Genetic deletion of the inflammation-regulated microRNA miR-146a increases exon-skipping mediated dystrophin restoration in Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a progressive muscle disease caused by loss of function mutations in the Dystrophin gene resulting in loss of dystrophin protein. Current DMD therapeutics use phosphorordiamidate morpholino oligomers (PMO) to skip over the frame-shifting exon during the splicing of the dystrophin pre-mRNA, resulting in translation of a truncated dystrophin protein product. While exon skipping therapies are promising, their potential has not been fully realized as increases in dystrophin protein have been minimal and highly variable in clinical trials. We previously described microRNAs that are upregulated in DMD and BMD muscle biopsies, bind to the dystrophin 3'UTR and inhibit dystrophin protein production. One of these dystrophin-targeting microRNAs, miR-146a, is regulated by the pro-inflammatory transcription factor NF-κB, is highly elevated in the muscles of dystrophin-deficient mice and is reduced by anti-inflammatory drugs. Here, we show that inflammation induces miR-146a expression in dystrophic myotubes. Using bioinformatics analysis, we validate previous findings that the dystrophin 3'UTR harbors a miR-146a binding site. An in vitro 3'UTR luciferase reporter assay further confirms that miR-146a inhibits dystrophin translation, while mutating the miR-146a binding site attenuates inhibition. In dystrophin-deficient mice we find that co-injection of an exon skipping PMO with miR-146a but not a control sequence reduces the extent of dystrophin positive fibers. To test the hypothesis that miR-146a is inhibitory to exon skipping dystrophin restoration, we generated a novel DMD double knockout mouse model with body-wide miR-146a deletion ( 146aX mice) and administered an exon 51 skipping PMO into the tibialis anterior (TA) muscles of mdx52 and 146aX mice. Excitingly, 146aX TAs showed increased dystrophin protein versus mdx52 as measured by capillary Western immunoassay and dystrophin-positive fiber quantification. Additionally, systemic PMO delivery increased dystrophin protein levels and increased the number of dystrophin-positive fibers in 146aX versus mdx52 muscles despite similar levels of skipped dystrophin transcripts in both groups. These data demonstrate genetic deletion of miR-146a is sufficient to increase dystrophin rescue via exon skipping. Our data suggests that antagomiR-mediated inhibition of miR-146a or other dystrophin targeting miRNAs could be a viable exon skipping DMD co-therapy and warrants further research.