153. Third Generation Antisense Targeted to Double Homeobox Protein 4 (DUX4) Reduced DUX4 Expression and Improved Differentiation of FSHD Myoblasts

Facioscapulohumeral muscular dystrophy (FSHD) is caused by aberrant expression of double homeobox protein 4 (DUX4) gene at chromosome 4q35. To date there is no effective treatment for the disease. In the current study, we have evaluated selected Third Generation Antisense compounds (3GAs) targeted to DUX4 for their effectiveness in knocking-down DUX4 in FSHD myoblasts. In addition, we examined the effects of the treatments on myogenic differentiation of FSHD myoblasts. Multiple 3GA compounds (3GA1-5) showed significant knock-down of DUX4 in the FSHD myoblasts, as examined by quantitative RT-PCR. We evaluated two 3GA candidates (3GA3 and 3GA5) at 4 different concentrations, 2.5 nM, 5nM, 25nM, and 50nM, and observed dose-response effects. One of the 3GA compounds completely knocked-down DUX4 expression at 50nM. The effects of 3GAs at25nM on cell differentiation were determined by fusion index, myotube size and number of atrophic myotubes after 7 days of muscle differentiation in culture. Our results showed that the treatments increased fusion index and reduced the number of atrophic myotubes. The studies showed that 3GAs successfully knocked-down DUX4, which improved the myogenic capacity of FSHD myoblasts. The findings demonstrated that the 3GAs are promising therapeutic molecules to be further developed for FSHD treatment.