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Dystrophin Restoration after Adeno-Associated Virus U7–Mediated Dmd Exon Skipping

#apaperaday: Dystrophin Restoration after Adeno-Associated Virus U7–Mediated Dmd Exon Skipping Is Modulated by Muscular Exercise in the Severe D2-Mdx Duchenne Muscular Dystrophy Murine Model

In today’s #apaperaday, Prof. Aartsma-Rus reads and comments on the paper titled: Dystrophin Restoration after Adeno-Associated Virus U7–Mediated Dmd Exon Skipping Is Modulated by Muscular Exercise in the Severe D2-Mdx Duchenne Muscular Dystrophy Murine Model

Today’s pick is from the American Journal of Pathology by Monceau et al on the effect of voluntary exercise on AAV U7 snRNA mediated exon skipping and treatment effects in the d2/mdx mouse model. doi 10.1016/j.ajpath.2022.07.016

Exon skipping can reframe dystrophin transcripts to allow production of partially functional dystrophins like those found in Becker patients. Normally exon skipping is achieved with antisense oligonucleotides, which give a transient effect.

An other option is to deliver an antisense gene. This needs specific genes that encode part protein & part antisense. The antisense gene mostly used for this approach is U7 snRNP, where the normal antisense part (targeting histone RNA) is replace by antisense sequence of choice

This antisense gene is delivered with AAV – which is also used to deliver the microdystrophin gene. Authors discuss that in the past people have shown that exercising mdx mice can increase the amount of microdystrophin that is produced.

Here they wanted to see the effect of exercise on antisense-gene mediated dystrophin restoration. However, instead of using the regular mdx mouse, they used the d2/mdx mouse, which supposedly is more severely affected. They started when mice were 5 months old & used only females.

Some concerns I had when I read this: while at a young age d2/mdx mice have more severe fibrosis (10 weeks), older d2/mdx mice recover (age 34 weeks see e.g. this article). Furthermore males are more severely affected than females (see same paper).

First authors subjected groups of female d2/mdx mice to 1 month of voluntary wheel running and compared the muscle to sedentary mice and analyzed tibialis anterior muscles. No differences were seen on centrally located nuclei, regeneration markers and fibrosis levels

Note that the levels of extracellular matrix/fibrosis (figure a top panels) are minimal anyway – this is mostly the endomysium surrounding the muscle fibers. No clear deposits of fibrosis can be seen. Authors quantify the levels (figure F) but it not clear what they mean.

In the legend they claim “percentage of the area occupied by ECM”, while the Y axis in Figure F says AU (arbitrary units). Due to the lack of wild type references we do not know whether the amount of ECM is increased at all or whether this is a ‘healthy amount’.

In either case, there is not a lot of fibrosis – which fits with the tibialis anterior generally being one of the less affected muscle, female d2/mdx mice showing less pathology AND older d2/mdx mice (these are 6 months old) recovering from earlier pathology.

The training did do SOMETHING though – it increased the number of type 1 muscle fibers and reduced the number of type 2 muscle fibers (so more endurance and less strength fibers  – which fits with chronic exercise).

Then authors injected a new set of mice (female 5 months old d2/mdx again) with AAV containing an antisense gene for exon 23 skipping (restoring dystrophin) and an antisense gene not targeting anything (not restoring dystrophin – negative control)

The mice injected with the exon 23 skipping AAV antisense gene were either sedentary or had a voluntary running wheel. They observed dystrophin restoration in all the exon 23 skipping AAV treated mice, but more in the sedentary mice than the wheel running mice (~80% vs 65%)

The numbers of mice they did the study with are rather low (4-6) however, so I do not know whether this is a real finding or whether it is chance. The number of viral genomes and exon skipping levels were the same between running and sedentary exon 23 antisense gene treated mice.

Authors outline that dystrophin transcripts show a 5′ to 3′ imbalance (ie there are more exon 1 containing dystrophin transcripts than exon 60 containing transcripts, see this paper) Authors wanted to investigate this in sedentary and running mice.

However, they analyzed exon 1 and exon 79 (the first and the last exon of the muscle dystrophin form). This is not informative, as Dp71, a smaller dystrophin that is expressed in all cells, also contains exon 63-79. So if you quantify exon 79 you have muscle dystrophin and Dp71.

Authors did not show a difference between sedentary and running mice for exon 1 and exon 79. HOWEVER, we do not know whether muscle dystrophin levels were indeed identical, or if muscle dystrophin levels went down, but Dp71 levels went up…

Authors discuss that the running seems to reduce the amount of dystrophin restoration in antisense treated mice compared to sedentary mice. However, they correctly point out that dystrophin restoration levels in both groups are well beyond the therapeutic levels.

They discuss the running might have a different effect on d2/mdx mice than on ‘regular’ mdx mice. I agree – regular mdx mice are very hypertrophic and regenerate continuously. For the 5-6 month old d2/mdx mice limited data suggest they regenerate less and have less pathology.

They also discuss the fact males are more severely affected and that future studies should be done with male mice (agreed!). Finally they say that clearly more studies are needed. I agree as well – this work shows that there are aspects of the d2/mdx mice we do not understand

While we heralded the d2/mdx mice as ‘a more severe model’ this study shows that at least 6 month old females have very little pathology in the tibialis anterior mice. This is supported by other studies – but so far limited data is available.

We had a meeting about this in 2017 & based on the consensus there currently Annamaria de Luca and my group (with Maaike van Putten) are conducting a HUGE natural history study in d2/mdx and regular mdx with funding of Duchenne UK and Charleys Fund.