1 2 aim

Mutation specific approaches

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).

Specific approaches:

  • Exon skipping
  • Drugs for stop codon readthrough

Current status

Four exon skipping drugs are approved in the USA (eteplirsen, golodirsen, casimersen and viltolarsen) and one in Japan (viltolarsen). These compounds received accelerated approval based on their ability to restore low levels of dystrophin protein. Whether these low levels can slow down disease progression is still being studied.

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 becomes unreadable, which results in premature truncation of the translation from gene into protein. In Becker patients, mutations maintain the genetic code, allowing for the production of 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 antisense oligonucleotides (AONs or ASOs). 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.

Making AONs drugs

AONs are small pieces of chemically modified DNA. The modifications are needed to give the AON druglike properties, e.g. making sure they are resistant to enzymes that breakdown DNA and giving them the ability to be taken up by tissues and preventing that they are filtered out by the kidney.

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. Finally, the DOVE tool helps assessing which exon needs to be skipped for which mutation.

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

 Summary of current situation

There are current 4 AONs approved. These are of the phosphorodiamidate morpholino oligomer (PMO) chemistry. This is a chemistry that is very efficiently filtered out by the kidney. As such frequent treatment with high doses of AONs are needed (weekly intravenous injections of 30-80 mg/kg) and uptake by skeletal muscle is suboptimal, resulting in only minimal increases in dystrophin.

To improve delivery to skeletal muscle and heart, different approaches are studied:

  1. Linking a positively charged peptide to PMOs. This will improve delivery to all tissues, including muscle and heart. The disadvantage is that the positively charged peptides can lead to toxicity especially in the kidney. The question is whether improved efficiency occurs at a dose that is manageable for the kidney.
  2. Linking a conjugate to PMOs that improve specific uptake by skeletal muscle. This can be achieved with antibodies to the transferrin receptor, which is enriched on skeletal muscle.
  3. Testing other chemical modifications that prevent clearance by the kidney and that show better efficacy and safety than similar compounds that were developed previously (see drisapersen).

Below I outline the current developments per exon.

Dystrophin assessment considerations:

As exon skipping aims to restore dystrophin, drug developers use analysis of dystrophin after treatment to assess whether the treatment worked as intended. This is usually done by a technique called Western blotting, which measures the amount and length of dystrophin compared to a reference sample (human control muscle). As it is known that the amounts of dystrophin in control muscles can vary, it is difficult to compare the dystrophin levels between different developers. Only when analysis is done by the same company can levels be compared.

Exon 51 skipping:
Since exon 51 skipping applies to the largest group of patients, AONs targeting exon 51 have been developed furthest. One exon skipping AON of the morpholino (PMO) chemistry called eteplirsen (exondys51) has received accelerated approval from the FDA.  This was based only on dystrophin restoration, the company developing eteplirsen (Sarepta) still needs to confirm that treatment slows down disease progression. The EMA did not approve eteplirsen; the committee for human medicinal products (CHMP) of the EMA gave a negative opinion in June 2018, after which Sarepta filed an appeal. The negative opinion was reconfirmed in September 2018.

Clinical trials with Eteplirsen:

Eteplirsen is a PMO AON targeting exon 51. It needs to be administered by intravenous infusion. Etepliresen was tested in 19 patients at different doses up to 20 mg/kg. Since not all patients in this trial responded equally well, a follow up trial testing two higher doses was done in a small trial involving 12 patients. In this study, dystrophin was restored for all patients after 24 weeks of eteplirsen treatment. Patients were analysed after 188 weeks of treatment 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).

FDA announced September 19 2016 that Eteplirsen was granted accelerated approval. This was based only on small increases in dystrophin observed in muscle biopsies of treated patients. FDA specified that functional effects are not yet confirmed. As such, Sarepta will have to confirm clinical benefit in additional clinical trials that are currently ongoing.

A phase 3 trial where weekly intravenous dosing with 30 mg/kg eteplirsen is tested for 96 weeks in ambulant patients has been completed in the USA and results have been published. This was an open label study, where patients with mutations amenable to exon 51 skipping are treated, while patients with non-amenable mutations were used as controls for functional tests and safety. Comparison between the groups was unfortunately not feasible, since most of the controls dropped out of the study before it was completed. A clinical trial in Europe in patients between 6 months and 4 years has been completed, suggesting treatment is tolerated also in younger patients.

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 to study safety, showing good tolerability in these populations as well. In the trial in young patients, again a group with non-amenable mutations is used as a control. Finally, Sarepta is performing a new clinical trial testing higher doses of eteplirsen (100 and 200 mg/kg/week) as requested by FDA.

Eteplirsen induces only small increases in dystrophin expression. As such there is room for improved AON compounds. Sarepta is currently testing a form of eteplirsen that is linked to a peptide-conjugate (SRP-5051 or vesleteplirsen)  that should improve AON uptake by tissues (so called pPMO). Results from biopsies analyses at 12 weeks for 4 Duchenne patients treated with monthly doses of 30 mg/kg vesleteplirsen revealed that patients produced ~2% dystrophin. Low levels of magnesium in the blood (hypomagnesemia) were found and managed with magnesium supplementation. Stage B of this clinical trial has now initiated where larger groups of patients are treated with 30 m/kg/month vesleteplirsen. Another case of hypomagnesemia occurred, which resulted in a temporary hold of this trial. After 7 monthly treatment results from biopsies from 20 patients revealed that patients produced ~5% of dystrophin. Low blood levels of magnesium and kalium were reported, but managed by supplementation.

The company PepGen is developing a similar compound (peptide linked to PMO) called PGN-EDO51. This compound has been tested in healthy volunteers while clinical trials for Duchenne patients are ongoing. Exon skipping was observed in the healthy volunteers and non-human primates, but for both healthy volunteers and primates cases of hypomagnesemia were reported.

Where the peptides used in vesleteplirsen and PGN-EDO051 increase general uptake of PMOs by all tissues, companies are also working on targeted delivery. Dyne Therapeutics is developing DYNE-251, a PMO targeting exon 51 linked to an antibody fragment recognizing the transferrin receptor. This receptor is expressed at high levels on skeletal muscle and thus this antibody should result in targeted delivery of the PMO to skeletal muscle. In mouse models this indeed results in improved levels of exon skipping and dystrophin restoration. A clinical  with DYNE-251 in Duchenne patients is currently ongoing. First results revealed that patients treated for 6 months with monthly doses of 5 mg/kg DYNE-251 produced 0.9% dystrophin. Results from patients in the higher dose groups are expected later in 2024.

Finally, companies are working on different chemical modifications of ASOs. BioMarin is developing BMN351, an ASO with a different backbone (phosphorothioate). This results in better bioavailability of the ASO allowing lower doses of ASO to be used. However, this backbone can also result in side effects (see drisapersen session below). BMN351 resulted in dystrophin restoration in a mouse model carrying a mutated version of the human dystrophin gene. Biomarin is now conducting a clinical trial for BMN351 in Duchenne patients.

Synthena is developing SQY51 an ASO with a ‘tricycloDNA’ chemistry that also targets exon 51. A clinical trial is ongoing in France for SQY51 in Duchenne patients.

Exon 53 skipping:

Sarepta has completed a trial for PMOs targeting exon 53 (golodirsen, collaboration with Francesco Muntoni in London). After 48 weeks of treatment an increase in dystrophin expression of ~1% was observed. Based on this, Sarepta has applied for FDA approval for golodirsen in Q4 of 2018. In August 2019, FDA informed Sarepta that golodirsen was not approved, due to safety concerns, relating to kidney damaged observed in preclinical models at high doses and infection risk of IV catheters needed for repeated IV infusions. Sarepta applied again with a plan to monitor potential risks resulting in FDA approving golodirsen in December 2019. As for eteplirsen FDA stressed that functional effects of golodirsen treatment have yet to be confirmed.

Nippon Shinyaku (Japan) and NS-Pharma have conducted clinical trials with PMOs for exon 53 skipping (viltolarsen) in Japan and in ambulatory patients in the USA. After 24 weeks of treatment with high doses (40 and 80 mg/kg) up to 5% dystrophin was observed in a muscle biopsy. NS-Pharma Received FDA approval for viltolarsen August 2020 and approval by the Japanese Ministry for Health, Labour and Welfare in March 2021.

Wave is performing clinical trials with WVE-N351, an ASO with better bioavailability than the PMO chemistry. First results revealed that patients produced 0.27% dystrophin after 3-4 bi-weekly doses of WVE-N351


Exon 45 skipping

Sarepta has initiated a placebo-controlled, 96 week phase 3 trial to evaluate exon 45 (casimersen) and 53 AONs. Interim analysis of a muscle biopsy of the casimersen treated patients revealed an increase in dystrophin levels from 0.9% (baseline) to 1.7% (1 year treatment). Based on these results FDA approved casimersen in February 2021. Also here, FDA stressed that functional effects of casimersen have yet to be confirmed.

Daiichi Sankyo was developing AONs with the ENA chemistry for exon 45 skipping in Japan. A first trial revealed the compound to be safe but increases in dystrophin were extremely modest. Daiichi Sankyo recently announced they will not develop this compound (renadirsen) further.

Exon 44 skipping

NS-Pharma has initiated a clinical trial with NS-089 in Japan. This is a PMO targeting exon 44.

Entrada is developing ENTR-601-44, a PMO targeting exon 44 that carries an EEV peptide. This peptide has similar amino acids as vesleteplirsen and PGN-EDO51 but is circular, which is expected to result in better delivery and lower toxicity. ENTR-601-44 is currently tested in healthy volunteers in the UK.

Avidity is running a clinical trial with AOC-1044, a PMO targeting exon 44, that is linked to an antibody targeting transferrin for improved delivery to muscle (similar to the Dyne approach).

Exon skipping for single exon duplications

Sarepta is conducting a clinical trial for exon 45, 51 and 53 skipping for patients with a duplication of exon 45 (casimersen treatment), exon 51 (eteplirsen treatment) and exon 53 (golodirsen treatment). Results have been presented at conferences showing dystrophin restoration after treatment.

Exon skipping genes

Exon skipping ASOs have to be delivered regularly (weekly or monthly), and delivery efficiency is currently relatively low. Researchers have investigated the possibility to deliver an antisense gene to muscle using AAV, thus combining gene therapy and exon skipping.

Kevin Flanigan (Columbus Ohio) has developed an antisense gene for patients with an exon 2 duplication. So far 3 patients have been treated, aged 7 months, ~9 years and ~14 years of age. Biopsies revealed dystrophin restoration at levels of 80%, 6% and 1%, respectively 12 months after treatment.

Audentes/Astellas was preparing for clinical trials with antisense genes for exon 51, 53 and 45 skipping. However, this development was recently deprioritized and terminated.

Other exons

Preclinical studies to identify exon skipping compounds for exon 50, 52, 54 and 55 are ongoing at several companies working in the exon skipping space.

Mutation specificity:

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 dialogue 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 with drisapersen (discontinued):

In addition, Wave therapeutics has completed a phase 1 trial dose-ascending safety test with an exon 51 skipping AON with a new modification, suvodirsen. This revealed that suvodirsen was tolerable at lower doses, but that the intensity of adverse events (fever, nausea and headaches) was more severe for higher doses. A placebo-controlled phase 2/3 trial to evaluate longer term treatment with lower doses of suvodirsen revealed that suvodirsen did not result in increases levels of dystrophin in biopsies. Therefore development of suvodirsen has been abolished.

The 2OMePS AON targeting exon 51 is called Drisapersen or Kyndrisa. All patients involved in an early subcutaneous trial were enrolled in an open label extension study where they receive weekly treatment with Drisapersen. Patients were 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 Drisapersen (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. They have discontinued the development of drisapersen and also other AONs that were in clinical development targeting exon 44, 45 and 53. They are currently working on the development of more effective and more save exon skipping compounds.

Ataluren and Gentamicin

Current status

Ataluren has received conditional approval from the European Medicines Agency (EMA) for treating ambulant Duchenne patients of 2 years and older with nonsense mutations. In 2023 the EMA indicated they would not renew the conditional approval. As such Ataluren will likely be taken off the market in EMA countries. This decision do not impact countries outside of the EU where ataluren is marketed.

These drugs only work for patients with a “stop signal” mutation. These mutations do not affect the genetic code, but introduce a stop signal in the middle of the gene in addition to the one at the end of the gene that signifies protein translation is complete. This is the case for ~10-15% of Duchenne patients. The drugs can also be beneficial for individuals with stop codons in other genes .

To force the cell to ignore the mutated stop codon and produce a complete dystrophin protein.

All genes have a start signal and a stop signal so the machinery that translates genes into proteins knows where to begin and where to end translation. Sometimes a small mutation can introduce a stop signal within the gene (in addition to the one at the end). This type of mutation is called a nonsense mutation. Normal stop signals generally differ slightly from these mutated stop signals (compare it to a stop signal at a busy intersection (normal stop signal) and one on a highway (mutated stop signal). Nevertheless, the cell will follow the aberrant stop signal and will stop the translation of the protein prematurely. There are drugs that suppress the usage of nonsense mutations, while they do not affect the normal stop codons. The first drug identified to do this in cultured cells and Duchenne mouse models was gentamicin (an antibiotic of the animoglycoside class).

Clinical trial:
Gentamicin has been tested in Duchenne patients, but has never convincingly shown dystrophin restoration.

Challenge 1:
In addition to its low efficiency, gentamicin is toxic when used for longer periods (it can damage the ears and the kidney).

Solution 1:
Screening a large number of drugs resulted in the identification of a drug that was also able to force cells to ignore mutated stop codons, without the toxic side effects. This drug is called PTC124 or ataluren or Translarna™ and is developed by PTC Therapeutics (USA). It can be taken orally and resulted in dystrophin restoration in cultured cells and the mdx mouse model. Ataluren is currently conditionally approved in Europe and Brazil for ambulant Duchenne patients with nonsense mutations aged 2 and older.

Clinical trials:
Ataluren was safe in healthy volunteers. A first trial in Duchenne patients where patients were treated with different daily doses of Ataluren for 4 weeks showed that treatment was well tolerated and that dystrophin expression was increased for treated patients. Trials to test whether this also results in functional improvement after long term treatment have been performed in multiple centers in the USA and Europe.

Unfortunately, treatment did not convincingly lead in to a functional improvement when compared to placebo treated patients using a 6 minute walk test and therefore the trials were put on hold. Patients involved in these trials in the USA and Europe can enrol in an open label trial.

After detailed analysis of the data and further optimization of dosing, a new confirmatory phase 3 trial in 220 DMD patients has been completed in North- and South-America, Asia, Australia and Europe. Ataluren treated patients on average walked 15 meters more in 6 minutes compared to placebo treated patients. In the pre-specified subgroup of patients (walking between 300 and 400 meter in 6 minutes at the start of the trial) ataluren treated patients walked 47 meters further than placebo treated patients. Ataluren treated patients also performed better in other functional tests. As before, Ataluren was well tolerated. Based on this data, EMA extended the conditional approval, requesting a new confirmatory study. In this trial patients were treated for 72 weeks with either ataluren or placebo. The treated patients walked 14 meters further than the placebo group in the 6 minute walk test (significantly different). During the 72 week trial, 12 ataluren treated patients lost ambulation vs 20 placebo treated patients. Treated patients performed better in the North Star Ambulatory Assessment and timed function tests. Furthermore, PTC initiated a clinical trial to assess dystrophin levels before and after treatment. Results are now available and suggest an increase in dystrophin expression after treatment, albeit very minor. Since ataluren was conditionally approved in Europe, PTC is collecting real world evidence in patients treated commercially with ataluren. Thus far comparison with natural history of untreated patients suggests ataluren treated patients have a later loss of ambulation and later onset of pulmonary problems.

Based on these results PTC has submitted an application to obtain full (instead of conditional) marketing with EMA and marketing authorization with the FDA. On September 15 2023 the EMA announced that the CHMP decided not to grant full marketing authorization and also did not extend the conditional approval for ataluren. PTC appealed this decision, after which the EMA re-evaluated the data. On January 26 2024 EMA announced that also after re-evaluation the CHMP recommends not to extend the conditional approval. This decision is pending confirmation from the European Commission.