BRAZILIAN RESEARCH CENTER - IMPORTANT INFORMATIONS
TAMOXIFEN INCREASES MUSCULAR STRENGTH OF THE MDX DYSTROPHIC MICE NEW
DECEMBER/2004
30/12/04: PTC Therapeutics: FAQ
30/12/04: (In Press: Biochimica et Biophysica Acta,2004) Up-regulation of mitogen activated protein kinases in mdx skeletal muscle following chronic treadmill exercise
Akinori Nakamura, Kunihiro Yoshida, Hideho Ueda, Shin’ichi Takeda, Shu-ichi Ikeda - Japan
Abstract
Dystrophin, a product of the Duchenne muscular dystrophy gene, is a cytoskeletal protein of skeletal and cardiac muscle fibers. Dystrophin-deficient muscle fibers are abnormally vulnerable to mechanical stress including physical exercise, which is a powerful stimulator of mitogen-activated protein kinases (MAPKs). To examine how treadmill exercise affects MAPK family members in dystrophin-deficient skeletal muscle, we subjected both mdx mice, an animal model for Duchenne muscular dystrophy, and C57BL/10 mice to treadmill exercise and examined the phosphorylated protein levels of extracellular-signal regulated kinase (ERK1/2), p38 MAPK and c-Jun N terminal kinase 1 and 2 (JNK1 and JNK2) in the gastrocnemius muscle. Phosphorylation of ERK1/2, p38 MAPK and JNK2, but not JNK1, increased more in the muscles of exercise trained mdx mice than in muscles of trained C57BL/10 or untrained mdx mice. These results show that physical exercise aberrantly up-regulates the phosphorylated form of ERK1/2, p38 MAPK and JNK2 in dystrophin-deficient skeletal muscle and that their up-regulation might play a role in the degeneration and regeneration process of dystrophic features.
22/12/04: Phase I study of dystrophin plasmid-based gene therapy in duchenne/becker muscular dystrophy
22/12/04: (In Press: J. Pediatr. 2005;25:95-7):Should Foot Surgery Be Performed for Children With Duchenne Muscular Dystrophy?
Khristinn Kellie Leitch,, Naweed Raza, Doug Biggar, Derek Stephen, James G. Wright and Benjamin Alman - Canada
Abstract: The authors conducted a retrospective study to determine the outcome of foot surgery in full-time wheelchair users with Duchenne muscular dystrophy. Medical records on all 88 teenaged boys with Duchenne muscular dystrophy treated at the authors’ institution were obtained and reviewed. Patients completed questions about shoe wear, pain, hypersensitivity, and cosmesis, and a foot examination was performed. There were no significant differences between patients who did and did not receive foot surgery with respect to shoe wear (p> . 0.05), pain (p> . 0.05), hypersensitivity (p> . 0.05), or cosmesis (p> . 0.05). Hindfoot motion was significantly better (p> . 0.05) but equinus contracture was significantly worse (p> . 0.05) in patients who had not had surgery.
18/12/04: Systemic delivery of antisense oligoribonucleotide restores dystrophin expression in body-wide skeletal muscles
Qi Long Lu,
17/12/04: (In Press: Brain & Development, 2004):Coagulation system activated in Duchenne muscular dystrophy patients with cardiac dysfunction
Toshio Saito, Yuko Yamamoto, Tsuyoshi Matsumura, Sonoko Nozaki,Harutoshi Fujimura, Susumu Shinno - Japan
Abstract
We investigated basic abnormalities of coagulation and fibrinolysis in Duchenne muscular dystrophy (DMD) patients with cardiac dysfunction. Forty seven patients with DMD, aged 13–37 years old, were enrolled. Based on left ventricular ejection fraction (LVEF) results determined by echocardiography, patients were divided into 3 groups: LVEF less than 30% (markedly depressed group), LVEF between 30 and 50% (slightly depressed), and LVEF greater than 50% (normal). We measured serum levels of total fibrin and fibrinogen degradation products (FDP), as well as plasma fibrinogen, thrombin–antithrombin complex (TAT), prothrombin fragment (F1C2), and D-dimer. The levels of TAT and F1C2 in the markedly depressed group were significantly elevated compared with the other groups, whereas FDP,
fibrinogen, and D-dimer levels did not differ among the groups. We concluded that activated coagulation is associated with cardiac dysfunction in patients with DMD.17/12/04: MUSCULAR DYSTROPHY GENE THERAPY TRIAL PASSES FIRST TEST BY NIH COMMITTEE
17/12/04: Duchenne Muscular Dystrophy: Stalled at the junction?
09/12/04:(In Press: Brain & Development, 2004): A novel approach to identify Duchenne muscular dystrophy patients for aminoglycoside antibiotics therapy
Shigemi Kimura, Kaori Ito, Toshihiko Miyagi, Takashi Hiranuma, Kowasi Yoshioka, Shirou Ozasa, Makoto Matsukura, Makoto Ikezawa, Masafumi Matsuo,Yasuhiro Takeshima, Teruhisa Miike - Japan
Abstract
Aminoglycoside antibiotics have been found to suppress nonsense mutations located in the defective dystophin gene in mdx mice, suggesting a possible treatment for Duchenne muscular dystrophy (DMD). However, it is very difficult to find patients that are applicable for this therapy, because: (1) only 5-13% of DMD patients have nonsense mutations in the dystrophin gene, (2) it is challenging to find nonsense mutations in the gene because dystrophin cDNA is very long (14 kb), and (3) the efficiency of aminoglycoside-induced read-through is dependent on the kind of nonsense mutation. In order to develop a system for identifying candidates that qualify for aminoglycoside therapy, fibroblasts from nine DMD patients with nonsense mutation of dystrophin gene were isolated, induced to differentiate to myogenic lineage by AdMyoD, and exposed with gentamicin. The dystrophin expression in gentamicin-exposed myotubes was monitored by in vitro dystrophin staining and western blotting analysis. The results showed that gentamicin was able to induce dystrophin expression in the differentiated myotubes by the read-through of the nonsense mutation TGA in the gene; a read-through of the nonsense mutations TAA and TAG did not occur and consequently did not lead to dystrophin expression. Therefore, it is speculated that the aminoglycoside treatment is far more effective for DMD patients that have nonsense mutation TGA than for patients that have nonsense mutation TAA and TAG. In this study, we introduce an easy system to identify patients for this therapy and report for the first time, that dystrophin expression was detected in myotubes of DMD patients using gentamicin.
04/12/04: HSCs for muscular dystrophy: a paradigm shift, back
04/12/04: (In Press: Neuromuscular Disorders, 2004): Report of a Muscular Dystrophy Campaign funded workshop Birmingham, UK, January 16th 2004. Osteoporosis in Duchenne muscular dystrophy; its prevalence, treatment and prevention
R. Quinlivan, H. Roper, M. Davie, N.J. Shaw, J. McDonagh, K. Bushby - UK
11. Conclusions and recommendations
11.1. DMD and osteoporosis
Duchenne muscular dystrophy leads to reduced mobility, which is associated per se with an increased chance of fractures and reduced bone mineral density. Bone mineral density, especially in the spine is further reduced by longterm steroid treatment. The development of vertebral fractures is dependent upon the cumulative steroid dose and the risk may be very high for those children receiving long-term steroids. The investigation of bone mineral density in children can be problematic and care is needed in the interpretation of data, however, individual children can act as their own controls. DXA scanning can therefore be used to monitor bone mineral density in an individual patient, but there is no clear relationship between a particular Z score and risk of fracture, and DXA results alone should not be used to change practice or lead to specific treatment. When developing randomised controlled treatment trials, different centres may not have compatible DXA machines or software but this can be often overcome using statistical methods for correction.
11.2. Prevention of complications
Fig. 1
is a schematic outline of the proposed management plan agreed by the group. Preserving muscle function will protect long bones, prolonging walking and standing is an important element of management. Sunshine and nutrition are extremely important in maintaining healthy bones and care must be taken to ensure optimal exposure. There is a lack of published evidence for the prevention of vertebral fractures using calcium and vitamin D supplements, there is no evidence based indication to prescribe supplements alongside steroids unless there is a demonstrated deficiency or unless dietary manipulation is not possible.When commencing steroids in children with DMD, it is prudent to check the 25OH vitamin D levels before treatment. All children should be referred to a dietician in order to optimise the dietary calcium and vitamin D intake. If the 25OH vitamin D levels are low, the clinician may wish to consider treatment with calcium and vitamin D supplements. A baseline DXA scan to assess bone mineral density is probably advisable with follow up scans every 1–2 years, however, at the moment there is no consensus on how to manage worsening bone mineral density in an individual child in the absence of a vertebral fracture. More research is required to establish the long-term effects of bisphosphonates in children, the most effective dose and frequency of treatment, especially oral treatment, before they can be recommended for prophylactic use.
11.3. Treatment of complications
Any patient receiving steroids who complain of back pain should have X-rays (Anterior, Posterior, and Lateral) of the spine taken and if a vertebral fracture is evident urgent referral to a metabolic bone specialist for intravenous bisphosphonate treatment is indicated. Under these circumstances it is not necessary to stop steroid treatment. In the presence of a long bone fracture, a common complication in DMD, no pharmacological treatment is required unless a silent vertebral fracture is discovered on a lateral spine X-ray.
01/12/04: Duchenne muscular dystrophy with associated growth hormone deficiency
01/12/04: Erythrocyte from Duchenne muscular dystrophy is fragile
NOVEMBER/2004
23/11/04: (Clinicial Nutrition, 2004; 23:1176-1183) Deleteriuos effects of immobilization upon rat skeletal muscle: role of creatine supplementation
M.S. Aoki, W.P. Lima, E.H. Miyabara, C.H.A. Gouveia, A.S. Moriscot - Brazil
Summary Aim: The aim of the study was to investigate the impact of creatine feeding (5 g kg1 body weight day1) upon the deleterious adaptations in skeletal muscle induced by immobilization. Methods: Male Wistar rats were submitted to hind limb immobilization together with three dietary manipulations: control, supplemented with creatine for 7 days (along with immobilization) and supplemented with creatine for 14 days (7 days before immobilization and together with immobilization). Muscle weight (wet/dry) was determined in the soleus (SOL) and gastrocnemius (GAS). The analysis of lean mass was performed by DEXA and myosin heavy chain (MHC) distribution by SDSPAGE. Results: After 14 days of creatine loading, immobilized SOL and GAS total creatine content were increased by 25% and 18%, respectively. Regardless of dietary manipulation, the immobilization protocol induced a decrease in the weight of SOL and GAS (Po0.001). However, creatine feeding for 14 days minimized mass loss in the SOL and GAS (Po0.05). Our findings also indicate that creatine supplementation maximizes the expected slow-to-fast MHC shift driven by immobilization (Po0.05). Conclusions: Previous creatine supplementation attenuates muscle wasting induced by immobilization. This effect is associated with the increment of intramuscular creatine content.
22/11/04: (Early view: Experimental Cell Research, 2005;302(2):170-179) CAPON expression in skeletal muscle is regulated by position, repair, NOS activity, and dystrophy
Laurent Ségalat, Karine Grisoni, Jonathan Archer, Cinthya Vargas, Anne Bertrand and Judy E. Anderson - France and Canada
In skeletal muscle, the localization of nNOS is destabilized in the absence of dystrophin, which impacts muscle function and satellite cell activation. In neurons, the adaptor protein, carboxy-terminal PDZ ligand of nNOS (CAPON), regulates the distribution of neuronal nitric oxide synthase (nNOS), which produces the key signaling molecule nitric oxide (NO). While a CAPON-like gene is known to compensate functionally for a dystrophic phenotype in muscle of Caenorhabditis elegans, CAPON expression has not been reported for mammalian muscle. Here, CAPON expression was identified in mouse muscle using Northern and Western blotting and in situ hybridization in combination with immunostaining for laminin. CAPON RNA was expressed in developing normal and dystrophic muscles near fiber junctions with tendons, and levels increased from 1 to 3 weeks. In regenerating normal muscle and also in dystrophic muscles in the mdx mouse, CAPON transcripts were prominent in satellite cells and new myotubes. Expression of CAPON RNA increased in diaphragm muscle of normal and mdx mice after treatment with L-arginine, the NOS substrate. Both CAPON and utrophin protein levels increased in dystrophic quadriceps muscle after treatment with the steroid deflazacort plus L-arginine, known to reduce the dystrophic phenotype. The identification of CAPON transcripts and protein in mammalian muscle and responses to L-arginine suggest CAPON may have a functional role in stabilizing neuronal NOS in skeletal muscle in the cytoskeletal complex associated with dystrophin/utrophin, with possible applications to therapy for human muscular dystrophy.
20/11/04: Abstract presented on Annual American Society of Anesthesiologists, october
Mivacurium in Children with Duchenne’s Muscular Dystrophy: A Comparison of Onset and Duration of Neuromuscular Block with Children without Neuromuscular Diseases
Joachim Schmidt, Tino Munster, Stefanie Wick, Hubert J. Schmitt - Germany
Introduction: Duchenne’s muscular dystrophy (DMD), an x-linked recessive disorder, is the most common myopathy seen in the pediatric population. Children afflicted with this disease may require anesthesia for muscle biopsy or orthopedic surgery. Of primary concern in children with this disorder is the choice of the neuromuscular blocking agent. Pharmacodynamic data of non-depolarising neuromuscular blocking agents are scarce 1, 2. Succinylcholine is not recommended due to its adverse side effects. Investigation in DMD children with advanced disease state showed prolonged recovery after a standard dosage of rocuronium 2. The aim of this study was to compare the response to 0,2 mg/kg mivacurium in children with DMD and children without neuromuscular diseases (control-group).
Discussion: The results of the present study demonstrate that the recovery of neuromuscular block following a standard dosage of 0,2 mg/kg mivacurium is significantly prolonged in children with DMD compared to controls. Our results confirm a retrospective study where an increased sensitivity to mivacurium in children with DMD was suggested 4. We therefore recommend the monitoring of the neuromuscular function if mivacurium is used in children with DMD.
20/11/04: ‘MOLECULAR PATCHES’ OR EXON SKIPPING AS A THERAPY FOR DUCHENNE MUSCULAR DYSTROPHY
18/11/04: (In Press: Molecular Therapy, 2004) Adeno-Associated Virus-Mediated Microdystrophin Expression Protects Young mdx Muscle from Contraction-Induced Injury
Mingju Liu, Yongping Yue, Scott Q. Harper, Robert W. Grange, Jeffrey S. Chamberlain, and Dongsheng Duan - USA
Duchenne muscular dystrophy (DMD) is the most common inherited lethal muscle degenerative disease. Currently there is no cure. Highly abbreviated microdystrophin cDNAs were developed recently for adeno-associated virus (AAV)-mediated DMD gene therapy. Among these, a C-terminaltruncated DR4-R23/DC microgene (DR4/DC) has been considered as a very promising therapeutic candidate gene. In this study, we packaged a CMV.DR4/DC cassette in AAV-5 and evaluated the transduction and muscle contractile profiles in the extensor digitorum longus muscles of young (7- week-old) and adult (9-month-old) mdx mice. At ~3 months post-gene transfer, 50–60% of the total myofibers were transduced in young mdx muscle and the percentage of centrally nucleated myofibers was reduced from ~70% in untreated mdx muscle to ~22% in microdystrophin-treated muscle. Importantly, this level of transduction protected mdx muscle from eccentric contractioninduced damage. In contrast, adult mdx muscle was more resistant to AAV-5 transduction, as only ~30% of the myofibers were transduced at 3 months postinfection. This transduction yielded marginal protection against eccentric contraction-induced injury. The extent of central nucleation was also more difficult to reverse in adult mdx muscle (from ~83% in untreated to ~58% in treated). Finally, we determined that the DR4/DC microdystrophin did not significantly alter the expression pattern of the endogenous full-length dystrophin in normal muscle. Neither did it have any adverse effects on normal muscle morphology or contractility. Taken together, our results suggest that AAVmediated DR4/DC microdystrophin expression represents a promising approach to rescue muscular dystrophy in young mdx skeletal muscle.
14/11/04: Abstracts presented on 14th Meeting of the European Neurological Society - Spain, june/2004:
S. Messina, P. Seminara, M. Aguennouz, M.C. Monici, H. Marini, F. Squadrito, G. Vita (Messina, I)
Previous studies provided evidences that generation of reactive oxygen species and activation of transcription factor NF-kB may play important roles in the pathogenesis of Duchenne muscular dystrophy. We tested whether IRFI 042, a vitamin E-like antioxidant, and PDTC, a NF-kB inhibitor, could have an effect on muscle weakness in mdx mice. We treated 48 5/6-week old mdx and wild type mice with intraperitoneal injections of PDTC (50 mg/kg), IRFI 042 (20 mg/kg), or vehicle, three times a week for five weeks. Data regarding weight, survival and forelimb strength and fatigue were collected. Motor performance measurements were carried out using a grip meter attached to a force transducer which measures peak force generated. Mdx mice treated with IRFI 042 or PDTC showed at the end of treatment a significantly higher forelimb strength than vehicle controls (IRFI 042: +53.6%, p<0.001; PDTC: +53.1%, p<0.05) as well as higher strength normalised to weight (IRFI 042: +57.8%, p<0.001; PDTC: +54%, p<0.05). Furthermore PDTC-treated mdx mice had significantly less fatigue than vehicle animals (-120%, p<0.004).
Our results suggest that PDTC and IRFI 042 might have a beneficial effect on weakness and fatigue in mdx mice. Further studies are needed to investigate the morphological and biochemical substrates of such encouraging preliminary results.
F. Chretien, P.A. Dreyfus, P. Caramelle, B. Chazaud, R.K. Gherardi (Créteil, F)
We have previously developed a murine model of bone marrow (BM)
transplantation from B6-TgGFP transgenic mice to normal irradiated B6 mice,
the cytoplasmic green fluorescent protein (GFP) being used as an unambiguous
marker of donor-derived cells in host muscle. Using this model we were able
to demonstrate that stem cell marker-expressing cells found in connective
tissue and myogenic precursor cells (satellite cells) may be derived from
bone-marrow in adulthood. To investigate the therapeutic potential of BM
transplantation in muscle diseases such as dystrophinopathies, we compared
the results obtained in C57Bl6 (B6) mice and mdx mice, a model for Duchenne
muscular dystrophy in which muscle regeneration is dramatically increased.
In mdx muscle compared to B6 muscle, we observed numerous GFP+ mononucleated
cells corresponding to macrophages and numerous necrotic fibers filled by
GFP+ macrophages and myoblasts. The number of GFP+ satellite cells number
was similar in mdx and B6 mice 6 months post transplantation. However, GFP+
muscle fibers were more numerous in mdx mice. They were from month 1 post
transplantation, a time point showing no GFP+ fiber in B6 mice. Six months
post transplantation, 4 fold more GFP+ muscle fibers were found in mdx mice
compared to B6 mice.
In contrast, dystrophin expression was only mildly increased (0,65% at month
6) and colocalizations between GFP and dystrophin was rarely found on serial
cross sections. We conclude that the nuclear domain of dystrophin is
probably much shorter than that of GFP.
F. Civati, M. Guglieri, M.G. D' Angelo, A. Tavano, F. Fabbro, M.L. Lorusso, M. Sironi, A.C. Turconi, G.P. Comi, N. Bresolin (Bosisio Parini, San Vito, Milan, I)
Background: Duchenne Muscular Dystrophy (DMD) is a fatal recessive
x-linked muscular disease caused by the absence of dystrophin.
Dystrophin isoforms are also expressed in the cerebral neocortex and in
the cerebellum. Cognitive impairment is described in 1/3 of DMD patients,
particularly in patients carrying deletions in the distal part of the
gene. Difficulties in verbal skills and reading abilities are more
frequently described in English speaking patients. According to the
literature children with DMD show linguistic deficits already in the
early phases of language development which mainly consist of poor
expressive verbal abilities and deficits in short-term memory. These
observations suggest that in some cases dystrophinopathy may be
associated to language disorders. Preliminary results in our group of
Italian speaking children showed deficit in Grammatical and Syntax
Comprehension.
Aims of the study are:
1) to describe the language and reading ability in a group of italian
DMD children
2) to clarify the nature of the language and reading deficits
Patients: 13 children with DMD (mean age 8,3 years; standard deviation
1,7) were diagnosed according to international standard criteria. Full
Intelligence Quotient(assessed using Wechsler Intelligence Scale) was
>70.
Methods: Language and Reading abilities were determined through: “Test
dello sviluppo Morfosintattico”, Battery of standardized tests, Battery
to evaluate Dyslexia and Dysortography and Tasks of correctness and
rapidity. To exclude additional cognitive deficits we evaluated
attention/executive functions domain and memory and learning domain
thought a Developmental Neuropsychological Assessment (NEPSY
particularly: Auditory attention and response set - Visual attention -
List learning - Memory for names).
Results: 8 patients out of 13 showed deficit in Syntax Comprehension. 7
patients out of 13 manifested deficits in Grammatical Comprehension. No
DMD patients (except one) presented disabilities in reading. Most of the
patients showed mild attention and memory deficits.
Discussion: An early identification of the language and eventually
attention/memory difficulties, would be important for an early
treatment, to support DMD children in their learning course.
13/11/04: Naked plasmid DNA for the treatment of muscular dystrophy
13/11/04: Compositional analysis of muscle in boys with Duchenne muscular dystrophy using MR imaging
12/11/04: Duchenne Muscular Dystrophy research focuses on early mechanisms
12/11/04: Key clues to muscle regeneration
12/11/04: RNA Treatment Used to Lower Cholesterol in Mice - RNA Interference can help in DMD
12/11/04: Current concepts in neuromuscular scoliosis
Lee S. Segal - USA
(Current Opinion in Orthopaedics Current Opinion in Orthopaedics 2004, 15:439–446)
Duchenne muscular dystrophy
Sussman provided an excellent overview of the treatment principles for patients with Duchenne muscular dystrophy (DMD) and spinal deformity. The onset of spinal deformity is usually noted between the ages of 11 and 13 years, which corresponds to the time most patients stop walking and become full-time sitters. All children should be screened because scoliosis develops in most patients with this condition. When the scoliosis magnitude reaches 20 to 30°, spine fusion should be done without delay. Nonoperative treatment for scoliosis, such as bracing, is not recommended for patients with DMD. The spine deformity is not responsive to nonsurgical modalities or preventable by them. Posterior spine fusion will not result in marked loss of truncal height or crankshaft deformity, because there is sufficient spinal growth in children with DMD by the time they are 10 or 11 years of age. With progression of the disease, the paraspinal muscles are progressively replaced by stiff fibrofatty tissue. This results in the dissection and exposure of the spine being more difficult, decreases flexibility and the ability to achieve correction, and increases intraoperative blood loss. The most notable complication from delaying surgical stabilization of the spinal deformity is postoperative pulmonary problems. There is a greater risk of postoperative pulmonary problems when the forced vital capacity (FVC) is less than 35%. All patients require full cardiac and pulmonary evaluation preoperatively. Posterior arthrodesis and instrumentation should extend proximally to the upper thoracic spine (T2–T4), ensuring that thoracic kyphosis is maintained and the center of mass of the head is forward. If this is not maintained, patients lose head control because they often lose strength in their neck flexor muscles while retaining strength in their neck extensors. The lower level of fusion distal remains controversial. Some favor ending fusion at L5, and others recommend extending it to the pelvis. Extension to the pelvis, however, increases blood loss, surgical time, and the risk of complications, especially with osteopenia of the pelvis. Sussman noted that patients who undergo stabilization of the spine have improved quality of life, better maintenance of pulmonary function, and a longer life span. Sengupta et al. evaluated the distal extent of posterior spine fusion in their retrospective series of 50 patients at two different centers. With a minimum follow-up time of 3 years, 31patients underwent fusion to the pelvis and 19 to L5. In these 19 patients, pedicle screws were used in the lumbar spine and sublaminar wires in the thoracic spine. In a comparison of the two groups (pelvic vs L5), the mean age was 14 versus 11.7 years, the mean Cobb angle was 48° versus 20°, the mean pelvic obliquity (PO) was 19.8° versus 9°, the mean estimated blood loss was 4.1L versus 3.3 L, the mean length of stay was 11.7 versus 7.7 days, and the FVC was 44% versus 58%. The PO was corrected and maintained in all but 2 patients with an initial PO greater than 20°. The authors concluded that lumbar instrumentation to L5 is adequate provided that surgery is performed early, soon after the patient becomes wheelchair bound, and with smaller preoperative curves and minimal PO. This selective approach was complemented when surgery was performed in younger patients and with the improvements made in spinal implants and techniques. Pedicle screw fixation into three or more levels achieved a solid distal foundation, allowing the spine to be upright, balanced, and without rotation. This allowed the PO to be corrected and prevented its progression in these patients with a relatively short life expectancy. This paper adds to the growing literature about and controversy over the distal extent of fusion in DMD patients. Mubarak et al. recommended fusion to L5 when the Cobb angles were less than 40° and the PO was less than 10°. Alman and Kim noted progression of the PO in 84% (32/38) of patients with lumbar fixation only, but none in the group of patients who underwent fusion to the pelvis. They recommended extending the fusion to the pelvis in general, particularly when the apex of the curve is below L1. However, they did recognize that sublaminar wire fixation may have contributed to the failure of lumbar fixation in controlling PO. In a review paper, Sussman noted an increased risk of postoperative pulmonary problems when the FVC was less than 35%. In a retrospective series of 30 patients, Marsh et al. evaluated the risks of surgery in patients with low FVC. Of the 30 patients, 13 had a FVC less than 30% of predicted values. The postoperative lengths of stay were similar, and complication rates were comparable to those in other series. The mean length of ventilator support was comparable. The authors emphasized the importance of a multidisciplinary team approach, and they supported the belief that DMD patients with a FVC less than 30% should not be denied posterior spine fusion for scoliosis for pulmonary factors alone. Iannaccone et al. examined the role of malnutrition in DMD patients undergoing spinal deformity correction. The authors identified nine boys who lost more than 5% of their body weight within 12 months of surgery, and they retrospectively compared this group with eight patients who gained weight after posterior spine fusion and eight patients of comparable age who did not undergo surgery and served as control individuals. The authors noted that weight loss was associated with the loss of self-feeding. This was not associated with loss of biceps strength. Possible factors contributing to this include the decrease in neck range of motion or head control, lack of adequate caregiving, and low cognitive function. One criticism of the paper was that the authors did not evaluate the preservation of thoracic kyphosis. Alman et al. analyzed the effect of steroid treatment and the development of scoliosis in DMD. The mutation in the dystrophin gene in DMD decreases production of the gene protein important in maintaining the integrity of the cell membrane, and steroids have been shown to maintain the integrity of the cell membrane and decrease the inflammation associated with myocyte cell death. In this nonrandomized prospective cohort study, the authors compared 30 boys (aged 7–10, and still able to walk) treated with deflazacort (a derivative of prednisone with decreased prevalence of side effects) with 24 boys who were not treated (control group). Muscle strength and pulmonary function were evaluated, and the two groups were matched for age and baseline pulmonary function. The patients were monitored for a minimum of 5 years, and Kaplan-Meier analysis was used to determine the risk of the development of scoliosis. Surgery was performed in 15 of 24 patients in the control group and only 5 of 30 in the treated group. This difference between the two groups was statistically significant (P < 0.001). The authors thought that steroid treatment slows the progression of scoliosis, but long-term evaluation is needed to determine whether treatment prevents the development of scoliosis or merely delays its onset. This study supports the hypothesis that the use of corticosteroids improves the natural history in DMD and that the benefits may be even more obvious when treatment is begun earlier in life.
07/11/04: (Early View:Pediatric Research, 2004) Evolving Therapeutic Strategies for Duchenne Muscular Dystrophy: Targeting Downstream Events
JAMES G. TIDBALL and MICHELLE WEHLING-HENRICKS - USA
E-mail: jtidball@physci.ucla.edu.
Duchenne muscular dystrophy (DMD) is a progressive, lethal, muscle wasting disease that affects 1 of 3500 boys born worldwide. The disease results from mutation of the dystrophin gene that encodes a cytoskeletal protein associated with the muscle cell membrane. Although gene therapy will likely provide the cure for DMD, it remains on the distant horizon, emphasizing the need for more rapid development of palliative treatments that build on improved understanding of the complex pathology of dystrophin deficiency. In this review, we have focused on therapeutic strategies that target downstream events in the pathologic progression of DMD. Much of this work has been developed initially using the dystrophin-deficient mdx mouse to explore basic features of the pathophysiology of dystrophin deficiency and to test potential therapeutic interventions to slow, reverse, or compensate for functional losses that occur in muscular dystrophy. In some cases, the initial findings in the mdx model have led to clinical treatments for DMD boys that have produced improvements in muscle function and quality of life. Many of these investigations have concerned interventions that can affect protein balance in muscle, by inhibiting specific proteases implicated in the DMD pathology, or by providing anabolic factors or depleting catabolic factors that can contribute to muscle wasting. Other investigations have exploited the use of anti-inflammatory agents that can reduce the contribution of leukocytes to promoting secondary damage to dystrophic muscle. A third general strategy is designed to increase the regenerative capacity of dystrophic muscle and thereby help retain functional muscle mass. Each of these general approaches to slowing the pathology of dystrophin deficiency has yielded encouragement and suggests that targeting downstream events in dystrophinopathy can yield worthwhile, functional improvements in DMD.
07/11/04: Effects of stretch-activated channel blockers on [Ca2+]i and muscle damage in the mdx mouse
05/11/2004: Development of an Innovative Technique of Gene Therapy: Exon Skipping
Rescue of Dystrophic Muscle Through U7 snRNA-Mediated Exon Skipping
05/11/2004: Correction of ATM gene function by aminoglycoside-induced read-through of premature termination codons
OCTOBER/2004
31/10/04: Researchers make muscular dystrophy breakthrough
31/10/04: Abstracts presented on Annual Meeting of The American Society of Human Genetics (Toronto, Canada - October 26-30/2004)
1) Body-wide delivery of a microdystrophin gene via intravascular
administration of rAAV6 vectors for treatment of muscular dystrophy. P.
Gregorevic, M. Blankinship, J. Allen, L. Meuse, S. Abmayr, J. Han, J.
Chamberlain. Senator Paul D. Wellstone Muscular Dystrophy Cooperative
Research Center, Dept. of Neurology, University of Washington, Seattle, WA.
Severe neuromuscular disorders, such as Duchenne muscular dystrophy (DMD),
lead to reduced quality of life and premature mortality. Historically, genetic
interventions for these diseases have been limited by an inability to achieve
widespread gene transfer to the affected tissues. Here we demonstrate a method
that, for the first time, enables transduction of the vast majority of both the
cardiac and skeletal musculature of adult mammals via a single intravenous
administration of pseudotype 6 recombinant adeno-associated virus (rAAV6)
vectors. As a means to enhance gene transfer, we have determined that IV
coadministration of vascular endothelium growth factor (VEGF) with rAAV6 vectors
can significantly increase vector accumulation and transgene expression in
striated muscles. Having established in reporter gene studies that this
technique enables tolerable, high-level transgene expression, we sought to
evaluate the potential of this technique for delivering a therapeutic transgene
to the musculature of the mdx dystrophic mouse, a model of DMD. Using this
technique, we have observed that treated mdx mice exhibit expression of
microdystrophin throughout the striated musculature in a manner that is
sufficient to reduce pathological features of the dystrophic phenotype. Compared
with untreated mice, the muscles of treated animals exhibit reduced
susceptibility to contraction-induced injury, and reduced serum creatine kinase
levels, reflecting a global reduction in muscle degeneration. These data are the
first to demonstrate body-wide amelioration of symptoms associated with
dystrophin deficiency in an adult animal following a genetic intervention. In
subsequent studies we have established that these techniques can be adapted to
express alternate transgenes for the treatment of other muscle diseases. We are
currently undertaking studies to determine whether the existing techniques are
effective in animals more similar in size and immunology to humans, and present
relevant data summarizing our progress
2) Therapeutic antisense-induced exon skipping for Duchenne muscular
dystrophy. A. Aartsma-Rus1, A. Janson1, G.
Platenburg2, M. Bremmer-Bout1, J.T. Den Dunnen1,
J.C.T. Van Deutekom1, G-J.B. Van Ommen1. 1) LUMC,
Leiden, the Netherlands; 2) Prosensa, Leiden, the Netherlands.
The severe Duchenne muscular dystrophy (DMD) is mostly caused by frame
disrupting mutations in the dystrophin gene, which result in non-functional
proteins. Mutations that keep the reading frame intact give rise to internally
deleted, semi-functional dystrophins and result in the milder Becker muscular
dystrophy (BMD). Antisense oligonucleotides (AONs) have the potential to
modulate the pre-mRNA splicing such that a specific exon is skipped. As a result,
the reading frame can be restored, which allows the synthesis of a BMD-like
dystrophin.
We have recently demonstrated the skipping of 20 different human DMD exons
using 2'-O-methyl RNA AONs with a full-length phosphorothioate backbone
(2OMePS), and confirmed the therapeutic applicability of this strategy in
cultures from 10 different DMD patients. We now compare the efficacy and
efficiency of 2OMePS AONs to morpholino, locked nucleic acid (LNA) and peptide
nucleic acid (PNA) AONs. While LNA was most efficient in inducing exon skipping,
we find that it acts in a less sequence-specific manner, probably due to its
extremely high RNA affinity. Awaiting further improvements in oligochemistry, we
therefore consider 2OMePS AONs currently the most favourable compounds.
To facilitate clinical application, multiple in vivo studies in animal
models are ongoing to develop safe and efficient AON-delivery methods. However,
as exon skipping is a sequence-specific therapy, it is desirable to directly
target the human DMD gene. We have therefore set up sequence-specific human exon
skipping in vivo in transgenic mice carrying the an intact copy of the
full-length 2.5 Mb human gene (hDMD). We injected 2OMePS AONs targeting
human exons 44, 46 and 49 into the m. gastrocnemicus of hDMD mice,
and showed skipping of the human (but not the murine) exons, which persisted for
at least 28 days. Based on pre-clinical data obtained by our group and others,
we are currently setting up a clinical trial aiming at local dystrophin
restoration following intramuscular injections of exon 46 and 51 specific AONs.
31/10/04: (In Press: Molecular Therapy, 2004) Optimization of Regional Intraarterial Naked DNA-Mediated Transgene Delivery to Skeletal Muscles in a Large Animal Model
Gawiyou Danialou,Alain S. Comtois,Stefan Matecki, Josephine Nalbantoglu, George Karpati, Renald Gilbert, Pascale Geoffroy, Sandra Gilligan, Jean-Francois Tanguay, and Basil J. Petrof - Canada
Effective gene therapy for muscular dystrophy will likely require intravascular administration. Although plasmid DNA (pDNA) contained within a large volume and rapidly infused into a major artery can achieve gene transfer within downstream muscles, this is associated with substantial muscle edema. Here we hypothesized that excessive edema-related increases in intramuscular pressure (IM pressure) developed during intraarterial pDNA injections could hinder successful gene delivery. Accordingly, we monitored IM pressure during injection of pDNA carrying a LacZ transgene into the femoral artery of rats and pigs. Large variations in IM pressure were found between different muscles. There was a significant inverse relationship between IM pressure and the subsequent level of gene transfer to muscle. Modification of the injection protocol to reduce IM pressure led to greatly increased pDNA-mediated gene expression and reduced muscle damage in pigs. Under the most optimized conditions, average transfection within eight different muscles of the pig hind limb amounted to 22% of all fibers, attaining a maximum of 60% in the gastrocnemius muscle. We conclude that IM pressure monitoring is a simple and useful procedure, which can be applied in both small and large animals to help optimize pDNA-mediated gene transfer to skeletal muscles by the intraarterial route.
28/10/04: First cord blood stem cells transplantation in DMD - more informations
Dr. Cheng Zhang answer my questions in this e-mail:
"Dear Dr. David Feder,
Thank you for your email. Yes, we used a nonrelated cord blood stem cell. Because the HLA match is very well so the immune reaction is not obvious, but we still use some immunossupresor drugs now. We are preparing another boys for transplantation.
Best wishes,
Cheng Zhang"
26/10/04: Stem cell transplantation in DMD - first report in China
This is the answer for Dr. Cheng Zhang about this first procedure in the world:
"Dear Dr. David Feder,
Thank you for your email. Today is 92 days that the DMD boy is transplanted by umbilical cord stem cells. He is twelve years old and he cannot walk for almost three years. The umbilical cord stem cells was transfer into his vein 92 days ago. The muscle biopsy was done after 76 days of the transplantation. We find that the dystrophin staining is weak positive in some muscle cells. We are conforming that the weak positive muscle cells come from the donor. The DMD boy has a little improvement in his locomotive function, for example, he touch his nose a little better than before. Now, the DMD boy is OK, he like watch TV, and sometimes play cards with nurse. We are still waiting and observing what happen on him.
Sincerely yours,
Cheng Zhang, MD, PhD - Professor and Chair, Department of Neurology
First Affiliated Hospital - Sun Yat-sen University - Guangzhou, Guangdong, 510080
P. R. China"
14/10/04: (Early View: Blood, 2004) Regional intravascular delivery of AAV-2-F.IX to skeletal muscle achieves longterm correction of hemophilia B in a large animal model
Valder R. Arruda, Hansell H. Stedman, Timothy C. Nichols, Mark E. Haskins, Matthew Nicholson, Roland W. Herzog, Linda B. Couto, Katherine A. High - USA
ABSTRACT
In earlier work, we showed that adeno-associated virus mediated delivery of a Factor IX gene to skeletal muscle by direct intramuscular injection resulted in therapeutic levels of circulating Factor IX in mice. However, achievement of target doses in humans proved impractical because of the large number of injections required. We used a novel intravascular delivery technique to achieve successful transduction of extensive areas of skeletal muscle in a large animal with hemophilia. We provide here the first report of long-term (>3 years, with observation ongoing), robust Factor IX expression (circulating levels of 4-14%) by muscle-directed gene transfer in a large animal, resulting in essentially complete correction of the bleeding disorder in hemophilic dogs. The results of this translational study establish an experimental basis for clinical studies of this delivery method in humans with hemophilia B. These findings also have immediate relevance for gene transfer in patients with muscular dystrophy.
14/10/04: Helpful Informations about Steroids in DMD
14/10/04: (EMBO reports, 2004; 5(9):872-6) Duchenne muscular dystrophy and dystrophin: pathogenesis and opportunities for treatment
Kristen J.Nowak & Kay E.Davies - MRC Functional Genetics Unit,University of Oxford,UK
Duchenne muscular dystrophy (DMD) is caused by mutations in the gene that encodes the 427-kDa cytoskeletal protein dystrophin. Increased knowledge of the function of dystrophin and its role in muscle has led to a greater understanding of the pathogenesis of DMD. This, together with advances in the genetic toolkit of the molecular biologist, are leading to many different approaches to treatment. Gene therapy can be achieved using plasmids or viruses, mutations can be corrected using chimaeraplasts and short DNA fragments, exon skipping of mutations can be induced using oligonucleotides and readthrough of nonsense mutations can be achieved using aminoglycoside antibiotics. Blocking the proteasome degradation pathway can stabilize any truncated dystrophin protein, and upregulation of other proteins can also prevent the dystrophic process. Muscle can be repopulated with myoblasts or stem cells. All, or a combination, of these approaches hold great promise for the treatment of this devastating disease.
14/10/04: Muscle ultrasound in children: Normal values and application to neuromuscular disorders
07/10/04: MY FIRST RESEARCH IN MUSCULAR DYSTROPHY - Tamoxifen increases muscular strength of the mdx dystrophic mice
(Abstract from 44th Annual Meeting - American Society for Cell Biology (December, 4-8, 2004, Washington)
A. Cavalsan,1
R. F. Vasquez,1 R. C. Lin,1 S. B. Zyngier,1 F.
J. Velloso,2 B. H. Santos,2 L. L. Fogaca,2 M.
Vainzof,2 D. Feder1
; 1 Pharmacology, ABC Faculty of Medicine, Santo Andre, Brazil,
2 Human Genome Research Center - IB USP, Sao Paulo, Brazil
The mdx mice is a well-known model of Xp21 dystrophin-deficient muscular
dystrophy. Although a good genetic and biochemical model, the mdx shows no
muscle weakness, but under physical exercise, a loss of muscular strength can be
detected. Here we have tested the possible therapeutic beneficial effect of
Tamoxifen in the degenerative process of the dystrophic muscle, analyzing muscle
strength of tamoxifen treated mdx mice, under intensive physical exercise. A
total of 22 mices aged 4 weeks were divided into 3 groups: control (n= 8)
treated with 0.5 mL saline ip, Tamoxifen 5 mg/Kg body weight ip (n= 8) daily and
Tamoxifen 10 mg/Kg ip (n=6) daily. The exercise protocol was done in a wheel
revolving at 18 cm/s, for 10 minutes, twice a day, 5 days/week, up to 12 weeks.
Whole-body strength was measured weekly using a force transducer coupled to a
computer. Mice tails were attached via a non-flexibile nylon cord to the
transducer, the animals were electricly stimulated to run, and the force to pull
the cord was registered continuously. Results were analyzed by the Kruskal-Wallis
test. The mice treated either with 5 mg/Kg (6.26+ 1.44 dynes/g body
weight) or 10 mg/Kg (6.46+ 2.52) showed a significant increase of
muscular strength (p<0.05) compared to the control group (3.66+ 0.77)
starting in the 5th week, and maintaining significance up to the end
of the experiment. Histological and histochemical analysis of the complex of the
gastrocnemius muscle are under analysis, but preliminary results suggest a less
degree of degeneration in the Tamoxifen treated groups.
Tamoxifen is an anti tumoral drug and act on TGF-beta. Its possible therapeutic
effect in the degenerative process of the dystrophic muscle could ameliorates
the clinical course of dystrophic patients.
FAPESP-CEPID, CNPq, PRONEX,
NEPAS-FMABC.
07/10/04: Early view: Abstracts from 44th Annual Meeting - American Society for Cell Biology (December, 4-8, 2004, Washington)
Human Retinal Dystrophin Transgene
Converts Lethal Muscular Dystrophy into Viable Mild Myopathy in
Dystrophin-Utrophin Null Mice
R. Gaedigk,1,2 D. J. Law,3 K. M. Fitzgerald-Gustafson,4,2
S. G. McNulty,1 N. N. Nsumu,1 A. C. Modrcin,5
R. J. Rinaldi,5 D. Pinson,6 S. C. Fowler,7 M.
Bilgen,4,8 J. Burns,9 S. D. Hauschka,10 R. A.
White1,2 ; 1 Medical Research, Children's Mercy Hospitals
& Clinics, Kansas City, MO, 2 Pediatrics, UMKC School of Medicine,
Kansas City, MO, 3 School of Biological Sciences, Univ. of
Missouri-Kansas City, Kansas City, MO, 4 Hoglund Brain Imaging
Center, Univ. of Kansas Medical Center, Kansas City, KS, 5
Rehabilitation Medicine, Children's Mercy Hospitals & Clinics, Kansas City, MO,
6 Pathology & Laboratory Medicine, Univ. of Kansas Medical Center,
Kansas City, KS, 7 Pharmacology & Toxicology, Univ. of Kansas,
Lawrence, KS, 8 Molecular & Integrative Physiology, Univ. of Kansas
Medical Center, Kansas City, KS, 9 Veterinary Imaging Services,
Topeka, KS, 10 Biochemistry, Univ. of Washington, Seattle, WA
Duchenne muscular dystrophy (DMD) is a progressive muscle disease caused by
severe dystrophin gene mutations that often result in death by the third decade.
The mdx mouse is the most commonly used DMD model. Although they lack
dystrophin and have underlying muscle disease, mdx mice appear physically
normal. This may be partially due to compensatory expression of the dystrophin-related
protein, utrophin. In contrast, double mutant mice (DM), deficient for both
dystrophin and utrophin (mdx/+, utrn -/-), die by 3 months of age
and suffer from severe muscle weakness, pronounced growth retardation, kyphosis,
weight loss, slack posture, and immobility (Deconinck et al. (1997) Cell 90:
717-727; Grady et al. (1997) Cell 90: 729-738). These features make them an
excellent physiological model for DMD research. The capacity of a naturally
occurring isoform, human retinal dystrophin (Dp260), to compensate for the
missing muscle isoform Dp427 was tested in DM mice. The expression of this
transgene prevents premature death and reduces the severe muscular dystrophy
phenotype to a mild myopathy. Electromyography (EMG), histology, radiography,
magnetic resonance imaging, and behavior studies show that DM transgenic mice
grow normally, have normal spinal curvature and locomotion, and have reduced
muscle pathology. EMG and histologic data from transgenic DM mice are typical of
mild myopathy, while the DM mice exhibit severe abnormalities commonly seen in
human dystrophinopathies. The expression of human Dp260 in DM mice converts a
severe and lethal muscular dystrophy into a non-lethal mild myopathy.
Muscle-specific expression of Dp260 could have several advantages over other
treatments of DMD
Visualization of
Ectopic Calcification in mdx Mouse Skeletal Muscle
N. Kikkawa, T. Ohno, M. Shiozuka, R. Matsuda; The Graduate School of Arts and
Sciences, The University of Tokyo, Tokyo, Japan
It has been demonstrated that osteogenic differentiation of skeletal muscle
cells are induced by osteogenic factors, such as bone morphogenetic proteins,
both in vitro and in vivo. Spontaneous ectopic bone formation in vivo has also
been reported in, for instance, fibrodysplasia ossificans progressiva,
which is a rare bone disorder. Another example of self-generating ectopic
calcification has been found in skeletal muscle of mdx mouse, a model of
Duchenne muscular dystrophy (DMD).
We observed the ectopic calcification in mdx mouse thigh muscle by using
x-ray micro CT-scanner SkyScan-1074, which gives resolution of 22um. The x-ray
images were reconstructed into three dimentional visions. Calcifications were
found as spicular structures running parallel to the muscle fiber. Paraffin
sections of the regions found by X-ray microtomography were Von Kossa stained to
confirm calcium deposits. We also detected ectopic calcifications of living mice
with mouse-whole-body x-ray CT-scanner of Aloka. No ectopic calcification was
observed in skeletal muscle of B10 mouse, which possesses normal dystrophin gene
and was used as a negative control.
Calcified regions of mdx mouse thigh did not overlap with the Evans
blue-positive areas but corresponded to some of the regenerating areas. Thus,
ectopic calcification can be a diagnostic marker of muscle regeneration in
mdx mice, and be available to determine the effects of drug or gene
therapies.
Muscle
Proteoglycan Levels in Duchenne Muscular Dystrophy Differ from Other Muscular
Dystrophies
S. Zanotti, E. Canioni, F. Cornelio, L. Morandi, M. Mora; Neuromuscular Diseases
and Neuroimmunology, National Neurological Institute C. Besta, Milano, Italy
Biglycan and decorin are small leucine-rich extracellular proteoglycans that
interact with several matrix proteins particularly collagens, and also with
cytokines whose activity they may modulate. To better understand the role of
these proteoglycans in muscle fibrosis in muscular dystrophies, we investigated
the expression of their transcripts and proteins in several forms of muscular
dystrophy compared to age-matched controls. mRNAs of both proteoglycans were
significantly downregulated in Duchenne muscular dystrophy and significantly
upregulated in Becker muscular dystrophy, sarcoglycanopathies and
dysferlinopathy. By immunohistochemistry, decorin and biglycan were mainly
localized in connective tissue and apparently increased with age. Their presence
increased in relation to increased fibrosis in all types of dystrophic muscle.
The increase in biglycan and decorin in most muscle diseases indicates a role of
these proteoglycans in the extracellular matrix organization. The significant
decrease of decorin and biglycan mRNA in DMD distinguishes this disease from the
other studied and may be related to the high levels of TGF-beta1 and TNF-alpha
found in this disease.
Sex Differences in
Muscle Stem Cell Transplantation Efficiency
B. Deasy,1 A. Lu,1 K. Urish,1 J. Tebbets,1
B. Gharaibeh,1 R. Rubin,2 J. Huard1 ; 1
University of Pittsburgh, Pittsburgh, PA, 2 Allegheny General
Hospital, Pittsburgh, PA
We previously demonstrated that muscle-derived stem cells (MDSCs) efficiently
regenerate skeletal muscle tissue after transplantation into dystrophic mdx
mice, which model muscular dystrophy. Here we show that transplantation of
female MDSC (F-MDSC) rather than male MDSCs (M-MDSC) significantly improves
skeletal muscle regeneration.
We transplanted F-MDSCs into the gastrocnemius muscles of female mdx mice and
the same number of M-MDSCs into the gastrocnemius muscles of male mdx mice. We
used a previously described protocol to measure the cells’ regeneration index (RI)_the
number of dystrophin-positive fibers in the host muscle per 105 donor
cells_two weeks after transplantation. The F-MDSCs elicited significantly
superior muscle regeneration equivalent to a 6-fold increase in efficiency (F-MDSC
RI=686±120 versus M-MDSCs RI=105±25, P< 0.01). Sex-crossed
transplantation also demonstrated the same trend for the donor cells. We
hypothesize that this difference may be related to reduced ability of the M-MDSC
to self-renew or to tolerate hypoxic stress. F-MDSCs maintained CD34 expression
(>80% of total cells) for up to 200 population doublings, whereas M-MDSCs
exhibited a rapid drop in CD34 expression (from >80% to <30% of total cells)
after only 45 population doublings. To support the notion that the F-MDSC are
more tolerant to stress, we also found that proliferation of F-MDSC was less
affected by low O2 conditions as compared with M-MDSC.
Our discovery of sex-related differences in muscle stem cell biology reveals
clear limitations to using male stem and progenitor cells for cell-tracking
purposes. This finding also warrants careful consideration by researchers
working to identify optimal populations of stem and progenitor cells for use in
cell therapy and tissue engineering, clinicians who perform bone marrow stem
cell transplantation, and basic scientists investigating cell and developmental
biology.
07/10/04: Effects of prednisone in canine muscular dystrophy
07/10/04: (Early view: Neuromuscular Disorders,2004) Albumin targeting of damaged muscle fibres in the mdx mouse can be monitored by MRI
Helge Amthor, Thomas Egelhof, Iain McKinnell, Mark E. Ladd, Isabelle Janssen,Johannes Weber, Hannsjo¨rg Sinn, Hans-Hermann Schrenk, Michael Forsting, Thomas Voit, Volker Straub - Germany
Increased sarcolemmal permeability has been implicated as a major pathological event in muscular dystrophies. In our study, we evaluated whether damaged muscle fibres can be specifically targeted using albumin as a carrier. We tagged human serum albumin (HSA) with Gadolinium (Gd) and systemically applied this compound (Gd-DTPA-HSA) to wildtype and dystrophin-deficient mdx mice. We performed magnetic resonance imaging before and after intravenous administration of Gd-DTPA-HSA and found localised signal enhancement only in mdx skeletal muscle. We also examined skeletal muscle after contrast enhanced magnetic resonance imaging using anti-human albumin antibodies and demonstrated intracellular accumulation of Gd-DTPA-HSA in clusters of damaged mdx muscle fibres. Comparison of magnetic resonance imaging and histological data emphasised the value of contrast agent enhanced magnetic resonance imaging for the in vivo assessment of fibre damage in muscular dystrophies. Furthermore, our data provide evidence that albumin can be used as a carrier to target covalently bound molecules to degenerating muscle fibres.
01/10/04:
Glucocorticoid treatment alleviates dystrophic myofiber pathology by activation
of the calcineurin/NF-AT pathway
SEPTEMBER/2004
25/09/04: Bone Marrow Cells Regenerate Heart in Brazil Test
25/09/04: Nonmuscle stem cells fail to significantly contribute to regeneration of normal muscle
23/09/04: (Early View:Muscle and Nerve, 2004): Localization and early time course of TGF-beta 1 mRNA expression in dystrophic muscle
Luc E. Gosselin, PhD, Jacqueline E. Williams, BS, Melissa Deering, MS, Daniel Brazeau, PhD, Stephen Koury, PhD, Daniel A. Martinez, PhD - USA
Fibrosis is a common pathological feature observed in muscle from patients with Duchenne muscular dystrophy (DMD). In the dystrophic (mdx) mouse model of DMD, the diaphragm is more severely affected than other skeletal muscles. The level of transforming growth factor-beta1 (TGF-beta1), an inflammatory cytokine, is significantly elevated in mdx diaphragm. However, little is known about the onset of TGF-beta1 messenger ribonucleic acid (mRNA) expression, or which cells express the mRNA. In this study, we characterized the location and time course of expression of TGF-beta1 mRNA in diaphragm from mdx mice. TGF-beta 1 mRNA was significantly elevated in mdx diaphragm at 6 and 9 but not 12 weeks of age, and these changes corresponded with changes in type I collagen mRNA and hydroxyproline concentration. Mononucleated cells localized to areas of fiber necrosis highly expressed the TGF-beta1 transcript in mdx diaphragm. Neutralization of TGF-beta 1 by decorin administration resulted in a 40% reduction in the level of diaphragm muscle type I collagen mRNA. These findings support a role for TGF-beta1 during the early stages of fibrogenesis in dystrophic diaphragm muscle. Therapeutic interventions aimed at neutralizing this cytokine may be beneficial in slowing the development of fibrosis in DMD.
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20/09/04: (In Press: International Journal of Obstetric Anesthesia, 2004) Anaesthetic management during labour of a manifesting carrier of Duchenne muscular dystrophy
M.K. Molyneux - UK
SUMMARY. We describe the peripartum anaesthetic management of a 36-year-old woman who was a manifesting carrier of Duchenne muscular dystrophy. Duchenne muscular dystrophy is an X-linked recessive disorder affecting young males associated with severe complications during anaesthesia if depolarising neuromuscular blocking drugs and volatile agents are used. A manifesting carrier is a heterozygous female who demonstrates the disease in a milder form than in males. This probably occurs because of skewed X-inactivation. We planned to establish regional anaesthesia should an operation be necessary during labour or delivery and to use propofol total intravenous anaesthesia and rocuronium if general anaesthesia became unavoidable. At 37 weeks, the woman went into spontaneous labour, but fetal distress necessitated caesarean section for which combined spinal-epidural anaesthesia was used.
18/09/04: Antisense Therapeutics: A Promise Waiting to Be Fulfilled
18/09/04: Regenerative capacity of the dystrophic (mdx) diaphragm after induced injury
18/09/04: (In Press: TRENDS in Molecular Medicine, 2004) New therapies for muscular dystrophy: cautious optimism
Giulio Cossu and Maurilio Sampaolesi - Italy
The quest for a therapy for muscular dystrophy has been the driving force behind the past 40 years of advances in this field. Numerous results, such as the identification of satellite cells and gene mutations that are responsible for most forms of dystrophies, advances in gene transfer and modification technology and, more recently, stem cells, have fueled hopes. However, administering corticosteroids still remains the only effective treatment available. Several recent advances have uncovered a diversity of possible therapeutic approaches, from pharmacological treatments to gene therapy (exonskipping and adeno-associated viruses) and cell therapy with different types of newly identified stem cells. Importantly, a combination of these strategies might greatly enhance the possibility of successful therapy.
15/09/04:'Mighty mouse' helping find ways to prevent osteoporosis
15/09/04: Heregulin ameliorates the dystrophic phenotype in mdx mice
15/09/04: (In Press: Neuromuscular Disorders, 2004) Characteristics of the increase in plasma brain natriuretic peptide level in left ventricular systolic dysfunction, associated with muscular dystrophy in comparison with idiopathic dilated cardiomyopathy
Jun Demachi, Yutaka Kagaya, Jun Watanabe, Masahito Sakuma, Jun Ikeda, Yasunori Kakuta, Iyoko Motoyoshi, Takeshi Kohnosu, Hiroaki Sakuma, Shigeru Shimazaki, Hideaki Sakai, Teiko Kimpara, Toshiaki Takahashi, Kiyoshi Omura, Miho Okada, Hiroshi Saito, Kunio Shirato - Japan
Abstract
To determine whether the plasma brain natriuretic peptide level increases differentially in muscular dystrophy and idiopathic dilated cardiomyopathy, we investigated the plasma brain natriuretic peptide level and echocardiographic parameters in patients with similarly low left ventricular ejection fraction. The plasma brain natriuretic peptide level was lower, and the left ventricular end-diastolic diameter was shorter in the patients with muscular dystrophy than in those with idiopathic dilated cardiomyopathy. The correlation between the plasma brain natriuretic peptide and left ventricular ejection fraction was shifted downward in the patients with muscular dystrophy compared with those with idiopathic dilated cardiomyopathy. Those between the brain natriuretic peptide and left ventricular end-diastolic diameter were superimposable, although the data from the muscular dystrophy patients were located at the shorter left ventricular end-diastolic diameter side. The plasma brain natriuretic peptide level may differentially increase in the two diseases with similar left ventricular systolic dysfunction. Differences in the left ventricular distension and in the physical activity might explain at least partially the different plasma brain natriuretic peptide levels.
08/09/04: Graffinity Pharmaceuticals AG and MyoContract AG Merge to Form Santhera Pharmaceuticals Ag
08/09/04:Mirus Bio Enters into Research Agreement with Genzyme
AUGUST/2004
29/08/04: (Early view: 34th Annual Meeting of Society for Neuroscience; October/2004, San Diego) FUNCTIONAL AND STRUCTURAL IMPROVEMENTS IN MDX SKELETAL MUSCLE FOLLOWING CHRONIC DAILY ADMINISTRATION OF PYRROLIDINE DITHIOCARBAMATE (PDTC)
C. Carlson, A. Siegel, A. Samadi, S. Vulcanar. - USA
The presence of severe dystrophy in the mdx
triangularis sterni (TS) indicates that passive stretch activates
pathogenic mechanisms that do not involve generalized increases in
resting Ca2+ influx (Carlson et al., Neurobiology of Disease, 14,
229,2003).To test whether nuclear activation of NFkB (Kumar and Boriek,
FASEB J., 17,386, 2003) is directly involved in dystrophic pathogenesis,
mdx mice were treated daily (29-82 days)with 50 mg/kg PDTC (ip),an
antioxidant which inhibits the nuclear activation of NFB by stabilizing
cytosolic IkB-a. Western blots indicated that a single dose increased
cytosolic IkB-a in mdx diaphragm for at least 5 hrs.Chronically treated
mdx mice exhibited modest increases in whole body tension (Carlson and
Makiejus, Muscle and Nerve, 13,480,1990),a significant (p<0.05) decrease
in fatigue,and small increases in gastrocnemius twitch tension.Untreated
mdx TS fibers (5-12 m)had an average RP
of -43 mV (+1.4 SE, N=77 fibers)which
was less (p<0.001) than that seen in nondystrophic TS fibers (5-17 m,-51
mV + 1.4,N=61).PDTC treated mdx
muscle fibers isolated at 8 to 9 months had more obvious striations and
an average RP (-52 +1.7 mV,N= 48)
that was identical to that seen in nondystrophic TS fibers.Older PDTC
treated mdx mice (11-22 m) had significantly improved RP
s
in both caudal (-41 mV+
2.8, N=52;p<0.001) and middle (-42 +
2.8 mV, N=44; p<0.05) regions of the TS in comparison to age matched
saline-injected mice (caudal: -23 +
2.0 mV, N=93; middle: -34.2 +2.5 mV,
N=50).These TS muscles also exhibited an increase in the appearance of
healthy intact fibers, particularly in the middle region of the muscle.These
results indicate that a stretch-induced inflammatory reaction involving
NFkB nuclear activation contributes significantly to dystrophic
pathology, and that agents which inhibit the nuclear activation of NFkB
should have pronounced value in the treatment of muscular dystrophy
C. Carlson, A. Siegel, A. Samadi, S. Vulcanar. FUNCTIONAL AND STRUCTURAL IMPROVEMENTS IN MDX SKELETAL MUSCLE FOLLOWING CHRONIC DAILY ADMINISTRATION OF PYRROLIDINE DITHIOCARBAMATE (PDTC) Program No. 684.18. 2004 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, 2004. Online
26/08/04: (Early view: Muscle and Nerve, 2004):Effects of exercise and steroid on skeletal muscle apoptosis in the mdx mouse
Jeong-Hoon Lim, MD, Dai-Youl Kim, MD, Moon Suk Bang, MD, PhD - Republic of Korea
26/08/04: (IN PRESS: Muscle and Nerve, 30:295-304, 2004): ADMINISTRATION OF INSULIN-LIKE GROWTH FACTOR-I IMPROVES FATIGUE RESISTANCE OF SKELETAL MUSCLES FROM DYSTROPHIC mdx MICE
PAUL GREGOREVIC, , DAVID R. PLANT, , and GORDON S. LYNCH, - Australia
ABSTRACT: Muscle fatigue occurs in many neuromuscular diseases, including the muscular dystrophies, and it contributes to a loss of functional capacity and reduced quality of life for affected patients. An improvement in fatigue resistance has been observed in diaphragm muscles of mdx mice following insulin-like growth factor-I (IGF-I) administration. Whether similar treatment can improve locomotor muscle function in mdx mice is not known. We examined the ef.cacy of IGF-I administration (1 mg/kg daily s.c. for 8 weeks) on structural, metabolic, and functional properties of extensor digitorum longus (EDL) and soleus muscles of mdx mice, and tested the hypothesis that IGF-I treatment would improve function in these muscles. After treatment, muscles were more resistant to fatigue during repeated maximal contractions than muscles from untreated mice, an improvement associated with increased muscle .ber succinate dehydrogenase activity in the absence of changes in cellular (single-.ber) contractile activation characteristics. The .ndings have important clinical implications, not just for the dystrophinopathies, but for all neuromuscular pathologies where fatigue of locomotor muscles limits functional capacity and decreases quality of life.
26/08/04: (IN PRESS: Transplantation, 2004) SUMMARY: IMMUNE PARAMETERS IN THREE DUCHENNE MUSCULAR DYSTROPHY PATIENTS FOLLOWING INTRAMUSCULAR TRANSPLANTATION OF NORMAL MYOGENIC CELLS UNDER FK506 IMMUNOSUPPRESSION.
D. Skuk, R. Roy, B. Roy, M. Goulet, F.J. Dugre´, J.P.Tremblay - Canada
Aims: To test the efficacy of FK506 immunosuppression to control acute rejection in the context of myoblast allotransplantation in Duchenne muscular dystrophy (DMD) patients. Methods: Three DMD patients received injections of myoblasts obtained from skeletal muscle biopsies of normal donors. The cells (30 x 106) were injected in 1 cm3 of the Tibialis anterior by 25 parallel injections. Similar patterns of saline injections were performed in the contralateral muscles. The patients received FK506 for immunosuppression, the doses being adjusted to maintain a blood concentration between 8 to 20 ug/L. Periodical monitoring of the patients included weight, hemogram, glycemia, lipid profile and serum levels of creatinine, urea, sodium, potassium and chlorides. Patients 1 and 2 were previously taking deflazacort to delay the progression of the disease and we maintained this treatment during the study. Muscle biopsies were performed at the injected sites 4 weeks later. Histological studies were done to analyze the success of the graft, and to characterize the immune cells present in the tissue. RT-PCR analysis of the biopsies were done to observe the expression of INFgamma, TNF-alpha, granzyme B, IL-6 and FasL. The presence of antibodies against the donor cells in blood samples taken before and at 2, 4 and 8 weeks after cell transplantation was analyzed by cytofluorometry. Results: We observed dystrophin-positive myofibers in the cellgrafted sites of the three patients: 9 % (patient 1), 6.8 % (patient 2), and 11 % (patient 3). Since patients 1 and 2 had identified dystrophin-gene deletions, these results were obtained using monoclonal antibodies specifically to the deleted exons. The grafted site exhibited some pockets of leukocyte infiltration composed of macrophages and CD4 and CD8 lymphocytes in patient 1. In patient 2, similar leukocyte infiltrations were smaller and scarce. No leukocyte accumulations were observed in patient 3. No antibodies against the donor-cells and the donor MHC class-I and class II were detected in the 3 cases. Only the IL-6 mRNA was significantly increased in one grafted site (patient 1). The only potential side effect of the immunosuppression was some weight increase in patient 3. Conclusions: Significant dystrophin expression can be obtained in the skeletal muscles of DMD patients following specific conditions of cell delivery and immunosuppression. Only the presence of some macrophage/T-lymphocyte accumulations in the vicinity of dystrophin-positive myofibers, and an increase in IL-6 expression could suggest to a moderate, non-completely controlled cellular acute rejection in patient 1. For the rest, the immunosuppression seems quite appropriate. Since patients 1 and 2 received chronic corticosteroid therapy, an additive effect of this treatment on the FK506-based immunosuppression is perhaps possible. The long-term benefit of a FK506-based immunosuppression for myoblast transplantation in DMD patients remains to be tested.
24/08/04: (IN PRESS: Molecular Therapy, 2004) Sustained Muscle Expression of Dystrophin from a High-Capacity Adenoviral Vector with Systemic Gene Transfer of T Cell Costimulatory Blockade
Zhilong Jiang, Gudrun Schiedner, Nico van Rooijen, Chau-Ching Liu, Stefan Kochanek, and Paula R. Clemens - USA
Adenoviral vector (Ad)-mediated gene delivery of normal, full-length dystrophin to skeletal muscle provides a promising strategy for the treatment of Duchenne muscular dystrophy (DMD). However, cellular and humoral immune responses induced by vector gene transfer limit the application of this approach. Blockade of the costimulatory interaction between naRve T cells and antigen-presenting cells has proven to be a successful means to diminish immunity induced by gene transfer. In this study we explore the potential of supplementing dystrophin gene delivery to dystrophin-deficient Dmd mouse skeletal muscle with systemic gene delivery of CTLA4Ig and CD40Ig molecules to effect costimulatory blockade. We found that systemic administration of a high-capacity Ad (HC-Ad) vector carrying murine CTLA4Ig (AdmCTLA4Ig) either alone or codelivered with an HC-Ad vector carrying murine CD40Ig (AdmCD40Ig) provided sustained expression of recombinant full-length murine dystrophin from an HC-Ad vector carrying the dystrophin cDNA (AdmDys). The level of AdmDys vector genomes remained stable in animals cotreated with systemic delivery of vectors carrying molecules to block costimulation. In addition, muscle CD4+ and CD8+ T cell infiltrates and Th1 cytokine production by splenocytes were reduced. The production of neutralizing antibody against Ad vector was significantly inhibited in mice receiving systemic codelivery of both AdmCTLA4Ig and AdmCD40Ig, but not in the mice treated with AdmCTLA4Ig alone. The results suggested that coblockade of both CD28/B7 and CD40L/CD40 costimulatory pathways is required for effective inhibition of the Ad vector-induced humoral immune response in Dmd mice, whereas blockade of CD28/B7 alone by murine CTLA4Ig would be sufficient for prolonged dystrophin expression in treated muscle.
24/08/04: Marathoning Mice Could Have Olympian Effects on Obesity
24/08/04: (IN PRESS: Neurobiology of Disease, 2004) The alteration of calcium homeostasis in adult dystrophic mdx muscle fibers is worsened by a chronic exercise in vivo
Bodvael Fraysse, Antonella Liantonio, Michela Cetrone,Rosa Burdi, Sabata Pierno, Antonio Frigeri, Michela Pisoni,Claudia Camerino, and Annamaria De Lucaa - Italy
Chronic exercise in vivo aggravates dystrophy in mdx mice. Calcium homeostasis was evaluated ex vivo by micro-spectrofluorometry on tendon-to-tendon dissected extensor digitorum longus (EDL) muscle fibers. Resting cytosolic calcium ([Ca2+]i) and sarcolemmal permeability through Gd3+-sensitive mechanosensitive calcium (MsCa) channel were significantly higher in mdx vs. wild-type fibers. The exercise further enhanced [Ca2+]i in mdx fibers and increased sarcolemmal permeability by activating nifedipine-sensitive leak calcium channels. The two genotypes did not differ in caffeine sensitivity and in the excitation-calcium release (ECaR) coupling mechanism by K+ depolarization. The exercise produced a similar adaptation of activation curve of ECaR and of sensitivity to caffeine. However, the inactivation of ECaR of mdx fibers did not adapt to exercise. No fiber phenotype transition occurred in exercised muscle. We provide the first evidence that an in vivo exercise worsens the impaired calcium homeostasis of dystrophic fibers, supporting the role of enhanced calcium entrance in dystrophic progression.
21/08/04: (IN PRESS: Molecular Therapy, 2004) AAV Vector-Mediated Microdystrophin Expression in a Relatively Small Percentage of mdx Myofibers Improved the mdx Phenotype
Madoka Yoshimura, Miki Sakamoto, Madoka Ikemoto, Yasushi Mochizuki, Katsutoshi Yuasa, Yuko Miyagoe-Suzuki, and Shin’ichi Takeda - Japan
Duchenne muscular dystrophy (DMD) is a lethal disorder of skeletal muscle caused by mutations in the dystrophin gene. Adeno-associated virus (AAV) vector-mediated gene therapy is a promising approach to the disease. Although a rod-truncated microdystrophin gene has been proven to ameliorate dystrophic phenotypes, the level of microdystrophin expression required for effective gene therapy by an AAV vector has not been determined yet. Here, we constructed a recombinant AAV type 2 vector, AAV2-MCKDCS1, expressing microdystrophin (DCS1) under the control of a muscle-specific MCK promoter and injected it into TA muscles of 10-day-old and 5-week-old mdx mice. AAV2-MCKDCS1-mediated gene transfer into 5-week-old mdx muscle resulted in extensive and long-term expression of microdystrophin and significantly improved force generation. Interestingly, 10-day-old injected muscle expressed microdystrophin in a limited number of myofibers but showed hypertrophy of microdystrophin-positive muscle fibers and considerable recovery of contractile force. Thus, we concluded that AAV2-MCKDCS1 could be a powerful tool for gene therapy of DMD.
20/08/04: Blood loss in pediatric spine surgery
More information about blood loss in DMD spine surgery
This article reviews the extent of blood loss in spine surgery for scoliosis corrections in the pediatric age group. An extensive literature review presents blood loss values in surgery for adolescent idiopathic scoliosis, cerebral palsy, Duchenne muscular dystrophy, spinal muscular atrophy, and myelomeningocoele. The underlying disorder plays a major role in determining the extent of blood loss. Blood loss is considerably higher in those patients with a neuromuscular scoliosis compared with adolescent idiopathic scoliosis. Within the neuromuscular group those with Duchenne muscular dystrophy demonstrate the highest mean levels of blood loss. Blood loss is also shown to be progressively greater with increasing numbers of vertebral levels incorporated into the fusion, with posterior fusions compared to anterior fusions, and in those patients having both anterior and posterior fusions
19/08/04: Newly discovered protein may be key to muscular dystrophy
(IN PRESS: Nature, 2004) SNF-6 is an acetylcholine transporter interacting with the dystrophin complex in Caenorhabditis elegans
Hongkyun Kim, Matthew J. Rogers, Janet E. Richmond & Steven L. McIntire - USA
Muscular dystrophies are among the most common human genetic diseases and are characterized by progressive muscle degeneration. Muscular dystrophies result from genetic defects in components of the dystrophin–glycoprotein complex (DGC), a multimeric complex found in the muscle cell plasma membrane. The DGC links the intracellular cytoskeleton to the extracellular matrix and is thought to be important for maintaining the mechanical integrity of muscles and organizing signalling molecules. The exact role of the DGC in the pathogenesis of disease has, however, remained uncertain. Mutations in Caenorhabditis elegans DGC genes lead to specific defects in coordinated movement and can also cause muscle degeneration. Here we show that mutations in the gene snf-6 result in phenotypes indistinguishable from those of the DGC mutants, and that snf-6 encodes a novel acetylcholine/choline transporter. SNF-6 mediates the uptake of acetylcholine at neuromuscular junctions during periods of increased synaptic activity. SNF-6 also interacts with the DGC, and mutations in DGC genes cause a loss of SNF-6 at neuromuscular junctions. Improper clearing of acetylcholine and prolonged excitation of muscles might contribute to the pathogenesis of muscular dystrophies.
17/08/04: (In Press: Molecular Therapy, 2004) Nucleofection of Muscle-Derived Stem Cells and Myoblasts with C31 Integrase: Stable Expression of a Full-Length-Dystrophin Fusion Gene by Human Myoblasts
Simon P. Quenneville,Pierre Chapdelaine,JoJl Rousseau, Jean Beaulieu, Nicolas J. Caron, Daniel Skuk, Philippe Mills, Eric C. Olivares, Michele P. Calos, and Jacques P. Tremblay - Canada
Ex vivo gene therapy offers a potential treatment for Duchenne muscular dystrophy by transfection of the dystrophin gene into the patient’s own myogenic precursor cells, followed by transplantation. We used nucleofection to introduce DNA plasmids coding for enhanced green fluorescent protein (eGFP) or eGFP-dystrophin fusion protein and the phage C31 integrase into myogenic cells and to integrate these genes into a limited number of sites in the genome. Using a plasmid expressing eGFP, we transfected 50% of a mouse muscle-derived stem cell line and 60% of normal human myoblasts. Co-nucleofection of a plasmid expressing the C31 integrase and an eGFP expression plasmid containing an attB sequence produced 15 times more frequent stable expression, because of site-specific integration of the transgene. Co-nucleofection of the C31 integrase plasmid and a large plasmid containing the attB sequence and the gene for an eGFP–full-length dystrophin fusion protein produced fluorescent human myoblasts that were able to form more intensely fluorescent myotubes after 1 month of culture. A nonviral approach combining nucleofection and the C31 integrase may eventually permit safe autotransplantation of genetically modified cells to patients.
13/08/04: Respiratory Care of the Patient with Duchenne Muscular Dystrophy - ATS Consensus Statement
03/08/04: 2004 PPMD Annual Conference Powerpoint Presentations
02/08/04: ( Molecular Therapy. 2004,10(2):386-398) A Facile Nonviral Method for Delivering Genes and siRNAs to Skeletal Muscle of Mammalian Limbs
James E. Hagstrom, Julia Hegge, Guofeng
Zhang, Mark Noble, Vladimir Budker, David L. Lewis, Hans Herweijer and Jon A.
Wolff - USA
Abstract
Delivery is increasingly being recognized as the critical hurdle holding back the tremendous promise of nucleic acid-based therapies that include gene therapy and more recently siRNA-based therapeutics. While numerous candidate genes (and siRNA sequences) with therapeutic potential have been identified, their utility has not yet been realized because of inefficient and/or unsafe delivery technologies. We now describe an intravascular, nonviral methodology that enables efficient and repeatable delivery of nucleic acids to muscle cells (myofibers) throughout the limb muscles of mammals. The procedure involves the injection of naked plasmid DNA or siRNA into a distal vein of a limb that is transiently isolated by a tourniquet or blood pressure cuff. Nucleic acid delivery to myofibers is facilitated by its rapid injection in sufficient volume to enable extravasation of the nucleic acid solution into muscle tissue. High levels of transgene expression in skeletal muscle were achieved in both small and large animals with minimal toxicity. Evidence of siRNA delivery to limb muscle was also obtained. The simplicity, effectiveness, and safety of the procedure make this methodology well suited to limb muscle gene therapy applications.
Mattie Bremmer-Bout, Annemieke Aartsma-Rus, Emile J. de Meijer, Wendy E. Kaman, Anneke A. M. Janson, Rolf H. A. M. Vossen, Gert-Jan B. van Ommen, Johan T. den Dunnen and Judith C. T. van Deutekom
- The NetherlandsThe therapeutic potential of frame-restoring exon skipping by antisense oligonucleotides (AONs) has recently been demonstrated in cultured muscle cells from a series of Duchenne muscular dystrophy (DMD) patients. To facilitate clinical application, in vivo studies in animal models are required to develop safe and efficient AON-delivery methods. However, since exon skipping is a sequence-specific therapy, it is desirable to target the human DMD gene directly. We therefore set up human sequence-specific exon skipping in transgenic mice carrying the full-size human gene (hDMD). We initially compared the efficiency and toxicity of intramuscular AON injections using different delivery reagents in wild-type mice. At a dose of 3.6 nmol AON and using polyethylenimine, the skipping levels accumulated up to 3% in the second week postinjection and lasted for 4 weeks. We observed a correlation of this long-term effect with the intramuscular persistence of the AON. In regenerating myofibers higher efficiencies (up to 9%) could be obtained. Finally, using the optimized protocols in hDMD mice, we were able to induce the specific skipping of human DMD exons without affecting the endogenous mouse gene. These data highlight the high sequence specificity of this therapy and present the hDMD mouse as a unique model to optimize human-specific exon skipping in vivo.
JULY/2004
29/07/04: (In Press: Neuromuscular Disorders, 2004) Prednisolone decreases cellular adhesion molecules required for inflammatory cell infiltration in dystrophin-deficient skeletal muscle
Michelle Wehling-Henricks, James J. Lee and James G. Tidball - USA
The mechanism of prednisolone's efficacy in the dystrophic pathology is unclear. Prednisolone's anti-inflammatory functions may be particularly important considering the significance of inflammatory cells in dystrophinopathy. In other pathologies, prednisolone's anti-inflammatory effects can be mediated by reducing cellular adhesion molecule (CAM) expression. The goal of this study was to examine the effects of prednisolone on inflammation and CAM expression in dystrophic muscle. Dystrophin-deficient, mdx mice were treated with 0.75 mg/kg prednisolone from 2 to 4 weeks of age. Prednisolone reduced macrophages (−59%, −57%), CD4+ T-cells (−50%, −60%), CD8+ T-cells (−58%, −48%), and eosinophils (−36%, −25%) in quadriceps and soleus muscles, respectively. Prednisolone-treated mice also exhibited decreased vascular P-selectin (−82%) and ICAM-1 (−52%) expression and fewer L-selectin (−79%) and ICAM-1 (−57%) expressing mononuclear cells in quadriceps. Prednisolone reduced sarcolemmal damage and degeneration as well. Our data show that prednisolone is an effective anti-inflammatory in dystrophic muscle and may function by modulating CAM expression.
28/07/04: Respiratory Care of the Patient With Neuromuscular Weakness: The (not so) New Paradigm
28/07/04: Powerpoint presentations (in PDF format) from Katie Bushby (http://www.parentprojectmd.org/news/latest/index.html):
General Principles of Management in DMD
Steroids — Speaking the Same Language
28/07/04:
(In Press:
Neuromuscular Disorders, 2004)
Deflazacort in Duchenne muscular dystrophy: a comparison
of two different protocols
W. D. Biggar L. Politano, V. A. Harris, L. Passamano, J. Vajsar, B. Alman, A. Palladino, L. I. Comi and G. Nigro - Canada and Italy
We compare the long-term benefits and side effects of deflazacort using two treatment protocols from Naples (N) and Toronto (T). Boys with Duchenne muscular dystrophy between the ages of 8 and 15 years and who had four or more years of deflazacort treatment were reviewed. Diagnostic criteria included males with proximal muscle weakness evident before 5 years, increased serum creatine kinase and genetic testing and/or a muscle biopsy consistent with Duchenne muscular dystrophy. Thirty-seven boys were treated with protocol-N using deflazacort at a dose of 0.6 mg/kg per day for the first 20 days of the month and no deflazacort for the remainder of the month. Boys with osteoporosis received daily vitamin D and calcium. Deflazacort treatment started between 4 and 8 years of age. Thirty-two were treated with protocol-T using deflazacort at a dose of 0.9 mg/kg per day, plus daily vitamin D and calcium. Treatment started between 6 and 8 years of age. All boys were monitored every 4–6 months. The results were compared with age-matched controls in the two groups (19 for protocol-N and 30 for protocol-T). For the boys treated with protocol-N, 97% were ambulatory at 9 years (control, 22%), 35% at 12 years (control, 0%), 25% at 15 years (control, 0%). For the 32 boys treated with protocol-T, 100% were ambulatory at 9 years (control, 48%), 83% at 12 years (control, 0%) and 77% at 15 years (control, 0%). No aids or leg braces were used for ambulation. In boys 13 years and older, a scoliosis of >20° developed in 30% of the boys on protocol-N, 16% on protocol-T and 90% of controls. For protocol-N, no cataracts were observed while in protocol-T, 30% of boys had asymptomatic cataracts that required no treatment. Fractures occurred in 19% (control 16%) of boys on protocol-N and 16% (control, 20%) of boys on protocol-T. This report illustrates: (a) the importance of collaborative studies in developing treatment protocols in Duchenne muscular dystrophy and (b) the long-term beneficial effects of deflazacort treatment in both protocols. However, the protocol-T seems to be more effective and frequently is associated with asymptomatic cataracts.
27/07/04: Prednisolone therapy in Duchenne muscular dystrophy prolongs ambulation and prevents scoliosis
25/07/04: Systemic delivery of genes to striated muscles using adeno-associated viral vectors
Gene therapy reaches muscles throughout the body and reverses muscular dystrophy in animal model
Gene Therapy Reverses Muscular Dystrophy in Animal Model
23/07/04: Anesthetic Implications of Neuromuscular Disease
Edwards D. Briggs and Jeffrey R. Kirschy -USA
(J Anesth, 17:177–185, 2003)
Even in healthy patients neuromuscular blocking agents must be administered and monitored with great vigilance to ensure that adverse drug interactions do not occur and that residual postanesthetic muscle paralysis is prevented. The use of muscle relaxants in patients with neuromuscular disease presents several additional potential hazards. The current article addresses the anesthetic implications of some of the more common neuromuscular diseases, including multiple sclerosis, myasthenia gravis, muscular dystrophy, critical illness polyneuropathy, spinal cord injury, and Guillain-Barré syndrome. When caring for patients with these conditions, the anesthesiologist must perform a thorough preoperative assessment, select an appropriate anesthetic technique and muscle relaxant (if needed), and provide careful monitoring of both hemodynamic parameters and the extent of neuromuscular blockade.
Comment by Jason Gregory and Kathryn E. McGoldrick, (Survey of Anesthesiology Volume 48, Number 4, August 2004)
Duchenne muscular dystrophy patients have a high incidence of cardiac abnormalities and often eventually develop a dilated cardiomyopathy that is frequently associated with mitral valve incompetence. Dysrhythmias and cardiac conduction defects are common. Respiratory compromise secondary to scoliosis is another major challenge complicating anesthetic management. Cardiac arrest owing to succinylcholine-induced hyperkalemia has been well documented. Muscular dystrophic patients are sensitive to nondepolarizing neuromuscular blocking agents, but these drugs can be used safely if their effects are properly monitored. Given the known association of Duchenne muscular dystrophy with malignant hyperthermia, the use of triggering agents should be avoided in patients with this condition.
23/07/04: (In Press: Molecular Therapy, 2004) Factors Influencing the Efficacy, Longevity, and Safety of Electroporation-Assisted Plasmid-Based Gene Transfer into Mouse Muscl