M. Eagle, M. MCallum, M. Guglieri V. Straub and K. Bushby - United Kingdom

Daily corticosteroids are the gold standard treatment for ambulant patients with Duchenne muscular dystrophy (DMD). Their efficacy in non-ambulant patients is unclear. The aim was to determine whether daily corticosteroids would improve (over the long term) forced vital capacity (FVC) in non-ambulatory patients with DMD. Eleven patients participated in a 6 months trial of prednisolone at 0.75 mg/kg/day with a ceiling dose of 40 mg/day. Six chose to continue steroids at the end of the study. FVC was monitored 6/monthly for two years and compared with an age and FVC matched group of patients who declined to participate in the steroid trial, and with the five patients who discontinued steroids at the end of the trial. Initially the mean FVC in the untreated matched group was 1.766 L and in the treated group it was 1.862 L (no significant difference). In both groups the age at loss of ambulation was 10.8 years. After two years FVC had improved to 2.13 L in the treated group and deteriorated in the untreated group to 1.32 L. Because the FVC in the patients who did not continue with the steroid treatment was significantly less than those who chose to continue it could not be directly compared, however it deteriorated over time from 1.17 to 0.8 L. Corticosteroids should be considered for non-ambulant patients with DMD to improve respiratory function.

2) 10 years follow-up of early corticosteroid treatment of Duchenne muscular dystrophy

L. Merlini, E. Malaspina, M. Gennari, A. Cicognani, E. Franzoni and B. Talim - Italy

Four patients with Duchenne muscular dystrophy (DMD) were started prednisone at the mean age of 3.4 years (2.4–4.0) and were regularly followed at least every 6 months for 10.2 years on average (9.7–11) up at the mean age of 13.15 years (12.2–14.9). At baseline, in addition to clinical signs, all had complete absence of dystrophin in muscle and three had an out-of-frame deletion in the dystrophin gene. They were given daily prednisone 0.75 mg/kg/day during the first month of treatment and then continued with 1.25 mg/kg AD. All received vitamin D and calcium rich diet and three were also given 500 mg/day calcium.

At the last follow-up all patients were ambulant, the two youngest still able to “run” 10 m. They were also able to climb 4 steps, the two youngest without using banister. The oldest one lost the ability to rise from the floor at the age of 12.5 years, while the other three retained this ability.

Currently their weight changes from 50 to 75 percentile; height was <3p in two, 10p and 25p in the two others. During treatment, they gradually became overweight, their final body mass index being 75–90p. Sexual maturation was delayed in all and three were started testosterone in their last visit for delayed puberty. Hypertension, diabetes, gastrointestinal bleeding, psychosis, compression fractures or cataracts has not developed in any case.

Early corticosteroid treatment (before 4 years of age) significantly changes the natural history of DMD, prolonging ambulation for at least 3–4 years on the average.

S. Pandya, E. Ciafaloni, D. Guntrum and R. Moxley - United States

We reported previously that daily prednisone therapy (1) prolongs independent ambulation to an average of 14.5 years and (2) delays decline in pulmonary function (forced vital capacity [FVC] < liter at an average age of 21.6) in a cohort of 30 DMD patients treated with daily prednisone and followed at our site since 1991. To describe the effect on (1) survival, (2) educational, occupational, marital status and (3) the emerging needs in the same cohort of patients. Patients received 0.75 mg/kg/d (40 mg/d maximum) of prednisone unless side effects required a reduction in dosage. Patients also received “standard care” including (1) instructions regarding diet, exercise and activities to maintain optimal function, (2) devices and equipment such as, night splints, long leg braces and wheelchair to optimize functional abilities, (3) surgery for treatment of contractures and scoliosis, (4) noninvasive nasal ventilation and (5) cardiac consultation and management. Patients have returned for follow up evaluations twice a year. We have measured vital signs, height, weight, FVC and side effects. During these visits we have inquired about, supported and assisted them in their educational, occupational and social aspirations. The average age of the cohort (N = 30) at initiation of prednisone was 10.5 years (5–15) and at last visit (N = 15) was 28.8 years (25–33). Five are ventilator dependent average age 31.5 years (27–33). Nine have died, average age 25.5 years (16–33), six are lost to follow up average age 17.5 years (12–20). The average dosage of prednisone at the last visit was 0.3 mg/kg/d. The main reason for reduction of dosage was weight gain. All patients have completed high school, 50% finished college, five worked/are working (teacher, architect, lawyer, editor, sales person), three drive/live independently, one is married. Patients as well as families describe independent living, care coverage, occupational and social opportunities as their primary concerns. Corticosteroids have changed the natural history of DMD and improved survival creating new challenges that will require comprehensive societal solutions.
 

C. Poulton, M. Kinali, S. Robb, M. Main, A. Manzur and F. Muntoni - United Kingdom

In 1993 a report was published on treatment of Duchenne dystrophy (DMD) with intermittent low-dosage prednisolone 0.75 mg/kg/day (10 days/month). Since its original publication at the Hammersmith we have used this regime (or 10 days on days off). In this study, we reviewed functional outcomes and steroid tolerance in DMD boys who have taken this regime (or 10 days on, 10 days off) for 18 months, from 2000 onwards. Fifty-one boys commenced, whilst independently ambulant, prednisolone. 24/51 boys lost ambulation (group 1) and 27/51 remain ambulant (group 2). Ages at commencement of prednisolone were similar in both groups (group 1: mean age 7.5 years, range [4–11 years]; group 2: 7.0 years [3.8–11.8 year]). Prednisolone regimes were similar in both groups with approximately 50% starting on 10 days on, 10 days off. Mean length of therapy was 2.6 years (group 1) and 3.3 years (group 2). Prednisolone was started at a motor ability score (MAS) of <30 (group 1) but at a MAS > 30 in group 2. Mean age of loss of independent ambulation was 10.0 years (group 1) and 10.44 years (group 2). Six boys remain independently ambulant aged >12 years. FVC was >70% at 18 months (N = 32) and at 24 months (N = 23) in both groups. Six boys discontinued prednisolone (4/6 due to weight gain, 1/6 because of epigastric pain; 1/6 due to parental choice). Data on behavioural changes were available for 36 boys. 19/36 (52%) boys had no behaviour changes, 8/17 had mild, 6/17 moderate and 2/17 severe, necessitating modification of therapy. One boys behaviour was improved. A vertebral fracture occurred in a patient with reduced mobility due to hydrocephalus. This open study suggests that intermittent regime has an overall positive functional effect maintains FVC and avoids major side effects such as with continuous steroid therapy. However the functional benefit conferred by this regime appears to be less. Our study provides for the first time information of limitations and advantages of a protracted intermittent steroid regime. While awaiting results of randomised studies the choice of existing corticosteroid regimes should be discussed with families.
 

K. Gorni, S. Orcesi, A. Berardinelli, A. Pini, M. Giannotta and E. Fazzi - Italy

Duchenne muscular dystrophy (DMD), an X-linked, recessive disorder, with onset before age 5 years, is the most common and severe form of childhood muscular dystrophy. At present, DMD therapy is based on symptomatic treatment and supportive care. Convincing evidence for clinical efficacy is available only for steroids. The precise way that glucocorticoids increase strength is unknown. Randomised controlled trials showed that corticosteroids improved muscle strength and function for 6 months to 2 years. The long-term benefit remains unclear and has to be weighed against the long-term side effects of these drugs. Two corticosteroids, prednisone (0.75 mg/kg per day) and deflazacort (0.9 mg/kg per day) have been used extensively. They appear to be equally effective in preserving skeletal muscle function. Both drugs are associated with side effects. Excessive weight gain can be particularly troublesome but deflazacort appears to be associated with less weight gain than prednisone. Many doubts remain about several important issues: when to begin the therapy, the best steroid to use, dose and regimen. An earlier beginning of therapy appears to be important in obtaining maximal benefit; at the moment there are just few studies on the use of steroids in early stages of the disease and the majority was referred to prednisone or prednisolone. We report here a study on the long-term functional performance and our clinical observations using deflazacort treatment in 31 boys who were between 3 and 14 years of age followed with controls every 3–6 months; the follow-up duration was at least 3 years. Twelve subjects started therapy before 6 years of age. The results confirm the importance of functional evaluation for children with DMD and show that deflazacort, like prednisone, improves functional ability in DMD within 6–9 months from the beginning of the treatment. In particular the improvement is more evident in subjects that started at earlier ages (<6 years).
 

J. Collins, M. Knue, C. Wang, K. Kinnett, M. Kalra, L. Cripe and B. Wong - United States

Corticosteroids are used in Duchenne muscular dystrophy (DMD) to improve strength and prolong ambulation. There is no consensus regarding a standard steroid regime. To evaluate outcome of DMD patients on different corticosteroid treatment regimes. This was a retrospective chart review of males with confirmed DMD by genetic testing or muscle biopsy. Patients were treated with daily deflazacort (dD; n = 14), daily prednisone (dP; n = 9), low dose 10 days on/10 days off prednisone (P10on/off; n = 18), or high dose weekend prednisone (HDW; n = 4). The primary outcome measure was treatment regime drop out rate. Secondary outcome measures in patients that remained on a treatment regime greater than 3 years (dD; n = 11, dP; n = 4, HDW; n = 2, P10on/off; n = 4) were motor function [ambulation, ability to stand from floor, and functional activity level (FAL)], body mass index (BMI), cushingnoid appearance, and forced vital capacity (FVC). The mean age for each group was similar (12 years) and the average age of treatment onset ranged from 6 to 8 years. The dropout rates were 21% for dD, 55% dP, 50% HDW, and 78% P10on/off. Reasons for dropout were – weight gain for dD group, weight gain or behavior changes for dP, decrease strength for HDW, and decrease strength or no benefit (57%) and weight gain for P10on/off. Percent of patients ambulating or arising independently at mean age 12 were dD 91%, dP 75% HDW 50% and P10on/off 25%. Median BMI percentiles were dD (88), dP (81), HDW (89), P10on/off (89). Cushingnoid appearance – dD (36%), dP (50%), HDW (50%), P10on/off (50%). Mean FVC (% predicted) – dD (98%), dP (110%), HDW (78.5%), P10on/off (54%). Daily steroid treatment use improves function over intermittent steroid dosing. Daily deflazacort was best tolerated and resulted in the most overall improved motor function. All steroid treatments elevate BMI.


7) Prednisone 10 days on/10 days off in 33 boys with Duchenne muscular dystrophy

C. Straathof, W. Overweg-Plandsoen, G. van der Burg, J. Verschuuren, A. van der Kooi and I. de Groot - Netherlands

A recent Cochrane review showed evidence that corticosteroids are effective in Duchenne muscular dystrophy (DMD) patients to improve muscle strength and to extend the ambulant phase. There is no consensus which treatment strategy (daily dose or alternating 10 days on/off scheme) is the best to minimize long term side effects. Awaiting the results of a forthcoming trial on treatment strategy we retrospectively analyzed data of DMD patients treated with prednisone in Rehabilitation Centre De Trappenberg. Thirty-three boys with DMD have been treated with prednisone 0.75 mg/kg/day 10 days on/10 days off. We evaluated loss of ambulation and the side effects of prednisone. Prednisone was started during ambulant phase at age 3.5–9.7 year (median 6.5 year). The median period of treatment was 27 months (range 3–123). The median age at which ambulation was lost was 10.8 year (mean 11 year; 95% CI 10.1–11.9 year). Fourteen patients had excessive weight gain on the length to weight percentiles during prednisone (5 patients 1 SD gain, 4 patients 1–2 SD, 5 patients 2 SD weight gain). This was reason to stop treatment in three boys. Seven boys (21%) had a bone fracture, in four of these patients this was the moment they lost ability to walk. Two boys had scoliosis surgery, one at age 13.5 year and one at age 8 year, 18 months after he had lost ambulation due to a femur fracture. Parents reported behavior complaints (hyperactivity, nycturia) in seven patients; treatment was stopped in one hyperactive boy. The prolongation of the ambulant phase in our cohort of DMD patients using prednisone 10 days on/10 days off was comparable to a similar group treated with daily dose prednisone. More than half of our patients did not have weight gain. The treatment did not increase fracture prevalence.

 

8) Steroids in Duchenne muscular dystrophy (DMD): Natural history and clinical evaluation using the North Star Ambulatory Assessment (NSAA)

M. Eagle, E. Scott, M. Main, J. Sheehan, M. Michelle, M. Guglieri, V. Straub and K. Bushby -  United Kingdom

Corticosteroids are the current gold standard treatment for ambulant boys with DMD. Because clinical trials to evaluate new compounds are imminent, accurate natural history data are required for steroid treated children. We describe the generation, validation and use of a scale for functional assessment in ambulant boys with DMD (the NSAA). We have used this scale to document the natural history of steroid treated DMD in the 4–12 age group. Intra and inter rater reliability studies were conducted in 4 groups of experienced and inexperienced evaluators both in the community and clinical setting. Reliability was good in all groups and excellent in experienced physiotherapists. Forty seven children started steroids over years (average age 6.6years). 21 children are ambulant between 9 and 13 years. 25% could jump before steroids and 57% afterwards, 38% could lift their head from the floor before steroids and 80% afterwards. In 4–7 year olds, motor ability measured by the NSAA, MRC% and timed tests improved after steroids and after 2–3 years remained better than the pre treatment ability. Eight year-old did not show such improvement and deteriorated more quickly. NSAA correlated with timed tests but not with manual muscle testing and was able to detect change in ability following increased dose of steroids. Forced vital capacity improved to more than the level expected in non-treated children. Ten children lost ambulation (average age 10.5). They tended to be older when steroids were initiated or had stopped steroids due to side effects. Side effects included high blood pressure, behaviour/emotional problems, cataracts, vertebral fractures, decreased height, weight gain but only six discontinued treatment due to side effects or inefficacy. The NSAA can be used to monitor motor function in ambulant DMD and can detect improvement due to modification of steroid dosage. Control of side effects is a major part of managing steroid treated DMD and should be dealt with proactively. The demonstration of the natural history of this age group of children using a standardised technique is an essential prerequisite for the design of future trials.

V. Kwinecki, V. Harris, J. Vajsar and D. Biggar - Canada

Objective: To examine a cohort of boys with DMD for patterns of weight gain and cardiac and pulmonary function at different ages. Design: Patterns of weight gain were reviewed retrospectively in 35 boys naïve to corticosteroids, 8–18 years of age with Duchenne muscular dystrophy (DMD). The mean weight for age of the boys was calculated and the boys were then organized into two groups, boys greater than the mean (heavy) and boys less than the mean (light). The cardiac and pulmonary functions of the heavy and light groups were then compared. Main Results: Between 8 and 13 years, the mean weight for boys was between the 75^th and 90^th centiles. Between 10 and 13 years, 42–45% of boys weighed more than the 90th and 10–14% less than the 10th centile. After 13 years, the mean weight declined and by 18 years it was at the 10th centile when only 16% of boys weighed more than the 90th centile and 52% weighed less than the 10th centile. Between 14 and 18 years, the pulmonary function (FVC-pp, FVC) in heavy boys was significantly greater than in the light boys. Three of the 11 heavy boys had impaired left ventricular function (LVEF < 45%) compared to 11 of 14 light boys. Conclusion: Our findings suggest that the design of clinical and therapeutic trials will need to accommodate for different weight profiles, different doses of pharmaceuticals when administered on a dose/kg body weight and possible differences in cardiopulmonary outcomes.

A. Berardinelli, K. Gorni, A. Pini, C. Motta and E. Fazzi -  Italy

It is well known that the prognosis of Duchenne muscular dystrophy is determined by respiratory and cardiac involvement. No therapies are available for the disease so far and the main goals of medical care are improvement of quality of life and management of respiratory and cardiac complications. In the last few years patients affected by DMD live much longer and their quality of life is globally better than it was. Use of steroids has becoming more and more common in the recent years: steroids are known to improve motor performances and some evidences suggest that they could be also useful in improving both respiratory and cardiac function in the years. The aim of our work is to describe the results of spirometric and cardiac evaluations in children affected by DMD treated with steroids. We will show data of a group of 20 DMD boys, with an age range 8–19 years, all treated with steroids for at least one year. 50% of patients stopped steroids mainly because children refused to continue the use in few cases due to some side effects. We found a quite good mean Vital Capacity (VC) in most of the children (about 1000 ml). Only one, 19 years old, needed mechanical ventilation and he was taking steroids for only one year. VC tended to be quite stable during the steroid therapy, while it decreased after stopping it. We had good heart studies results for all of the children. So, our data seem to confirm a positive effects of steroids not only on motor functions in children and we suggest to continue its use also after loss of ambulation unless serious side effects occur.

C. Melo e Souza, M. Magario and A. Godoy  - Brazil

Several reports suggest the benefits of corticosteroids (CS) for Duchenne muscular dystrophy patients (DMDp). In 2004 we founded a clinical center in the interior of Sao Paulo State, Brazil, to follow only DMDp. Since then we have seen individuals from all over the country. Six out of 35 DMDp came to us with the prescription of CS (0.5–1  mg/Kg). The age they began the use varied from 3 to 14 years. The period they took CS varied from to 7 years. Three of them need ventilatory assistance; one is wheelchair dependent and two can walk. The levels of creatine kinase were as high as 150 times normal. They were reduced to up to three times normal. One patient, 21 years of age, using CS for 7 years, got an increase on his muscle power (pectoral girdle and wrist flexors). The only patient not taking CS at the present time is now 20, used CS from 6 to 10 years of age. He has severe weakness in all four limbs. Our clinical data point to the need of a standardization of the use of CS in DMDp. Ages 5–7 seem to be good ones to start the use. We should not interrupt the prescription as soon as the patient stop walking, as many physicians have done. Additional benefits regarding ventilatory function seem to occur for those who take CS for many years. The side effects were minimal.

M. Yoshimura, A. Nakamura, M. Kobayashi and S. Takeda Japan

Oral administration of corticosteroids such as prednisolone or deflazacort is a preferable treatment in Duchenne muscular dystrophy (DMD), and motor deteriorations and cardiac dysfunction can be at least temporally improved. However, the mechanisms of action of these drugs on dystrophic muscles have not been fully understood. We have established and maintained a colony of Beagle-based canine X-linked muscular dystrophy in Japan (CXMDJ), a suitable animal model for DMD. Before the investigation in CXMDJ, we administered deflazacort, a newly developed corticosteroid, to normal Beagle dogs and evaluated the adverse effects. We orally administered deflazacort (Carcort(R)) 0.9 mg/kg/day for 3 months to 4 normal Beagle dogs of 1 month of age. We regularly examined clinical status, complete blood cell counts (CBC), serum chemistry, and evaluated the temporal, neck, thigh and lower leg muscles by 3.0T magnetic resonance imaging (MRI) at 4 month of age. After euthanasia, the histopathology of skeletal muscles from whole body was extensively examined. We observed severe developmental delay and atrophy of the temporal, truncal and leg muscles compartments in all dogs examined after 1-month of administration. Three of the 4 dogs showed repetitive inguinal or abdominal wall herniations and needed surgical operation. No other abnormal findings were detected on CBC or serum chemistry examination. MRI revealed an extensive loss of muscle mass without abnormal signal intensity in all muscles examined, and an increase in subcutaneous fat tissue was noticed. The muscle pathology showed that marked muscle atrophic change in the temporal, thoracic, paraspinal, abdominal and thigh flexors and lower leg muscles. Deflazacort induced striking general muscle atrophy and severe inguinal herniation in the normal Beagle dogs. This severe adverse effect might be taken into consideration in the treatment of DMD by deflazacort. Molecular mechanism of muscle atrophy by deflazacort treatment should be more extensively investigated.

S. Chaouch, D. Furling, A. Goyenvalle, L. Garcia, J. Di Santo, Y. Torrente, G. Butler-Browne and V. Mouly - France, United Kingdom and Italy
We are now at a point in time where gene therapy is becoming a reality. However, in order to validate these strategies it is essential to have in vitro human cell culture models. The use of patient myoblasts is not always possible due to their drastically decreased proliferative capacity induced by the repeated cycles of degeneration and regeneration. Therefore it is necessary to envisage new in vitro models. In the pioneering studies of Weintraub et al. it had been shown that the forced expression of the myogenic transcription factor myoD was able to convert fibroblasts into myoblasts. In the present study we have developed a universal in vitro model from skin fibroblasts which have been immoralised using hTERT and then converted into myoblasts by a lentivirus containing an inducible myoD contruct. We have then used this model to validate a strategy for exon skipping using fibroblasts isolated from a DMD patient. These fibroblasts were immortalised and then transduced using an inducible myoD construct. We first confirmed the expression of myoD in vitro and the potential of these cells to form differentiated myotubes. These cells were then transfected with an U7 construct to promote exon skipping in the patient. In order to test if these cells could reconstitue muscle fibres in vivo expressing human dystrophin they were injected into cryodamaged TA muscles of immunodeficient RAG−/− gammaC−/− C5−/− mice. Muscle were analysed after 27 days of regeneration and fibres expressing human dystrophin were observed. Therefore this cellular model provides us with an ideal model system to test different therapeutic strategies for various neuromuscular diseases when patient myoblasts are not available.

14) In vivo biodistribution of non-viral systems for oligoribonucleotides delivery

P. Rimessi,  P. Sabatelli, F. Gualandi, P. Spitali, M. Bovolenta, E. Martoni, M. Fabris, V. Nigro, E. Nusco, E. Calzolari and A. Ferlini -  Italy

Successful approaches of single exon skipping and dystrophin protein synthesis restoring have been described upon antisense oligoribonucleotides (AONs) treatment of both patients’ derived cells and mdx mice. The main obstacle to be still overcome is to find a AONs delivery system able to efficiently reach both skeletal and cardiac muscle. In fact, in the mdx model the cardiac muscle can be reached effectively only by using recombinant adeno-associated viruses (AAV) as vehicles. One hypothesis for explaining the difficulty for naked AONs in entering into cardiomyocytes could be the absence of cardiomyopathy in the mdx animal model. In order to test this hypothesis we performed a biodistribution analysis of novel delivery systems both in normal (F1B) and cardiomyopathic hamster (delta-sarcoglycan deficient BIO14.6), which represents a suitable animal model for dilated cardiomyopathy, at variance from mdx. We had obtained previous evidences that our nanoparticles were able to efficiently bind AONs. We tested two kinds of fluorescent polymethyl methacrylate core-shell-type nanospheres, T1Fluo and Z2Fluo, 500 and 200 nm, respectively, presenting on their surface cationic groups originally designed for the reversible adsorption of DNA oligonucleotides. The two nanoparticles differ both in their size and in the presence on the surface of Z2Fluo of polyethylene glycol (PEG). A total of 20 six weeks-old hamsters, 10 F1B and 10 BIO14.6, were treated via intraperitoneal injection and sacrificed 48–72 h after treatment. Fluorescence and electron microscope analysis of different tissues from treated hamsters indicate that both nanoparticles enter into several cell types, including cardiomyocytes, independently from the presence of the cardiomyopathy and myopathy. In conclusion the novel non viral delivery systems we tested do enter both in cardiac and skeletal muscle, and may represent suitable vehicles for in vivo delivery of antisense oligoribonucleotides.
 

K. Wee , Z. Pramono, J. Wang, K. MacDorman, W. Yee and P. Lai -  Singapore

Antisense oligonucleotide (AON) induction of exon skipping offers a potential therapeutic strategy for Duchenne muscular dystrophy (DMD). Although clinical trials of AON-mediated DMD therapy have begun, identification of effective AON target sites remains empirical for lack of a more precise method to predict their binding accessibility. Because splicing and transcription occur in tandem, AONs must bind to their target sites before splicing factors do. Furthermore, co-transcriptional pre-mRNA folding forms transient secondary structures, which redistributes accessible binding sites, thereby complicating the identification of accessible target sites during transcription. Our study included the dynamics of pre-mRNA secondary structures in an evaluation of published AONs and their ability to induce exon skipping. Correlation of the number of nucleotides having entirely inaccessible windows with AON efficacy and efficiency accounted for up to 94% of published AONs where their localizations proved to be most critical. Only one nucleotide with entirely-inaccessible windows is sufficient at its 3′ and 5′ end to block AON efficacy, but three or more in succession are necessary further from the flanks. The efficiency of an AON is more vulnerable to the presence of these nucleotides at the site’s 3′ end than its 5′ end. Our results show that co-transcriptional pre-mRNA folding is important in predicting AON targets. Using these results, a software tool, Dynamic AON was developed to select AON target sites. Three novel AONs designed to skip exon 51 with this tool showed efficient and selective skipping of the targeted exon


16) Rescue of human dystrophin after transplantation of exon skipping-engineered DMD stem cells in a dystrophic animal model

R. Benchaouir, A. Goyenvalle, M. Meregalli, M. Belicchi, A. Farini, M. Battistelli, N. Bresolin, L. Garcia and Y. Torrente - Italy and France

Duchenne muscular dystrophy (DMD) is a hereditary disease caused by genomic mutations that disrupt the dystrophin mRNA reading frame. This destabilizes the dystrophin and its associated complex proteins, provoking progressive and irreversible muscle degeneration. In some cases, forced exclusion (skipping) of a single or multiple exons can restore the reading frame, giving rise to a shorter, but still functional protein. Most of DMD mutations are localized into the central rod domain of the dystrophin gene; for this particular reason, this protein is well adapted for exon-skipping application since in frame removing of central spectrin-like repeats, was demonstrated to conserve its functionality. In a cell therapy perspective, exon skipping approach was used to treat a subpopulation of adult stem cells extracted from DMD patients. We previously shown that one population of human stem cells, harbouring the CD133 surface antigen, was able to efficiently participate in muscle regeneration in vivo. To extend this work, we evaluated the muscle regeneration potentiality of blood and muscle-derived CD133+ cells after in vitro exon skipping treatment. Lentiviral vectors were constructed to convey specific antisense oligonucleotides able to induce an efficient exon-skipping and to correct the initial frameshift caused by the DMD deletion. In our case, DMD cells yielding deletion of exons 49 and 50 were treated with vectors able to perform skipping of the exon 51, rendering in frame the dystrophin mRNA sequence. The skipped blood and muscle-derived stem cells were able to fuse in vivo with scid/mdx mice regenerative fibers and, not only perform expression of a functional human dystrophin, but also restructure the dystrophin-associated complex such as alpha and beta-sarcoglycans proteins. These data demonstrate that autologous engrafting of blood or muscle-derived CD133+ cells, preliminary genetically modified to re-express a functional dystrophin, seems to represent a promising approach for DMD.


17) Phase 2 study of PTC124 for nonsense mutation suppression therapy of Duchenne muscular dystrophy (DMD)

C. Bönnemann, R. Finkel,  B. Wong, K. Flanigan, J. Sampson, L. Sweeney, A. Reha, G. Elfring, L. Miller and S. Hirawat -  United States

PTC124 is a novel, nonantibiotic, drug that promotes ribosomal readthrough of mRNA containing a nonsense (premature stop codon) mutation. This Phase 2 study is evaluating PTC124 safety, compliance, PK, effects on full-length muscle dystrophin protein expression, and clinical activity in patients with nonsense-mutation-mediated DMD. Patients receive PTC124 administered orally for 28 days at dose levels of 4, 4, 8 mg/kg (low dose); 10, 10, 20 mg/kg (mid dose); and 20, 20, 40 mg/kg (high dose) after breakfast, lunch, and dinner, respectively. 26 boys (ages: 5–13 years; stop codons: 15 UGA, 6 UAG, 5 UAA; baseline serum CK: 8645–49500 IU; steroid use: 19/26) completed PTC124 at the low (n = 6) or mid (n = 20) dose levels. All adverse events and laboratory abnormalities were mild-moderate with no dose-related changes in frequency or severity. Compliance was >98% for both dose levels. Day 1 and Day 28 PK indicate stable plasma exposures over time; however, exposures were lower than in PTC124-treated adult healthy adult volunteers and cystic fibrosis patients. Myotube cultures from pre-treatment muscle biopsies showed dose-dependent increases in dystrophin expression with in vitro PTC124 treatment in 24/24 evaluable patients. Relative to baseline, visually appreciable post-treatment qualitative increase of in vivo dystrophin expression is noted in 4/6 and 10/20 boys at low and mid doses, respectively. Within the 28 days of treatment, serum CK, AST, and ALT levels decreased significantly but changes in muscle strength and timed functions were small and not significant. Preliminary evidence indicates that PTC124 safely induces full-length dystrophin expression in vitro and in vivo and decreases serum muscle enzyme levels in boys with nonsense-mutation-mediated DMD. Although low and mid dose levels demonstrated these effects, subjects did not achieve plasma exposures associated with maximal preclinical activity. Evaluation at the high dose level in 12 additional boys is ongoing.

18) Identification and characterization of small molecules for the treatment of Duchenne muscular dystrophy

W. Friesen, Y. Tomizawa, J. Zhuo, R. Baiazitov, S. Lee, T. Nadarajan, Y. Moon, H. Sweeney and E. Welch -  United States

PTC Therapeutics, Inc. (PTC) and Parent Project Muscular Dystrophy (PPMD) are collaborating to discover new drugs to treat Duchenne muscular dystrophy (DMD). Four targets were selected to enter the drug discovery program based on functional validation from animal studies. The targets selected for high throughput screening (HTS) included targets representing growth factors and proteins involved in muscle membrane stabilization. Using a proprietary drug discovery platform technology, referred to as GEMS (Gene Expression Modulation by Small-molecules), we sought to identify small molecules that up- or down-regulate the production of proteins that have the potential to treat DMD. Stable muscle or kidney cell lines containing the firefly luciferase (fLuc) reporter gene flanked by the 5 and 3 untranslated regions (UTR) for each of the targets were constructed and used in HTS. The activities of the hits identified from these HTSs were confirmed in the cell-based reporter assays as well as in assays to monitor protein levels. For several molecular scaffolds of hits against each target, the activities are dose-dependent and target specific. A number of molecules exhibit good pharmacological properties (e.g., low cytotoxicity and microsome metabolic stability). We are in the process of establishing structure–activity relationships for the molecules in each of the chemical classes to optimize their pharmaceutical properties. The ultimate goal of this drug discovery and development effort is to identify small molecules that can specifically modulate the production of a number of proteins that can be ultimately used as monotherapies or as part of a combination therapy to treat muscular dystrophy.

19) MicroRNA expression in Duchenne and Becker muscular dystrophy

M. Aguennouz , O. Musumeci, N. Lanzano, S. Soufiani, R. Crupi, C. Rodolico, A. Toscano and G. Vita¹ - Italy

Duchenne and Becker muscular dystrophies (DMD, BMD) are progressive disorders due to dystrophin deficiency that results in severe skeletal muscle degeneration. The pathological mechanisms underlying these diseases are not fully understood. MicroRNAs (miRNA) are endogenous RNAs of 22 nucleotides that can play important regulatory roles in animals by targeting mRNAs for cleavage or translational repression. miRNA-1 (miR-1), miRNA-133 (miR-133a and miR-133b) and miRNA-206 (miR-206) are transcribed together in a tissue-specific manner during development and have distinct roles in modulating skeletal muscle proliferation and differentiation in cultured myoblasts. miR-1 promotes myogenesis by targeting transcriptional repressor of muscle gene expression; miR-133 enhances myoblast proliferation by repressing serum response factor (SRF); miR-206 regulates connexin 43 expression during skeletal muscle development. We evaluated the presence and the level of expression of miR-1, miR-133a, miR-133b and miR-206 in muscle biopsies obtained from 10 DMD and 10 BMD patients (age range respectively, 2–8 years and 6–12 years), and 5 normal subjects using a relative quantification (RQ) RealTime PCR and Northern blot analysis. Our preliminary results evidenced presence of miRNAs in all DMD, BMD and normal biopsies. RQ showed an increased expression of miR-1 in DMD muscles versus controls. Our findings suggest that miRNAs could be involved in regeneration and maturation of DMD muscles, acting as key regulators of different processes such as early development, cell proliferation, apoptosis, metabolism, and cell differentiation.


20) CTGF expression in normal and dystrophic muscles: Correlation to fibrosis

P. Noirez, I. Ambrosi, M. Fiszman, C. Dubois and H. Alameddine - France

Duchenne muscular dystrophy (DMD) is not a fibrotic disease per se yet muscle biopsies of DMD patients are generally characterized by excessive production, deposition, and contraction of extracellular matrix similar to the one observed in fibrotic diseases. To understand the molecular basis leading to the accumulation of extracellular matrix (ECM) components in muscular dystrophy, it is essential to determine the factor(s) influencing dysregulation of the normal balance between production and/or hydrolysis of ECM components. A growing body of evidence indicates that CTGF (Connective Tissue Growth Factor), a protein of CCN’s family (Cysteine-rich 61/Ctgf/Nephroblastoma Overexpressed), is involved in different experimental and pathological situations of fibrosis in skin, liver, kidney, lung, and heart. Whether CTGF plays a role in the development of fibrosis in dystrophic muscles has not been assessed. In this study, we have investigated the existence of a correlation between CTGF expression and the extent of fibrosis in mdx muscles. Diaphragm and limb muscles of 21 days-, 6 weeks-, 3, 6, 12 and 18 months old normal and mdx mice, were examined. Serial cross-sections were stained with Hematoxylin–Eosin and Sirius Red to quantify fibrosis. CTGF expression was monitored by immunohistochemistry, Western blotting and quantitative RT-PCR. Quantification of fibrosis confirmed previous results indicating that limb muscles of mdx mice are less fibrotic than the diaphragms and that fibrosis increases with age. In mdx muscles, immunoreactivity to CTGF was increased in comparison to age matched controls, an observation that favours the existence of a correlation between CTGF expression levels with the extent of fibrosis in muscles. This is now being confirmed by quantitative RT-PCR, and in biopsies of dystrophic patients. If our results were to be confirmed, CTGF would represent a potential therapeutic target to slow down disease progression or functional deterioration. Acknowledgments: The authors thank the AFM and INSERM for financial support.


21) Novel myostatin inhibitors increase muscle mass in wild-type and mdx mice

J. Lachey, A. Pullen, R. Pearsall and J. Seehra - United States

Myostatin, or GDF-8, is a well-characterized negative regulator of muscle growth. Myostatin overexpression reduces skeletal muscle mass whereas myostatin inhibition causes a dramatic muscle mass increase. Myostatin is a member of the transforming growth factor-B superfamily and binds to the activin type llB receptor (ActRllB) with high affinity. Consistent with myostatin acting through ActRllB to elicit its muscle effects, treatment with a soluble form of ActRllB increases muscle mass in wild-type mice. Additional unidentified ActRllB ligands that inhibit muscle growth have also been proposed. Therefore use of a soluble ActRIIb to promote muscle growth is advantageous as it will inhibit the uncharacterized ligands as well as myostatin. Here we describe two novel molecules comprised of the ActRllB extracellular region (ACE031) or a mutant thereof (ACE032) fused to a human IgG1 Fc domain. ACE032 has a 10-fold greater binding affinity as defined by cellular assays and was designed to increase efficacy of the molecule. Whole body NMR analysis revealed ACE031-treated mice had a 3-fold increase in lean tissue compared to the vehicle-treated controls over a month. Treatment with ACE032 caused a significantly increased lean tissue mass although to a lesser extent than ACE031. Consistent with a selective muscle effect, gastrocnemius, pectoralis and femoris muscle weights were significantly increased in the ACE031 (33.0–45.8%) and ACE032 (29.5–46.0%) groups. To assess the possible benefit of a soluble ActRllB molecule in muscle disease, we tested RAP031 (ActRllB extracellular region fused to a mouse IgG1 Fc) in mdx mice, the mouse model of Duchenne muscular dystrophy. We found that gastrocnemius, femoris and pectoralis weights of ACE031-treated mdx mice were significantly increased (27.0–84.7%) compared to the vehicle-treated group. Further, we report ACE031-treated mdx mice exhibited functional improvements in the dystrophic phenotype compared controls, providing support that our molecules potentially have important clinical applications.

22) No evidence for increased muscle regeneration in myostatin deficient mdx mice (mstn-/-mdx)

H. Amthor, M. Friedrichs and T. Voit - Germany and France
 

At the 11th WMS congress in Brugge we reported that dystrophic muscle from mdx mice that lack myostatin (mstn−/−mdx) contained no increased number of satellite cells. Preliminary histological analysis furthermore revealed no increase in number of revertant fibres. These results did not support the current view that blockade of myostatin can stimulate regeneration of dystrophic muscle.

We further explored the muscle phenotype of mstn−/−mdx mice and investigated the histological properties of extensor digitorum longus (EDL) muscle from 1 1/2 old mice. We first determined the proportion of fibres containing central nuclei and found no difference between muscle from mstn−/−mdx (32.1 ± 7.33) and mstn+/+mdx (37.5 ± 7.44), (p = 0.25). We than counted the total number of myofibres at the midbelly cross section. Although there was an increase in fibre number in mstn−/−mdx (1582 ± 233) compared to mstn+/+mdx (1213 ± 350), this increase was not statistically significant (p = 0.078).

Fourth, the generation of revertant dystrophin positive fibres was compared in muscle from mstn−/−mdx and mstn+/+mdx on serial sections from EDL muscle. It has been previously suggested that the extent of muscle regeneration is proportional to the number of revertant fibres especially to the number of revertant fibres per cluster. This results from the clonal expansion of satellite cells during the muscle regeneration that experienced a second mutation allowing for the re-expression of dystrophin. As more vigorously muscle regenerates as more revertant fibres will develop within a cluster, because revertant fibres are more resistant to muscle degeneration than neighbouring dystrophin negative fibres and thus will accumulate over time. Here we show that mstn−/−mdx contained a higher number of revertant fibres (38.4 ± 15.7) compared to mstn+/+mdx mice (20.82 ± 6.7), (p = 0.045). Mstn−/−mdx also contained a higher number of clusters of revertant fibres (16.2 ± 4.45) compared to mstn+/+mdx mice (9.26 ± 3.65), (p = 0.020). However, when normalized for the total number of myofibres per cross section, the increase in the number of revertant fibres was not statistically significant (p = 0.101), nor was the number of clusters (p = 0.065). We next compared the number of revertant fibres per cluster. Clusters were considered that contained at least three revertant fibres. We found slightly less revertant fibres per cluster in muscle from mstn−/−mdx mouse (5.5 ± 2.59) compared to mstn+/+mdx mice (6.4 ± 4.08), however, the decrease was not statistically significant (0.658).

In summary, these data suggest that lack of myostatin does not improve regeneration of dystrophic muscle of mdx mouse. This is an unexpected finding and it dissents with the current view.

23) DLK1 as a candidate for booster gene therapy in muscular dystrophy

L. Joergensen, C. Jensen, E. Davis, C. Charlier, M. Georges and H. Schroeder - Denmark
 

The callipyge (CLPG) phenotype is a muscular hypertrophy in sheep, which manifests in heterozygous animals inheriting the CLPG mutation from their father. The causative mutation enhances expression of genes in the DLK1-GTL2 locus in cis. Recently, we demonstrated a perfect correlation between the CLPG phenotype and ectopic DLK1 expression in hypertrophied muscles. Furthermore, transgenic mice ectopically expressing ovine DLK1 exhibited a generalized muscular hypertrophy. Taken together, these factors imply a role for DLK1 in muscle growth. Therefore, we speculated that DLK1 could be a candidate for muscular dystrophy booster gene therapy. Here, we propose to up-regulate proteins actively participating in muscle development and regeneration. Enhancement of endogenous proteins could strengthen the muscle against the constant damage occurring in muscular dystrophies and also avoid the potential immune rejection of conventional gene therapy. To analyze this possibility, we investigated skeletal muscle regeneration in DLK1 transgenic mice and littermate controls following injury. H&E and Sirius stainings showed that DLK1 mice initiated muscle regeneration earlier than wild-type controls. However, when gene expression of myogenic factors was investigated using qPCR there were no statistically significant differences in Pax7, Myf5, MyoD, Myogenin, or Mef2a expression during regeneration of DLK1 and control muscle. The protein expression patterns were investigated by counting the number of cells expressing Pax7, Myogenin, and p27 and no statistically significant differences were observed. Interestingly, ADAM12, Utrophin and Integrin β1 mRNA expression was up-regulated, while Myostatin mRNA expression was significantly lower in DLK1 mice. This could in part explain the hypertrophic phenotype of DLK1 muscle since lower myostatin expression, a known inhibitor of muscle growth, increases muscle mass. Presently, DLK1 is still a potential booster gene candidate given its ability to increase muscle mass and up-regulate structural proteins, despite an inability to significantly induce or enhance the regenerative potential.


24) NPC1 overexpression attenuates muscular dystrophy in mdx and α-dystrobrevin-null mice

M. Steen, Y. Tesch, M. Adams and S. Froehner - United States
 

The loss or alteration of certain proteins from the dystrophin complex results in muscular dystrophies. Understanding the mechanism by which α-dystrobrevin (αDb) loss causes muscular dystrophy in mice may provide new therapeutic approaches to the human diseases. Our objective was to understand regulatory pathways required for maintenance of healthy muscle. We hypothesized that by comparing gene expression of αDb-null muscles to healthy muscles, we would identify genes whose misregulation results in muscular dystrophy. (1) We used Affymetrix microarrays to compare gene expression levels in muscles of αDb-null mice with littermate controls. RT-PCR and immunoblotting confirmed changes in levels of Niemann-Pick Type C1 (NPC1) transcript and protein. (2) We generated transgenic mice expressing NPC1 specifically in skeletal muscle and bred these mice onto αDb-null and dystrophin-null (mdx) backgrounds. We assessed the number of regenerating fibers in various muscles and compared serum creatine kinase levels in transgenic and control mice. NPC1 transcript and protein levels are reduced 50% in skeletal muscle of αDb-null mice. NPC1 facilitates the trafficking of free cholesterol from late endosomes and lysosomes to other compartments. Mutations in NPC1 cause a progressive neurodegenerative disorder. Transgenic overexpression of NPC1 reduced both the percentage of regenerating muscle fibers and serum creatine kinase levels in αDb-null and mdx mice. Therefore, transgenic overexpression of NPC1 in skeletal muscle ameliorates the dystrophic phenotype of αDb-null and mdx mice. Our results suggest the involvement of a cholesterol-trafficking protein, NPC1, in two forms of muscular dystrophy. Interventions that alter cholesterol trafficking may represent a new therapeutic target for diseases of muscle degeneration.


25) In vitro activities and in vivo pharmacokinetics of dual cysteine proteases inhibitors and antioxidant

B. Pignol, S. Auvin, D. Carre and P. Chabrier - France
 

Cell death observed in neuromuscular disorders such as Duchenne muscular dystrophy (DMD) was often associated with calpain activation and overproduction of reactive oxygen species. The aim of the study was (1) to compare on human skeletal muscle cell protection by BN82270, which inhibits calpain1-2/cathepsinB-L and lipid peroxidation, with its analog and methylprednisolone (MP), used in treatment of DMD patients. (2) To compare the distribution of this analog in muscles with the BN82270 which reduces the dystrophic progression in mdx mice (10th WMS TP3.05). In vitro, we observed that maitotoxin (MTX) which induced a massive influx of calcium, increased in a concentration-dependent manner calpain activity, lipid peroxidation and induced cell death. In this model, calpain/cathepsin L inhibitors and antioxidants act synergistically to inhibit maitotoxin-induced necrosis (J Neurochem. 2006). A new synthetic analog chemically designed to possess both calpain/cathepsin inhibitory and antioxidant activities totally protect human skeletal muscle myoblast cells differentiated into myotubes. Moreover, this water soluble compound was significantly more potent than BN82270 and MP (2 and 20 times, respectively) to protect HSM cells against death induced by MTX. After administration of synthetic products in vivo, the Area under the curve (AUC 0–18 h) was increased 4 times in muscles with this new analog (1630 μg/g min) compared to the BN82270 (436.05 μg/g min, p = 0.0002, ***). These results indicate that analog of BN82270 enhances the beneficial effect already demonstrated with BN82270 to prevent cell death in vitro. The higher quantity of this analog in muscles quantified by AUC 0–18 h suggests that inhibition of dystrophic progression in vivo already demonstrated with BN82270 (Neuromuscul Disord. 2006) could be enhanced.

 

M. Grounds and H. Radley - Australia
 

Background: Dystrophic myofibres of Duchenne muscular dystrophy (DMD) boys are susceptible to sarcolemma damage. Little is known about the balance between myofibre repair and the alternative fate of necrosis. Using the mdx mouse model of DMD we have shown reduced necrosis of dystrophic muscles in vivo using highly specific drugs to silence the pro-inflammatory cytokine tumour necrosis factor alpha (TNF); specifically using antibodies to block TNF (human Remicade and mouse-specific cV1q) or soluble TNF receptors (Enbrel). Both Remicade and Enbrel are in wide clinical use to treat inflammatory diseases such as rheumatoid arthritis and Crohn’s disease, thus such drugs are immediately attractive for potential application to DMD. Aim: To test long-term benefits of cV1q treatment combined with voluntary exercise in mdx mice. Hypotheses: We propose that inflammatory cytokines, specifically TNF, increase initial sarcolemmal damage and exacerbate necrosis of dystrophic myofibres. Methods: mdx mice were injected weekly with cV1q antibody from 19 days of age, exposed to voluntary exercise on a running wheel (measured) and sampled at 90 days (3 months) for detailed histological muscle analysis and serum creatine kinase (CK) measurements. Results: Benefits of cV1q treatment were only demonstrated in exercised (not un-exercised) mdx mice. The cV1q treated mice ran more (indicating improved muscle function) and had much lower CK levels and reduced dystropathology. Conclusions: These long-term studies with cV1q in mdx mice, (i) emphasise the importance of exercise for drug testing in this mouse model and (ii) confirm the benefits of anti- TNF-alpha drugs on dystrophic muscle. These data support an important role for inflammation in exacerbation of muscular dystrophy and suggest new drug interventions to reduce the clinical severity of DMD and related neuromuscular disorders. Understanding the molecular basis of the adverse effects of TNF-alpha is a central focus of our research.

27) Imatinib mesilate (Gleevec^®) ameliorates the dystrophic phenotype in exercised mdx mice

J. Bizario, D. Cerri, F. Matioli, P. Morales, L. Couto, F. Castro and M. Costa - Bazil


Duchenne muscular dystrophy (DMD) is a neuromuscular disorder caused by mutations in the dystrophin gene. It is characterized by progressive skeletal muscle degeneration that leads to weakness and early death by respiratory and cardiac breakdown. There is no specific treatment to DMD. Preclinical tests to find new drugs that can stop or retard DMD progression are usually performed in exercised mdx mouse. One important feature in DMD is the massive muscle infiltration by immune cells and the replacement by fibrous or fatty tissue. Immunomodulators have recently emerged for DMD trials. Imatinib mesilate is a specific inhibitor of tyrosine kinases, such as Bcr-Abl, PDGFR-β and c-Kit receptors. It also inhibits the profibrogenic activity of TGF-β. The present study aimed to evaluate imatinib mesilate in mdx mice submitted to treadmill exercise. Four-week old mice were analyzed in the beginning and in the end of a physical activity program during six weeks considering histopathological evaluation of gastrocnemius and diaphragm muscles, serum creatine kinase dosage and whole body strength increment. Comparative analyses showed that 0.125 mg/mouse/day resulted in amelioration of the muscular conditions, increased force increment (p < 0.0001, unpaired t-test, n = 15 and 21 for the untreated and treated group, respectively) and decreased CK levels (p = 0.0022, n = 7). Histological analyses of the gastrocnemius showed abrupt decreasing of the area occupied by injured myofibers infiltrated by Blue Evans dye (p = 0.0009, n = 3), while diaphragm showed no significant difference. Taken together, these data suggest that Gleevec^® can ameliorate the dystrophic phenotype in mdx mice, and could be used as potential drug to future clinical tests.

 

N. Whitehead, C. Pham and D. Allen - Australia
 

Duchenne muscular dystrophy (DMD) is a degenerative muscle disease caused by the absence of the protein, dystrophin. Recently, we have shown that increased Ca²⁺ entry through stretch-activated channels (SACs) contributes to muscle damage in mdx mice both in isolated muscles subjected to eccentric (stretched) contractions and in vivo. For many years it has been postulated that reactive oxygen species (ROS) contribute to damage in dystrophic muscle. Since elevated intracellular Ca²⁺ is known to accelerate ROS production, this could be one pathway by which Ca²⁺ entry through SACs leads to muscle damage. Therefore, in this study we investigated whether the antioxidant N-acetylcysteine (NAC) could reduce stretch-induced muscle damage in mdx muscle. Extensor digitorum longus muscles from mdx and wild type mice were perfused with or without 20 mM NAC. Solutions also contained 0.02% Evans Blue Dye (EBD) for assessment of membrane permeability. Muscles underwent 3 eccentric (stretched) contractions at 35 °C. Tetanic force was measured before and 60 min after eccentric contractions and then muscles were frozen and sectioned for EBD uptake. Following the eccentric contractions, force fell to 35 ± 3% for mdx muscles and NAC significantly improved force to 51 ± 2% (P < 0.01). As expected, force was much greater for wild-type muscles (69 ± 5%) and NAC had no additional effect. The area of EBD uptake was 8.6 ± 1.8% in mdx muscle cross-sections and this was significantly reduced by NAC to 2.6 ± 0.8% (P < 0.01). Wild-type muscles had a value of 1.8 ± 0.7%. The results of this study show that the antioxidant, NAC, significantly reduces stretch-induced muscle damage in isolated muscles from mdx mice. We now aim to investigate the source(s) of ROS production in mdx muscles and to determine the key proteins targeted by ROS, which impair muscle function and contribute to muscle damage.


29) Treatment with the proteasomal inhibitor Velcade rescues the dystrophin complex in experimental and pathological models of muscular dystrophies

E. Gazzerro, S. Assereto,  F. Sotgia, F. Zara, R. Biancheri, C. Bruno, M. Lisanti and C. Minetti - Italy and United States
 

Background: Activation of the ubiquitin/proteasome proteolytic systems plays a key role in the muscular dystrophic process. We demonstrated that local and systemic treatment of mdx mice with MG-132, a well-characterized proteasomal inhibitor, rescues the expression of the dystrophin–glycoprotein complex (DGC), and improves the histopathological signs of muscular dystrophy. These results were confirmed in skeletal muscle biopsies from patients affected by Duchenne and Becker muscular dystrophies. Objectives: Our aim was to test the efficiency of Velcade, a selective proteasomal inhibitor FDA-approved for treatment of multiple myeloma, and whose side effects have been explored and managed. Methods: Velcade at 5 and 10 μM was injected into gastrocnemius muscles of mdx mice. After 24 h, skeletal muscle tissues from treated and untreated hindlimbs, these last injected with PBS only, were collected. In addition, Velcade at 0.1–50 μM was administered on explants from freshly-isolated skeletal muscle biopsies of dystrophin deficient patients. Dystrophin, alpha-, beta-dystroglycan and alpha-sarcoglycan expression were examined by immunofluorescence and western immunoblot. Results: As expected, all the proteins of the DGC complex were reduced in skeletal muscle fibers from untreated mdx mice. Remarkably, Velcade either at 5 or 10 μM, rescued the expression level and subcellular localization of alpha-, beta-dystroglycan, alpha-sarcoglycan and dystrophin. Moreover, a decrease of the activated form of NF-kbeta was observed in the Velcade-treated mice when compared to untreated controls. This transcription factor is involved in the inflammatory reaction of DMD. In accordance, Velcade up-regulated the expression levels of dystrophin, alpha-sarcoglycan and beta-dystroglycan in muscle explants from the dystrophin deficient patients examined. Conclusions: Administration of the proteasomal inhibitor Velcade in the skeletal muscles from mdx mice, rescues the expression and plasma membrane localization of the DGC complex, and reduces the activation of the pro-inflammatory molecule NF-kbeta. These results are confirmed in muscle explants obtained from dystrophin deficient patients.

 

S. Messina, A. Mazzeo, A. Bitto, M. Aguennouz, A. Migliorato, M. Monici, M. De Pasquale, F. Squadrito and G. Vita - Italy
 

Soy isoflavones have been reported to have antioxidant bioactivities, scavenging free radicals and increasing antioxidant protein expression, and also to inhibit the transcription factor NF-κB. We showed in previous studies that the inhibition of the transcription factor NF-κB through drugs with also antioxidant properties, have beneficial effects in mdx mice. The drugs used are not available for clinical studies. We tested whether genistein and flavocoxid, supplements with known antioxidant and antinflammatory properties readily available for clinical use, could have a beneficial effect on muscle function, morphology and biochemical pattern in mdx mice. Five-week old mdx mice received for five weeks either genistein (2 mg/kg i.p. daily), flavocoxid (5 mg/kg i.p. daily or vehicle; flavonoids treatment 1)‘increased forelimb strength (p < 0.05) and strength normalized to weight (p < 0.05) and decreased fatigue (p < 0.05; 2) reduced serum creatine-kinase levels (p < 0.01; 3) increased GPX activity and reduced markers of oxidative stress (p < 0.05; 4) blunted NF-κB DNA-binding activity (p < 0.05; 5) reduces muscle necrosis (p < 0.01) and enhances regeneration (p < 0.05) with an augmented number of myogenin-positive satellite cells and myonuclei, and of developmental myosin heavy chain-positive fibers. Our results suggest that these flavonoids might have a beneficial effect on muscle function and morphology in mdx mice. Further studies are needed to investigate the biochemical substrates of such encouraging preliminary results taking into account that these supplements could be easily introduced in the daily diet of patients with DMD
 

A. Amenta, B. McKechnie, M. Abedi and J. Fallon  - United States
 

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


34) T- and B-lymphocytes depletion has a great effect on the fibrosis of the dystrophic skeletal muscles in the scid/mdx mouse

A. Farini, M. Meregalli, M. Belicchi, M. Battistelli, D. Parolini, G. D’Antona, M. Gavina, R. Bottinelli and Y. Torrente - Italy

The abnormal connective tissue proliferation following the muscle degeneration is a major pathologic feature of Duchenne muscular dystrophy (DMD), a genetic myopathy due to a lack of the sarcolemmal dystrophin protein. Since this fibrotic proliferation is likely to be a major obstacle to the efficacy of future therapies, the interventions to understand and prevent it will be necessary for an effective treatment. The murine animal model of DMD (mdx) reproduces the histopatological alterations of the muscles of patients with DMD. To investigate the role of T- and B-lymphocytes in the development of fibrosis, we created a new animal model, the scid/mdx mouse, by breeding the mdx mouse with the scid immunodepressed mouse. We assessed histological analysis for fibrosis and used ELISA analysis to determine TGF-beta1 expression. In scid/mdx mice, we observed several dystrophic features as centrally located nuclei, necrosis, muscle degeneration, similar to the mdx animals. Moreover, the scid/mdx mice show similar muscle force compared to the mdx mice. The muscle fibrosis reduction in T- and B-lymphocytes-depleted scid/mdx mice is correlated to low expression of TGF-beta1. These data demonstrate a correlation between the absence of B- and T-lymphocytes and the loss of fibrosis accompanied by the reduction of TGF-beta1 suggesting the importance of the immunomodulation of the immune system in the Duchenne muscular dystrophy.