J. Morgan

Muscle histology vs MRI in Duchenne muscular dystrophy

Objective: There are currently no effective treatments to halt the muscle breakdown in Duchenne muscular dystrophy (DMD), although genetic-based clinical trials are being piloted. Most of these trials have as an endpoint the restoration of dystrophin in muscle fibers, hence requiring sufficiently well-preserved muscle of recruited patients. The choice of the muscles to be studied and the role of noninvasive methods to assess muscle preservation therefore require further evaluation. Methods: We studied the degree of muscle involvement in the lower leg muscles of 34 patients with DMD >8 years, using muscle MRI. In a subgroup of 15 patients we correlated the muscle MRI findings with the histology of open extensor digitorum brevis (EDB) muscle biopsies. Muscle MRI involvement was assigned using a scale 0–4 (normal–severe). Results: In all patients we documented a gradient of involvement of the lower leg muscles: the posterior compartment (gastrocnemius > soleus) was most severely affected; the anterior compartment (tibialis anterior/posterior, popliteus, extensor digitorum longus) least affected. Muscle MRI showed EDB involvement that correlated with the patients age (p = 0.055). We show a correlation between the MRI and EDB histopathologic changes, with MRI 3–4 grades associated with a more severe fibro-adipose tissue replacement. The EDB was sufficiently preserved for bulk and signal intensity in 18/22 wheelchair users aged 10–16.6 years. Conclusion: This study provides a detailed correlation between muscle histology and MRI changes in DMD and demonstrates the value of this imaging technique as a reliable tool for the selection of muscles in patients recruited into clinical trials.

Alveolar rhabdomyosarcoma-associated proteins PAX3/FOXO1A and PAX7/FOXO1A suppress the transcriptional activity of MyoD-target genes in muscle stem cells.

Rhabdomyosarcoma (RMS) is the commonest soft-tissue sarcoma in childhood and is characterized by expression of myogenic proteins, including the transcription factors MyoD and myogenin. There are two main subgroups, embryonal RMS and alveolar RMS (ARMS). Most ARMS are associated with chromosomal translocations that have breakpoints in introns of either PAX3 or PAX7, and FOXO1A. These translocations create chimeric transcription factors termed PAX3/FOXO1A and PAX7/FOXO1A respectively. Upon ectopic PAX3/FOXO1A expression, together with other genetic manipulation in mice, both differentiating myoblasts and satellite cells (the resident stem cells of postnatal muscle) can give rise to tumours with ARMS characteristics. As PAX3 and PAX7 are part of transcriptional networks that regulate muscle stem cell function in utero and during early postnatal life, PAX3/FOXO1A and PAX7/FOXO1A may subvert normal PAX3 and PAX7 functions. Here we examined how PAX3/FOXO1A and PAX7/FOXO1A affect myogenesis in satellite cells. PAX3/FOXO1A or PAX7/FOXO1A inhibited myogenin expression and prevented terminal differentiation in murine satellite cells: the same effect as dominant-negative (DN) Pax3 or Pax7 constructs. The transcription of MyoD-target genes myogenin and muscle creatine kinase were suppressed by PAX3/FOXO1A or PAX7/FOXO1A in C2C12 myogenic cells again as seen with Pax3/7DN. PAX3/FOXO1A or PAX7/FOXO1A did not inhibit the transcriptional activity of MyoD by perturbing MyoD expression, localization, phosphorylation or interaction with E-proteins. Chromatin immunoprecipitation on the myogenin promoter showed that PAX3/FOXO1A or PAX7/FOXO1A did not prevent MyoD from binding. However, PAX3/FOXO1A or PAX7/FOXO1A reduced occupation of the myogenin promoter by RNA polymerase II and decreased acetylation of histone H4, but did not directly bind to the myogenin promoter. Together, these observations reveal that PAX3/FOXO1A and PAX7/FOXO1A act to prevent myogenic differentiation via suppression of the transcriptional activation of MyoD-target genes.

Biochemical characterization of patients with in-frame or out-of-frame DMD deletions pertinent to exon 44 or 45 skipping

IMPORTANCEnIn Duchenne muscular dystrophy (DMD), the reading frame of an out-of-frame DMD deletion can be repaired by antisense oligonucleotide (AO)-mediated exon skipping. This creates a shorter dystrophin protein, similar to those expressed in the milder Becker muscular dystrophy (BMD). The skipping of some exons may be more efficacious than others. Patients with exon 44 or 45 skippable deletions (AOs in clinical development) have a less predictable phenotype than those skippable for exon 51, a group in advanced clinical trials. A way to predict the potential of AOs is the study of patients with BMD who have deletions that naturally mimic those that would be achieved by exon skipping.nnnOBJECTIVEnTo quantify dystrophin messenger RNA (mRNA) and protein expression in patients with DMD deletions treatable by, or mimicking, exon 44 or 45 skipping.nnnDESIGN, SETTING, AND PARTICIPANTSnRetrospective study of nondystrophic controls (nu2009=u20092), patients with DMD (nu2009=u20095), patients with intermediate muscular dystrophy (nu2009=u20093), and patients with BMD (nu2009=u200913) at 4 university-based academic centers and pediatric hospitals. Biochemical analysis of existing muscle biopsies was correlated with the severity of the skeletal muscle phenotype.nnnMAIN OUTCOMES AND MEASURESnDystrophin mRNA and protein expression.nnnRESULTSnPatients with DMD who have out-of-frame deletions skippable for exon 44 or 45 had an elevated number of revertant and trace dystrophin expression (approximately 19% of control, using quantitative immunohistochemistry) with 4 of 9 patients presenting with an intermediate muscular dystrophy phenotype (3 patients) or a BMD-like phenotype (1 patient). Corresponding in-frame deletions presented with predominantly mild BMD phenotypes and lower dystrophin levels (approximately 42% of control) than patients with BMD modeling exon 51 skipping (approximately 80% of control). All 12 patients with in-frame deletions had a stable transcript compared with 2 of 9 patients with out-of-frame deletions (who had intermediate muscular dystrophy and BMD phenotypes).nnnCONCLUSIONS AND RELEVANCEnExon 44 or 45 skipping will likely yield lower levels of dystrophin than exon 51 skipping, although the resulting protein is functional enough to often maintain a mild BMD phenotype. Dystrophin transcript stability is an important indicator of dystrophin expression, and transcript instability in DMD compared with BMD should be explored as a potential biomarker of response to AOs. This study is beneficial for the planning, execution, and analysis of clinical trials for exon 44 and 45 skipping.

P03 Exon skipping and dystrophin restoration in Duchenne muscular dystrophy patients after systemic phosphorodiamidate morpholino oligomer treatment

1FC2.1 Exon skipping and dystrophin restoration in Duchenne Muscular Dystrophy patients after systemic phosphorodiamidate morpholino oligomer treatment S. Cirak1 *, V. Arechavala-Gomeza1, M. Guglieri2, L. Feng1, S. Torelli1, K. Anthony1, M.E. Garralda3, D.J. Wells4, G. Dickson5, M.J.A. Wood6, S. Wilton7, V. Straub2, S.B. Shrewsbury8, C. Sewry9, J.E. Morgan1, K. Bushby2, F. Muntoni1. 1UCL Institute of Child Health The Dubowitz Neuromuscular Centre, London, United Kingdom, 2Institute of Human Genetics, Newcastle University, Newcastle, United Kingdom, 3Academic Unit of Child and Adolescent Psychiatry, Imperial College St Mary’s Campus, London, United Kingdom, 4Royal Veterinary College, London, United Kingdom, 5Royal Holloway University of London, United Kingdom, 6Department of Physiology, Anatomy and Genetics, University of Oxford, United Kingdom, 7Centre for Neuromuscular and Neurological Disorders, University of Western Australia, Perth, Australia, 8AVI BioPharma, Bothell, WA, United States, 9Centre for Inherited Neuromuscular Disorders, Robert Jones and Agnes Hunt NHS Trust, Oswestry, United Kingdom

P5 Detection of circulating miRNAs in serum in a mouse model of Collagen VI Deficiency

Collagen VI is the main extracellular matrix (ECM) protein synthesized and secreted mainly by fibroblasts that forms a structurally network of microfilaments. nCollagen VI binds several cell surface receptors and ECM components, suggesting that collagen VI is an important mechanical link between muscle cells and surrounding ECM. [1] nMutations related to COL6A1, COL6A2 and COL6A3 genes encoding for the three main collagen VI chains, cause the collagen VI-related myopathy, a specific subtype of congenital muscular dystrophy (CMD). nIn particular Ullrich congenital muscular dystrophy (UCMD) is a severe form characterized by progressive muscle wasting leading to loss of ambulation around ten years of age and respiratory problems in late childhood or young adulthood. nBethlem Miopathy (BM) is a mild-form characterized by a slow course usually without respiratory symptoms where around 50% of patients lose ambulation in the fifth decade. [2] nBeing the genetic test aimed to find mutations in COL6A genes expensive and time consuming mainly because the large size of the three genes, (106 coding exons in total [2]) the diagnosis of collagen VI-related myopathies is based on the clinical signs assessment coupled to the muscular biopsy which is useful to highlight any dystrophic or myopathic changes in the muscle fibers of the patient. nThe aim of the present work has been to identify a panel of miRNAs significantly dysregulated in col6a1-/- mice, the animal model of Bethlem myopathy and Ullrich congenital muscular dystrophy. nmiRNAs represent an ideal biomarker because they are easy to detect, well preserved in serum and non invasive. nThe findings of this work can be translated, in a further step, in a cohort of affected patients in order to become a powerful tool for the diagnosis and the follow-up of collagen VI-related myopathy. n nResearch projects aimed to link the differential miRNA expression to pathological conditions are increasing and this suggests how miRNAs could be used as powerful tool to track disease related changes on a large scale in the forthcoming years. nIn order to identify differentially expressed miRNAs in knock-out col6a1-/- (KO) mice serum, a miRNAs expression profiling analysis by SYBR Green-based real-time PCR of 752 miRNAs in 5 col6a1+/+(WT) and 6 col6a1-/- has been performed. nFifty one (6.5%) of the 333 miRNAs expressed in col6a1-/- mice serum, showed a significant dysregulation between col6a1-/- and WT mice serum. nThe statistical analysis revealed 17/51 miRNAs up-regulated and 34/51 down-regulated. nTo further characterized the dysregulated miRNAs we checked their involvement in disease related pathways coupled with literature analysis. nThese analyses showed that mmu-miR-195a-5p, mmu-miR-26a-5p, mmu-let-7c-5p and mmu-let-7b-5p are up-regulated in col6a1-/- mice serum, whereas mmu-miR-29b-3p and mmu-miR-29a-3p are downregulated ofd of. These observations are quite interesting because these miRNAs could play an important role in the collagen VI-related myopathies linking genes involved in essential signalling pathway for skeletal muscle cells development, differentiation or coding for ECM proteins. nIn particular miRNAs targets interfere with ERBb, MAPK, PIK3/Akt, mTOR, ECM-receptors, actin cytoskeleton, focal adhesion and calcium signalling. nIn conclusion, this project has identified 6 dysregulated microRNAs (mmu-miR-195a-5p, mmu-miR-26a-5p, mmu-let-7c-5p, mmu-let-7b-5p, mmu-miR-29b-3p and mmu-miR-29a-3p) that could be used in future studies as biomarkers and possibly as a tool for the diagnosis and the follow-up of collagen VI-related myopathies. nFurther analyses in larger cohorts of BM and UCMD patients are needed in order to validate the data obtained in mice models.

G.O.4 Search for SNPs modifiers in DMD with different corticosteroids response by candidate genes targeted resequencing

Abstract Corticosteroid treatment is an established therapy for DMD patients, with proven efficacy in delaying the loss of the ambulation. However the response to corticosteroids is not the same for all DMD individuals, some patients having no appreciable benefit. Considering the considerable side effects of this therapy, it would be desirable to identify biomarkers able to predict the response to corticosteroids. We have carried out a targeted exon sequencing in 243 genes (identified within the BIO-NMD consortium) which are connected to DMD pathways (identified using the MedScan informatics tools developed by ARIADNE), preceded by a targeted specific enrichment step. For target enrichment Agilent’s SureSelect in solution approach was selected. Sixteen DMD patients (eight high responders and eight low responders) were processed using 5500xl SOLiD™ Sequencer. Quality filtering was carried out on the SNP calls returned by LifeScope using quality filters, SNPs in introns were discarded. After quality filtering, 735 SNPs were identified across all samples. We applied two novel statistical methods for analysing variance in the two DMD groups: the multidimensional scaling, and the discriminant analysis, both used to determine which variables discriminate between two (or more) naturally occurring groups. Using both tools we were able to identify 34 SNPs which are differently represented in the two categories (responders/low responders) and do discriminate patients. These SNPs belong to 23 genes, several of which encode for the structural proteins vinculins, integrins, and laminins. In order to validate this data, and to confer robustness and specificity to our identified SNPs, 84 additional DMD patients as well as other non-DMD individuals are now being studied to increase the statistical power of the data. Validated SNPs may represent pharmacogenetic biomarkers for corticosteroid therapy response.

P1.52 BIO-NMD: Discovery and validation of biomarkers for neuromuscular diseases (NMDs) – An EU funded FP7 project

www.bio-nmd.eu Volker Straub1, Annemieke Aartsma-Rus2, Cristina Al-Khalili Szigyarto3, Christophe Beroud4, Paolo Bonaldo5, Paola Borgiani6, Paola Braghetta5, Amina Chaouch1, Sebahattin Cirak7, Roseline Favresse8, Nikolai Daraselia9, Cecilia Gelfi10, Peter A.C. ’t Hoen2, Ekaterina Kotelnikova9, Yannick Le Priol9, Hanns Lochmuller1, Jenny Morgan7, Francesco Muntoni7, Giuseppe Novelli6, Nicoletta Paolillo6, Raimo Tanzi11, Cathy Turner1, Mathias Uhlen3, Alessandra Ferlini12 On behalf of the BIO-NMD consortium

P08 Optimal dystrophin mini-construct for gene delivery to skeletal muscle

Selective removal of exons flanking an out-of-frame DMD mutation can result in an in-frame mRNA transcript that may be translated into an internally-deleted, Becker muscular dystrophy (BMD)-like but functionally active dystrophin protein with therapeutic activity. Antisense oligonucleotides (AOs) have been designed to bind to complementary sequences in the targeted mRNA and modify premRNA splicing to correct the reading frame of a mutated transcript so that gene expression is restored. The rapid steady advances made in this field suggest that it is likely that AO-induced exon skipping will be the first gene therapy for DMD to reach the clinic. Indeed two different chemistries of AO continue to show encouraging results in clinical trials targeted at skipping exon 51 of the DMD gene, skipping of which would have the potential to treat 13% of DMD patients. However, the different deletions that cause DMD would require skipping of different exons, and the clinical workup of other AOs. A major UK consortium is currently developing a peptide-conjugated AO for the targeted skipping of exon 53 for use in the next clinical trial. This AO would have the potential to treat 8% of DMD patients. Detailed comparative analysis of an array of overlapping conjugated and naked AOs has been performed in DMD patient cells and the choice of AO for the next clinical trial will be presented.

P04 Lentivirus-mediated stem cell therapy for Duchenne muscular dystrophy

Duchenne muscular dystrophy is a genetic disorder characterized by loss of dystrophin leading to progressive muscle fibre degeneration, finally resulting in failed muscle regeneration. One possible therapeutic approach is to deliver viral vectors that can transduce skeletal muscle and replace dystrophin. Satellite cells (SCs), stem cells residing underneath the basal lamina of myofibres, play a central role in skeletal muscle regeneration. Long-term therapeutic benefit could only be achieved by restoring dystrophin protein expression not only in muscle fibres but also in SCs, thereby maintaining healthy muscle fibres throughout life. Lentiviruses hold great potential as a gene therapy tool for skeletal muscle, as they can stably integrate their genomes into dividing and non-dividing cells, and provide long-term expression. However, low transduction efficiencies in muscle and early promoter silencing in vivo have been discouraging. Here, we show that primary SC cultures can be transduced with lentiviral vectors requiring moderate MOIs. Lentiviral transduction does not affect SC myogenicity, and transgene expression is maintained for at least 3 weeks in culture. Interestingly, transduction of single myofibres in vitro revealed GFP expression in both associated SCs and the myofibre syncytium. Next, we will compare muscle-specific (e.g. Desmin) and silencingresistant (e.g. 2AUCOE) promoters with our results of a strong viral promoter, and determine the level and longevity of transgene expression in SCs and myofibres in vitro and in vivo. Finally, we will investigate the potential of transduced SCs engrafted into immunodeficient mice to repair and regenerate skeletal muscle in vivo.