M. van Putten

Serum matrix metalloproteinase-9 (MMP-9) as a biomarker for monitoring disease progression in Duchenne muscular dystrophy (DMD)

To identify serum biomarkers that allow monitoring of disease progression and treatment effects in Duchenne muscular dystrophy (DMD) patients, levels of matrix metalloproteinase-9 (MMP-9), tissue inhibitors of metalloproteinase-1 (TIMP-1) and osteopontin (OPN) were determined in 63 DMD patients on corticosteroid therapy. These proteins were selected for their role in the pathogenesis of muscular dystrophy. Levels of MMP-9 and TIMP-1 were significantly higher in sera of DMD patients compared to healthy controls, whereas the OPN levels showed no significant difference. MMP-9 levels were also observed to be significantly higher in older, nonambulant patients, compared to ambulant patients. Longitudinal data from a smaller cohort of DMD patients followed up for over 4years showed that MMP-9, but not TIMP-1 increased significantly with age. Hence, MMP-9 is a potential DMD biomarker for disease progression. Future studies have to confirm whether serum MMP-9 levels can be used to monitor therapeutic response.

Natural disease history of mouse models for limb girdle muscular dystrophy types 2D and 2F

Limb-girdle muscular dystrophy types 2D and 2F (LGMD 2D and 2F) are autosomal recessive disorders caused by mutations in the alpha- and delta sarcoglycan genes, respectively, leading to severe muscle weakness and degeneration. The cause of the disease has been well characterized and a number of animal models are available for pre-clinical studies to test potential therapeutic interventions. To facilitate transition from drug discovery to clinical trials, standardized procedures and natural disease history data were collected for these mouse models. Implementing the TREAD-NMD standardized operating procedures, we here subjected LGMD2D (SGCA-null), LGMD2F (SGCD-null) and wild type (C57BL/6J) mice to five functional tests from the age of 4 to 32 weeks. To assess whether the functional test regime interfered with disease pathology, sedentary groups were taken along. Muscle physiology testing of tibialis anterior muscle was performed at the age of 34 weeks. Muscle histopathology and gene expression was analysed in skeletal muscles and heart. Muscle histopathology and gene expression was analysed in skeletal muscles and heart. Mice successfully accomplished the functional tests, which did not interfere with disease pathology. Muscle function of SGCA- and SGCD-null mice was impaired and declined over time. Interestingly, female SGCD-null mice outperformed males in the two and four limb hanging tests, which proved the most suitable non-invasive tests to assess muscle function. Muscle physiology testing of tibialis anterior muscle revealed lower specific force and higher susceptibility to eccentric-induced damage in LGMD mice. Analyzing muscle histopathology and gene expression, we identified the diaphragm as the most affected muscle in LGMD strains. Cardiac fibrosis was found in SGCD-null mice, being more severe in males than in females. Our study offers a comprehensive natural history dataset which will be useful to design standardized tests and future pre-clinical studies in LGMD2D and 2F mice.

G.P.109

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder caused by out of frame mutations in the dystrophin gene. Restoration of the reading frame would in theory allow the production of a shorter but partly functional dystrophin protein as seen in patients with Becker muscular dystrophy (BMD). This can be achieved with antisense oligonucleotides (AONs) that induce skipping of specific exons in the pre-mRNA. Over the years chemical modifications have been developed to increase AON binding affinity to the target RNA, increase resistance against nuclease degradation and improve cellular uptake. The 2 ′ -deoxy-2 ′ -fluoro AON (2FPS) is an attractive candidate chemistry for exon skipping. It has been shown that ILF2/3 proteins are recruited to the 2FPS/pre-mRNA duplex, resulting in (undesired) enhanced exon skipping in a model of spinal muscular atrophy (SMA). In this study we compared 2 ′ -F phosphorothioate (2FPS) and 2 ′ -O-methyl phosphorothioate AONs (2OMePS) targeting different human and mouse dystrophin exons in vitro and in vivo . In vitro , the 2FPS AONs induced higher exon skipping levels than their 2OMePS AON counterparts, and were effective at concentrations where their 2OMePS AON counterparts were not. Surprisingly, a 2FPS AON targeting mouse exon 23 appeared to be ineffective after local intra muscular administration (2 times 2.9nmol) and after 8weeks of systemic treatment in mdx mice (4 times 50mg/kg/week). To assess whether this is a generalized effect of 2FPS AON or specific for mouse exon 23 skip, we are currently evaluating a 2FPS AON targeting a human exon in our hDMD mouse model, which contains the complete human DMD gene integrated into the mouse genome.

Natural disease history of the dy2J mouse model of laminin α2 (merosin)-deficient congenital muscular dystrophy

Merosin deficient congenital muscular dystrophy 1A (MDC1A) is a very rare autosomal recessive disorder caused by mutations in the LAMA2 gene leading to severe and progressive muscle weakness and atrophy. Although over 350 causative mutations have been identified for MDC1A, no treatment is yet available. There are many therapeutic approaches in development, but the lack of natural history data of the mouse model and standardized outcome measures makes it difficult to transit these pre-clinical findings to clinical trials. Therefore, in the present study, we collected natural history data and assessed pre-clinical outcome measures for the dy2J/dy2J mouse model using standardized operating procedures available from the TREAT-NMD Alliance. Wild type and dy2J/dy2J mice were subjected to five different functional tests from the age of four to 32 weeks. Non-tested control groups were taken along to assess whether the functional test regime interfered with muscle pathology. Respiratory function, body weights and creatine kinase levels were recorded. Lastly, skeletal muscles were collected for further histopathological and gene expression analyses. Muscle function of dy2J/dy2J mice was severely impaired at four weeks of age and all mice lost the ability to use their hind limbs. Moreover, respiratory function was altered in dy2J/dy2J mice. Interestingly, the respiration rate was decreased and declined with age, whereas the respiration amplitude was increased in dy2J/dy2J mice when compared to wild type mice. Creatine kinase levels were comparable to wild type mice. Muscle histopathology and gene expression analysis revealed that there was a specific regional distribution pattern of muscle damage in dy2J/dy2J mice. Gastrocnemius appeared to be the most severely affected muscle with a high proportion of atrophic fibers, increased fibrosis and inflammation. By contrast, triceps was affected moderately and diaphragm only mildly. Our study presents a complete natural history dataset which can be used in setting up standardized studies in dy2J/dy2J mice.