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

## TL;DR

This study shows that subcutaneous delivery of gapmer antisense oligonucleotides can reduce DUX4 levels and improve muscle function in mouse models of FSHD.

## Contribution

The study demonstrates the efficacy of systemic subcutaneous gapmer delivery for DUX4 reduction in FSHD models.

## Key findings

- Subcutaneous gapmer treatment reduced DUX4 mRNA and improved muscle function in FLExDUX4 mice.
- Muscle fibrosis and TGFβ1 levels were significantly reduced after long-term treatment.
- 2′MOE gapmers effectively reduced DUX4 and inflammation in ACTA1-MCM;FLExDUX4 mice.

## Abstract

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

2′MOE-AO dose-dependently inhibits DUX4, and the dosage correlates to muscle uptake in FLExDUX4 mice. Long term subcutaneous administration significantly reduced DUX4 in several muscles of forelimb and hindlimb, reduced muscle pathology, and improved muscle function in FLExDUX4 and ACTA1-MCM;FLExDUX4 FSHD mice.