Body-wide deletion of miR-146a improves exon skipping-mediated dystrophin restoration in mdx mice


Pre-Clinical Research

Poster Number: 235


Kelsey Calabrese, University of Maryland, Nikki McCormack, PhD, Children's National Research Institute, Chris Heier, PhD, Children's National Medical Center, Alyson Fiorillo, PhD, Children's National Medical Center (Washington, DC)

Duchenne Muscular Dystrophy (DMD) is an X-linked genetic disease where patients experience progressive muscle weakness. DMD is caused by a lack of dystrophin protein most commonly caused by a deletion or frameshift mutation in the dystrophin gene. Exon skipping is a promising therapy where delivery of antisense oligonucleotides (AOs) “skips over” a frame-shifting exon resulting in production of a truncated dystrophin protein. Our lab has shown miR-146a downregulates dystrophin protein levels and is found at high levels in DMD model (mdx) mice. We hypothesized deletion of miR-146a in mdx would increase levels of dystrophin after treatment with exon skipping oligos. mdx52 mice were crossed to mice with body-wide deletion of miR-146a to generate mdx:146a-/- (146aX) mice. 8-week-old mdx52 and miR-146aX mice were injected 2x with an exon 51-skipping PMO (400 mg/kg) 2 weeks apart and muscles were collected 2 weeks after the second injection. We quantified dystrophin levels in the fibers of 5 different muscle groups (tibialis anterior, triceps, quadriceps, gastrocnemius, diaphragm) in mdx52 and 146aX mice. Overall, there was an 81% increase in dystrophin protein observed in miR-146aX muscles. We observed variable dystrophin staining intensity and performed a secondary analysis where we stratified data into bright dystrophin+ fibers and faint dystrophin+ fibers. The criteria for this analysis were based on a reference set of fibers. This revealed that 146aX mice had a 50% increase in bright dystrophin+ fibers as compared to mdx but had a 122% increase in light dystrophin+ fibers suggesting that genetic deletion of miR-146a may not only increase overall dystrophin rescue, it may also increase the number of fibers that express therapeutic dystrophin and enable more protein production from each skipped dystrophin transcript. Given this, inhibition of miR-146a may serve as a viable therapeutic target for a DMD therapeutic to be used in combination with exon skipping AOs.