#apaperaday: Deletion of miR-146a enhances therapeutic protein restoration in model of dystrophin exon skipping

In today’s #apaperaday, Prof. Aartsma-Rus reads and comments on the paper titled: Deletion of miR-146a enhances therapeutic protein restoration in model of dystrophin exon skipping

It is from MolTherapy nucleic acids by McCormack et al on dystrophin exon skipping with and without miR-146a. DOI: 10.1016/j.omtn.2024.102228

Duchenne patients do not make functional dystrophin, while Becker patients make internally deleted partially functional dystrophins. Exon skipping can allow Duchenne patients to make Becker-like proteins. This uses antisense oligonucleotides (ASOs).

Authors argue that a specific percentage of exon skipping does not lead to the same percentage of dystrophin. There are many reasons for this, e.g. the dynamics of the transcript (short half-life) and protein (long half-life, but takes time to produce and accumulate).

Authors focus on the fact that not all transcripts will be translated, as micro-RNAs (mIRs) can bind transcripts to cause them to be cleaved and cleared or to prevent translation. Authors here focus on mIR-146a, which is elevated in Duchenne and Becker patients and mouse models.

In Becker patients, higher levels of mIR-146a meant lower levels of dystrophin. mIR-146a is stimulated by inflammation and steroid treatment can reduce levels. Authors here studied where mIR-146a can bind the dystrophin transcripts and found 2 strong predicted sites.

For the strongest, they made a luciferase reporter, showing miR-146a could decrease expression, while mutating the binding site prevented this decrease. Injecting miR-146a and ASO in an mdx52 mouse (deletion of mouse dystrophin exon 52) resulted in less dystrophin than ASO alone.

Less dystrophin here was less dystrophin positive fibers – no Western blot was done. Authors then made a deletion miR-146a mouse which produced roughly the same amount of dystrophin as wild types. The miR-146a mouse was then crossed with mdx52.

Treating this mouse with intramuscular injections of ASOs resulted in more dystrophin than in an mdx52 with miR-146a. Now authors did both a western (wes) and counted positive fibers, showing more dystrophin levels and more positive fibers.

After systemic treatment with 2 (very) high doses of ASOs, authors found the same thing in all skeletal muscles tested but not in the heart (as expected because this ASO chemistry does not reach the heart). I understand why authors find higher dystrophin levels by western blot.

I also understand that authors see more intense staining in positive fibers. What I do not understand is why authors see more dystrophin positive fibers. The mechanism of miR-146a would be that it binds to transcripts and prevents translation.

So when there is skipping, not having miR-146a would lead to more dystrophin per transcript. Authors show exon skipping levels are the same, so how is it possible that additional nuclei have skipped transcripts and produce dystrophin? Authors do not discuss this.

Authors do show that the strength and endurance in the mdx52 mice without miR-146a is higher after exon skipping, than regular mdx52 mice. However, they do not take along wild types or miR-146a deleted mice so we do not know whether this is not a consequence of the model per se.

Authors discuss that targeting miR-146a may help improve the dystrophin levels after treatment with approved ASOs, as these are currently quite low. Note that the increases in the mdx52 miR-146a were not THAT high (about 1.5 times more), but every little bit will help.

However, authors do not discuss that the patients treated with these ASOs will be on steroids, while they already know that steroid treatment will reduce the amount of miR-146a that is produced. So the question is whether additional reduction is even possible.

I like the idea, and I like the mechanistic work the authors did to confirm that the miR-146a does indeed seem to target dystrophin transcript. I like that authors tested things both ways (adding miR-146a reduces dystrophin and removing it increases).

However, given the context and the steroid response, I do not know whether there will be a merit in Duchenne patients of the combined treatment. Authors outline that more work is needed, with antagomirs – agreed, but they ideally should also test this in a steroid-treated model.