P.50An in situ hybridization-based method for detecting DUX4 RNA expression in vitro

Abstract

Facioscapulohumeral muscular dystrophy (FSHD) is among the most common forms of muscular dystrophy. FSHD is caused by aberrant expression of the toxic DUX4 gene in muscle. Detecting endogenous DUX4 in patient tissue using conventional methods can be challenging, due to the low level of DUX4 expression. In addition to impacting basic research, the difficulty to reliably detect DUX4 expression could pose challenging for future prospective clinical trials involving DUX4 inhibition therapies, where DUX4 levels would be useful as a therapeutic outcome measure. Developing simple and trustworthy DUX4 detection methods is an important need in the FSHD field. Our objective is to develop a new strategy to detect endogenous DUX4 mRNA. Here, we describe a novel and efficient technique for detecting over-expressed and endogenous DUX4 mRNA in human cells, which employs the RNAscope assay, a RNA in situ hybridization (ISH) technology. We show that a custom-designed RNAscope assay can detect DUX4 mRNA in transfected HEK293 cells, and importantly, it was highly sensitive for detecting lower amounts of endogenous DUX4 mRNA in FSHD patient-derived myotubes. Moreover, the RNAscope assay was able to track reductions in DUX4 mRNA following treatment with our RNAi therapy. In conclusion, we found that RNAscope was a highly sensitive method for detecting DUX4 mRNA in vitro, and may enable us to develop a new, rapid RNA ISH-based molecular diagnostic assay for FSHD. This study suggests that RNAscope-based DUX4 expression assays may be useful for development as a prospective clinical outcome measure.