What Can We Learn From Clinical Trials of Exon Skipping for DMD?

On 20 September 2013, GlaxoSmithKline (GSK) and Prosensa announced that GSK's Phase III clinical trial (NCT01254019) of Drisapersen, an exon skipping drug for Duchenne muscular dystrophy (DMD), failed to meet the primary endpoint of a statistically significant improvement in the 6 Minute Walking Distance Test (6MWT) compared to placebo.1http://www.gsk.com/media/press-releases/2013/gsk-and-prosensa-announce-primary-endpoint-not-met-in-phase-iii-.htmlGoogle Scholar On 12 November 2013, Sarepta Therapeutics announced that the US Food and Drug Administration (FDA) has considered its application for accelerated approval of Eteplirsen as a DMD drug to be premature.2Sarepta Therapeutics http://investorrelations.sareptatherapeutics.com/phoenix.zhtml?c=64231&p=irol-newsArticle&ID=1875187&highlight=Google Scholar The news came as a great disappointment to the scientific community and more specifically to the families and foundations that had followed the trail of research articles and press announcements. Moreover, the clinical trial results and the FDA's view of the relationship between the dystrophin biomarker and functional endpoint of 6MWT in clinic will have a far reaching impact beyond exon skipping therapy in DMD. Currently, the most promising therapies for DMD are arguably gene therapy and exon skipping, both aiming to restore the expression of dystrophin. For exon skipping, the general strategy of restoring expression of the mutated dystrophin gene by excluding frame-disrupting mutations was vindicated by early experiments in dystrophic animal models.3Heemskerk HA de Winter CL de Kimpe SJ van Kuik-Romeijn P Heuvelmans N Platenburg GJ et al.In vivo comparison of 2’-O-methyl phosphorothioate and morpholino antisense oligonucleotides for Duchenne muscular dystrophy exon skipping.J Gene Med. 2009; 11: 257-266Crossref PubMed Scopus (153) Google Scholar,4Tanganyika-de Winter CL Heemskerk H Karnaoukh TG van Putten M de Kimpe SJ van Deutekom J et al.Long-term exon skipping studies with 2’-O-methyl phosphorothioate antisense oligonucleotides in dystrophic mouse models.Mol Ther Nucleic Acids. 2012; 1: e44Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar,5Lu QL Rabinowitz A Chen YC Yokota T Yin H Alter J et al.Systemic delivery of antisense oligoribonucleotide restores dystrophin expression in body-wide skeletal muscles.Proc Natl Acad Sci USA. 2005; 102: 198-203Crossref PubMed Scopus (356) Google Scholar,6Wu B Lu P Benrashid E Malik S Ashar J Doran TJ 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 (102) Google Scholar This principle has been substantiated for DMD by clinical trials over the last 7 years with two chemistries, the 2’O-methyl phosphorothioate backbone (2OMePS, named PRO051/Drisapersen initiated by Prosensa/GSK) and the morpholino backbone (PMO, named Eteplirsen initiated by AVI Biopharma, now Sarepta Therapeutics).7Kinali M Arechavala-Gomeza V Feng L Cirak S Hunt D Adkin C 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 (572) Google Scholar,8Cirak S Arechavala-Gomeza V Guglieri M Feng L Torelli S Anthony K 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 (699) Google Scholar,9van Deutekom JC Janson AA Ginjaar IB Frankhuizen WS Aartsma-Rus A Bremmer-Bout M et al.Local dystrophin restoration with antisense oligonucleotide PRO051.N Engl J Med. 2007; 357: 2677-2686Crossref PubMed Scopus (699) Google Scholar,10Goemans NM Tulinius M van den Akker JT Burm BE Ekhart PF Heuvelmans N et al.Systemic administration of PRO051 in Duchenne's muscular dystrophy.N Engl J Med. 2011; 364: 1513-1522Crossref PubMed Scopus (596) Google Scholar Both drugs target dystrophin exon 51 and both elicited the expected skipping of exon 51 and production of dystrophin protein following intramuscular injections. In a subsequent open-label, dose-escalation systemic study, five weekly subcutaneous injections of Prosensa/GSK's PRO051 at 0.5, 2, 4, or 6 mg/kg induced skipping of exon 51 accompanied by low levels of dystrophin in 12 DMD boys. But, importantly, this data lacked pretreatment controls.10Goemans NM Tulinius M van den Akker JT Burm BE Ekhart PF Heuvelmans N et al.Systemic administration of PRO051 in Duchenne's muscular dystrophy.N Engl J Med. 2011; 364: 1513-1522Crossref PubMed Scopus (596) Google Scholar This lack of pretreatment controls in combination with the use of the highly sensitive dystrophin antibody MANDYS106 led to reports of expansion of dystrophin positive fibre counts to up to 100%. The follow-up extension for 12-weeks at 6 mg/kg of the PRO051 reported stabilisation of motor function in the boys.10Goemans NM Tulinius M van den Akker JT Burm BE Ekhart PF Heuvelmans N et al.Systemic administration of PRO051 in Duchenne's muscular dystrophy.N Engl J Med. 2011; 364: 1513-1522Crossref PubMed Scopus (596) Google Scholar However, the study included several boys below 7 years of age where natural history would predict improved motor function within the experimental period. A subsequent Phase IIb placebo-controlled 6 mg/kg/week study (NCT01153932) of Drisapersen on 53 DMD patients again reported a significant benefit in 6MWT in the treatment over the placebo group. Clinical benefits were maintained, but with reduced significance, after 48 weeks of treatment. However, no muscle biopsy results have yet been reported.11http://clinicaltrials.gov/show/NCTO1153932Google Scholar This was followed by the phase III trial, with 186 patients, that failed to show statistically significant improvements in the primary outcome measure of the 6MWT. The difference in conclusion between the Phase III and earlier studies with the exact same treatment regime is therefore attributed almost solely to the high variability of the 6MWT endpoint measurement within the time-frame of the subject population and difference in sample sizes. What have we learnt from the Prosensa/GSK trials? According to the comment from the FDA in response to Sarepta's application for Eteplirsen targeting the same dystrophin exon 51, the failure of the Drisapersen trial indicates a “disconnect between increased expression of dystrophin and clinical efficacy.” This criticism has serious implications far beyond exon skipping in DMD raising questions about our assessment of all experimental therapies that aim to restore or produce functional dystrophins in DMD, including gene replacement therapies that deliver a known functional gene product as a drug. So, is there clear evidence indicating a disconnect between the levels of dystrophin expression and clinical efficacy? Fortunately, the answer is no. Results, both from animal models6Wu B Lu P Benrashid E Malik S Ashar J Doran TJ 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 (102) Google Scholar and clinical studies on Becker muscular dystrophy (BMD) patients, all point to a positive connection. Much of the confusion about levels of dystrophin expression come from over emphasizing assessment of dystrophin expression solely by immunohistochemistry (IHC) without pretreatment control biopsies for each patient and the use of highly sensitive antibody MANDYS106. Prosensa reported that up to 100% of fibers were dystrophin positive in the muscle samples of some Drisapersen-treated patients in its phase II study.10Goemans NM Tulinius M van den Akker JT Burm BE Ekhart PF Heuvelmans N et al.Systemic administration of PRO051 in Duchenne's muscular dystrophy.N Engl J Med. 2011; 364: 1513-1522Crossref PubMed Scopus (596) Google Scholar IHC is critical to provide us with a rough estimate of the levels of protein expression, the proportion of cells expressing the protein, and especially patterns of distribution. However, one must be aware that judgment of positivity of dystrophin staining with IHC is highly subjective and especially troublesome in DMD samples due to many factors, including the degeneration-related background staining, and highly variable distribution of both revertant fibers (spontaneously expressing dystrophin) and antisense oligomer-induced dystrophin expression, all of which call for comparison with a pretreatment biopsy. Small areas of clustered revertant fibers, reaching a few dozen or more, could easily be misinterpreted to result from treatment. For these reasons, IHC, even though very valuable for assessing distribution and localization, should never be the sole source of evidence for levels of dystrophin expression and require confirmatory backup. Currently, the most generally available and reliable assessment of dystrophin levels in muscles is by western blots although the data are still, arguably semiquantitative, and it is generally difficult to convincingly demonstrate levels of dystrophin below 10% of normal levels. However, dystrophin levels higher than 10% of normal level can be demonstrated without much difficulty in most laboratories as observed from western blots using tenfold dilutions of positive control protein.7Kinali M Arechavala-Gomeza V Feng L Cirak S Hunt D Adkin C 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 (572) Google Scholar,8Cirak S Arechavala-Gomeza V Guglieri M Feng L Torelli S Anthony K 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 (699) Google Scholar,10Goemans NM Tulinius M van den Akker JT Burm BE Ekhart PF Heuvelmans N et al.Systemic administration of PRO051 in Duchenne's muscular dystrophy.N Engl J Med. 2011; 364: 1513-1522Crossref PubMed Scopus (596) Google Scholar Unfortunately, data from western blots in the Drisapersen systemic trials showed only trace amounts of dystrophin in the treated muscle biopsy samples.10Goemans NM Tulinius M van den Akker JT Burm BE Ekhart PF Heuvelmans N et al.Systemic administration of PRO051 in Duchenne's muscular dystrophy.N Engl J Med. 2011; 364: 1513-1522Crossref PubMed Scopus (596) Google Scholar In fact, the image presented in the publication shows no clear difference in signal intensity between the baseline samples and those from 2/7 weeks after treatment.10Goemans NM Tulinius M van den Akker JT Burm BE Ekhart PF Heuvelmans N et al.Systemic administration of PRO051 in Duchenne's muscular dystrophy.N Engl J Med. 2011; 364: 1513-1522Crossref PubMed Scopus (596) Google Scholar The FDA also cited the failure of PTC Therapeutics's Ataluren trial as another example of the disconnect between levels of dystrophin and clinic outcomes. Ataluren has been selected to induce significant read-through of nonsense mutations of DMD gene, resulting in restoration of dystrophin expression in patients carrying such mutations. However, the screening assay method initially used to identify the specificity of PTC124 has been contradicted by more recent study.12Auld DS Lovell S Thorne N Lea WA Maloney DJ Shen M et al.Molecular basis for the high-affinity binding and stabilization of firefly luciferase by PTC124.Proc Natl Acad Sci USA. 2010; 107: 4878-4883Crossref PubMed Scopus (146) Google Scholar Furthermore, studies to validate the effect of PTC124 in vitro did not demonstrate any significant read-through of nonsense mutations.13McElroy SP Nomura T Torrie LS Warbrick E Gartner U Wood G et al.A lack of premature termination codon read-through efficacy of PTC124 (Ataluren) in a diverse array of reporter assays. 2013; (http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001593)Google Scholar More importantly, clinical trials of Ataluren have shown only the phase IIa results with little evidence of dystrophin restoration by IHC with a very subjective scoring method.14Finke RS Flanigan KM Wong B Bönnemann C Sampson J Sweeney HL et al.Phase 2a study of ataluren-mediated dystrophin production in patients with nonsense mutation duchenne muscular dystrophy.PLOS ONE. 2013; 8: e81302Crossref PubMed Scopus (179) Google Scholar Unfortunately, the phase IIb muscle biopsy results have not been presented and reported to the public. Therefore, the disappointing functional data from the Ataluren and Drisapersen trials are, in fact, entirely consistent with the failure of either agent to induce production of significant amounts of dystrophin protein and do not support the notion of the “disconnect” proclaimed in the FDA report. The rationale of both exon-skipping and stop-codon read-through is that any benefit derives from production of significant amounts of dystrophin and the degree of efficacy should be related to the level of dystrophin. This has been clearly demonstrated in animal models of DMD.6Wu B Lu P Benrashid E Malik S Ashar J Doran TJ 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 (102) Google Scholar,15Wu B Xiao B Cloer C Shaban M Sali A Lu P et al.One-year treatment of morpholino antisense oligomer improves skeletal and cardiac muscle functions in dystrophic mdx mice.Mol Ther. 2011; 19: 576-583Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar This conclusion is also consistent with the data from Sarepta's clinical trials of the exon skipping drug Eteplirsen (formerly AVI-4658). The first open label, dose escalation (cohorts 1–6: 0·5, 1, 2, 4, 10, and 20 mg/kg bodyweight respectively), repeated intravenous administration study showed that Eteplirsen was well tolerated. There was a statistically significant dose-response as well as remarkable variability in dystrophin production in patient muscles. In the low dose cohorts 1–4, there was no increase in dystrophin expression, with the exception of one subject in cohort 3. However, 6 of 8 subjects in the two highest dose cohorts 5 and 6 showed an increase in dystrophin expression. Three patients, one in each of cohorts 3, 5, and 6, showed a substantial number of dystrophin-positive fibers, 21, 15, and 55%, respectively. Western blot analysis of these patients also showed an increase following treatment of protein levels from 2 to 18%, from 0.9 to 17% and from 0 to 7.7% of normal muscle values, respectively.8Cirak S Arechavala-Gomeza V Guglieri M Feng L Torelli S Anthony K 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 (699) Google Scholar This was followed by a placebo-controlled phase IIb trial using higher doses (30 and 50 mg/kg/week, each cohort had n = 4 subjects) of Eteplirsen.16Mendell JR Rodino-Klapac LR Sahenk Z Roush K Bird L Lowes LP et al.Eteplirsen for the treatment of Duchenne muscular dystrophy.Ann Neurol. 2013; 74: 637-647Crossref PubMed Scopus (539) Google Scholar Ambulant boys were treated for 24 weeks and primary outcome was the percentage of increase in the number of dystrophin positive fibres in comparison to baseline biopsies. The secondary outcome was safety and the 6MWT. After another 24 weeks of open label extension, a final muscle biopsy was taken at 48 weeks. The results showed the number of dystrophin positive fibres up to 52 and 43% in the 30 and 50 mg/kg cohorts respectively.16Mendell JR Rodino-Klapac LR Sahenk Z Roush K Bird L Lowes LP et al.Eteplirsen for the treatment of Duchenne muscular dystrophy.Ann Neurol. 2013; 74: 637-647Crossref PubMed Scopus (539) Google Scholar Overall, the Eteplirsen trials showed a clear trend of dose-dependent increase in dystrophin expression. Clearly, one major difference between the two of exon skipping clinic trials is that Eteplirsen has been systemically at up to 50 more than higher than The dose of 6 mg/kg for Drisapersen was the result of no clear has far been for studies have reported higher of exon skipping with the than with amounts of on the Winter CL Heemskerk H Karnaoukh TG van Putten M de Kimpe SJ van Deutekom J et al.Long-term exon skipping studies with 2’-O-methyl phosphorothioate antisense oligonucleotides in dystrophic mouse models.Mol Ther Nucleic Acids. 2012; 1: e44Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar,5Lu QL Rabinowitz A Chen YC Yokota T Yin H Alter J et al.Systemic delivery of antisense oligoribonucleotide restores dystrophin expression in body-wide skeletal muscles.Proc Natl Acad Sci USA. 2005; 102: 198-203Crossref PubMed Scopus (356) Google Scholar,6Wu B Lu P Benrashid E Malik S Ashar J Doran TJ 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 (102) Google Scholar is therefore not that the Eteplirsen trial reported dystrophin by western The Eteplirsen treated patients also showed a benefit of in 6MWT over the placebo JR Rodino-Klapac LR Sahenk Z Roush K Bird L Lowes LP et al.Eteplirsen for the treatment of Duchenne muscular dystrophy.Ann Neurol. 2013; 74: 637-647Crossref PubMed Scopus (539) Google Scholar of the motor function in DMD boys in Eteplirsen extension study has now observed over 2 one to be aware of the sample 12 subjects) and the with the open label of the extension the levels of dystrophin were clearly below 10% by western and from only one sample from the image presented in the the high percentage of dystrophin positive fibers JR Rodino-Klapac LR Sahenk Z Roush K Bird L Lowes LP et al.Eteplirsen for the treatment of Duchenne muscular dystrophy.Ann Neurol. 2013; 74: 637-647Crossref PubMed Scopus (539) Google Scholar This again the of using IHC and number of dystrophin positive fibers as a of levels of dystrophin expression. also to be aware that results of 6MWT stabilization have been reported from the Drisapersen open label extension studies and natural history although the is of age and M S L et in duchenne patients with 12 ONE. PubMed Scopus Google M E et and in Duchenne muscular dystrophy: natural history over 48 weeks from a 2013; PubMed Scopus Google Scholar therefore to be the reported levels of dystrophin the Eteplirsen treatment regime can be placebo phase III studies will be to show Eteplirsen can the There should be no that dystrophin is critical for normal muscle for its lack is the of DMD and the protein product of the Duchenne muscular dystrophy Full Text PDF PubMed Scopus Google Scholar could that increase of any of dystrophin benefit the dystrophic However, the of low amounts of dystrophin in these trials is with distribution of the dystrophin positive The number of these fibers in dystrophic muscles is to provide and functional improvement to the general of a view by studies in animal Winter CL Heemskerk H Karnaoukh TG van Putten M de Kimpe SJ van Deutekom J et al.Long-term exon skipping studies with 2’-O-methyl phosphorothioate antisense oligonucleotides in dystrophic mouse models.Mol Ther Nucleic Acids. 2012; 1: e44Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar,5Lu QL Rabinowitz A Chen YC Yokota T Yin H Alter J et al.Systemic delivery of antisense oligoribonucleotide restores dystrophin expression in body-wide skeletal muscles.Proc Natl Acad Sci USA. 2005; 102: 198-203Crossref PubMed Scopus (356) Google Scholar,6Wu B Lu P Benrashid E Malik S Ashar J Doran TJ 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 (102) Google Scholar Therefore, therapy on dystrophin restoration must the can the treatment produce amounts of by both IHC and western and to the answer this one would first much dystrophin is to have a significant impact on the of biopsies from patients provide some is with natural from which reduced amounts of dystrophins are The aim of exon skipping therapy in DMD patients is to by production of from the dystrophin on levels in Becker patients that this degree of can result from of normal dystrophin levels in western M Torelli S S P L et levels as low as are to muscular dystrophy in the 2007; 17: Full Text Full Text PDF PubMed Scopus Google Scholar patients of clinical have been with dystrophin levels of between and of normal of Duchenne and Becker muscular dystrophy: on improved Google Scholar in patients with DMD have been with than 10% dystrophin. this it is to that significant improvement in the by exon skipping will require expression of dystrophin to at 10% of normal levels with distribution. levels of dystrophin have not far been convincingly demonstrated in any of the systemic trials of exon skipping and read-through higher levels of dystrophin are to of with muscle and higher in dystrophin distribution. This not benefit to muscle of levels of but the of patients with low levels of dystrophin indicates that this is all the data available from animal studies of exon skipping and gene therapy and clinic trials of exon skipping, we that dystrophin protein far from is a biomarker to be taken assessing experimental therapies that aim to produce and restore the expression of dystrophin in DMD. This principle is to any therapy that to produce are in the early of clinical trials for the exon skipping is therefore critical to such data as as that be for clinical trials targeting or for gene replacement The by several of and in the and of these clinical trials be as we have learnt about the natural history of DMD, the and of clinical trial and outcome and have a clinical for clinical The most learnt is clinical such as 6MWT are for assessing the but of this principle to have led to the that clinical benefit can be without the production of a of protein valuable and from the two exon skipping trials is that the of a antisense in the mouse and models to provide a to it will do in DMD. The relationship of dose to exon-skipping and dystrophin production with the in animal models has been in the rationale for of the for a in with a that the general of dose and regime be to In of dystrophin is to exon-skipping therapy in DMD and it is to reliable data levels of dystrophin expression to clinic This will provide for trials antisense and targeting as well as for gene replacement studies in the dystrophic mouse and have shown a dose dystrophin and effect on dystrophic muscles. In fact, clinical and outcome from the treatment of the two are from animal from the Ataluren and Drisapersen clinical trials of exon skipping far demonstrated to our little dystrophin to the notion of a between the levels of dystrophin expression and clinical outcome The Ataluren phase III trial be to clinical benefit to DMD patients, but no muscle biopsies have been in this phase III of dystrophin levels should more on western the most available with data from IHC, although more reliable and are AA S DS et of dystrophin protein in skeletal muscle using 2012; 7 Google Scholar we would the FDA and that dystrophin is not a biomarker for lack of dystrophin is the primary in DMD and of the of the principle of substantial dystrophin restoration in DMD would have impact on all of