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#apaperaday: Exon skipping induces uniform dystrophin rescue with dose-dependent restoration of serum miRNA biomarkers and muscle biophysical properties

In today’s #apaperaday, Prof. Aartsma-Rus reads and comments on the paper titled: Exon skipping induces uniform dystrophin rescue with dose-dependent restoration of serum miRNA biomarkers and muscle biophysical properties

Today’s pick is from Molecular Therapy nucleic acids by Chwalenia et al on studying how serum miRNA biomarkers change upon restoring dystrophin at different levels in the mdx mouse model. Doi 10.1016/j.omtn.2022.08.033

When muscles are damaged, components leak out, e.g. the famous creatine kinase (CK), which is elevated in patients with muscular dystrophy due to chronic muscle damage, but also in healthy individuals if we do too strenuous exercise or have a muscle injury.

miRNAs, small double-stranded RNAs that specifically target mRNA transcripts to regulate gene expression, can leak out as well. Different tissues make different miRNA and the miRNAs that have been found in individuals with muscle damage are called ‘myomiRs’

Like CK these markers are not disease specific, they are a marker for muscle damage. Here authors explore whether myomiRs can also be used as a pharmacodynamic marker for dystrophin restoration. For this they use male mdx mice and treat them with peptide-conjugated morpholinos

The peptide is called Pip9b2 and is able to deliver the morpholino efficiently to muscle. Authors use a single IV injection at 3, 6 and 9 mg/kg to induce different levels of dystrophin. Animals are treated at 8 weeks of age and analyzed 2 weeks later. Wild types are included!

Authors analyzed blood (for the myomirs) and the tibialis anterior (for dystrophin and muscle stiffness analysis). They see a dose dependent increase in dystrophin: 3 mg/kg: 4%, 6 mg/kg 17% and 9 mg/kg 45% – no dystrophin was found in untreated mice, 100% in wild types.

Authors first validated the myomir analysis tool – as some myomirs are very similar in sequence, not all assays can discriminate properly. After optimization authors analyzed myomir levels in the different groups, showing a dystrophin level dependent decrease.

Note that there is a lot of variability within the different dosing groups. miR-133a-3p levels correlated most closely with dystrophin levels, followed by CK. Authors also looked at distribution of dystrophin, showing that at 6 and 9 mg/kg expression was uniform.

This is important as these authors have shown earlier that for a given dystrophin level uniform expression is better than patchy expression (e.g. it is better to have all fibers express 10% of dystrophin than to have 10% of fibers produce 100% dystrophin https://pubmed.ncbi.nlm.nih.gov/31849191/)

Finally authors studied muscle stiffness with atomic force microscopy, showing that dystrophin restoration improves stiffness to beyond wild type levels. Authors discuss this is likely due to the hypertrophy in mdx mice. It would be interesting to see longer term data.

I would expect that the hypertrophy will reduce with time. Authors also discuss that the myomirs are markers for muscle turnover and that surprisingly CK was performing rather well, while in Duchenne patients it is more variable than the myomirs.

They also propose the myomirs as biomarkers for human trials, given that in mice they respond to therapeutic approaches (dystrophin restoration but also those addressing muscle quality). Some notes/considerations about this:

Myomirs appear to have the same pattern as CK: they are higher in early stages of the disease because they leak out of the muscle. However, with time as disease progresses, muscle mass is reduced so there is less CK and less myomir to leak into the blood.

Thus levels will go down when patients age. This is not a sign that disease is getting better but rather that it is getting worse. Because the mdx mouse model is very good at muscle remodeling CK levels and myomir levels remain high for most of the mouse lifespan.

Thus in mdx mice, when muscle quality improves (due to dystrophin restoration, less inflammation, less fibrosis etc etc), the levels of CK and myomir will go down and indeed they will be a pharmacodynamic marker. The question is whether this will also be the case in humans.

I think studies in more severely affected models would be informative to shed some light on this (e.g. the DMD rat model and young d2/mdx mice). Having said all this: I like that the authors use a systematic set up and include all relevant controls.

As always, more work needs to be done to assess whether the findings translate between mouse and humans. However, you need to start somewhere. Looking forward to future studies and results.