA) and vectorized them in AAV vectors. To determine efficient AO, we tested in particular exon splicing enhancer (ESE) motifs, which target internal exon sequences, crucial to pre-mRNA splicing. We designed different 2′O-methyl oligoribonucleotides which were tested first in vitro. Then we have designed AAV vector harboring chimeric U7 snRNA carrying efficient antisense sequences against exons 6 and 8 of the dog dystrophin gene, which were injected in GRMD muscles. Among different antisense oligonucleotides tested in GRMD, the combination of 2 AONs targeting, respectively, ESE of exon 6 and 8 induced delta5–9 in-frame skipping. In this study, we show the efficacy of these ESE sequences for exon-skipping in culture myotubes and in vivo. We also detected an almost complete restoration of dystrophin expression after intramuscular injections and systemic delivery of AAV-[U7-ESE6/U7-ESE8] vectors in GRMD. In this study, we provide evidence that efficient skipping of two exons can be achieved in GRMD through U7snRNA shuttles. Theoretically over 75% of Duchenne patients could benefit from skipping of a single exon and multi-exon skipping would significantly extend this percentage to most DMD mutations

24) Vascular endothelial growth factor gene transfer using adeno-associated viral vectors stimulates skeletal muscle regeneration and enhances muscle function in mdx mice

S. Messina, M. Aguennouz, A. Bitto, A. Migliorato, M. Giacca, F. Squadrito and G. Vita - Italy

Vascular endothelial growth factor (VEGF) is a major regulator of blood vessel formation during development and in the adult organism. Several evidences support its role in myogenesis and in myoblast migration and survival. Recently, it has been reported that the delivery of VEGF using adeno-associated-virus (AAV) vectors reduces muscle damage and promotes muscle regeneration in experimental models of muscle necrosis in mouse. We tested whether this effect was reproducible in mdx mice evaluating and we also measured the effect on muscle function by using a grip meter, CK level and morphological and biochemical parameters. Ten 4-week old mdx and ten wild-type mice were treated with intramuscular administration of AAV-VEGF (3 × 108 viral particles) or AAV-LacZ (3 × 1011 viral particles) into the biceps and tibialis anterior (TA) muscles. Evaluations were performed one month after injection. VEGF-AAV treatment increased the expression of VEGF. Mdx mice treated with AAV-VEGF showed a significantly higher forelimb strength than AAV-LacZ controls (+19,5%, p < 0.05) as well as higher strength normalised to weight (+14,9%, p < 0.05). There were no differences in CK levels between VEGF- and LacZ-treated mdx mice. Quantitative morphological evaluation of VEGF-treated biceps and TA muscles showed a reduction of necrotic area (p < 0.05) and an increase of regenerating area (p < 0.05). We report herein the novel observation of a pro-regenerative role of VEGF in mdx mice and of its beneficial effects on functional parameters. Further studies are needed to better clarify the mechanisms underlying the VEGF-induced benefit in mdx mice and to investigate possible therapeutic implications in Duchenne muscular dystrophy.