DUX4 reduction and muscle function improvement by subcutaneous delivery of gapmer antisense oligonucleotides

Facioscapulohumeral muscular dystrophy (FSHD) is caused by aberrant expression of double homeodomain protein 4 (<i>DUX4</i>). The disease has no effective treatment. Previously we demonstrated effective <i>DUX4</i> knockdown <i>in vitro</i> and <i>in vivo</i> using 2'-O-methoxyethyl (2'MOE) and locked nucleic acid (LNA) gapmer antisense oligonucleotides delivered via intramuscular injections. This study aimed to evaluate <i>in vivo</i> efficacy of the gapmers via systemic delivery using mouse models expressing <i>DUX4</i> at different levels. First, we injected the gapmers subcutaneously to <i>FLExDUX4</i> mice at 20 mg/kg twice a week for 10 weeks. Results showed significant reduction in <i>DUX4</i> mRNA and improved muscle function, assessed by grip strength. Muscle fibrosis and circulating TGFβ1 levels were significantly reduced, approaching baseline level. A dose-dependent <i>DUX4</i> reduction was observed in 2'MOE gapmer treated. In the <i>ACTA1-MCM;FLExDUX4</i> model, where DUX4 expression was induced by tamoxifen (5 mg/kg), treatment with 2'MOE gapmers effectively reduced <i>DUX4</i>, improved muscle function, and decreased inflammation. These findings highlight the therapeutic potential of gapmer-based <i>DUX4</i> reduction, leading to phenotypic improvement and restoration of muscle function in FSHD mouse models.