#apaperaday: Targeted Antisense Oligonucleotide-Mediated Skipping of Murine Postn Exon 17 Partially Addresses Fibrosis in D2.mdx Mice

In today’s #apaperaday, Prof. Aartsma-Rus reads and comments on the paper titled: Targeted Antisense Oligonucleotide-Mediated Skipping of Murine Postn Exon 17 Partially Addresses Fibrosis in D2.mdx Mice

A pick from IJMS MDPI by Trundle et al on skipping exon 17 in periostin as a treatment for fibrosis in skeletal muscular dystrophy in d2/mdx mice. DOI: 10.3390/ijms25116113

Due to lack of dystrophin, muscles undergo chronic damage and inflammation, which inhibits repair and results in fibrosis (mice) and fibro-adiposis (human) formation. Periostin is a protein that influences the key regulator of fibrosis TGFbeta (my second least favorite protein)

It has been shown that periostin increases fibrosis formation and collagen deposition n dystrophic animal models. Here authors analyzed periostin transcripts, seeing one isoform that lacked exon 17, which was predicted to form a periostin that binds TGFbeta less well.

Note that this is not actually shown, only predicted by the models. Authors decide to skip exon 17, speculating that having the skipped periostin likely will mean less TGFbeta activity and therefore less fibrosis formation in dystrophic mouse models.

First, they show in d2/mdx mice that indeed periostin transcripts with and without exon 17 exist and that when stimulating mouse fibroblasts with TGFbeta, the amount of transcripts with exon 17 (the TGFbeta binding version) increase.

They designed 3 antisense oligonucleotides targeting exon 17, which each resulted in an increased amount of exon 17 skipped isoform. The one that worked best was then taken along for studies in the d2/mdx mouse. Authors show that periostin levels peaked at 6 weeks.

Afterward, levels decline. This fits with fibrosis levels that we see peak at week 10-12 and then decline (the periostin likely precedes the deposition of the collagen). Authors treated 2-week-old d2/mdx mice for 10 weeks with weekly 10 mg/kg of the best ASO intraperitoneally.

They use wild-type mice as controls & treat them with control ASOs (hooray). 4 per group. They treated 5 d2/mdx with Periostin ASOs & 6 with control ASOs. We will get back to the importance of the numbers later. Authors did intraperitoneal injection as this delivers well to the diaphragm.

Authors see increased exon 17 skipping in periostin ASO-treated mice and see a decrease in full-length periostin protein. They see less fibrosis in the diaphragm of these mice (not fully normal) and increased grip (as wild type). No difference was seen in fatigue (not shown).

Authors discuss that periostin is also involved in cardiac fibrosis and may have therapeutic effects there too. However, they could not study this in this model as the d2/mdx and the wild-type both have cardiac issues (though not at the age used here by authors).

Authors also discuss that the treatment reduces fibrosis formation, but that likely combined treatment will be better. What authors do not discuss is that their evidence that periostin lacking exon 17 cannot bind TGFbeta is only from predictions. No evidence for this is provided.

Results may show a reduction in fibrosis, so there is indirect evidence, but the exact mechanism is unknown. This caveat should at least have been mentioned. Furthermore, authors use both male and female mice, while we know that males do  worse functionally.

This means if you compare groups of 3+3 control ASO injected with 2+3 periostin injected mice, the mere fact that 1 group has 1 male or female less will influence your outcomes! If you have fewer females, the control group will do better; if you have fewer males, it will do worse.

Authors do not discuss this and ideally should have used only males (largest deficit, easier to measure a treatment effect, especially given the fact that most Duchenne patients are also male). Finally, authors focus a lot on fibrosis, while Duchenne patients have mainly adiposis.

Authors do not discuss this, or whether periostin levels are also increased in patient muscles. Without this information, it is difficult to assess whether the approach is just for the mouse model or also for the patients.