Improving antisense oligonucleotide therapeutic applications: Development of a novel antibody-mediated delivery system

A number of therapeutic antisense oligonucleotides (ASOs) applications have been developed as promising candidates for severe and devastating conditions such as Duchenne muscular dystrophy (DMD). DMD is a lethal, X-linked inherited disorder characterized by progressive muscle weakness, wasting and degeneration. ASOs can be used to modulate dystrophin pre-mRNA splicing in a manner that restores the reading frame of the DMD transcript, producing a shorter but functional dystrophin protein (approach approved by FDA in 2016). Nonetheless, one of the major challenges for successful ASOs therapy in patients remains poor delivery and uptake in targeted tissues and cellular compartments. In order to address this issue, we designed and developed a novel antibody-mediated ASO delivery system. mAb 3E10, a cell penetrating anti-dsDNA antibody derived from MRL/mpj/lpr mice, was used as a carrier molecule for phosphordiamidate morpholino oligomer (PMO) ASOs for targeted muscle specific delivery.

We have explored and optimised several conjugation strategies and conducted in vitro assessments of antibody-PMO conjugate functionality in several murine muscle cell lines. The three main conjugate designs explored were: covalent (non-reversible) 3E10 Fab’ fragment and PMO conjugation, covalent 3E10 Fab’ fragment and double stranded sOligo-PMO hybrid conjugation and Fab-X-Y-PMO antigen binding based complex.
Covalent Fab-PMO conjugate retained 3E10 antibody properties of dsDNA binding and myotube penetration, but lacked efficacious targeted exon skipping. The latter two conjugation strategies were designed to facilitate PMO release following myotube penetration. However, Fab-sOligo-PMO hybrid conjugate exhibited reduced myotube penetration and no detectable exon skipping, most likely due to deficient PMO release by nucleases. The 3E10 Fab-X-Y-PMO compound demonstrated the proof-of-concept efficacy results for both myotube penetration and targeted exon skipping in vitro. Nonetheless, further in vivo 3E10 Fab-X-Y-PMO compound screening is required for a comprehensive efficacy evaluation in comparison to unconjugated PMO. Overall, 3E10 antibody-PMO delivery system presents an innovative approach for targeted PMO delivery to muscle tissues.