Workshop report194th ENMC international workshop. 3rd ENMC workshop on exon skipping: Towards clinical application of antisense-mediated exon skipping for Duchenne muscular dystrophy 8–10 December 2012, Naarden, The Netherlands
Introduction
Twenty-seven participants from 9 countries (Australia; Belgium; England; France; Germany; Italy; Japan; The Netherlands; USA) attended the third ENMC workshop on exon skipping “Towards clinical application of antisense-mediated exon skipping for Duchenne muscular dystrophy.” The topic of this workshop was on ‘Streamlining the development path of exon skipping compounds’ and followed the formula of similar workshops held in 2004 on ‘Antisense oligonucleotides in DMD’, which focused on intramuscular administration of antisense oligonucleotides and in 2007 on ‘Planning Phase I/II Clinical Trials using systemically delivered Antisense Oligonucleotides in Duchenne Muscular Dystrophy (DMD)’.
The workshop was organized with the support of Parent Project Muscular Dystrophy (PPMD), Duchenne Parent Project (the Netherlands) and Parent Project Onlus (Italy), in addition to the ENMC, and was attended by representatives of the three companies involved in the clinical development of exon skipping, Prosensa and GlaxoSmithKline for the 2′-O-methyl phosphorothioate RNA modified (2OMePS) AONs and Sarepta (previously AVI Biopharma) for the phosphorodiamidate morpholino oligomers (PMOs), as well as patient representatives, academics (scientists and clinicians) and an expert associated to the European Medicine Agency (EMA) who, while participating as an individual brought broad regulatory perspective to the workshop.
Duchenne muscular dystrophy (DMD) is a severe, progressive muscle-wasting disorder that affects ∼1 in 5000 newborn males [1], [2]. The disease is caused by mutations in the dystrophin encoding DMD gene. The dystrophin protein normally provides muscle fibers with stability during contraction by linking the cytoskeleton to the extracellular matrix. In DMD patients mutations disrupt the open reading frame, leading to a prematurely truncated dystrophin protein that cannot fulfill its linker function. By contrast, the less severe Becker muscular dystrophy (BMD) is caused by mutations that maintain the reading frame, allowing the production of internally deleted proteins that have (partially) maintained their linker function [3], [4]. Corticosteroids are the only drugs that have shown a beneficial effect, however treatment is only symptomatic [5], [6] acting to slow disease progression. Despite improved care, most DMD patients die by the third or fourth decade of life due to respiratory or cardiac complications, and in the latter stage of their condition are highly affected by severe and generalized muscle weakness that precludes almost all voluntary movement.
In the absence of curative treatment, the exon skipping approach aims to convert the severe DMD into a milder BMD phenotype by modulating the pre-mRNA splicing of the dystrophin transcript. This can be achieved with antisense oligonucleotides (AONs), pieces of chemically modified DNA or RNA that target specific exons, interfere with the splicing machinery and cause them to be excluded (skipped) from the mature mRNA. In this manner the open reading frame can be restored, allowing the production of a partially functional BMD-like dystrophin rather than a non-functional DMD-like dystrophin [7]. After extensive optimization in patient-derived cell cultures and multiple mouse and dog models (reviewed in [7]), proof of concept (dystrophin restoration) has been achieved after local intramuscular injections and systemic treatment for both 2OMePS and PMO AONs [8], [9], [10], [11]. Currently, AON-induced exon skipping is being tested in hundreds of DMD patients in late phase clinical trials and is viewed by many as the most promising approach for DMD.
Annemieke Aartsma-Rus introduced the issue of applicability and mutation specificity. Exon skipping aims to restore the reading frame. Therefore, different exons have to be skipped for patients with different mutations. Exon skipping will not apply to all mutations, as a dystrophin protein lacking the dystroglycan binding domain (encoded by exon 64–70), all its actin binding domains (encoded by exon 2–8 and 35–44) or over 75% of its central rod domain is not functional. Fortunately, DMD mutations (mostly deletions) cluster in a hotspot region (between exon 42 and 55), and these are generally amenable to exon skipping. Due to the clustering of mutations, skipping certain exons applies to larger groups of patients, e.g. exon 51 skipping would apply to the largest group of patients (13%) and is currently being tested in clinical trials. However, the majority of patients theoretically eligible for exon skipping requires the skipping of an exon that would only benefit a very small subset of patients (less than 0.5% of all patients) [12]. As each AON may be considered as a separate drug, the clinical development of AONs targeting these exons will be relatively lengthy and expensive, but especially challenging when the issue of demonstrating clinical efficacy for very small population of affected individuals is considered. The aim of the ENMC workshop was to discuss strategies to allow a streamlined way forward involving all key stakeholders (patient representatives, academic researchers, clinicians, industry representatives, with the insight of the regulatory authorities).
Section snippets
Currently ongoing clinical trials for exon 51 skipping
Multiple clinical trials are currently ongoing for drisapersen (a subcutaneously delivered 2OMePS AON targeting exon 51, previously known as GSK2402968/PRO051, developed by GSK and Prosensa) and eteplirsen (an intravenously delivered PMO targeting exon 51, previously known as AVI4658, developed by Sarepta, previously AVI Biopharma).
Padraig Wright (GSK) indicated that drisapersen development is being done thoroughly, since it is potentially the first AON drug intended for lifelong treatment of a
Response to treatment
Francesco Muntoni presented the published eteplirsen dose escalation trial that took place in the UK in which 6 groups of children received ascending doses of the PMO to induce exon 51 skipping. In the 2 higher doses (10 and 20 mg/kg for 12 weeks) 80% of patients appeared to show a response in terms of protein and skipping although there was variability, with some good responders and some poor responders [8]. Various causes for the difference in response after this 12 weeks trial were assessed.
Outcome measures
Especially for future trials that of necessity will have to be conducted in smaller groups of patients, good outcome measures will be crucial.
Towards developing exon skipping for small subsets of patients
At the moment each AON is observed by the EMA as a different entity, and thus has to be tested separately. For exons or combinations of exons that would only benefit small numbers of patients, however, clinical development will be very challenging. With current regulations it will not be feasible to test AONs targeting different exons in a single trial (e.g. in a group of patients with small mutations in in-frame exons). However, once multiple AONs of a certain class chemistry have been shown
Developing improved chemistries and delivery methods
Additional AON chemistries and delivery methods are currently in development. Dominic Wells presented data for peptide conjugated PMO (Pip6a-PMO) studies in mdx mice. Unlike their non-conjugated counterparts, Pip6a-PMOs are able to induce relatively high levels of exon skipping in both skeletal muscle and heart. Repeated systemic injections with Pip6a-PMO improved specific force and normalized force drop after eccentric concentration of the tibialis anterior. Volker Straub presented the effects
Cardiac issues
Francesco Muntoni presented an analysis of cardiac issues in DMD patients. With improved ventilatory support more patients now die from heart pathology (∼40%), and almost all patients (at least 90%) have cardiac symptoms). Cardiac problems arise earlier than usually assumed; some patients already show signs of cardiomyopathy at 10 year old, but these often remain undetected because the patients do not show symptoms due to their physical limitations. It is clear that early detection and treatment
Patient perspective
Three patient representatives (Kathi Kinnett, Parent Project Muscular Dystrophy, USA, Elizabeth Vroom, Duchenne Parent Project, the Netherlands and United Parent Projects Muscular Dystrophy and Francesca Ceradini, Parent Project Onlus, Italy) attended the meeting. They stressed that the development of exon skipping will require a combined effort of Industry, clinicians, scientists, regulators and patient representatives. Communication and regular updates to the patients will be of key
Future outlook
These are exciting times for the AON-mediated exon skipping field. However, it is clear that combined efforts of all key stakeholders are needed to develop this approach for as many patients as possible.
Communication will be key and the patient organizations hope to obtain commitment from the companies involved in exon skipping to provide correct and timely updates (e.g. 3–4 times per year) to patient community representatives, who can then translate this information (if needed) and distribute
Workshop participants
Dr. Annemieke Aartsma-Rus, Leiden University Medical Center, The Netherlands.
Dr. Pavel Balabanov, European Medicines Agency, United Kingdom.
Dr. Prisca Boisguerin, Université Montpellier 2, Montpellier Cedex, France.
Dr. Giles Campion, Prosensa Therapeutics, Leiden, The Netherlands.
Dr. Francesca Ceradini, Italian Parent Project, Onlus, Italy.
Dr. Judith van Deutekom, Prosensa Therapeutics, Leiden, The Netherlands.
Prof. George Dickson, Biological Sciences, Royal Holloway College, London, United
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