DMD – ANIMAL MODELS: EP.81 Enhanced exon skipping and Dystrophin production in a mouse model of Duchenne muscular dystrophy with EEV-PMO treatment

Abstract

Antisense oligonucleotide-induced (ASOs) exon skipping is a promising treatment strategy for Duchenne muscular dystrophy (DMD), an X-linked recessive and fatal neuromuscular disease. DMD is caused by mutations in the dystrophin gene and affects approximately 1 in 3,500-5,000 male births. By modulating the splicing of dystrophin pre-mRNA, ASOs can restore the mRNA reading frame and generate a truncated, functional dystrophin protein. This exon skipping approach can address ∼80% of the mutations that cause DMD. However, the poor exposure and endosomal escape in target tissue after systemic administration of ASOs remains the biggest hurdle for the treatment of patients with DMD and results in insufficient dystrophin protein restoration, especially in non-skeletal muscle tissues such as the heart. We have developed a novel ASO delivery system by conjugation of Entrada's proprietary Endosomal Escape Vehicle (EEVTM) to a phosphorodiamidate morpholino oligomer (PMO), an effective ASO for DMD treatment. The EEV is composed of a cyclic cell-penetrating peptide coupled with a nuclear localization sequence, which is rationally designed to facilitate intracellular delivery, endosomal escape and finally localization in the nucleus. The EEV-PMO significantly enhances the systematic delivery of PMOs and results in robust exon skipping and restoration of dystrophin protein in the mdx mouse model skeletal as well as cardiac muscle. A single dose (20 mg/kg) by intravenous administration of EEV-PMO led to >50% exon skipping and >15% correction of dystrophin protein in the heart seven days post injection, and dystrophin protein levels were sustained for up to four weeks. Repeat dosing (10 mg/kg, 4 QW) resulted in >30% of wild-type dystrophin protein in the heart and normalized serum creatine kinase level seven days post last injection. These results have confirmed the significant therapeutic potential of the EEV-PMO delivery system for DMD. Furthermore, these results demonstrate that EEVs can serve as a general and highly efficient delivery platform that possesses great potential for the delivery of other therapeutic ASOs beyond DMD.