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 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. 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.
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. 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 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).
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 by 2021 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 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. Finally, Sarepta is planning a new clinical trial testing higher doses of eteplirsen 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 that should improve AON uptake by tissues (so called pPMO). 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. Currently a placebo-controlled phase 2/3 trial to evaluate longer term treatment with lower doses of suvodirsen is ongoing.
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 development for AONs targeting other exons
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 damage observed in preclinical models at high doses and infection risk of IV catheters needed for repeated IV infusions.
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).
Nippon Shinyaku (Japan) and NS-Pharma has conducted clinical trials with PMOs for exon 53 (viltolarsen) skipping 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 is preparing to file a new drug application with the FDA in 2019.
Daiichi Sankyo also is 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 aims to continue developing this compound.
Preclinical studies to identify exon skipping compounds for exon 44, 52, 54 and 55 are ongoing at several companies working in the exon skipping space.
Clinical trials with drisapersen (discontinued)
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 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. 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.