194th 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

Abstract

1. IntroductionTwenty-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.1.1 Aim of the workshopDuchenne muscular dystrophy (DMD) is a severe, progressive muscle-wasting disorder that affects ∼1 in 5000 newborn males [1Mendell J.R. Shilling C. Leslie N.D. et al.Evidence-based path to newborn screening for Duchenne muscular dystrophy.Ann Neurol. 2012; 71: 304-313Crossref PubMed Scopus (527) Google Scholar, 2Moat S.J. Bradley D.M. Salmon R. Clarke A. Hartley L. Newborn bloodspot screening for Duchenne muscular dystrophy: 21 years experience in Wales (UK).Eur J Hum Genet. 2013; ([epub Jan 23])PubMed Google Scholar]. 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 [3Hoffman E.P. Fischbeck K.H. Brown R.H. et al.Characterization of dystrophin in muscle-biopsy specimens from patients with Duchenne’s or Becker’s muscular dystrophy.N Engl J Med. 1988; 318: 1363-1368Crossref PubMed Scopus (754) Google Scholar, 4Monaco A.P. Bertelson C.J. Liechti-Gallati S. Moser H. Kunkel L.M. An explanation for the phenotypic differences between patients bearing partial deletions of the DMD locus.Genomics. 1988; 2: 90-95Crossref PubMed Scopus (923) Google Scholar]. Corticosteroids are the only drugs that have shown a beneficial effect, however treatment is only symptomatic [5Bushby K. Finkel R. Birnkrant D.J. et al.Diagnosis and management of Duchenne muscular dystrophy, part 1: diagnosis, and pharmacological and psychosocial management.Lancet Neurol. 2010; 9: 77-93Abstract Full Text Full Text PDF PubMed Scopus (1318) Google Scholar, 6Bushby K. Finkel R. Birnkrant D.J. et al.Diagnosis and management of Duchenne muscular dystrophy, part 2: implementation of multidisciplinary care.Lancet Neurol. 2010; 9: 177-189Abstract Full Text Full Text PDF PubMed Scopus (811) Google Scholar] 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]Aartsma-Rus A. Antisense-mediated modulation of splicing: therapeutic implications for Duchenne muscular dystrophy.RNA Biol. 2010; 7Crossref PubMed Scopus (62) Google Scholar]. After extensive optimization in patient-derived cell cultures and multiple mouse and dog models (reviewed in [[7]Aartsma-Rus A. Antisense-mediated modulation of splicing: therapeutic implications for Duchenne muscular dystrophy.RNA Biol. 2010; 7Crossref PubMed Scopus (62) Google Scholar]), proof of concept (dystrophin restoration) has been achieved after local intramuscular injections and systemic treatment for both 2OMePS and PMO AONs [8Cirak S. Arechavala-Gomeza V. Guglieri M. et al.Exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: an open-label, phase 2, dose-escalation study.Lancet. 2011; 378: 595-605Abstract Full Text Full Text PDF PubMed Scopus (680) Google Scholar, 9Goemans N.M. Tulinius M. van den Akker J.T. et al.Systemic administration of PRO051 in Duchenne’s muscular dystrophy.N Engl J Med. 2011; 364: 1513-1522Crossref PubMed Scopus (583) Google Scholar, 10Kinali M. Rechavala-Gomeza V. Feng L. et al.Local restoration of dystrophin expression with the morpholino oligomer AVI-4658 in Duchenne muscular dystrophy: a single-blind, placebo-controlled, dose-escalation, proof-of-concept study.Lancet Neurol. 2009; 8: 918-928Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar, 11van Deutekom J.C. Janson A.A. Ginjaar I.B. et al.Local dystrophin restoration with antisense oligonucleotide PRO051.N Engl J Med. 2007; 357: 2677-2686Crossref PubMed Scopus (688) Google Scholar]. 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.1.2 The issue of mutation specificityAnnemieke 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]Aartsma-Rus A. Fokkema I. Verschuuren J. et al.Theoretic applicability of antisense-mediated exon skipping for Duchenne muscular dystrophy mutations.Hum Mutat. 2009; 30: 293-299Crossref PubMed Scopus (407) Google Scholar]. 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).2. State of the art2.1 Currently ongoing clinical trials for exon 51 skippingMultiple 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 childhood onset disorder. Furthermore, due to the mutation specificity of the approach, it is also potentially the first of a family of AON drugs for DMD patients. It has been postulated previously that after the first two AON compounds are developed fully, a faster development path for AONs of the same chemistry could be considered [[13]Muntoni F. 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.Neuromuscul Disord. 2010; 20: 355-362Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar]. Multiple drisapersen trials are currently ongoing (see Table 1). Furthermore, GSK is planning additional trials for drisapersen in non-ambulant boys and adults and in very young children.Table 1Clinical trials ongoing with drisapersen at the time of the workshop.Trial numberTestWeekly doseDurationNumber of patientsStatusDMD114673OLE following phase I/II dose escalation study6 mg/kgNA12Ongoing>150 weeksDMD114117Compare weekly vs intermittent dosing vs placebo6 mg/kg48 weeks53Complete, results presentedDMD114044Double-blind placebo controlled phase III trial6 mg/kg48 weeks186Recruitment completeDMD114876Dose comparison study3 vs 6 mg/kg48 weeks⁎24Weeks treatment followed by 24weeks follow up.51Recruitment completeDMD114349OLE for DMD114117 and DMD114044 studies6 mg/kgNA150OngoingOLE is Open Label Extension; NA is not applicable. 24 Weeks treatment followed by 24 weeks follow up. Open table in a new tab For the open label extension study following the escalating dose study [[9]Goemans N.M. Tulinius M. van den Akker J.T. et al.Systemic administration of PRO051 in Duchenne’s muscular dystrophy.N Engl J Med. 2011; 364: 1513-1522Crossref PubMed Scopus (583) Google Scholar] all 12 patients have now been treated for over 3 years, providing a wealth of information on long term safety and tolerability. Patients were treated with weekly subcutaneous injections for the first 72 weeks, after which they were off treatment for 8 weeks, followed by an intermittent dosing scheme of 8 weekly injections and a treatment free period of 4 weeks. Injection site reactions were the most frequently observed side effect. This is a common observation for 2′-O-modified PS AONs that are delivered to human subjects though the subcutaneous route. Proteinuria was observed in all patients, but at levels that do not cause clinical concern (renal function is unaffected); indeed the proteinuria is reversible during off treatment periods and although it reappears following treatment re-initiation, its severity does not appear to increase on the longer term. Transient thrombocytopenia has been observed in the open label extension study following the published dose escalation study [[9]Goemans N.M. Tulinius M. van den Akker J.T. et al.Systemic administration of PRO051 in Duchenne’s muscular dystrophy.N Engl J Med. 2011; 364: 1513-1522Crossref PubMed Scopus (583) Google Scholar], but no levels reached <75 × 109/L.The patients involved in this trial have been followed with the 6 min walk test. Between the end of the dose escalation trial and the onset of the OLE trial, 2 boys had lost ambulation. Of the remaining 10, 2 have lost ambulation during the OLE trial, while the remaining 8 have remained ambulant; in 7 of these boys the distance walked in 6 min has been maintained or improved for over 3 years compared to the baseline. Despite the fact that a placebo group was not present in this study, the data are encouraging as they diverge significantly from the natural history studies.Recruitment for the other studies has been completed and results of the phase III study are expected in Q4 of 2013. The results of the dosing regimen study have recently been presented at a meeting (April 11, Cold Spring Harbor, NY, USA). In exploratory study, the continuous treatment arm (6 mg/kg/weeks, n = 18) showed a statistically significant difference from placebo (n = 18) on 6MWD at 24 weeks (mean, 35.09 m; [95% CI, 7.59–62.60 m], p = 0.014), with trends supportive of efficacy in other timed function tests and the North Star Ambulatory Assessment (NSAA). Also at 48 weeks an encouraging trend differentiating from placebo (35.84 m [−0.11 to 71.78 m], p = 0.051) was observed. The intermittent treatment arm (n = 17) did not separate from placebo at week 24, though by week 48 there was a clinically meaningful trend from placebo on 6MWD (27.08 m [−9.83 to 63.99 m], p = 0.147), supported by trends in timed function tests and the NSAA. There was little change in muscle strength at either time point for either treatment arm. Drisapersen was generally well tolerated, with the majority of adverse events related to injection site reactions and proteinuria (John Kraus, personal communication).The dose comparison study is ongoing in the USA. Results from this study are anticipated in Q1 2014. More than 100 patients have been enrolled in the second open label extension study. Notably, out of the over 300 patients currently involved in drisapersen trials the retention rate has been very high (96%).Jerry Mendell presented on ongoing eteplirsen trials (coordinated by Sarepta). Following the dose escalation study which tested doses of up to 20 mg/kg eteplirsen, [[8]Cirak S. Arechavala-Gomeza V. Guglieri M. et al.Exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: an open-label, phase 2, dose-escalation study.Lancet. 2011; 378: 595-605Abstract Full Text Full Text PDF PubMed Scopus (680) Google Scholar] the current trial compares 30 and 50 mg/kg doses. For the first 24 weeks groups of 4 patients received 30 or 50 mg/kg eteplirsen or placebo. Then, the placebo group was stopped and split into two groups of 2 patients receiving either 30 or 50 mg/kg. Biopsies were obtained before treatment, after 12 weeks for the 50 mg/kg group and 2 placebo treated patients, after 24 weeks for the 30 mg/kg group and 2 placebo treated patients and after 48 weeks for all patients. Patients were unblinded to the arm in which they participated in week 36.Dystrophin analysis was performed by immunofluorescent analysis and the percentage of dystrophin positive cells was assessed for each patient based on at least 1000 muscle fibers by blinded investigators. In the patients treated for 48 weeks, the percentage of dystrophin positive fibers had increased significantly to 52% and 42% for the 30 and 50 mg/kg groups, respectively. For the patients receiving first placebo and then treatment for 24 weeks, the percentage increased to 34% and 43% for 30 and 50 mg/kg groups. No dystrophin was observed for the 50 mg/kg group after 12 weeks of treatment, while a significant increase was observed for the 30 mg/kg group after 24 weeks of treatment. These data, which will need to be supported by detailed analysis of the level of dystrophin produced by the fibers, suggest that chronic administration of a lower (30 mg/kg) dose is more efficient than a higher (50 mg/kg) dose administered over a shorter period of time, echoing data from preclinical models [[14]Malerba A. Sharp P.S. Graham I.R. et al.Chronic systemic therapy with low-dose morpholino oligomers ameliorates the pathology and normalizes locomotor behavior in mdx mice.Mol Ther. 2011; 19: 345-354Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar].These patients were followed with the 6 min walk test as well. For patients receiving 50 mg/kg the distance they were able to walk stabilized, while it decreased for the group receiving placebo (24 weeks) and decreased further for the first 12 weeks of treatment (until week 36) and then stabilized. For the 30 mg/kg group 2 patients (identical twin brothers) quickly lost ambulation in the early phase of the study and were removed from further analysis. Aside from a single proteinuria event no drug related adverse events were reported.2.2 What is known for 2OMePS and PMOs from preclinical and clinical trialsIn preclinical and clinical pharmacokinetic/pharmacodynamic studies AON plasma and tissue levels are monitored and related to molecular and clinical outcome measures. Sjef de Kimpe presented the various 2OMePS AON detection systems used at Prosensa to assess plasma and tissue levels, whole body distribution, tissue localization, and metabolites, such as an hybridization ELISA-like assay, radioactive screening, in situ hybridization, and HPLC (with mass spectrometry). Similar systems are also in place (HPLC) or being developed (ELISA-like system) to detect PMOs. Each system has advantages and limitations. Notably, these systems cannot reveal in which compartment of the tissue the AON is located (e.g. in the muscle fibers or in the interstitial space or fibro-adipose tissue or cytoplasmic versus nuclear location).Pete Sazani presented pharmacokinetic studies with different PMOs. Studies in animal models show very good tolerability even at very high doses of PMOs [[15]Wu B. Lu P. Benrashid E. et al.Dose-dependent restoration of dystrophin expression in cardiac muscle of dystrophic mice by systemically delivered morpholino.Gene Ther. 2010; 17: 132-140Crossref PubMed Scopus (101) Google Scholar] and experience so far suggest there does not seem to be dose limitations due to safety issues in humans either. As PMOs are non-charged and small enough to be filtered out by kidney they have a very short serum half-life (2–3 h) and the vast majority is excreted via urine on the first pass (∼95% of PMO is present in urine within 30 min). Only minimal amounts are taken up by muscle.Studies in primates using intravenous or subcutaneous doses of 5–320 mg/kg/kg for 12 weeks revealed good tolerability of PMOs. On histology, some basophilic material (probably the PMO) could be observed in kidney tubular epithelium, but this did not affect kidney function and disappeared with turnover of kidney epithelial cells. When comparing data in rodents, non-human primates and humans it appears that the dosing needs to be scaled by weight (i.e. the same dose in mg/kg is used for different models regardless of their size and there is no allometric conversion as is the case for 2OMePS AONs).Ed Kaye presented data on PMOs tested in healthy volunteers and patients suffering from other diseases such as cancer and hepatitis. These PMOs were 25–30mers and the PK properties were similar for different PMOs regardless of size and sequence composition. Furthermore, PK was similar for different species, as also already observed for DMD PMOs. For each PMO higher doses resulted in higher half-lives, although the doses tested were relatively low (up to 90 mg in total). Excretion takes place primarily through urine for all PMOs tested so far.In addition to regular PMOs some preclinical development has been done for peptide linked PMOs (p-PMOs). Most advanced studies have been performed with the B-peptide and the PMO-plus compound, which unfortunately both resulted in significant kidney toxicity in non-human primates. While the development of these p-PMOs has been discontinued, both Sarepta and the MDEX consortium in UK are pursuing the search of other p-PMOs with an acceptable therapeutic index.Sjef de Kimpe presented PK and absorption, distribution, metabolism and excretion (ADME) data on 2OMePS AONs. Also here profiles were similar for different AONs. However, unlike the PMO, this chemistry is charged and binds serum proteins with low affinity, which prolongs their circulation time, half-lives, and uptake by muscle. The length of the AON typically influences the strength of serum protein binding.Biodistribution studies revealed that except for the brain and the spinal cord, 2OMePS AONs are taken up by all tissues upon systemic delivery. They could still be detected 28 days after delivery, albeit at lower levels. The half-life appears to be 4 weeks for most tissues, while clearance is quicker from kidney. PK analysis in patients revealed that 2OMePS AONs are quickly cleared from the plasma and then distributed to muscle and other tissues. Multiple dosing leads to increasing pre-dose trough levels. The serum half-life in the second phase (after uptake by tissues) in patients is also ∼4 weeks.Metabolite analysis with HPLC for 2OMePS AONs revealed that after 24 h mainly the parent AON was present, while after 7 days some metabolites were evident (missing e.g. n−1, n−2, n−3 etc.). The behavior of the metabolites is very predictable: the n−1 and n−2 AONs still induce some exon skipping, while those with more nucleotides missing are inactive.Data were presented on 2OMePS AONs to induce DMD exon 45 and 53 skipping. The safety analysis gave very similar results for both AONs and when compared to PRO051. In monkeys pro-inflammatory class effects are typically observed at high doses of 2OMePS AONs, e.g. complement activation, lymphoid proliferation, release of cytokines and chemokines and the detection of split complement factors. However, this appears to be a species specific effect. In humans mainly injection site reactions are observed, while a rise in systemic inflammatory response factors or a decrease in complement are not characteristic findings. 2OMePS AONs accumulate in kidney (tubular epithelium) and liver. Proteinuria is often observed in 2OMePS AON treated humans, but liver and kidney function parameters are generally normal.Sjef de Kimpe also presented on preclinical safety studies for 2OMePS AONs targeting exon 51 and exon 44 (up to 9 months data available) and exon 45 and exon 53 (3 month data available). These did not reveal novel toxicity and confirmed that each 2OMePS AON behaved in a similar fashion. When discussing the possibility of an expedited development path for additional 2OMePS AONs, the concern of regulators is toxicity outside the well documented class effects. However, for DMD the risk for this is minimal as (1) exaggerated pharmacology (the drug working too well) is not an issue, since the effect of the AONs is limited by the number of dystrophin transcripts (2) off target effects have not been observed and remain unlikely since the AONs are designed with high DMD gene sequence-specificity and 2OMePS chemistry is sensitive to mismatches (3) the structural diversity of 2OMePS AONs is limited compared to small molecules (4) the 2OMePS compound closely resembles natural RNA nucleotides (5) the AONs have predictable metabolites with similar structural constraints and (6) AON metabolites have either reduced biological activity (n−1, n−2) or no biological activity (when more nucleotides are removed), so derivatives are not expected to be a safety concern. Thus for future 2OMePS compounds 3 month toxicity studies would give adequate information for risk assessment. This developmental plan has been considered acceptable.Art Levin presented the comments of the FDA on the NDA/safety data on the mipomersen study done by ISIS Pharmaceuticals and future implications for AON therapeutics. Mipomersen is a 20mer 2’-O-methoxyethyl phosphorothioate/phosphorothioate gapmer AON that works through RNase H cleavage of ApoB transcripts of patients with familial hypercholesterolemia (a rare disease leading to death in the 3rd decade of life due to cardiovascular complications). The FDA advisory panel has recommended approval and this has resulted in approval in the USA, while approval in EU has not been granted.The data from the clinical trial revealed that the compound is active: LDL levels were lower after 5 weeks of treatment. Some side effects were observed: proteinuria (as anticipated for this class of compounds) and increased ALT levels and in liver fat (as a consequence of ApoB inhibition). Other reported side effects were injection site reactions and flu-like symptoms, which resulted in discontinuation of 10% of patients involved in the trial. No activation of complement or increases in complement split products were detected in any of the patients, while this was observed for monkeys in preclinical safety tests (at 7 times the clinical exposure tested in patients). Malignant neoplasms were observed in some treated patients, but the incidence was similar to that observed in analogous patient populations. In many patients antibodies to mipomersen (or antibodies able to bind mipomersen) were observed, especially in patients with high plasma trough concentrations. There was no apparent effect of these antibodies on safety or efficacy.3. Response to treatmentFrancesco 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]Cirak S. Arechavala-Gomeza V. Guglieri M. et al.Exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: an open-label, phase 2, dose-escalation study.Lancet. 2011; 378: 595-605Abstract Full Text Full Text PDF PubMed Scopus (680) Google Scholar]. Various causes for the difference in response after this 12 weeks trial were assessed. Plasma levels per se or genotype did not appear to be a major contributory factor. To assess whether non responsiveness was caused by intrinsic differences between patients, an in vitro test on patient-derived cell cultures was performed. Fibroblasts were isolated from a skin biopsy and forced into myogenesis by MyoD transduction and then treated with AONs and studied using a quantitative PCR test. No difference in exon skipping levels was observed between responsive and non-responsive patients. This further underlines that all patients had the potential to respond and that their non-responsiveness was probably due to a lack of PMO in the muscle that was biopsied. Indeed differences in the amount of dystrophin positive fibers in different fascicles of the same muscle were noted in this study, and have been previously well documented in the mdx mice, indicating the limitation of considering patients as “responders or non-responders” based on a single and limited muscle biopsy sample. Indeed it has been reported in the mdx mouse model that multiple treatments over time with a low PMO dose results in more dystrophin positive cells than a single high dose [[16]Malerba A. Thorogood F.C. Dickson G. Graham I.R. Dosing regimen has a significant impact on the efficiency of morpholino oligomer-induced exon skipping in mdx mice.Hum Gene Ther. 2009; 20: 955-965Crossref PubMed Scopus (50) Google Scholar]. This indirectly suggests that a number of non-responders in this trial might have responded if they had been treated for longer. However, it is clear that response to treatment is a multifactorial issue, of which drug exposure is most likely one component, but for which many components are still unknown.3.1 Anticipated responseThe exon skipping approach aims to convert the DMD into a BMD phenotype. Thus, more information on the natural history of BMD patients is warranted to allow better prediction of the possible beneficial effects of exon skipping.Jan Verschuuren presented a comprehensive study in a cohort of 27 BMD patients, from whom a biopsy was taken at the time of muscle function assessment. Dystrophin levels were assessed with quantitative western blotting and varied between 17% and 74% of normal. No correlation was found between dystrophin levels and muscle strength, the mean fat fraction (assessed by MRI), or disease progression (as defined by the age of disease milestones). A significant subset of patients carried an exon 45–47 deletion (13 patients). Also for these patients, who expressed varying levels of an identical internally deleted dystrophin, no correlation was observed between dystrophin levels and muscle function, quality or strength. However, for this subgroup a significant correlation between age and muscle strength and between age and mean fat fraction was observed. This suggests that the disease progresses at a relatively homogeneous way in patients with a similar mutation, but that disease progression is not directly correlated with dystrophin levels, provided these levels exceed a minimal threshold.Francesco Muntoni presented work on quantification of dystrophin levels assessed with immunofluorescent staining and western blot for DMD patients needing exon 51 skipping and their BMD counterparts. For these patients, a good correlation between dystrophin levels and severity was found [[17]Anthony K. Cirak S. Torelli S. et al.Dystrophin quantification and clinical correlations in Becker muscular dystrophy: implications for clinical trials.Brain. 2011; 134: 3547-3559Crossref PubMed Scopus (104) Google Scholar]. However, for BMD mutations as those that would result from exon 4