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Synthetic antisense oligonucleotides (ASOs) have emerged as one of the most promising therapeutic approaches. So far, nine ASO drugs have received approval for clinical use, and four of them are based on splice-switching principles demonstrating the impact of ASO-mediated splice modulation. Notably, three among them (Exondys 51, Vyondys 53 and Viltepso) are based on phosphorodiamidate morpholino (PMO) chemistry whereas Spinraza is based on 2′-O-methoxyethyl phosphorothioate (2′-MOE PS) chemistry. Although systemic delivery of PMOs has displayed a good safety profile even at high doses, the 2′-O-methyl phosphorothioate modified (2′-OMe PS) ASO drug candidate (drisapersen) failed due to safety issues. The potency of 2′-modified RNA for splice-switching needs to be further improved by novel design strategies for broad applicability. Towards this goal, in this study, we evaluated the potential of incorporating DNA segments at appropriate sites in 2′-OMe PS and 2′-MOE PS ASOs to induce exon skipping. For this purpose, a four-nucleotide DNA segment was systematically incorporated into a 20-mer 2′-OMe PS and 2′-MOE PS ASO designed to skip exon 23 in mdx mouse myotubes in vitro. Our results demonstrated that 2′-modified RNA PS ASOs containing four or less PS DNA nucleotides at the 3′-end yielded improved exon 23 skipping efficacy in line with fully modified ASO controls. Based on these results, we firmly believe that the present study opens new avenues towards designing splice modulating ASOs with limited chemical modifications for enhanced safety and therapeutic efficacy.

Delivery to the target site and adversities related to off-target exposure have made the road to clinical success and approval of antisense oligonucleotide (AON) therapies challenging. Various classes of AONs have distinct chemical features and pharmacological properties. Understanding the similarities and differences in pharmacokinetics (PKs) among AON classes is important to make future development more efficient and may facilitate regulatory guidance of AON development programs. For the class of 2'-O-methyl phosphorothioate (2OMe PS) RNA AONs, most nonclinical and clinical PK data available today are derived from development of exon skipping therapies for Duchenne muscular dystrophy (DMD). While some publications have featured PK aspects of these AONs, no comprehensive overview is available to date. This article presents a detailed review of absorption, distribution, metabolism, and excretion of 2OMe PS AONs, compiled from publicly available data and previously unpublished internal data on drisapersen and related exon skipping candidates in preclinical species and DMD patients. Considerations regarding drug-drug interactions, toxicokinetics, and pharmacodynamics are also discussed. From the data presented, the picture emerges of consistent PK properties within the 2OMe PS class, predictable behavior across species, and a considerable overlap with other single-stranded PS AONs. A level of detail on muscle as a target tissue is provided, which was not previously available. Furthermore, muscle biopsy samples taken in DMD clinical trials allowed confirmation of the applicability of interspecies scaling approaches commonly applied in the absence of clinical target tissue data.

Duchenne muscular dystrophy (DMD) is an X-linked condition caused by a deficiency of functional dystrophin protein. Patients experience progressive muscle weakness, cardiomyopathy and have a decreased life expectancy. Standards of care, including treatment with steroids, and multidisciplinary approaches have extended the life expectancy and improved the quality of life of patients. In the last 30 years, several compounds have been assessed in preclinical and clinical studies for their ability to restore functional dystrophin levels or to modify pathways involved in DMD pathophysiology. However, there is still an unmet need with regards to a disease-modifying treatment for DMD and the attrition rate between early-phase and late-phase clinical development remains high. Currently, there are 40 compounds in clinical development for DMD, including gene therapy and antisense oligonucleotides for exon skipping. Only five of them have received conditional approval in one jurisdiction subject to further proof of efficacy. In this review, we present data of another 16 compounds that failed to complete clinical development, despite positive results in early phases of development in some cases. We examine the reasons for the high attrition rate and we suggest solutions to avoid similar mistakes in the future. ispartof: FRONTIERS IN PHARMACOLOGY vol:12 ispartof: location:Switzerland status: published

BACKGROUND: Studies 4658-201/202 (201/202) evaluated treatment effects of eteplirsen over 4 years in patients with Duchenne muscular dystrophy and confirmed exon-51 amenable genetic mutations. Chart review Study 4658-405 (405) further followed these patients while receiving eteplirsen during usual clinical care. OBJECTIVE: To compare long-term clinical outcomes of eteplirsen-treated patients from Studies 201/202/405 with those of external controls. METHODS: Median total follow-up time was approximately 6 years of eteplirsen treatment. Outcomes included loss of ambulation (LOA) and percent-predicted forced vital capacity (FVC%p). Time to LOA was compared between eteplirsen-treated patients and standard of care (SOC) external controls and was measured from eteplirsen initiation in 201/202 or, in the SOC group, from the first study visit. Comparisons were conducted using univariate Kaplan-Meier analyses and log-rank tests, and multivariate Cox proportional hazards models with regression adjustment for baseline characteristics. Annual change in FVC%p was compared between eteplirsen-treated patients and natural history study patients using linear mixed models with repeated measures. RESULTS: Data were included from all 12 patients in Studies 201/202 and the 10 patients with available data from 405. Median age at LOA was 15.16 years. Eteplirsen-treated patients experienced a statistically significant longer median time to LOA by 2.09 years (5.09 vs. 3.00 years, p

Duchenne muscular dystrophy (DMD) afflicts 1 in 5000 newborn males, leading to progressive muscle weakening and the loss of ambulation between the ages of 8 and 12. Typically, DMD patients pass away from heart failure or respiratory failure. Currently, there is no cure, though exon-skipping therapy including eteplirsen (brand name Exondys 51), a synthetic antisense oligonucleotide designed to skip exon 51 of the dystrophin gene, is considered especially promising. Applicable to approximately 14% of DMD patients, a phosphorodiamidate morpholino oligomer (PMO) antisense oligonucleotide eteplirsen received accelerated approval by the US Food and Drug Administration (FDA) in 2016. Throughout clinical trials, eteplirsen has been well tolerated by patients with no serious drug-related adverse events. The most common events observed are balance disorder, vomiting, and skin rash. Despite its safety and promise of functional benefits, eteplirsen remains controversial due to its low production of dystrophin. In addition, unmodified PMOs have limited efficacy in the heart. To address these concerns of efficacy, eteplirsen has been conjugated to a proprietary cell-penetrating peptide; the conjugate is called SRP-5051. Compared to eteplirsen, SRP-5051 aims to better prompt exon-skipping and dystrophin production but may have greater toxicity concerns. This paper reviews and discusses the available information on the efficacy, safety, and tolerability data of eteplirsen and SRP-5051 from preclinical and clinical trials. Issues faced by eteplirsen and SRP-5051, including efficacy and safety, are identified. Lastly, the current state of eteplirsen and exon-skipping therapy in general as a strategy for the treatment of DMD are discussed.

Duchenne muscular dystrophy is a severe, progressive muscle-wasting disease that is caused by mutations that abolish the production of functional dystrophin protein. The exon skipping approach aims to restore the disrupted dystrophin reading frame, to allow the production of partially functional dystrophins, such as found in the less severe Becker muscular dystrophy. Exon skipping is achieved by antisense oligonucleotides (AONs). Several chemical modifications have been tested in nonclinical and clinical trials. The morpholino phosphorodiamidate oligomer eteplirsen has been approved by the Food and Drug Administration, whereas clinical development with the 2 '-O-methyl phosphorothioate (2OMePS) AON drisapersen was recently stopped. In this study, we aimed to study various aspects of 2OMePS AONs in nonclinical animal studies. We show that while efficiency of exon skipping restoration is comparable in young and older C57BL/10ScSn-Dmd(mdx)/J (mdx/BL10) mice, functional improvement was only observed for younger treated mice. Muscle quality did not affect exon skipping efficiency as exon skip and dystrophin levels were similar between mdx/BL10 and more severely affected, age-matched D2-mdx mice. We further report that treadmill running increases AON uptake and dystrophin levels in mdx/BL10 mice. Finally, we show that even low levels of exon skipping and dystrophin restoration are sufficient to significantly increase the survival of mdx-utrn-/- mice from 70 to 97 days.

A retrospective study in which we reviewed the hospital files of a subset of 7 patients with Duchenne muscular dystrophy participating in the open-label phase I/II PRO051-02 study in Leuven. The objective of this study was to describe in detail the injection site reactions in these children treated with drisapersen (PRO-051), a 2'-O-methyl phosphorothioate RNA antisense oligonucleotide, that induces exon 51 skipping in Duchenne muscular dystrophy. Antisense oligonucleotides, restoring the reading frame by skipping of exons, have become a potential treatment of Duchenne muscular dystrophy and other monogenetic diseases. Erythema followed by hyperpigmentation, fibrosis, and calcification were seen at the injection sites in all children. Ulcerations, which were difficult to heal, occurred in 5 of 7 children. Progression still occurred after switching to intravenous administration of drisapersen or even after stopping therapy. Systemic reactions included a reversible proteinuria and α1-microglobulinuria. Moreover, hypotrichosis was a common feature.Conclusion: Subcutaneous administration of drisapersen causes severe and progressive injection site effects. What is known: • Antisense oligonucleotides offer the possibility to convert Duchenne muscular dystrophy to the less severe Becker type. This can potentially be achieved by targeting and skipping specific exons of the Duchenne muscular dystrophy gene to restore the disrupted reading frame and to induce the production of a semi functional dystrophin protein. • Drisapersen is such an antisense oligonucleotides which can be administered subcutaneously. Its use has been tested extensively in the escalating dose pilot study (PRO051-02). What is new: • This report describes the injection site reactions caused by this type of agent in detail which has never been done before. We therefore reviewed the hospital files of 7 patients with Duchenne muscular dystrophy participating in the phase I/II open-label, escalating dose pilot study (PRO051-02) with drisapersen. • Severe side effects starting with erythema, hyperpigmentation, and later fibrosis, calcification, and difficult to treat ulcerations developed in all patients, and these continued to progress even after cessation of drisapersen. We discuss some possible underlying mechanisms. The exact mechanism however is still not known.

The timed 4-stair climb (4SC) assessment has been used to measure function in Duchenne muscular dystrophy (DMD) practice and research. We sought to identify prognostic factors for changes in 4SC, assess their consistency across data sources, and the extent to which prognostic scores could be useful in DMD clinical trial design and analysis. Data from patients with DMD in the placebo arm of a phase 3 trial (Tadalafil DMD trial) and two real-world sources (Universitaire Ziekenhuizen, Leuven, Belgium [Leuven] and Cincinnati Children's Hospital Medical Center [CCHMC]) were analyzed. One-year changes in 4SC completion time and velocity (stairs/second) were analyzed. Prognostic models included age, height, weight, steroid use, and multiple timed function tests and were developed using multivariable regression, separately in each data source. Simulations were used to quantify impacts on trial sample size requirements. Data on 1-year changes in 4SC were available from the Tadalafil DMD trial (n = 92) Leuven (n = 67), and CCHMC (n = 212). Models incorporating multiple timed function tests, height, and weight significantly improved prognostic accuracy for 1-year change in 4SC (R2: 29%-36% for 4SC velocity, and 29%-34% for 4SC time) compared to models including only age, baseline 4SC and steroid duration (R2:8%-17% for 4SC velocity and 2%-13% for 4SC time). Measures of walking and rising ability contributed important prognostic information for changes in 4SC. In a randomized trial with equal allocation to treatment and placebo, adjustment for such a prognostic score would enable detection (at 80% power) of a treatment effect of 0.25 stairs/second with 100-120 patients, compared to 170-190 patients without prognostic score adjustment. Combining measures of ambulatory function doubled prognostic accuracy for 1-year changes in 4SC completion time and velocity. Randomized clinical trials incorporating a validated prognostic score could reduce sample size requirements by approximately 40%. Knowledge of important prognostic factors can also inform adjusted comparisons to external controls. ispartof: PLOS ONE vol:15 issue:6 ispartof: location:United States status: published

Treball Final de Grau en Medicina. Codi: MD1158. Curs acadèmic: 2019/2020 Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy. Current treatment for this disease focuses on a symptomatic approach, however, mutation-specific therapies have gained regulatory approval, offering the potential of a long-term correction. This systematic review’s aim is to compare the efficacy and safety profile of exon skipping therapy for DMD. To achieve this, 5 clinical trials were analyzed on two exon skipping therapies, selected after a structured Pubmed and Embase search; eteplirsen (an already approved treatment) and drisapersen. For this purpose, the variables of motor function, and adverse events were studied, among others. To evaluate the methodological quality, the Cochrane Manual of Systematic Reviews (version 5.1.0) was used, as well as the Scottish Intercollegiate Guidelines Network (SIGN). Both therapies had limitations, for example, eteplirsen studies displayed a moderate risk of bias and drisapersen had safety issues that caused its rejection by the FDA. Even though they both showed clinical benefits in the right subgroups of population and demonstrated to improve the survival and motor function of DMD patients, the heterogenicity between the samples and design of the studies of both therapies prevents a well-founded comparison.

Duchenne muscular dystrophy (DMD) is a degenerative muscle disease with over 65% of patients having a deletion of one or more exons within the DMD gene.1 Exon deletions that disrupt the reading frame cause rapid mRNA degradation with no functional dystrophin protein produced.

Objective This double‐blind, randomized, placebo‐controlled Phase 2 study (NCT01462292) assessed the 24‐week efficacy, safety, tolerability, and pharmacokinetics of two different subcutaneous drisapersen doses, and the 24‐week off‐dose persistent effect, in ambulant Duchenne muscular dystrophy (DMD) patients. Methods Male DMD patients (≥5 years; time to rise from floor ≤15 s) were randomized to drisapersen 3 mg/kg/week, 6 mg/kg/week or placebo. The primary efficacy endpoint was change from baseline in 6‐minute walking distance (6MWD) at week 24. Secondary endpoints included changes in timed function tests, muscle strength, and pulmonary function tests. Results Fifty‐one patients were randomized to placebo (N = 16), drisapersen 3 mg/kg/week (N = 17) or 6 mg/kg/week (N = 18). All but 2 patients had baseline rise from floor time

Background Exon skipping has been considered a promising therapeutic approach for Duchenne muscular dystrophy (DMD). Eteplirsen received conditional approval in the United States in 2016. To date, no systematic reviews or meta-analyses of randomized controlled trials (RCTs) of exon skipping drugs have been published to determine the pooled estimates for the effect of exon skipping in treating DMD. Methods A systematic review and meta-analysis of double-blind RCTs comparing exon-skipping drugs with placebo in DMD was performed. Trials were identified by searching published and unpublished studies from electronically available databases and clinical trial registries through October 2017. The primary outcomes were changes in the 6-min walk test (6MWT) distance, North Star Ambulatory Assessment (NSAA) scores, and adverse events. Random-effects meta-analysis and assessment of risk of bias were performed. This systematic review was registered at PROSPERO (CRD42016037504). Results Five studies involving 322 participants were included, investigating eteplirsen in one and drisapersen in four studies. There were no changes in 6MWT distance (mean difference [MD] − 9.16, 95% confidence interval [CI] − 21.94 to 3.62) or NSAA scores (MD 1.20, 95% CI − 2.35 to 4.75) after 24 weeks of treatment in the exon-skipping group compared with placebo. Subgroup analysis for a 6 mg/kg weekly injection of drisapersen showed significant changes in the 6MWT, favoring drisapersen after 24 weeks (MD − 20.24; 95% CI − 39.59 to − 0.89). However, drisapersen resulted in a significant increase in injection site reactions (risk ratio [RR] 3.67, 95% CI 1.96 to 6.89, p

International audience; Duchenne muscular dystrophy (DMD), the most common lethal genetic disorder, is caused by mutations in the dystrophin (DMD) gene. Exon skipping is a therapeutic approach that uses antisense oligonucleotides (AOs) to modulate splicing and restore the reading frame, leading to truncated, yet functional protein expression. In 2016, the US Food and Drug Administration (FDA) conditionally approved the first phosphorodiamidate morpholino oligomer (morpholino)-based AO drug, eteplirsen, developed for DMD exon 51 skipping. Eteplirsen remains controversial with insufficient evidence of its therapeutic effect in patients. We recently developed an in silico tool to design antisense morpholino sequences for exon skipping. Here, we designed morpholino AOs targeting DMD exon 51 using the in silico tool and quantitatively evaluated the effects in immortalized DMD muscle cells in vitro. To our surprise, most of the newly designed morpholinos induced exon 51 skipping more efficiently compared with the eteplirsen sequence. The efficacy of exon 51 skipping and rescue of dystrophin protein expression were increased by up to more than 12-fold and 7-fold, respectively, compared with the eteplirsen sequence. Significant in vivo efficacy of the most effective morpholino, determined in vitro, was confirmed in mice carrying the human DMD gene. These findings underscore the importance of AO sequence optimization for exon skipping.

Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disease that primarily affects young males. Patients with DMD are unable to produce dystrophin, a crucial protein found in myocytes, leading to a loss of muscle support and integrity. Corticosteroids are the standard supportive treatment for DMD; however, there is a high demand to expand the number of safe, effective pharmacologic options. Recently a surge of new therapeutics for DMD is offering hope to patients. A variety of these new medications, such as stop codon readthrough agents, exon-skipping agents, and utrophin modulators, aim to replace dystrophin in myocytes. Other new therapeutics aim to prevent or repair muscle damage caused by the absence of dystrophin. This review provides an update on the medications being investigated in DMD.

Antisense oligonucleotide therapy has been reported to be associated with renal injury. Here, the mechanism of reversible proteinuria was investigated by combining clinical, pre-clinical, and in vitro data. Urine samples were obtained from Duchenne muscular dystrophy (DMD) patients treated with drisapersen, a modified 2′O-methyl phosphorothioate antisense oligonucleotide (6 mg/kg). Urine and kidney tissue samples were collected from cynomolgus monkeys (Macaca fascicularis) dosed with drisapersen (39 weeks). Cell viability and protein uptake were evaluated in vitro using human conditionally immortalized proximal tubule epithelial cells (ciPTECs). Oligonucleotide treatment in DMD patients was associated with an increase in urinary alpha-1-microglobulin (A1M), which returned to baseline following treatment interruptions. In monkeys, increased urinary A1M correlated with dose-dependent accumulation of oligonucleotide in kidney tissue without evidence of tubular damage. Furthermore, oligonucleotides accumulated in the lysosomes of ciPTECs and reduced the absorption of A1M, albumin, and receptor-associated protein, but did not affect cell viability when incubated for up to 7 days. In conclusion, phosphorothioate oligonucleotides appear to directly compete for receptor-mediated endocytosis in proximal tubules. We postulate that oligonucleotide-induced low molecular weight proteinuria in patients is therefore a transient functional change and not indicative of tubular damage. Keywords: Antisense oligonucleotide therapy, low molecular weight proteinuria, megalin, receptor-mediated endocytosis, proximal tubule epithelial cell

Duchenne Muscular Dystrophy (DMD) is a severe muscle disorder caused by lack of dystrophin. Predictive biomarkers able to anticipate response to the therapeutic treatments aiming at dystrophin re-expression are lacking. The objective of this study is to investigate Matrix Metalloproteinase-9 (MMP-9) as predictive biomarker for Duchenne. Two natural history cohorts were studied including 168 longitudinal samples belonging to 66 patients. We further studied 1536 samples obtained from 3 independent clinical trials with drisapersen, an antisense oligonucleotide targeting exon 51: an open label study including 12 patients; a phase 3 randomized, double blind, placebo controlled study involving 186 patients; an open label extension study performed after the phase 3. Analysis of natural history cohorts showed elevated MMP-9 levels in patients and a significant increase over time in longitudinal samples. MMP-9 decreased in parallel to clinical stabilization in the 12 patients involved in the open label study. The phase 3 study and subsequent extension study clarified that the decrease in MMP-9 levels was not predictive of treatment response. These data do not support the inclusion of serum MMP-9 as predictive biomarker for DMD patients.

What is the current status of Duchenne muscular dystrophy therapy? Duchenne muscular dystrophy (DMD) is a devastating disease and the most prevalent lethal genetic disorder worldwide [1]. DMD is an X-linked recessive genetic disorder mostly caused by frame-shift mutations in the DMD gene, which disrupts the translational reading frame, resulting in a lack of dystrophin protein [2]. The lack of dystrophin, a muscle supporting protein, leads to lethal muscle wasting in DMD patients [3]. Due to advances in clinical management, specifically with pulmonary intervention, DMD patient survival has been prolonged, although most patients develop dilated cardiomyopathy eventually, and die from cardiac or respiratory failure by 30 years of age. Currently there is no cure for DMD patients, with treatment strategies being limited to glucocorticoid corticosteroids. DMD trials using corticosteroids found that there was an overall improvement in muscle strength as well as prolonged ambulation in the short term (6 months to 2 years) [4]. However, this treatment strategy also presented various limitations with its use. Presently, glucocorticoids have harsh side effects such as impaired growth and increased susceptibility to infection. Although glucocorticoid treatment has shown potential value in DMD, other treatment options are heavily being explored. Does antisense therapy hold the key? Recently, antisense-mediated exon-skipping therapy has emerged as a promising strategy for the treatment of DMD [5]. Antisense therapy is a strategy that uses short DNAlike molecules called antisense oligonucleotides (AONs) to selectively hybridize to nascent pre-mRNA, in order to correct various genetic diseases via exon skipping, splice modulation or by inhibiting gene expression [5]. In DMD, antisense-mediated exon skipping is currently one of the most promising treatment options. AONs can be used to restore the open reading frame, by establishing expression of a truncated but functional dystrophin, seen similarly in Becker muscular dystrophy, its milder counterpart [5]. Becker muscular dystrophy although commonly arising from mutations in the DMD gene retains its ability to produce partially functional dystrophin protein, due to inframe mutations that maintain the reading frame [5]. Antisense therapy, although currently promising as a treatment option for various disorders, has had slow success. Antisensemediated RNA regulation, which was first observed in nature, has greatly improved since the late 1970s, after Zamecnik et al. demonstrated that a gene expression can be inhibited by transfection of a short antisense (complementary) DNA sequence [6]. Antisense oligonucleotide drugs for Duchenne muscular dystrophy: how far have we come and what does the future hold?

Corrado Angelini, Elisabetta Tasca Neuromuscular Laboratory, Fondazione San Camillo Hospital IRCCS, Venice, Italy Abstract: Therapeutic trials studying Duchenne muscular dystrophy (DMD) in Europe and the USA have been done using a protocol that includes manual muscle testing and functional testing, and have shown the efficacy of steroid drugs in various doses and regimens. Further, drisapersen and eteplirsen (exon skipping drugs) and ataluren (a drug to overcome stop codon mutations) have achieved some clinical improvement. Cardioprotective drugs are efficacious in DMD, and eplerenone, an aldosterone inhibitor and diuretic, is now being used to treat the disease. The dietary approach should be used in wheelchair-bound DMD children in combination with respiratory assistance. The importance of some of the treatments proposed is that they might also be useful in other genetic disorders where stop codon mutations are present; moreover, it is possible that these new treatments will improve quality of life for many patients. Keywords: Duchenne muscular dystrophy, steroids, ataluren, drisapersen, eplerenone, eteplirsen

Duchenne muscular dystrophy (DMD) is caused mostly by internal deletions in the gene for dystrophin, a protein essential for maintaining muscle cell membrane integrity. These deletions abrogate the reading frame and the lack of dystrophin results in progressive muscle deterioration. DMD patients experience progressive loss of ambulation, followed by a need for assisted ventilation, and eventual death in mid-twenties. By the method of exon skipping in dystrophin pre-mRNA the reading frame is restored and the internally deleted but functional dystrophin is produced. Two oligonucleotide drugs that induce desired exon skipping are currently in advanced clinical trials.

MMP-9 serum levels increase over time in Duchenne muscular dystrophy patients and decrease upon treatment with drisapersen

This 48-week, randomized, placebo-controlled phase 3 study (DMD114044; NCT01254019) evaluated efficacy and safety of subcutaneous drisapersen 6 mg/kg/week in 186 ambulant boys aged >=5 years, with Duchenne muscular dystrophy (DMD) resulting from an exon 51 skipping amenable mutation. Drisapersen was generally well tolerated, with injection-site reactions and renal events as most commonly reported adverse events. A nonsignificant treatment difference (P = 0.415) in the change from baseline in six-minute walk distance (6MWD; primary efficacy endpoint) of 10.3 meters in favor of drisapersen was observed at week 48. Key secondary efficacy endpoints (North Star Ambulatory Assessment, 4-stair climb ascent velocity, and 10-meter walk/run velocity) gave consistent findings. Lack of statistical significance was thought to be largely due to greater data variability and subgroup heterogeneity. The increased standard deviation alone, due to less stringent inclusion/exclusion criteria, reduced the statistical power from pre-specified 90% to actual 53%. Therefore, a post-hoc analysis was performed in 80 subjects with a baseline 6MWD 300-400 meters and ability to rise from floor. A statistically significant improvement in 6MWD of 35.4 meters (P = 0.039) in favor of drisapersen was observed in this subpopulation. Results suggest that drisapersen could have benefit in a less impaired population of DMD subjects.

Duchenne muscular dystrophy is a rare genetic disorder with life-limiting pathology. Drisapersen induces exon 51 skipping, thereby producing a shorter but functional dystrophin protein. The longest available data are from an open-label extension study (PRO051-02) treating 12 boys with drisapersen (6 mg/kg/week subcutaneously). The median change (range) from baseline to week 177 in six-minute walking distance (6MWD) was 8 (-263, 163) metres. The current analysis aimed to put the results from PRO051-02 in the context of natural progression by comparing the functional trajectory of drisapersen-treated subjects to a matched natural history (NH) cohort, treated by standard of care. Subjects were matched individually by age and 6MWD, as the primary analysis, and by age and rise from floor (RFF), as sensitivity analysis. A total of 75 NH subjects were available for 6MWD analysis, of which matching was possible for 9 ambulant drisapersen-treated subjects. None of the 6 "stable" (baseline 6MWD ≥330 metres) drisapersen-treated subjects lost ambulation vs 4 out of 10 matched NH subjects over a comparable timeframe (~3.4 years), compared with 2 out of 3 ambulant "in decline" drisapersen-treated subjects vs all 6 matched NH subjects. A total of 79 NH subjects were available for RFF analysis. For continuous ambulatory subjects (N = 4), the RFF decline was more pronounced in the NH cohort than in the drisapersen-treated subjects. In conclusion, a comparison of ambulant drisapersen-treated subjects with matched NH subjects showed a difference in functional trajectories over a timeframe of up to 3.4 years in favour of drisapersen. publisher: Elsevier articletitle: Comparison of ambulatory capacity and disease progression of Duchenne muscular dystrophy subjects enrolled in the drisapersen DMD114673 study with a matched natural history cohort of subjects on daily corticosteroids journaltitle: Neuromuscular Disorders articlelink: http://dx.doi.org/10.1016/j.nmd.2016.11.013 content_type: article copyright: © 2016 The Authors. Published by Elsevier B.V. ispartof: Neuromuscular Disorders vol:27 issue:3 pages:203-213 ispartof: location:England status: published

Summary Background Duchenne muscular dystrophy is caused by dystrophin deficiency and muscle deterioration and preferentially affects boys. Antisense-oligonucleotide-induced exon skipping allows synthesis of partially functional dystrophin. We investigated the efficacy and safety of drisapersen, a 2′-O-methyl-phosphorothioate antisense oligonucleotide, given for 48 weeks. Methods In this exploratory, double-blind, placebo-controlled study we recruited male patients (≥5 years of age; time to rise from floor ≤7 s) with Duchenne muscular dystrophy from 13 specialist centres in nine countries between Sept 1, 2010, and Sept 12, 2012. By use of a computer-generated randomisation sequence, we randomly allocated patients (2:2:1:1; block size of six; no stratification) to drisapersen 6 mg/kg or placebo, each given subcutaneously and either continuously (once weekly) or intermittently (nine doses over 10 weeks). The primary endpoint was change in 6-min walk distance (6MWD) at week 25 in patients in the intention-to-treat population for whom data were available. Safety assessments included renal, hepatic, and haematological monitoring and recording of adverse events. This trial is registered with ClinicalTrials.gov, number NCT01153932. Findings We recruited 53 patients: 18 were given continuous drisapersen, 17 were given intermittent drisapersen, and 18 were given placebo (continuous and intermittent groups combined). At week 25, mean 6MWD had increased by 31·5 m (SE 9·8) from baseline for continuous drisapersen, with a mean difference in change from baseline of 35·09 m (95% CI 7·59 to 62·60; p=0·014) versus placebo. We recorded no difference in 6MWD changes from baseline between intermittent drisapersen (mean change −0·1 [SE 10·3]) and placebo (mean difference 3·51 m [−24·34 to 31·35]) at week 25. The most common adverse events in drisapersen-treated patients were injection-site reactions (14 patients given continuous drisapersen, 15 patients given intermittent drisapersen, and six given placebo) and renal events (13 for continuous drisapersen, 12 for intermittent drisapersen, and seven for placebo), most of which were subclinical proteinuria. None of the serious adverse events reported (one for continuous, two for intermittent, and two for placebo) resulted in withdrawal from the study. Interpretation Continuous drisapersen resulted in some benefit in 6MWD versus placebo at week 25. The safety findings are similar to those from previous studies. Ambulation improvements in this young population with early-stage Duchenne muscular dystrophy are encouraging but need to be confirmed in larger studies. Funding GlaxoSmithKline, Prosensa Therapeutics BV (a subsidiary of Prosensa Holding NV).

Antisense therapy was hailed as a therapeutic approach with almost limitless opportunities three decades ago, and expectations had remained high—even recently, as exemplified by Merck's acquisition of Sirna Therapeutics for US$1.1 billion in 2006. Unfortunately, the antisense approach was unable to live up to its early hype and the unrealistic time frame posited for its translational and commercial development. However, the field abided and appears to have now matured, with more than 130 clinical trials listed on ClinicalTrials.gov, three approved antisense drugs, and several others under consideration for market approval in the United States and Europe.Several aspects of antisense technology make it attractive for drug development. First, the approach is relatively specific: it modulates target gene expression via Watson-Crick base pairing. The versatility of antisense technology permits its use to decrease or increase gene expression and to modulate splicing or transcript processing. Moreover, immune system modulation is possible with antisense oligonucleotides. At the same time, aptamers can serve as RNA-based antibodies to specifically bind to proteins in a manner unlike traditional Watson-Crick base pairing. Because oligonucleotides are synthetic pieces of chemically modified RNA or DNA, scaling up for GMP production is easier than for viral vectors or cell therapies. Side effects of antisense approaches are relatively limited. Exaggerated pharmacology can occur, but the majority of side effects are due to the chemical modifications of the oligonucleotides; e.g., subclinical increases in clotting time, a reduction in platelets, and, in some patients, thrombocytopenia have been observed for phosphorothioate-modified oligonucleotides.The introduction of the phosphorothioate modification by Fritz Eckstein in 1966 is arguably one of the more important contributions to the field of therapeutic oligonucleotides. This modification not only increases oligonucleotide stability but also enables low-affinity binding to serum proteins, which prevents renal clearance and facilitates uptake by most tissues in the body. The importance of this contribution was recently recognized by the Oligonucleotide Therapeutics Society, which awarded Fritz a lifetime achievement award during their 11th annual meeting held in Leiden, the Netherlands (11–14 October 2015).This meeting highlighted the versatility of the oligonucleotide approach and also showcased how the early promise is finally coming to fruition. Numerous oligonucleotide-based therapeutics are being tested in clinical trials. Two of the three US Food and Drug Administration (FDA)–approved oligonucleotides work via RNase H-mediated cleavage of the targeted RNA (fomivirsen (Vitravene) and mipomersen (Kynamro), both developed by Isis Pharmaceuticals); the third is pegaptanib (Macugen), an aptamer developed by OSI Pharmaceuticals and Pfizer. Targeted knockdown of gene expression can also be achieved with short interfering RNA (siRNA), which basically hijacks the endogenous microRNA (miRNA) system by providing RNA input for the RNA-induced silencing complex. siRNA is effective both in vitro and upon local delivery in animal models. The power of this approach was recognized by the Nobel Prize for Physiology or Medicine in 2006. Although siRNA is very potent, delivery of double-stranded RNA is much more challenging than single-stranded RNA. Nevertheless, with the introduction of the GalNAc conjugate (derived from N-acetylgalactosamine and targeting the asialoglycoprotein receptor that is highly expressed on liver), efficient targeting of the liver is now possible. Multiple clinical trials are currently ongoing, e.g. Alnylam's program targeting antithrombin 3 in hemophilia patients. Whereas siRNA targets a single transcript, endogenous miRNAs often target multiple transcripts. Artificial miRNA mimics can be used to simultaneously bring about knockdown of several target genes, e.g., the mimic for miR-34 that is in clinical development by Mirna Therapeutics for the treatment of cancer.Oligonucleotides can also be used to modulate pre-mRNA splicing. This approach was first reported by Ryszard Kole in 1993, whose group showed that RNase H-resistant, single-stranded antisense oligonucleotides could restore normal splicing for a cryptic splicing mutation in the β-globin gene in an in vitro splicing system. This approach was exploited for the development of therapies targeting Duchenne muscular dystrophy, where the aim is to avoid the inclusion of a target exon into the dystrophin transcript, so as to restore the correct reading frame and allow the production of partially functional protein instead of the nonfunctional one generated by the mutation. Two oligonucleotides have recently been reviewed by the FDA for Duchenne muscular dystrophy: drisapersen (2′-O-methyl phosphorothioate modification, BioMarin) and eteplirsen (phosphorodiamidate morpholino oligomer, Sarepta). The FDA concluded that drisapersen was not ready for approval in its current form, and the briefing document on eteplirsen recently published by FDA suggests this will also not be approved. Despite these apparent setbacks, the fact that the field has advanced to the point at which applications are being made to drug regulatory agencies indicates its maturation and augurs well for future success. Finally, Ionis Pharmaceuticals is developing a splice-modulating oligonucleotide for spinal muscular atrophy. Here, intrathecal injections are used to deliver the oligonucleotides to the nervous system. Notably, systemically delivered oligonucleotides will not enter the brain or the spinal cord. However, upon local delivery to the central nervous system, uptake by neurons is efficient and long lasting. Thus, oligonucleotide therapy is an attractive option to treat neurological diseases, as underlined by preclinical work focusing on such disorders as Huntington disease and amyotrophic lateral sclerosis.It is clear that the field of oligonucleotide therapeutics has recently gained new momentum, and I anticipate that the number of approved oligonucleotide drugs will increase measurably over the next decade. Hopefully, these advances will also include therapies for patients with rare genetic disorders who have unmet medical need.

Currently, antisense oligonucleotides (AONs)-mediated exon skipping therapy is the most promising therapy for Duchenne muscular dystrophy. We performed preclinical studies using dystrophic mdx52 mice and CXMD J dystrophic dogs to obtain proof of concept of AONs-mediated exon skipping therapy. Then, Japan participated in the worldwide clinical trials of drisapersen (Phase III). Finally, we developed a new AON targeting exon 53 in collaboration with Nippon Shinyaku Co., Ltd. in Japan and started an investigator-initiated clinical study at the National Center Hospital of NCNP (Phase I). This early phase exploratory study is the first clinical trial of an AON developed in Japan and a good example of translational research for treatment of DMD in Japan. In this chapter, we summarize the long development of the drug and discuss the future direction of splicing therapy for DMD.

Background Currently, the most promising therapies for Duchenne muscular dystrophy (DMD) are exon skipping and stop codon read-through, two strategies aimed at restoring the expression of dystrophin. A phase 3 clinical trial with drisapersen, a drug designed to induce exon 51-skipping, has failed to show significant improvement of the primary outcome measure, the six-minute walk test. Discussion Here, we review some key points that should be considered when designing clinical trials for these new therapies. First, younger patients have more functional abilities and more muscle fibers to preserve than older patients and therefore are better subjects for trials designed to demonstrate the success of new treatments. Second, the inclusion of patients on corticosteroids both in the treatment and placebo groups is of concern because the positive effect of corticosteroids might mask the effect of the treatment being tested. Additionally, the reasonable expectation from these therapies is the slowing of disease progression rather than improvement. Therefore, the appropriate clinical endpoints are the prolongation of the ability to stand from the floor, climb stairs, and walk, not an increase in muscle strength or function. Hence, the time frames for the detection of new dystrophin, which occurs within months, and the ability to demonstrate a slowing of disease progression, which requires years, are strikingly different. Finally, placebo-controlled trials are difficult to manage if years of blindness are required to demonstrate a slowing of disease progression. Thus, accelerated/conditional approval for new therapies should be based on surrogate biochemical outcomes: the demonstration of de novo dystrophin production and of its beneficial effect on the functional recovery of muscle fiber. Summary These data suggest that clinical trials for DMD patients must be adapted to the particular characteristics of the disease in order to demonstrate the expected positive effect of new treatments.

Antisense oligonucleotide (AO)-mediated exon-skipping therapeutics shows great promise for Duchenne muscular dystrophy (DMD) patients. However, recent failure with drisapersen, an AO candidate drug in phase 3 trial, highlights the importance of exploring other effective AO chemistries for DMD. Previously, we demonstrated the appreciable biological activity of peptide nucleic acid (PNA) AOs in restoring dystrophin expression in dystrophin-deficient mdx mice intramuscularly. Here, we further explore the systemic potential and feasibility of PNA AOs in mediating exon skipping in mdx mice as a comprehensive systemic evaluation remains lacking. Systemic delivery of PNA AOs resulted in therapeutic level of dystrophin expression in body-wide peripheral muscles and improved dystrophic pathology in mdx mice without any detectable toxicity. Up to 40% of dystrophin restoration was achieved in gastrocnemius, to a less extent with other skeletal muscles, with no dystrophin in heart. Notably, comparable systemic activity was obtained between PNA AOs and phosphorodiamidate morpholino oligomer, a DMD AO chemistry in phase 3 clinical trial, under an identical dosing regimen. Overall, our data demonstrate that PNA is viable for DMD exon-skipping therapeutics with 20 mer showing the best combination of activity, solubility, and safety and further modifications to increase PNA aqueous solubility can enable longer, more effective therapeutics without the associated toxicity.

Muscular dystrophies are a group of hereditary disorders caused by mutations in genes that have essential roles in muscle function and are generally characterized by progressive muscle weakness and degeneration. Among those, Duchenne muscular dystrophy (DMD) is the most common and severe. DMD is a fatal, X-linked recessive disorder which affects 1 in 3500 boys and is caused by mutations in the dystrophin gene. Currently there is no cure for the disorder and treatment options are limited to physiotherapy and corticosteroids that slow the course of the disorder, but are also associated with severe side effects when administered over prolonged periods of time. This chapter will provide an overview of the approaches currently being pursued for the treatment of DMD and the state of the art strategies that are in clinical trials. Emphasis will be placed on describing the progress made over the years to reach the current state, as well as the challenges that the field of gene and molecular therapies face to ensure that the approach is safe to use in patients and effective.

Sarepta will pay BioMarin $35 million to settle a lengthy patent dispute over treatments for Duchenne muscular dystrophy (DMD). The companies’ legal tangle centered on patents related to antisense oligonucleotides that allow a missing muscular protein to be made in boys with DMD. BioMarin also scores a 5% royalty on U.S. sales of exon-skipping compounds 51, 45, and 53 and an 8% royalty on European sales. The deal helps BioMarin recoup some of the roughly $840 million it paid in 2014 to acquire Prosensa, a Dutch biotech firm whose lead DMD drug, drisapersen, was later rejected by FDA.

BackgroundDrisapersen induces exon 51 skipping during dystrophin pre-mRNA splicing and allows synthesis of partially functional dystrophin in Duchenne muscular dystrophy (DMD) patients with amenable mutations.MethodsThis 188-week open-label extension of the dose-escalation study assessed the long-term efficacy, safety, and pharmacokinetics of drisapersen (PRO051/GSK2402968), 6 mg/kg subcutaneously, in 12 DMD subjects. Dosing was once weekly for 72 weeks. All subjects had a planned treatment interruption (weeks 73-80), followed by intermittent dosing (weeks 81-188).ResultsSubjects received a median (range) total dose of 5.93 (5.10 to 6.02) mg/kg drisapersen. After 177 weeks (last efficacy assessment), median (mean [SD]) six-minute walk distance (6MWD) improved by 8 (-24.5 [161]) meters for the 10 subjects able to complete the 6MWD at baseline (mean age [SD]: 9.5 [1.9] years). These statistics include 2 subjects unable to complete the test at later visits and who scored "zero". When only the 8 ambulant subjects at week 177 were taken into account, a median (mean [SD]) increase of 64 (33 [121]) meters in 6MWD was observed. Of 7 subjects walking ≥330 m at extension baseline, 5 walked farther at week 177. Of 3 subjects walking ConclusionDrisapersen was generally well tolerated over 188 weeks. Possible renal effects, thrombocytopenia and injection-site reactions warrant continued monitoring. Improvements in the 6MWD at 12 weeks were sustained after 3.4 years of dosing for most patients. For a small, uncontrolled study, the outcomes are encouraging, as natural history studies would anticipate a decline of over 100 meters over a 3-year period in a comparable cohort.Trial registrationClinicalTrials.gov NCT01910649.

Highly anticipated as new disease-modifying treatments for Duchenne muscular dystrophy (DMD), therapeutics by BioMarin Pharmaceutical (Kyndrisa™; drisapersen) and Sarepta Therapeutics (eteplirsen; AVI-4658) both recently received negative FDA reviews and are now facing battles for approval in the U.S. At present, BioMarin is committed to working with the FDA to forge a pathway to approval following the failure of its NDA, while Sarepta awaits the formal decision on its NDA, which is expected by late May 2016. Despite the critical nature of both reviews, analysts consider that there is still a narrow possibility of approval of both drugs. According to Consensus forecasts from Thomson Reuters Cortellis for Competitive Intelligence, Kyndrisa is forecast to achieve sales of USD 533.71 million in 2021.

Drisapersen (DRIS) is a 2’-O-methyl-phosphorothioate antisense oligonucleotide that induces skipping of exon 51 to correct Duchenne muscular dystrophy (DMD) mutations. This open-label extension (OLE) to a 5-week dose-escalation study assessed the long-term safety and efficacy of 6 mg/kg DRIS in 12 boys (5–16 years) with DRIS-correctable mutations. Dosing was once weekly for 72 weeks, with a planned interruption (weeks 73–80), followed by intermittent dosing (Weeks 81–188). Median (mean [SD]) 6-min walking distance (6MWD) declined between original baseline and OLE baseline (6–15 months) from 394 (402 [73]) to 362 (384 [121]) m at OLE baseline (n = 10). At Week 177, the mean (range) age of boys who completed the 6MWD at OLE baseline (n = 10) was 12.9 (9.3–15.4) years. The median (mean [SD]) change from OLE baseline was +7.5 (−24.5 [161]) m for these 10 boys; 2 boys (baseline 6MWD 140 m further than at OLE baseline. The mean (SD) change from OLE baseline to Week 177 in rise from floor was 1.0 (0.9) s for boys (n = 6) who attempted the test, and 6.9 (10.7) s when missing data for boys unable to attempt the test were replaced by 30s (n = 11). There was a mean (SD) change from OLE baseline to Week 177 in absolute forced vital capacity of 0.06 (0.29) L (n = 12). There were no clear trends in other timed tests. Over 188 weeks, all 12 boys had ⩾1 adverse event (study drug-related injection-site reactions, n = 12; gastrointestinal disorders, n = 6; decreased platelet counts, n = 5). All had increases in urine α-1-microglobulin levels and transient mild proteinuria; these were non-progressive and reversible in drug-free periods. No boys permanently withdrew from treatment. Data from this OLE are encouraging, as the natural history of DMD in steroid-treated boys receiving standard care at one of the study sites shows a median (mean [SD]) 6MWD decline of −106 (−125 [139])m over 104 weeks.

Exon skipping in Duchenne muscular dystrophy (DMD) aims to restore dystrophin expression and maintain muscle function. The largest RCT so far targeting exon 51 failed to reach its primary endpoint despite promising results from preceding studies. It underscores the need for biomarkers. Quantitative MRI (qMRI) is promising as it is a non-invasive method which can be repeated easily. qMRI was used in 5 boys from the 48week RCT (DMD114044) and open label extension (OLE) study (DMD114349) assessing exon 51 skipping with weekly subcutaneous dosing at 6mg/kg drisapersen. T1w, 3-point Dixon and quantitative fat suppressed T2 scans were acquired from the left lower leg at 3T at 3 occasions: n =5), around the start of the OLE phase ( n =5) and n =4). Two boys were on placebo during RCT. Outcome measures were % fatty infiltration, contractile cross-sectional area (cCSA), and mean and standard deviation (SD) of T2 relaxation times in five lower leg muscles. Results were compared to three DMD controls from our natural history study. Interval between scans on treatment varied between 322 and 825days. cCSA and fat% did not show a clear pattern. However, in treated boys, a reduction in mean value and SD of the T2 relaxation time was visible especially in the tibialis anterior muscle. In contrast, T2 values of controls increased. The oldest boy with the highest initial fat% showed a large reduction in fat% and increase in cCSA for all lower leg muscles during 10.5months on treatment. Nonetheless he lost ambulation during the extension phase. The observed shift in T2 values opposite to that of controls may point towards less damaged muscle tissue. Subclinical improvement in muscle structure can provide a base for developing more potent drugs or studying prolonged treatment in younger patients as disease progression may lead to a point of no return regarding ambulation. qMRI should be considered as a biomarker in DMD trials.

Antisense oligonucleotide (AON) therapies for Duchenne muscular dystrophy (DMD) are aimed at inducing skipping of specific exons during pre-mRNA splicing. This results in restoration of the reading frame and consequently synthesis of a shorter yet functional version of the dystrophin protein, as has been demonstrated in healthy control and DMD patient-derived muscle cell cultures, various DMD mouse and dog models, and recently in DMD patients treated with eteplirsen or drisapersen. As in many DMD patients a low level of spontaneous, rescuing background exon skipping occurs, accurate quantification of exon skip levels before and after AON treatment is required to monitor molecular AON drug effect in clinical studies. So far exon skip levels have generally been assessed using conventional 1st generation RT-PCR, in which non-skipped and skipped transcripts are co-amplified followed by visual comparison or densitometry of exon skip levels by gel electrophoresis or lab-on-chip. As dystrophin is not a highly abundant protein, underlying DMD transcript levels are relatively low and two rounds of PCR amplification (primary and nested) are required, often resulting in more efficient amplification of the shorter ‘skipped’ fragment compared to the longer ‘non-skipped’ fragment and end-stage saturation of the amplified products. With the recent introduction of 3rd generation digital droplet RT-PCR (ddPCR), a state-of-the-art technology became available which allows absolute quantification of copy numbers of transcripts with and without exon skip in a single PCR reaction, with high precision, sensitivity and reproducibility. We have developed ddPCR TaqMan probes targeting specific exon–exon junctions in control, BMD, DMD and mdx samples and will present data demonstrating its suitability for assessing DMD transcript copy numbers and exon skip levels. We recommend ddPCR for quantifying exon skip efficiencies in the various stages of (pre-) clinical development of AONs.

Prosensa Therapeutics is developing 2 ′ O -modified phosphorothioate antisense oligonucleotides (AONs) to provide a mutation-specific therapy in the treatment of Duchenne muscular dystrophy (DMD). Drisapersen is the lead compound in development for treatment of a subset of DMD patients amenable to skip exon 51. Chronic treatment with 6 mg/kg subcutaneous has been associated with subclinical elevations in urinary protein. These elevations are mild (generally less than 1 g/L) and concern mainly low molecular weight proteins (LMWP). Safety data show that there is no progression with prolonged treatment and that values return to baseline either spontaneously or following treatment interruption. These mild and transient elevations in urinary LMWP in patients receiving drisapersen are considered to reflect interference with the tubular re-absorption of proteins from the glomerular filtrate rather than indications of overt renal toxicity. AONs demonstrate class predictive kinetic and distribution profiles, with the kidney being the main organ of accumulation, primarily in proximal tubular cells. A dose related appearance of basophilic granules in proximal tubular cells is commonly observed in animals treated with AONs. These granules represent endosomal accumulation of AONs and are not regarded as toxicologically relevant in the absence of associated degenerative pathology. The exact mechanism (s) and receptor (s) involved in renal uptake of AONs have not yet been established, but are likely to be similar to those associated with reabsorption of LMWP, i.e. endocytosis via the megalin–cubulin receptors. In vitro experiments using a human tubular cell line, ciPTEC, could be a suitable tool to investigate the mechanism of tubular oligonucleotide absorption and the potential interference with tubular protein reabsorption.