FocusDuchenne Muscular Dystrophy

Exon-Skipping Therapy: A Roadblock, Detour, or Bump in the Road?

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Science Translational Medicine  02 Apr 2014:
Vol. 6, Issue 230, pp. 230fs14
DOI: 10.1126/scitranslmed.3008873


Exon skipping is a promising therapeutic for Duchenne muscular dystrophy patients, but the road to drug approvals is foggy and may require more early-stage derisking and regulatory guidance.

Duchenne muscular dystrophy (DMD) affects 1 in 5000 newborn males. These boys appear healthy as infants and young children but then experience a heartbreaking decline, as muscle tissue gradually wastes away, leaving patients nonambulant by their late teens. The heart and respiratory muscles are similarly weakened, compromising life span. DMD has served as a test bed for population genetics in the 1950s, gene identification methods (reverse genetics) in the 1980s, and the integration of molecular diagnostics into patient diagnosis and family counseling. Additional landmarks are held by the dystrophin gene: It is the largest in the human genome—2.3 million base pairs—and has one of the highest known spontaneous mutation rates of all human genes. Although we have made great progress in understanding the molecular basis of DMD, translation of these advances to improvements in patient care has been painfully slow. Early hints of success with stem cell transplantation and replacement viral gene therapy hit technical delivery roadblocks and immunological barriers that have remained persistently impassable.

With this backdrop, the innovative exon-skipping therapy for DMD, which rescues dystrophin production, caught the rapt attention of scientists, physicians, and patients. This approach treats a DMD patient systemically with antisense oligonucleotides directed against the dystrophin mRNA transcript. These oligos are designed to rescue the target mRNA, restoring the proper reading frame and thus protein production; in contrast, antisense therapy for other disorders focuses on destroying or inhibiting the target mRNA. Dystrophin rescue in DMD is accomplished by excluding an exon that neighbors a deletion mutation, effectively transforming an out-of-frame (nonsense, loss-of-function) transcript to an in-frame transcript capable of de novo dystrophin production in patient muscle (Fig. 1). The newly produced dystrophin protein lacks amino acids but retains some of its cytoskeletal function. The concept that internally truncated dystrophin could be functionally derived from observations in patients that similar mutations underlie the milder Becker muscular dystrophy.

Fig. 1 Repairing disrupted dystrophin.

In exon-skipping therapy, DMD patients are treated systemically with antisense oligonucleotides (Oligo, reddish-brown bars) directed against the dystrophin mRNA transcript (exons 47 to 52 of the dystrophin gene are shown in purple inset). These oligos rescue the target mRNA (also shown in purple inset), restoring the proper reading frame and thus production of dystrophin protein (blue twisted structure). Dystrophin rescue is accomplished by excluding an exon that neighbors a deletion mutation (illustrated by [ ] in the dystrophin protein shown in the “After exon skipping” panel), effectively transforming an out-of-frame (nonsense, loss-of-function) transcript to an in-frame transcript capable of de novo dystrophin production in patient muscle. This dystrophin protein lacks some amino acids but retains cytoskeletal function. The dark red square in the muscle myofiber is amplified in three insets (at the left, outlined in dark red) that represent normal muscle (Normal), untreated Duchenne muscle (Duchenne), and Duchenne muscle after exon-skipping treatment (After exon skipping).


Since the first report of spontaneous exon skipping in patient cells in 1991 (1), more than 350 publications have validated this corrective strategy. Impressive systemic rescue of dystrophin has been seen in the mdx mouse model of DMD in many independent laboratories (25) and was further supported by preclinical efficacy in a DMD large animal model (6). Early clinical trials in DMD patients first used local intramuscular injection of antisense oligos and have been followed by intravenous and subcutaneous injections focused on systemic dystrophin rescue (79).


In late 2013, a series of public reports caused a pregnant pause in the progress. A phase 3 trial of 186 DMD patients (125 on drug and 61 placebo) carried out by GlaxoSmithKline (GSK) in partnership with Prosensa failed to show significant improvement of the primary outcome measure—the 6-minute walk test. GSK has since terminated its relationship with Prosensa on the exon-skipping program in DMD. Simultaneous with this late-stage failure, an exon-skipping oligo generated by an alternative oligonucleotide chemistry [morpholinos (PMO) made by Sarepta] showed impressive safety profiles and a wide therapeutic index in two small trials in DMD patients. However, when Sarepta sought accelerated approval, the U.S. Food and Drug Administration (FDA) did not find the data compelling enough to grant conditional approval.

What happened? Is this yet another case in which impressive preclinical data and an enticing mechanism of action failed to translate into human patient efficacy? This is possible, but the breadth and depth of preclinical studies suggest that the problem may be simply a matter of optimizing and improving the clinical approach. However, the current situation also serves to illustrate the shifting sands and blind alleys that may disorient the modern orphan drug developer as well as the families anxious for a drug that can slow the tragic progression of DMD. The problems faced by the two programs (GSK-Prosensa and Sarepta) are quite different, and these differences are instructive both for other drugs in the orphan pipeline and for the path forward in exon-skipping therapeutics.

GSK-Prosensa used a traditional chemistry for their oligonucleotide drug 2-O-methyl phosphorothioate (2OMePS). Similar chemistry has been used in about 60 clinical trials for many different disorders, and nearly all studies showed challenges with a sufficient therapeutic window—namely, difficulty in delivering enough of the oligo inside the cells to achieve potent modulation of RNA metabolism without hitting a toxicity ceiling. The toxicity limit on 2OMePS chemistry is about 6 mg/kg before activation of the complement and innate immune systems directed against the DNA-like drug. It was never clear that the 6 mg/kg/wk subcutaneous dose could produce sufficient truncated dystrophin to anticipate clinical benefit. A subset of patients in the phase 2 trials showed an impressive stabilization in the six-minute walk test, and this was used as a rationale for moving ahead to phase 3 studies. But the 6-minute walk test, especially in children, is far from perfect as an outcome measure. Recent public meetings between FDA and DMD stakeholder groups suggested that alternatives to the 6-minute walk test (such as measures of strength) may be more acute surrogate measures of potential drug efficacy. Ideally, the GSK-Prosensa program could have been derisked at phase 2 by altering dose and chemistry until greater and more consistent de novo dystrophin production was observed in patient muscle; de novo dystrophin production as a surrogate biochemical outcome measure might have served as a basis for accelerated approval. Prosensa continues to test additional exon-skipping drugs using the 2OMePS chemistry, likely with this intent.

Morpholinos, such as that used by Sarepta in its DMD trials, are chemically less similar to DNA than 2OMePS but retain strong sequence-specific base pairing to the mRNA target. Morpholinos appear capable of evading many of the toxicities plaguing the 2OMePS chemistry. Whereas 2OMePS hit its toxicity ceiling at 6 mg/kg, morpholinos have been taken to much higher levels in both humans and animals (960 mg/kg in mice, 320 mg/kg in monkeys, and 50 mg/kg in human trials) with few adverse events (9). Preclinical studies in multiple laboratories have shown a clear dose-dependence of morpholino oligo rescue of dystrophin, and more appears to be better. Furthermore, morpholinos are cleared quickly by the kidney and excreted intact. However, morpholinos are costly; dosed at 50 mg/kg/wk, raw drug production costs may exceed $100,000 per patient per year, making morpholinos potentially cost prohibitive unless scale-up is able to reduce costs.

Morpholino chemistry is safer, is capable of yielding higher dystrophin production in preclinical studies, and has shown promise in initial human trials. So why has the FDA seemed hesitant regarding accelerated approval? First, dystrophin’s validity as a biomarker is not yet well established. Determining what constitutes an “adequate” dystrophin level to predict later clinical benefit is complicated by difficulties in obtaining representative muscle biopsies. Furthermore, dystrophin rescue levels are likely quite variable across a DMD patient’s muscle (on the basis of dog and mouse exon-skipping data). These issues coupled with technical challenges in quantitating levels of the large (427 kD) dystrophin protein highlight the substantial amount of work that remains to be done before dystrophin is readily accepted as a surrogate biomarker that predicts later clinical benefit.

The approach to clinical trials may also need optimization. As a DMD boy ages, his myofibers are replaced in a patchy fashion by fibrofatty connective tissue, leaving less and less muscle to rescue by exon skipping or any other approach and possibly impairing regeneration. Exon skipping likely needs to take place in a preexisting myofiber (Fig. 1). Many exon-skipping trials have focused on older boys, because these patients, with their low 6-minute walk distances, could show the greatest benefit. However, older patients may be harder to treat than younger patients, because older boys have fewer myofibers to rescue. On the other hand, younger patients display greater 6-minute walk performance, which may remain stable over the course of a clinical trial—a natural history potentially seen in untreated patients. Thus, the 6-minute walk test might not be a suitable primary end point in young patients. It is clearly important to be able to draw strong correlations between the amount of de novo dystrophin production and clinical outcomes. However, the significant variability in dystrophin expression in the muscles of both exon-skipping and Becker muscular dystrophy patients serves as a barrier to establishing biochemistry–clinical cause–clinical effect relationships. Understanding this variability is necessary to make the therapeutic approach more robust and dystrophin production data more compelling.


Understandably, DMD parents who have seen impressive stabilization in 6-minute walk distance in a subset of morpholino-treated patients have pressed FDA for accelerated approval. FDA has been very attentive, holding many meetings with parents and other stakeholders. But on what data can an accelerated approval be based—clinical (6-minute walk test) or surrogate biochemical (de novo dystrophin in patient muscle)? Dystrophin is the primary site of the biochemical defect in DMD, and restoration of dystrophin by an experimental treatment is a logical basis for accelerated approval. However, the Sarepta trial results have shown dystrophin levels to be highly variable among patients and significantly increased in very few patients. Although stabilization in 6-minute walk distance in a subset of patients is indeed impressive, it also has been observed in a very small number of patients (N = 10) in an unblinded phase 2b trial. Stabilization of walk distance was used by GSK-Prosensa to justify the resource-intensive phase 3 trial that then failed, and control groups in other trials have also shown impressive stabilization in subsets of patients. Thus, FDA may be hesitant to run that same play again.

So, has exon skipping hit a roadblock, detour, or bump in the road? Perhaps the best analogy is a very large pothole—one that gives a driver pause. There is a strong feeling in both the scientific and patient communities that exon skipping works, but a more compelling case must be built in the DMD clinical development program. There are a number of routes toward building such a case and traversing the pothole. The potency of exon-skipping drugs could be increased with the use of adjuvants or modified oligos, but these regimens will take time to develop and implement. A quicker pothole patch would be to optimize aspects of the clinical trial design, including younger age of treatment, dose optimization, and improvements in dystrophin protein measurements in patient muscle biopsies. These tweaks could pave the way to a more compelling accelerated package (for example, dose optimization leading to greater dystrophin detection, using robust detection methods). There are emerging international consensus standards for dystrophin measurements in human muscle as well as new applications of mass spectrometry that can be applied to very small amounts of muscle. These manipulations may help scientists steer around the pothole.

For the subset of DMD patients with “skip friendly” gene mutations, exon skipping remains one of the most promising approaches for dystrophin restoration in the near term. The road to drug approvals for orphan diseases is rarely smooth, but emphasis on derisking and regulatory changes that encourage accelerated approvals has everyone working to improve driving conditions.


  1. Competing interests: E.P.H. is principal investigator of NIH and Department of Defense grants focused on PMO-based development of exon skipping for DMD and co-founder of ReveraGen Biopharma and Agada Biosciences.
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