Annemieke Aartsma-Rus has provided the following explanation to the recently published article in The FASEB Journal:
Since exon skipping modulates the amounts of dystrophin RNA transcripts (i.e. the DNA copies that are translated into protein), we wanted to study this in more detail. What we see is that much more dystrophin RNA and protein is produced in heart than skeletal muscle, and slightly more dystrophin in diaphragm than skeletal muscle. This may in part explain why exon skipping works less in heart than other muscles: if there is more transcripts, skipping levels will be lower even when similar amounts of antisense oligonucleotide (the exon skipping compound) end up in heart and a limb muscle (example: 10 AONs vs 100 dystrophin RNAs in e.g. calf muscle = 10% skip, but 10 AONs vs 1000 dystrophin RNAs in heart = 1% skip) (note that another part of the explanation is that antisense oligonucleotides reach the heart with lower efficiency, so this is part of the explanation, not the full explanation).
The next thing we show is that the dystrophin transcripts are not all complete, so the amount of transcripts containing exon 1/the beginning is higher than the amount of transcripts containing later exons/the end (and in order for the transcripts to be translated into a functional dystrophin, they have to be complete from start to end). The cause of this phenomenon (them not being complete) is not known. In healthy mouse muscle, there is a slight imbalance, in mdx muscle (without dystrophin) there is a much bigger imbalance (so the percentage of transcripts being incomplete is higher). When the reading frame is restored in mdx muscle by exon skipping, the imbalance is not restored. Also, in Becker patient muscles we see this imbalance - the level of imbalance is between that found in healthy muscle and muscle without dystrophin and varies a lot between Becker patients. To put it very simple, we find in Becker patients that the amount of dystrophin they produce relates not to the amount of dystrophin transcripts but to the amount of COMPLETE transcripts (this is very logical, but the imbalance of dystrophin has not been well studied, so we did not know this before).
What this means:
Because not all dystrophin transcripts are complete, this means that not every transcript from which you skip an exon, can be translated into a dystrophin protein. The level of imbalance will impact on how patients respond to exon skipping (if the imbalance is mild, there can be more dystrophin formed that when the imbalance is very severe). Note that this does not mean exon skipping will not restore dystrophin (we've seen in GKS and Prosensa and Sarepta trial that dystrophin is formed after treatment - so apparently there are sufficient numbers of transcripts skipped to allow some to be translated. It means that things are more complicated than we thought and that perhaps we have another explanation for why some patients fail to produce dystrophin after treatment and why some patients produce more than others. (note that we still need to proof that the amounts of dystrophin currently produced lead to a functional benefit in placebo-controlled trials as are planned by Sarepta and ongoing for GSK).
We do not know the mechanism of the transcript imbalance (why the dystrophin transcript is less stable in DMD patients and BMD patients, and why there are differences between patients). This is something we would like to study further. However, by publishing this now, we allow other researchers to also work this out (it will go quicker when multiple people work on it).
When we can shed more light on this, we can also study ways to correct the imbalance (i.e. leading to more complete dystrophin transcripts and to more dystrophin protein). If we could achieve this, that would most likely improve the effect of exon skipping, since the same amount of skip would be expected to lead to more dystrophin. It would also be expected to improve the working of ataluren, as also there more stable transcripts would mean more dystrophin. However, we will need to do more work to identify the cause of this imbalance, in order to find a way to improve things.
Feel free to put this on a website, or to ask for further elucidation if things are not yet clear (we're talking hardcore genetic research here).