Link between MHC Fiber Type and Restoration of Dystrophin Expression and Key Components of the DAPC by Tricyclo-DNA-Mediated Exon Skipping

Abstract

Exon skipping mediated by tricyclo-DNA (tc-DNA) antisense oligonucleotides has been shown to induce significant levels of dystrophin restoration in mdx, a mouse model of Duchenne muscular dystrophy. This translates into significant improvement in key disease indicators in muscle, cardio-respiratory function, heart, and the CNS. Here we examine the relationship between muscle fiber type, based on myosin heavy chain (MHC) profile, and the ability of tc-DNA to restore not only dystrophin but also other members of the dystrophin-associated glycoprotein complex (DAPC). We first profiled this relationship in untreated mdx muscle, and we found that all fiber types support reversion events to a dystrophin-positive state, in an unbiased manner. Importantly, we show that only a small fraction of revertant fibers expressed other members of the DAPC. Immunoblot analysis of protein levels, however, revealed robust expression of these components, which failed to correctly localize to the sarcolemma. We then show that tc-DNA treatment leads to nearly all fibers expressing not only dystrophin but also other key components of the DAPC. Of significance, our work shows that MHC fiber type does not bias the expression of any of these important proteins. This work also highlights that the improved muscle physiology following tc-DNA treatment reported previously results from the complete restoration of the dystrophin complex in all MHCII fibers with equal efficiencies.

Keywords

• muscle
• Duchenne
• mdx
• antisense oilgo
• revertant
• tricyclo