In vivo delivery of third generation oligonucleotides targeting DUX4 using a new mouse model of FSHD

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disorder caused by aberrant expression of the double homeobox 4 (DUX4) gene. Aberrant expression of DUX4 has been shown to affect critical molecular pathways, which results in muscle pathologies and weakness in FSHD. Knock down of DUX4 using antisense oligonucleotides is a viable treatment for FSHD. In an in vitro study, we showed that several third generation antisense oligonucleotides (3GAs) targeting DUX4 reduced expression of DUX4 mRNA effectively. In addition, the treatments partially corrected defects in myogenesis in cell culture. The purpose of this study is to determine whether 3GA can be systemically delivered into skeletal muscles and reduce expression of DUX4 in vivo. A newly developed FSHD mouse model (FlexD mice) was used for the study. The FlexD mice were treated with fluorescein tagged 3GA (3GA-F) via intramuscular (IM) and subcutaneous (SC) injections, respectively. Mice treated intramuscularly received 20µg of 3GA-F into the tibialis anterior (TA) muscles (n=3). Mice treated subcutaneously received one injection of 30mg/kg 3GA-F (n=2). The TA muscles were harvested 24 hours afterwards and stained for hematoxylin and eosin to determine muscle pathology. In addition, the muscles were immunofluorescently stained for a sarcolemmal protein, dystrophin, to determine the localization of the 3GA-F. In addition to single injection with 3GA-F to determine localization of the 3GA-F, a separate group (n=3) of mice were treated with 30mg/kg of two different 3GAs, which targeted different regions of DUX4, via subcutaneous injections every other day for total 3 injections. Our results showed that 3GA entered muscles after both local and systematic delivery. One of the 3GAs slightly reduced DUX4 and a DUX4 regulated gene, Trim36, after 3 SC injections, however the change did not reach significant level. Delivery of antisense oligonucleotides to intact muscles for therapeutic purpose has been very challenging. Our findings showed that 3GA were able to enter muscles by local and systemic delivery, which is important for future therapeutic development for FSHD. Experiments to optimize the dosing regimen of 3GA to further enhance the knockdown of DUX4 and its downstream target genes are currently underway