Mutation specific approaches

Exon skipping and stop codon readthrough are mutation specific therapeutic approaches. This means that they will only work for subsets of patients who have specific mutations (see the following pages for more information). To know whether a Duchenne patient is eligible for exon skipping or stop codon readthrough it is important to have a full genetic diagnosis of the disease (i.e. the disease causing mutation in the dystrophin gene needs to be identified).

Exon skipping


To correct the genetic code and allow the production of a partially functional dystrophin.


The genetic code of genes is dispersed over so called exons. When a protein needs to be made, genes make a temporary copy (called RNA). Before this RNA can be translated into protein the exons first need to be joined and the intermittent pieces that do not contain the genetic code (introns) need to be removed. This is a process that is called "splicing".

In Duchenne patients the genetic code of the dystrophin gene is disrupted, meaning that the code become unreadable, which results in premature truncation of the translation from gene into protein. In Becker patients mutations maintain the genetic code, allowing for a protein that maintains the functional domains.

Exon skipping aims to restore the genetic code from Duchenne patients, so a partially functional, Becker-like dystrophin protein can be made, rather than a non-functional Duchenne protein. This is achieved by AONs (antisense oligonucleotides). AONs are small pieces of modified RNA that recognize a target exon, bind to it and hide it from the splicing machinery. This results in the skipping of said exon and restoration of the genetic code.

Annemieke Aartsma-Rus explains exon skipping in this movie.

Exon skipping is also explained in this 'Dance your PhD' video.

AON treatment has induced exon skipping resulting in the production of Becker-like dystrophins in patient-derived cultured cells and the mdx mouse model. In the mouse model, this was accompanied by functional improvement.

There are different types of AONs (chemistries).


For different mutations and types of mutations different exons need to be skipped to restore the genetic code. As most patients have a deletion and these cluster in a hotspot, the skipping of some exons applies to more patients than others. An image representation of the exons in the dystrophin gene is available here. A much more comprehensive discussion of exon skipping, including images that help to visualize the way it works, is available here.

While exon skipping would be beneficial to the majority of mutations, there are some exceptions.

Challenge 1:

Exon skipping cannot be tested in healthy volunteers, since skipping an exon that restores the genetic code in patients disrupts the genetic code in healthy people (thus potentially converting them into Duchenne patients).

Clinical trials:

The first clinical trial tested local injection of exon 51 AONs in the shin muscle 4 Duchenne patients (Prosensa (now BioMarin) and LUMC). Dystrophin was restored locally in each patient.

A second trial using a different chemistry took place in London (Muntoni/AVI Biopharma (now called Sarepta)).

Challenge 2:

Over 30% of the human body consists of muscle, so it is impossible to perform local injections of each and every muscle.

Solution 2:

As a result of the disease, patients and mouse muscles are leaky (damaged). While AONs are not taken up from blood by healthy muscles, they are by the leaky damaged muscle. Thus in this case, the disease helps the treatment. Intravenous and subcutaneous injections in mouse models have resulted in body wide exon skipping and dystrophin restoration.

Clinical trials:

Clinical trials have been completed in which different doses of
2OMePS (Prosensa Therapeutics/GSK) or PMO (AVI-Biopharma (now called Sarepta)) AONs were tested through subcutaneous injection or intravenous injection. Both 2OMePS and PMO treatment appears to result in dystrophin restoration.

The PMO AON targeting exon 51 is currently called Eteplirsen. Since not all patients in the systemic trial responded equally well, a follow up trial testing two higher doses was done in a small trial involving 12 patients. Dystrophin was restored for all patients after 24 weeks of eteplirsen treatment. Patients have now been treated for over 188 weeks and for the 10 patients who are still ambulant the 6 minute walk distance declined less than would be anticipated from the natural history (although this should be interpreted with caution given the small group size).

A phase 3 trial where weekly intravenous dosing with 30 mg/kg Eteplirsen is tested for 96 weeks in ambulant patients is currently ongoing in the USA. This is an open label study, where patients with mutations amenable to exon 51 skipping are treated, while patients with non-amenable mutations are used as controls for functional tests and safety. In addition, open label trials have been initiated in the USA in young patients (less than 6 years old) and in patients with limited or no ambulation. In the trial in young patients, again a group with non-amenable mutations is used as a control.

Sarepta has filed Accelerated approval with the Food and Drug Administration in the US based on the data of their long term treatment of 12 patients. FDA requested in June 2016 that Sarepta provided them with specific data obtained in the ongoing phase 3 study (dystrophin restoration in biopsies with a technique called Western blotting). Based on the analysis of these biopsies, FDA announced September 19 2016 that Eteplirsen was granted accelerated approval. Sarepta will have to confirm clinical benefit in additional clinical trials that are currently ongoing and will be planned. In December 2016 Sarepta filed for approval with the European Medicines Agency. Evaluation is currently pending.

The 2OMePS AON targeting exon 51 is currently called Drisapersen or Kyndrisa. All patients involved in the early subcutaneous trial were enrolled in an open label extension study where they receive weekly treatment with Drisapersen. Patients have been treated for more than 6 years (including treatment breaks). For 8/10 patients still ambulant at the start of the extension study the 6 minute walk distance has stabilized, while the natural history would predict a decrease. However, lacking a placebo group, these results should be interpreted with caution.

GlaxoSmithKline (GSK) had in-licensed Drisapersen, from Prosensa and has coordinated several trials. In all trials using subcutaneous injection of Drisapersen injection site reactions and proteinuria were more frequently observed in Drisapersen treated patients than placebo treated patients. A trial comparing different dosing regimens has been completed in patients who were at a relatively early stage of the disease. This study involved 54 patients receiving either placebo, weekly subcutaneous treatment with Drisapersen or an intermittent regimen for 48 weeks. Both treated groups walked ~35 meters more than placebo-treated patients in the 6 minute walk test.

A trial comparing different doses has been completed in patients who were in an early disease stage (able to rise from floor in 15 seconds). Patients received placebo, 3 or 6 mg/kg Drisapersen for 24 weeks. Patients treated with 6 mg/kg walked 27 meter more than patients treated with placebo or 3 mg/kg after 24 weeks.

A Phase III placebo-controlled trial was initiated in 2011, to assess the safety and effectiveness of treatment with Drisapersen in 186 ambulant patients. No significant difference in the distance walked in 6 minutes was observed between placebo and Drisapersen treated patients at 48 weeks. Meanwhile, GSK has returned the license to develop Drisapersen to Prosensa and Prosensa has been acquired by BioMarin.

Prosensa/Biomarin have analysed the compiled data of the systemic trials and extension studies. Results are suggestive of a slower disease progression in treated younger patients but also older patients who are treated for 24 months. Based on these data they have filed for Accelerated Approval with the Food and Drug Administration and for Marketing Autorization with the European Medicine Agency in 2015.  Furthermore, they have started the phased redosing of patients in open label extension studies with Drisaperson (which were stopped after the phase III trial results were reported). The FDA reported on Jan 14 2016 that Drisapersen is currently not ready for approval.

On May 31 2016 BioMarin announced withdrawal of their application with EMA.

Challenge 3:

AONs to skip different exons are considered different drugs by the regulatory agencies. This means that developing AONs for different exons is very costly and time consuming, as each has to go through all stages of preclinical and clinical development.


Hopefully, AON development will become faster after the first 2 or 3. TREAT-NMD is coordinating a discussion about this with regulatory agencies on behalf of exon skipping scientists, clinicians and industry and the patient community. The most recent meeting was held on April 29 2015. The resulting publication is now available (free copy can be found here).

Clinical trials:

Sarepta has started a trial for PMOs targeting exon 53 (collaboration with Francesco Muntoni in London). They are planning a placebo-controlled, 96 week phase 3 trial to evaluate exon 45 and 53 AONs. Nippon Shinyaku (Japan) is currently conducting a clinical trial with PMOs for exon 53 skipping in Japan. A placebo-controlled safety and dose finding trial followed by an open label phase with this compound is ongoing in ambulatory patients in the USA. Nippon Shinyaku also is evaluating and with AONs with the ENA chemistry for exon 45 in Japan. In addition, Wave therapeutics is preparing for an exon 51 skipping trial with a new AON modification.

Prosensa/BioMarin has completed a phase I/II trial where different doses of 2OMePS AONs targeting exon 44 were tested using intravenous and subcutaneous injections. Trials testing 2OMePS AONs targeting exon 45 and exon 53 have been started by Prosnesa/BioMarin. However, the clinical development of these compounds has now stopped and BioMarin will focus on the development of next generation exon skipping compounds.

Drugs for stop codon read-through>
18 Sep 2017