CORDIS - EU research results

A phase I/IIa clinical trial in Duchenne muscular dystrophy using systemically delivered morpholino antisense oligomer to skip exon 53

Final Report Summary - SKIP-NMD (A phase I/IIa clinical trial in Duchenne muscular dystrophy using systemically delivered morpholino antisense oligomer to skip exon 53)

Executive Summary:
DMD is a lethal and incurable muscle wasting disease due to the inability to produce dystrophin in muscle due to mutations in the dystrophin gene. The DMD incidence spans a range of 2.2 to 5.5 per 10 000 newborn males. Loss of dystrophin in DMD results in muscle degeneration, inflammation and replacement of muscle with adipo-fibrous tissue. The dystrophin gene is composed of different sections called exons that are joined together like a puzzle. In some patients with DMD one or more exons are “deleted” resulting in a non-functional protein as the missing parts in the puzzle do not match with each other (for a visualisation of the effect of different deletions on the DMD gene visit: In the milder form of Becker muscular dystrophy (BMD), also due to mutations in the dystrophin gene, a shortened but functional dystrophin protein is produced as the missing pieces of the puzzle allow the two parts to be joined together ( Most BMD patients are able to walk into late adulthood and have a normal lifespan. Therefore, therapeutic strategies that would genetically aim to create a milder BMD form of the disease is an attractive possibility for DMD patients. One of the therapeutic strategies is the skipping of some specific exons of the dystrophin gene using antisense oligonucleotides (AOs). A consortium was created to develop a particular AO for exon 53 (SRP-4053, which can induce a change in the way the unfavourable DMD deletion is processed by the transcriptional machinery in the muscle in DMD boys with specific mutations. In addition the consortium designed and performed a randomized control clinical trial in 4 sites of 3 EU Member state nations (UK; France; Italy) to address the safety, biochemical efficacy and exploratory clinical efficacy of SRP-4053 and to validate novel outcome measures in DMD such as muscle magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS). Treated boys are compared to DMD boys with DMD deletions who cannot receive the SRP-4053 due to their different mutations. Originally designed to recruit 12 children, the study exceeded the recruitment target and currently has 25 treated children who are receiving the weekly SRP-4053. In view of the larger study, and the decision to continue the treatment period for longer than the originally planned 48 weeks, the study is currently ongoing. Safety of the drug has been demonstrated so far with no drug related severe adverse event, and all children continuing to receive regularly the medication. The study is currently ongoing according to the plan of the most recent protocol.
Project Context and Objectives:
Antisense oligonucleotides (AOs) designed to target RNA and modulate pre-mRNA splicing to restore functional protein offer great promise as a therapeutic intervention for human diseases including rare orphan diseases (ROD) such as Duchenne muscular dystrophy (DMD). The development of novel, effective medications for treating such conditions is hampered by their individual rarity. Few patient numbers, frequently distributed over a wide geographical area, lack of natural history data, poor understanding of the patho-mechanisms and difficulty in finding a drug sponsor, collectively impede progress in finding a cure for these conditions.
DMD is a lethal and incurable muscle wasting disease due to the inability to produce dystrophin in muscle, secondary to mutations which affect the X-linked DMD gene open reading frame (ORF). The DMD incidence spans a range of 2.2 to 5.5 per 10 000 newborn males, with an average incidence of 1: 5,000 and each year approximately 800 affected boys are born in EU, despite prenatal diagnostic screening efforts. This is due to the fact that there is a high incidence of de novo mutations (because of the large gene size), so even if screening operates at maximum efficiency, new patients are still being born, emphasising the necessity to identify a therapy. Loss of dystrophin in DMD results in muscle degeneration, inflammation and replacement of muscle with adipo-fibrous tissue. In the milder allelic Becker muscular dystrophy (BMD), DMD mutations do not disrupt the ORF, a shortened but functional dystrophin protein is produced, and most patients are able to walk into late adulthood and have a normal lifespan. Therefore, inducing exon skipping by using antisense AOs in order to restore the ORF is an attractive treatment strategy for DMD patients, as this will genetically create the milder BMD form of the disease. Most of the efforts so far have concentrated on skipping one particular exon of DMD pre-mRNA (exon 51), as its removal achieves restoration of the ORF in ~ 13% of DMD boys and the trial results have fulfilled proof-of principle: exon skipping is a viable therapeutic option for DMD patients. Indeed, the company which is taking forward in USA the AO that our UK consortium (MDEX Consortium) had previously developed to skip exon 51(Kinali et al, Lancet Neurology 2009; Cirak et al, Lancet 2011) has been seeking accelerated approval by the FDA and a final decision is currently awaited. This is a morpholino (PMO) AO which was trialled in a phase I (Kinali et al, Lancet Neurology 2009) and subsequently in a phase IIa study in UK (Cirak et al, Lancet, 2011, Muntoni PI of the studies).
In order to make AO available also to DMD children carrying different mutations, but also to increase the experience around AOs in DMD so that in the future it could be possible to obtain regulatory approval of different AOs based on the platform technology as opposed to each individual AO, rigorous safety and efficacy data is required and so a second clinical trial using a different AO with the same chemistry across a different exon is the next step. We chose exon 53 for our study and the novel AO was termed SRP-4053.
We assembled a consortium - SKIP-NMD - to allow synergy between academia, industry, DMD parental associations and advocacy groups to perform the exon 53 skipping trial. This consortium includes: 1) key EU academic opinion leaders in the translational research field of DMD; 2) four industrial companies including SMEs, including Sarepta Therapeutics a world-leader in PMO technology who will provide the drug and 3) six parental organisations, involved as interested parties.
The SKIP-NMD project objectives were to: (i) finalise the lead PMO sequence to induce exon 53 skipping (WP1); (ii) perform the required pre-clinical toxicology (WP2); (iii) design and perform a randomised control clinical trial at 4 locations in 3 EU countries (UK, France and Italy), that addresses the safety, biochemical efficacy and exploratory clinical efficacy of SRP-4053 (WP3 & 4); (iv) validate novel outcome measures that for the first time will provide a continuous link between the ambulant and non-ambulant phases of the condition in DMD boys and assess the role of non-invasive biomarkers, such as muscle magnetic resonance imaging (MRI) and spectroscopy (MRS) and serum micro RNA (miRNA) determination, in monitoring the response to therapeutic intervention (WP5) and (v) harmonise the approach to ethical procedures across the consortium (WP6).

Project Results:
Work progressed on schedule during the 42-months of the project. Based on the advances in the field, namely a better knowledge of the DMD natural history and the lessons learned from the results of previous exon skipping clinical trials, some changes were introduced in the planned clinical trial and natural history study (Work package 4 and 5). Specifically data from the previous exon 51 clinical trial in which the drug eteplirsen was administered to a group of DMD boys and results compared to a matched population of untreated children for a period of 4 years, demonstrated that a clear, statistical significant separation in the clinical course and primary outcome measure 6 Minute Walking test (6MWT) between the untreated and treated children could only be achieved at week 144 (Mendell et al, Ann Neurol. 2016 Feb;79(2):257-71). As a result, the SKIP-NMD clinical trial has been extended to 144 months of follow-up for all treated patients. This was achieved by additional resources made available by the industrial partner Sarepta. Moreover, in order to have more robust data on the safety and the exploratory efficacy, the decision was also made to recruit more children in the treatement arm compared to the original plan of 12 DMD boys. Indeed 25 children were recruited into the treatment arm. Consequently, the work related with these WP will continue beyond the 42 months of the project. A summary of the results from each work package (WP) is described below:
WP1. Eight AOs were blindly tested on different cell lines including immortalized myoblasts derived from DMD patients with different exon 53 skippable mutations. All induced specific efficient exon 53 skipping, in a concentration-dependent manner with no detectable toxicity. Low dose response curves were used to elucidate the optimal PMO for exon 53 skipping. The efficacy of this PMO to restore dystrophin protein was confirmed by western blot analysis of protein from treated patient cells carrying relevant mutations. This PMO is referred to as SRP-4053.
WP2. GLP industry-standard toxicology and safety pharmacology studies were performed by SRPT and CRL in accordance with regulatory requirements to ensure protocol submission for the clinical trial. SRPT developed the manufacturing process and analytical assays to safeguard the compound purity. Large scale GMP production of SRP-4053 has been assured in order to guarantee the preliminary manufacturing of two of the six batches required for the clinical trial.
WP3. Finalization of the clinical trial design and the Standard Operative Procedures for all the outcomes including the novel clinical exploratory measures has involved the formation of the Clinical Trial Management Group led by partner GOSH to oversee and organize the clinical trial. Five meetings attended by all scientific partners, Sarepta and representatives of 5 advocacy groups belonging to the 3 participating countries facilitated design of the clinical trial protocol. The necessary safety toxicology reports, the related ethics documents and the full Clinical Trial Application for the SRP-4053 First-in-Man clinical trial (4053-101) was submitted for review to the regulatory authorities following the European Medicine Agency Voluntary Harmonization Procedure for multinational trials in June 2014, with subsequent positive opinion. Favourable ethical opinions for the research study were obtained in all participating centres. (See:
The study was structured into: a dose escalation component followed by a maintenance/treatment part. In the dose escalation study (part 1), twelve patients amenable to exon-53-skipping (i.e. deletions of exons such as 42-52, 45-52, 47-52, 48-52, 49-52, 50-52, 52, or 54-58) of ≥6 years of age and above and ambulant (able to walk ≥ 250 metres in 6 minutes) were randomized to receive SRP 4053 (n=8) or placebo (n=4). Patients received a weekly IV infusion of SRP 4053/placebo at escalating dose levels, each for at least 2 weeks: 4 mg/kg/week in weeks 1-2; 10 mg/kg/week in weeks 3-4; 20 mg/kg/week in weeks 5-6; and 30 mg/kg/week beginning on week 7. After this, all patients (including the placebo treated boys) entered the open-label phase of the study. This was an open-label treatment with SRP-4053 at the highest tolerated dose as determined by the safety data in Part 1. In addition, a group of DMD patients with mutations not amendable to exon 53 skipping (13 untreated patients), along with up to 24 DMD patients with mutations not amenable to exon-53-skipping, matched for age and motor function. Treated patients underwent routine safety evaluations and exploratory efficacy assessments.
Specifically, all subjects had lower limb MRI scans and, in selected sites, also phosphorus MRS, at least at 3 time points. Physiotherapy assessment included validated and widely used outcome measures such as the 6-minute walk test , the North Star Ambulatory assessment , timed 4-step test; newly validated upper limb outcome measures, including tools measuring strength and fatigability, such as the Myotools moviplate, myopinch and handgrip, and a functional scale: the performance of upper limb (PUL). As an exploratory endpoint, all patients did wear an accelerometer (actimetry) device, developed by SME SYSNAV and Institut de Myologie, to monitor daily activity for the duration of the study. Quality of life data was collected by means of parental questionnaires. Finally, blood samples was collected for exploratory serum biomarker analyses and a comparison will be carried out between the treated and untreated cohorts. The primary biological endpoint will be the quantification of dystrophin detected by published methods which were refined considerably as part of the work of the consortium. The biochemical outcome measure will consist in comparing muscle biopsy samples collected at baseline and after 48 weeks of PMO treatment. The primary functional endpoint will be the change from baseline in the 6-minute walk test at the end of the treatment period.
WP4. As a result of the seamless collaboration and efforts put forth by the SKIP-NMD consortium partners directly involved with this work package, the clinical trial completed enrolment of Part 1 that was followed by a DSMB review that allowed proceeding with the open label phase of the study with the highest tolerated dose of SRP 4053 (30mg/Kg). The recruitment for the treated cohort for part 2 was completed on the 17th of February 2016, exceeding the recruitment goal of 24 subjects with a total enrolment of 25 treated subjects. The first 12 treated patients enrolled in the study have already or will shortly complete 48 weeks of treatment and will have a second muscle biopsy. As mentioned above, taking into account the developments in the field, in particular the results of previous exon-skipping clinical trials, a decision was made to extend the clinical trial to 144 weeks for the treated cohort. This decision was made in agreement with Sarepta that will sponsor this study extension. The results for the primary biological endpoint will be available soon after all the treated patients have the second muscle biopsy and the appropriate analyses are performed. The primary functional endpoint will be analysed and the results will be publicly available at the end of the clinical trial.
WP5. As of April 14th 2016, 11 patients were enrolled in the untreated control part 2 of the trial and recruitment is projected to remain open until end of June 2016 (13 patients recruited at the end of June). This untreated population of patients have been performing all the assessments as per protocol mentioned above in WP3 including all the well-known and validated but also the new/exploratory outcome measures.WP6. This aims to harmonise the approach to ethical procedures for all the partners involved following highest standard requirements in accordance with EU regulations. An Ethical Board has been established, chaired by an independent clinician, to promote, support and assure the highest standard for the process of consenting families in the clinical trial. Child-friendly cartoons to explain the condition have been developed for each centre ( and a train-set, which allows exon skipping to be explained in a playful and comprehensible way, has been included in the consent process. The SKIP-NMD website ( containing multi-lingual webpages, is part of this educational plan and has a central role in promoting an educational webinar (
WP7. We have disseminated SKIP-NMD project results to date via the SKIP-NMD website, via presentations at national and international meetings (including the prestigious 2015 Frontiers in Neuroscience Academy of Neurology meeting in Washington, and the International Neuromuscular Congress in Nice in 2014); 2 publications on dystrophin quantification and biochemical characterization of skippable mutations which are at the final stage of preparation, and poster presentations on dystrophin quantification methodologies at National and International meetings including the 2015 and 2016 MRC Neuromuscular translational Research meeting in UK; the 2014 International Neuromuscular Congress in Nice; the 2014 and 2015 World Muscle Society meetings in Berlin and Brighton, and with accepted posters in Granada 2016, also on and on the role of new functional outcome measures such as the ActiMyo and the Myotools.

Summary. The various activities described above have been achieved on time by mutual cooperation of the various partners involved in the different work packages, and refined both at the regular face-to-face consortium meetings of the entire consortium (London 2012; London 2013; Paris 2013; Newcastle 2014; Rome 2014; Cagliari 2015; London 2016), in addition to the regular teleconferences focused on the different work packages.

Potential Impact:
RNA therapeutics have been in clinical trials for over 15 years and in this time more than 3,000 patients and healthy volunteers have received AO therapy for up to and beyond one year. Currently about 60 AOs are in clinical trials, but despite this AOs remain relatively unknown in the market place. The greatest progress has occurred in developing therapies for ROD, particularly neuromuscular (e.g. DMD and myotonic dystrophy) and neurodegenerative (e.g. motor neuron disease; ataxias; Spinal Muscular Atrophy) disorders, where other effective treatments are unavailable. For the case of DMD, 100 new cases/60 million population arise annually with a European total of ~800. Since the European health burden for DMD healthcare is €150,000/annum/patient, this amounts to an annual bill of €120 million. Developing a successful AO therapeutic paradigm for DMD will contribute to reducing the corresponding health burden and generate novel marketable products, which together will realize socio-economic benefits to the EU community. By gaining more experience in the delivery, efficacy and safety profile of AO to DMD patients, a proven disease model, with validated outcome measures and in which there is a much wider window of therapeutic response compared to available other models, is therefore likely to influence the entire field of AO therapeutics across a variety of human ROD. DMD is a good disease model for optimizing AO delivery and efficacy mechanisms for two reasons. Firstly, ~65-70% of DMD cases have out-of-frame DMD deletions and secondly a much milder allelic form of the disease called Becker (BMD) is typically due to in-frame DMD deletions. It is therefore expected that the in-frame products following AO treatment of DMD cases should slow overall disease progression, improving both life span and quality. Secondly dystrophic skeletal muscle is targeted at appreciable levels by PMO AOs, as suggested by previous preclinical and clinical data.
The recent outcome of the long term (>144 weeks) systemic administration of eteplirsen (the PMO targeting exon 51) to DMD boys with eligible deletions, reported at several recent international meetings and in peer review publications (Mendell et al, Ann Neurol. 2016 Feb;79(2):257-71), clearly demonstrated stabilisation of the decline in boys receiving the PMO. After 3 years the boys receiving the PMO remained essentially stable in their 6 minute walk functional test, while controls declined at least 150 metres during the same period. Similarly, the respiratory function tests exhibited lack of disease progression in the treated boys. Based on these results, and more recent data presented at the FDA AdCom meeting in April 2016, in which the 4 year treatment data demonstrate not only a separation of more than 160 metres between treated and untreated patients, but also the steady increase number of children who lost the ability to walk in the untreated group at 4 years (85%) while only 17% of treated boys had lost the ability to walk during the same interval (data accessible on the Sarepta Therapeutics website), the FDA is considering an accelerated approval for this PMO and a final decision is awaited.
As for DMD boys skippable for exon 53, we recently demonstrated that the phenotype of the equivalent BMD patients is mild, with higher level of dystrophin protein production compared to BMD with deletions in other regions for example around exon 45, in whom the phenotype is also often more severe (Anthony et al, Neurology 2014). On the whole therefore we are confident that the BMD-like molecule induced by skipping exon 53 in DMD boys with eligible deletions will result in the production of an effective protein, which can be expected to slow down the disease progression in the treated boys, as for eteplirsen in boys skippable for exon 51.
Also, the current trial for exon 53 introduces several improvements that will answer to some of the criticisms raised by regulatory authorities for recently completed exon skipping trials. In particular, the current trial has a control (untreated) group, a higher number of treated patients compared to the previous Sarepta trial on eteplirsen (only 12 DMD boys received the active drug), longer follow-up, better defined outcome measures including some new ones such as muscle MRI which holds promises to capture disease progression in a quantifiable way, improved and standardized laboratorial techniques developed after feedback from FDA in the 2015 meeting organised by them, and also following the experience of the FDA filing for eteplirsen. In addition, more literature on DMD natural history is being published and becoming available to help interpreting clinical trials results in general and ours in particular.
Following on from our clinical trial we need to ensure that exon-skipping therapy is applicable to the wider DMD community as expediently possible across Europe. Regulatory approval for this AO chemistry as a class will be sort, which requires safety/toxicology data from several different individual AOs studies. Our project will generate data contributing to this stipulation, thus have considerable impact on gaining AO class approval. It will also contribute significantly with invaluable information regarding new outcome measures in DMD.

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