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.
Aim:
To correct the genetic code and allow the production of a partially functional dystrophin.
Background:
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 break down DNA and giving them the ability to be taken up by tissues and preventing that they are filtered out by the kidney.
Applicability:
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 currently 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:
- 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).
- 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.
- 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.
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. Because most Duchenne patients produce low, but individually variable levels of dystrophin, the comparison has to be done between dystrophin after treatment and dystrophin levels at baseline, to assess whether levels increased. Dystrophin quantification 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.. Furthermore, the time of obtaining a biopsy will influence how much dystrophin is produced and this varies between trials. Finally, some exons are easier to skip than others. As such it is difficult to compare dystrophin levels between AONs targeting different exons and between trials done by different companies.
Confirmatory studies for the approved exon skipping compounds (PMOs)
Eteplirsen (exon 51 skipping)
The 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.
Golodirsen (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. A clinical trial to assess functional effects of golodirsen is currently ongoing.
Viltolarsen (exon 53 skipping)
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. A confirmatory study was performed where patients received weekly injections of viltolarsen (80 mg/kg) or placebo. This trial failed to meet its primary endpoint, time to rise from floor velocity did not differ between treated patients and the placebo group. The trial is still ongoing and additional data for up to 2 years of treatment will be collected.
Meanwhile, a clinical trial where viltolarsen treatment is evaluated in ambulant and non-ambulant patients is ongoing.
Casimersen (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 (for casimersen this is part of the 96 week study).
Additional trials with PMOs
Brogidirsen (exon 44 skipping) and NS-50/NCNP-03 (exon 50 skipping)
NS-Pharma has initiated a clinical trial with Brogidirsen (NS-089) in Japan. This is a PMO targeting exon 44. An open label clinical trial was done in 6 ambulant patients in Japan, where patients received weekly intravenous infusions of 40 mg/kg or 80 mg/kg brogidirsen. Results have been published). Patients showed an increase in dystrophin levels of 10% and 15% for the 40 mg/kg and 80 mg/kg dose, respectively.
Notably, patients who require exon 44 skipping have higher baseline dystrophin levels, as exon 44 skipping occurs spontaneously. It is speculated that this facilitates an increase in dystrophin expression after induced exon 44 skipping (see also AOC1044).
NS Pharma is also conducting a clinical trial with a PMO targeting exon 50 (NS-50/NCNP-03). No results are yet reported.
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.
Improving exon skipping with chemical modifications
BMN351 (exon 51 skipping)
BioMarin is developing BMN351, an ASO with a different backbone than the approved exon skipping drugs (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.
SQY51 (exon 51 skipping)
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.
WVE-N531 (exon 53 skipping)
Wave is performing clinical trials with WVE-N531, an ASO with better bioavailability than the PMO chemistry. An interim analysis showed that after 24 weeks of biweekly treatment with 10 mg/kg WVE-N351 patients produced 5.5% more dystrophin. Treatment was tolerated well and patients are currently enrolled in an open label study.
Renadirsen (exon 45 skipping)
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.
Increasing general tissue uptake with short peptides
Vesleteplirsen
Eteplirsen (exon 51 skipping) induces only small increases in dystrophin expression. As such there is room for improved AON compounds. One way to achieve this is by linking a short, positively charged, peptide to an AON. This peptide will increase delivery of AONs to all tissues, so also to skeletal muscle.
Sarepta has tested a form of eteplirsen that is linked to a peptide-conjugate (SRP-5051 or vesleteplirsen). 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.
Sarepta announced in October 2024 that they will discontinue the development of vesleteplirsen due to safety issues, with kidney damage not restoring between treatment breaks and kidney function declining for some patients.
PGN-EDO51
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.
Preliminary results from the dose finding trial shows exon 51 skipping and minor increases in dystrophin (0.3%) for patients treated with monthly doses of 5 mg/kg for 3 months. Dosing of patients with 10 mg/kg revealed a similar increase in dystrophin (0.4%), after which the company decided in May 2025 to discontinue development of PGN-EDO51.
ENTR-601-44
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 has a lower positive charge and it is circular, which is expected to result in lower toxicity. ENTR-601-44 has tested their compound in healthy volunteers in the UK, which revealed exon 44 skipping in a muscle biopsy and good tolerability. Currently a clinical trial is ongoing in Duchenne patients.
ENTR-601-45
Entrada has also developed ENTR-601-45, which is a PMO targeting exon 45 with the same EEV peptide as ENTR-601-44. Information about the clinical trial can be found here.
Increasing muscle-specific uptake with transferrin receptor antibodies
AONs can also be delivered more specifically to skeletal muscle and heart by conjugating them to transferrin receptor antibodies. The transferrin receptor (TfR1) is an iron transporter that is expressed on most cells in the body, but highly expressed on skeletal muscle and. The antibodies will bind to the receptor and facilitate uptake of the AON, without impacting the iron transport function.
DYNE-251 (exon 51 skipping)
Dyne Therapeutics is developing DYNE-251, a PMO targeting exon 51 linked to an antibody fragment recognizing the transferrin receptor. A clinical with DYNE-251 in Duchenne patients is currently ongoing. Results revealed that patients treated for 6 months with monthly doses of 5 mg/kg DYNE-251 produced 0.9% more dystrophin, while those treated with 10 and 20 mg/kg produced 3% and 3.7% more. Patients treated with 40 mg/kg experienced side effects, likely related to DYNE-251, from which they have fully recovered. Patients treated for longer times with 10 mg/kg and 20 mg/kg showed improvement in function as assessed by multiple outcome measures.
Delpacibart zotadirsen (exon 44 skipping)
Avidity is running a clinical trial with delpacibart zotadirsen (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 but with a larger antibody). Preliminary results showed an increase of 25% of dystrophin after 3 6-weekly intravenous doses of 5 mg/kg delpacibart zotadirsen. No further increase of dystrophin restoration was observed for patients receiving 10 mg/kg every 8 weeks. Currently, patients in the clinical trial are switched to a regimen of 5 mg/kg every 6 weeks.
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 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.
