Delivery of a chemically modified noncoding RNA domain improves dystrophic myotube function

Abstract

Fast twitch, type II muscle fibers are particularly prone to degradation in skeletal muscle pathologies, such as sarcopenia and muscular dystrophies. We previously showed that endogenous activation of the exercise-induced long noncoding RNA CYTOR promotes fast-twitch myogenesis. In the present study, we identify an independent pro-myogenic element within human CYTOR and optimize its RNA delivery. In human primary myoblasts exogenous, vector-based CYTORexon 2 recapitulates the effect of full-length CYTOR by enhancing fast-twitch myogenic differentiation. Furthermore, chemically modified CYTORexon 2 RNA{Psi}U (N1-me-PseudoU, 7-methyl guanosine 5Cap, polyA tail) enhanced RNA stability and reduced the immunogenic response to CYTORexon 2 RNA. We demonstrate that viral- or chemically optimized RNA-mediated CYTORexon 2 administration enhances the commitment towards myogenic maturation in Duchenne muscular dystrophy-derived primary myoblasts, induced myogenic progenitor cells and mouse embryonic stem cells. Furthermore, chemically optimized CYTORexon 2 improves key disease characteristics in dystrophic myotubes, including calcium handling and mitochondrial bioenergetics. In summary, our findings identify CYTOR exon 2 as the pro-myogenic domain of CYTOR that can be delivered in a disease context using chemical modifications. This is of particular importance given the susceptibility of type II muscle fibers in different muscle pathologies such as aging and dystrophies, and the reported oncogenic effect of CYTOR exon 1. Our study, therefore, highlights the potential of identifying functional domains in noncoding RNAs. Delivery, or targeting of such RNA domains could constitute next-generation RNA therapeutics.