Association Francaise Contre Les Myopathies Research Articles

143. Off-Target Analysis of a rAAV-U7snRNA Vector Used for the Treatment of Duchenne Patients By Exon Skipping

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder affecting 1 male in 5000 and caused by mutations in the gene encoding for the Dystrophin protein. So far, there are no strongly effective treatments but gene-based therapies are currently being developed. Among them, the selective removal by exon skipping of exons flanking an out-of frame mutation in the dystrophin messenger can result in shorter in-frame transcripts that are translated into functionally active Dystrophin. Exon skipping can be achieved using modified U7-small nuclear RNA (snRNA) in which a therapeutic antisense oligonucleotide (AO) is cloned. Such optimized U7snRNA, called U7.OPT, can be packaged in recombinant vectors derived from Adeno-Associated Virus (rAAV), allowing a long-lasting repair of the dystrophin messenger after one single administration.We recently published the high ability of a rAAV8.U7.OPT vector to transduce the skeletal muscle but also the liver and restore the open reading frame of the dystrophin transcript in GRMD dogs muscles injected in one forelimb. We are now moving forward to a phase I/II clinical trial in DMD patients treatable by skipping of the exon 53 of the dystrophin messenger.Because an abundant presence of the therapeutic U7.OPT RNA can in theory lead to partial hybridization to non-targeted messenger(s) with unwanted side effects, we looked for possible off-target events. Primary human myotubes and hepatocytes, untransduced or transduced with our clinical rAAV.U7.OPT vector, served as our in vitro model system. The skipping of exon 53 of the human dystrophin messenger upon vector transduction was validated. In silico analysis using the miRanda algorithm identified some gene candidates expressed in liver and muscle, i.e. messengers which could be the target of our dystrophin-specific AO. Three of them were analyzed by RT-PCR and RTqPCR and showed not detectable exon skipping events or differential expression. Other candidates are currently analyzed. Simultaneously, further investigation included a full transcriptomic analysis by RNA sequencing comparing the samples obtained from untransduced vs transduced primary cells. Preliminary results revealed differential gene expression patterns between the two types of samples. We are now exploring the respective impact of the rAAV transduction itself vs the AO effect per se to finally identity potential off-target events.The complete data will be presented and discussed in the context of our future clinical trial in DMD patients.This project is supported by AFM (Association Francaise contre les Myopathies) and ADNA (Advanced Diagnostics for New Therapeutic Approaches), a program dedicated to personalized medicine, coordinated by Institut Merieux and supported by research and innovation aid from the French public agency, BpiFrance.

Correction: New Findings in a Global Approach to Dissect the Whole Phenotype of PLA2G6 Gene Mutations

There were errors in the Funding section. The correct funding information is as follows: MAS and co-authors are thankful to the Deanship of Scientific Research, King Saud University, Riyadh, Saudi Arabia, for supporting the work through the research group project number RGP-VPP-301. RC was supported by the Gebert Ruf Foundation (grant number GRS-046/09). GS was supported by the French National Agency for Research(ANR), Association Contre les Syndromes Cerebelleux, France, The Verum Foundation, the European Union (Omics call, Neuromics), The Fondation Roger de Spoelberch and the program Investissements d'avenir ANR-10-IAIHU-06 (to the Brain and Spine Institute). HA was supported by Association Francaise contre les Myopathies (AFM), France. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

EURORDIS Summer School for patient advocates in clinical trials and drug development

EURORDIS, the European Organisation for Rare Diseases launched this new initiative in 2008 with the support of the Drug Information Association (DIA) Philanthropy programme, the Association Francaise contre les Myopathies (AFM), Fundacio Dr. Robert and the European Agency for Health and Consumers (EAHC). Many of EURORDIS’ members are extensively involved in EU decision-making processes in the European Medicines Agency (EMA), including as experts, patients’ representatives and/or members of scientific committees and working parties. The aim of the EURORDIS Summer School is to assist these and future patient advocates to i) better promote drug development, ii) be involved in regulatory affairs, and to iii) guarantee access to treatments and iv) improve the quality of orphan drug information to patients. The 4-day programme consists of: 1) a description of the drug development process from clinical trials to regulatory stages and includes statistics, ethics and post-marketing aspects. 2) a description of the committees and working parties at the EMA with particular emphasis on the role of patients’ representatives and 3) the role of EURORDIS and in particular of its “Task Forces” whose volunteers can also be involved in the above activities. The aim of the Summer School is to: 1. to establish a forum for exchange of information, education and training for these high-level selected patient advocates from a wide range of rare diseases across Europe; 2. to enhance the experience they have gained and their leadership capacities. As patient advocates are increasingly involved in all aspects of drug development (from research to economic aspects), there is a growing need to provide support and training in these areas. The EURORDIS Summer School not only addresses these needs but also provides the means for advocates to interact with each other as well as with regulators, academic partners and industry. In addition to the learning experience, the Summer School provides an excellent forum for in-depth exchange of information and experiences, all in the beautiful city of Barcelona.

The development of antisense oligonucleotide therapies for Duchenne muscular dystrophy: Report on a TREAT-NMD workshop hosted by the European Medicines Agency (EMA), on September 25th 2009

A workshop entitled The Development of Antisense Oligonucleotide Therapies for Duchenne Muscular Dystrophy, sponsored by the EU funded Clinical Network TREAT-NMD (Translational Research in Europe for the Assessment and Treatment of Neuromuscular Diseases; www.treat-nmd.eu), Association Francaise contre les Myopathies, Duchenne Parent Project, European Medicines Agency, Muscular Dystrophy Association and Patient Project Muscular Dystrophy, was held on 25th September 2009 at the European Medicines Agency (EMA), London, UK.

The Ciona intestinalis MyoD homolog is essential for myogenesis

We used gene over expression and gene knockdown to study the functional properties of CiMDF, the single myogenic regulatory factor gene (MRF) of Ciona intestinalis. Overexpression was achieved by injecting eggs with synthetic CiMDF mRNA. RNA-injected eggs did not develop typical tadpole morphology, but instead formed flattened balls of cells. Expression of the four muscle-specific genes assayed was strongly upregulated in muscle cells of injected embryos. Notably, these muscle-specific genes were also expressed in non-muscle lineage cells of injected embryos. This was especially apparent for troponin I, which was typically detected in all vegetal hemisphere cells of mRNA-injected embryos. Animal hemisphere cells of mRNA-injected embryos did not express muscle genes. We knocked down CiMDF expression by injecting eggs with antisense morpholino oligonucleotides (MO). MO-injected eggs often developed into quite normal tadpoles with clearly recognizable heads and tails; nevertheless, muscle-specific gene expression was reduced in these embryos compared to controls. Moreover, MO-treated larvae were almost always paralyzed and never showed the frenzied tail movements of normal larvae. Electron microscopy revealed that ‘‘muscle’’ cells of MO-treated larvae lacked the myofibrils seen in normal larval muscle. We conclude that CiMDF is a bona fide MRF because it elicits ectopic myogenesis, and that it is essential for tail muscle development. Awards from the NIH (1 R15 HD047357-01) and Rhode Island College to THM, and the Association Francaise contre les Myopathies (AFM) to HY supported this work.

FC‐27 A spontaneous dog model for in vivo gene therapy of junctional epidermolysis bullosa

Junctional epidermolysis bullosa (JEB) is a mechanobullous skin disease associated with mutations in the basement membrane components of the dermo‐epidermal junction. This condition is a suitable prototype for proving the feasibility of a gene therapy approach to genodermatoses. As successful genetic treatment requires immunocompetent animal models to study the possible host immune response to the transgene, we have characterized a breed of short‐haired pointers suffering from a mild form of JEB. These animals exhibit skin blistering and erosions following minor trauma. Immunohistochemical analysis of the skin biopsies and immunoprecipitation of spent medium from cultured JEB keratinocytes showed reduced expression and secretion of the α3 chain of laminin 5 (α3, β3, γ2), the major adhesion ligand of basal keratinocytes. The search for genetic mutations detected a homozygous insertional mutation (4818 + 207 ins6.5 kb) in intron 35 of the lama3 gene. Consequently, keratinocytes from dogs with JEB secrete reduced amounts of wild‐type laminin 5, and adhesive properties of the keratinocytes are compromised. Retroviral transfer of wild‐type dog α3 cDNA into JEB keratinocytes enhanced secretion of laminin 5 in the extracellular matrix and restored the adhesion, differentiation and proliferative capacity of the transduced cells. Transplantable fibrin‐based skin equivalents made with transduced JEB keratinocytes and grafted onto SCID mice generated normal cohesive and stratified epithelia and showed stable localized deposition of laminin 5 at the dermo‐epidermal junction. Our results form the basis for preclinical assays of gene therapy on a unique immunocompetent animal model for an inherited skin disease. Funding: DEBRA (UK) Foundation, Association Francaise Contre les Myopathies.

The Ability of the Association Française Contre les Myopathies to Promote Research in Neuromuscular Diseases

The Association Francaise contre les Myopathies (AFM) is now one of the most powerful patient groups in the field of human genetics. This paper tells the history of this successful association, which supports research in genetics and muscle physiology or pathophysiology, and has raised awareness of the plight of neuromuscular patients. AFM has acted as a catalyst in promoting and boosting scientific advances by identifying bottlenecks, filling gaps, providing instant resources for innovative projects, and lobbying for new legislation.