Knocking Down DUX4 in Immortalized Facioscapulohumeral Muscular Dystrophy Patient-Derived Muscle Cells.

The third most common muscular dystrophy in the world, facioscapulohumeral muscular dystrophy (FSHD), is an inherited disorder characterized by distinct asymmetric, progressive skeletal muscle weakness that begins in the upper body and spreads to other regions with age. It is caused by mutations that induce aberrant expression of the DUX4 gene in skeletal muscle. DUX4 is highly cytotoxic in skeletal muscle, dysregulating numerous signaling pathways as a result of its transcription factor activity. A promising set of approaches being developed to treat FSHD uses antisense oligonucleotides (AOs) to inhibit DUX4 transcript expression. Both steric-blocking and gapmer AOs have been shown to induce efficient DUX4 transcript knockdown in vitro and in vivo. Here, we describe a protocol that allows reliable screening of DUX4-targeting AOs through the evaluation of DUX4 transcript expression by quantitative real-time polymerase chain reaction. We also describe methods to assess the efficacy of these AOs by looking at their effect on the expression of DUX4 downstream target and potential off-target genes, as well as on the amelioration of in vitro muscle cell phenotypes.