Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder caused by the absence of functional dystrophin. Ongoing research aims to continually improve the pharmacological properties of oligonucleotides for DMD. In this regard, we have developed stereopure antisense oligonucleotides featuring chimeric phosphodiester (PO), phosphorothioate (PS) and phosphoryl guanidine-containing (PN) backbones.
This study evaluated the exon skipping activity and dystrophin protein restoration of stereopure PN-containing oligonucleotides designed to skip exons 52, 51, and 44. We obtained immortalized myoblasts or immortalized fibroblasts engineered to express MYOD1 from patients with DMD who have mutations amenable to exon 51, 52, or 44 skipping. These cells were differentiated in vitro and treated with oligonucleotides designed to skip the respective exons and restore the translational reading frame. Percent exon skipping relative to total transcript was measured by qRT-PCR using exon specific primers. Using capillary western blot assays, percent dystrophin protein restoration after treatment was quantified against a standard curve generated from control (wild-type) cell lysates.
Our results demonstrated that PN-containing oligonucleotides designed to skip exons 52, 51, or 44 yielded high levels of exon skipping and dystrophin protein restoration in vitro. Across all exons assessed, we observed 50%-95% exon skipping and noted substantial dystrophin protein restoration, ranging from 65% to near control levels. These preclinical data highlight the promise of PN-containing exon skipping oligonucleotides as potential investigational treatments for patients with DMD, and raise the potential for the advancement of molecules designed for patients with a variety of mutations amenable to exon skipping. Notably, a PN-containing oligonucleotide called WVE-N531 is being developed for patients with DMD amenable to exon 53 skipping. In a Phase 1b/2 trial, three boys were administered three biweekly doses of WVE-N531, which resulted in substantial accumulation and exon-skipping in skeletal muscle.