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#apaperaday: Increase in Full-Length Dystrophin by Exon Skipping in Duchenne Muscular Dystrophy Patients with Single Exon Duplications

In today’s #apaperaday, Prof. Aartsma-Rus reads and comments on the paper titled:  Increase in Full-Length Dystrophin by Exon Skipping in Duchenne Muscular Dystrophy Patients with Single Exon Duplications: An Open-label Study

Today’s pick is from @journal_nd by Nicolau and @KevinMFlanigan on a clinical trial for exon skipping with approved antisense oligonucleotides (ASOs) in Duchenne patients with single exon duplications DOI: 10.3233/JND-230107

Exon skipping aims to make the dystrophin gene code readable by skipping an exon for Duchenne patients. In this way instead of no functional protein, a partially functional dystrophin can be produced as is also found in Becker patients.

So far this has been tested and applied only to Duchenne patients with deletion mutations (i.e. the most common mutation type). For patients with duplications, things are more challenging as for duplications involving multiple exons you need to target a specific exon.

In other words, skipping the original exon can e.g. restore the gene code, while skipping the duplicated one will not do this. Thus you get a dilution of your effect, as the ASOs will be equally inclined to bind the original and the duplicated exon.

For single exon duplications, things are different however. Now skipping either the original or the duplicated exon will restore the normal gene code. The only problem is that skipping them BOTH with disrupt the gene code. So you aim for limited efficiency here.

We’ve tested exon skipping for single exon duplications years ago in cultured patient cells (https://pubmed.ncbi.nlm.nih.gov/17612397/). What we saw was that in cultured cells, we were too efficient and generally primarily skipped both exons, though we did achieve some dystrophin restoration.

Since delivery in humans is less effective than transfecting ASOs in a 2D layer of cells, multiple people (including me) have speculated that perhaps this would work out in patients with single exon duplications. Here the authors have tested this in collaboration with Sarepta.

They have done a clinical trial in patients with single exon duplications of exon 51, 45 or 53, i.e. the exons for which Sarepta has FDA approval in the USA (eteplirsen, casimersen and golodirsen). As these variants are very rare, authors managed to include 3 patients.

Patients were treated with the respective ASO at 30 mg/kg for 48 weeks. All patients received all infusions, except patient 3 who missed a single infusion. Two patients were non-ambulant, and PROMIS upper extremity and forced vital capacity remained stable for the year.

One individual reported improved trunk control. Biopsies were obtained before and after treatment. RT-PCR analysis revealed an increase in normal transcripts for each subject (we will get back to that). More importantly, there was an increase in dystrophin seen by western blot.

Treatment was well tolerated, with some infusion-related side effects reported. Authors discuss that it was difficult to recruit patients due to the rarity of duplications and single exon duplications. The study was not powered to find efficacy – just to show proof-of-concept.

Authors say that the baseline dystrophin levels were higher than expected, which might have been due to the high level of normal transcripts they detected by RT-PCR at baseline. However, this might in part also have been an artifact of the PCR:

As there is a single exon duplication, during the PCR reaction the two exons can hybridize to the ‘wrong’ counterpart, duplicated to original and vice versa. This can lead to there seemingly being no duplication. Due to wobbles, this is not very efficiently amplified.

However, we found already in our 2007 paper that there were normal transcripts detected for the duplicated patients with (in our hands) no detectable dystrophin. It is possible that this is playing a role here as well, and therefore I do not fully trust the exon skipping levels.

What I trust are the dystrophin restoration data: it is clear dystrophin is increased in each case and this seems to be more efficient than for deletions (note that patient 1 was golodirsen and patients 2 and 3 were casimersen treated: deletions have ~1% dystrophin after 1 year).

This can be because the ASOs can skip either the original or duplicated exon (with deletions they have only one target). Furthermore, the restored dystrophin is full-length so probably more stable than the Becker-type dystrophins produced for patients with a deletion.

Authors discuss that patient 2 was the only ambulant, younger patient, and he appears to show the least dystrophin. They speculate this might be due to muscle turnover being higher as he is younger and growing. It might also be variability (difficult with n=3).

As this is an open label study with only three patients, it is impossible to draw conclusions at a functional level. However, it is clear that exon skipping can restore dystrophin for single exon duplications so these can be included in the trials and the label.

Kudos to the authors and Sarepta for coordinating and facilitating this trial. Even though it applies to a very small group of patients, it is important data to this small group.