Identifying and Avoiding tcDNA-ASO Sequence-Specific Toxicity for the Development of DMD Exon 51 Skipping Therapy

Abstract

Tricyclo-DNA (tcDNA) antisense oligonucleotides (ASOs) hold promise for therapeutic splice-switching applications and the treatment of Duchenne muscular dystrophy (DMD) in particular. We have previously reported the therapeutic potential of tcDNA-ASO in mouse models of DMD, highlighting their unique pharmaceutical properties and unprecedented uptake in many tissues after systemic delivery, including the heart and central nervous system. Following these encouraging results, we developed phosphorothioate (PS)-modified tcDNA-ASOs targeting the human dystrophin exon 51 (H51). Preliminary evaluation of H51 PS-tcDNA in mice resulted in unexpected acute toxicity following intravenous administration of the selected candidate. In vivo and in vitro assays revealed complement activation, prolonged coagulation times, and platelet activation, correlating with the observed toxicity. In this study, we identify a novel PS-tcDNA sequence-specific toxicity induced by the formation of homodimer-like structures and investigate the therapeutic potential of a detoxified PS-tcDNA targeting exon 51. Modification of the H51-PS-tcDNA sequence, while maintaining target specificity through wobble pairing, abolished the observed toxicity by preventing homodimer formation. The resulting detoxified wobble-tcDNA candidate did not affect coagulation or complement pathways any longer nor activated platelets in vitro and was well tolerated in vivo in mice, confirming the possibility to detoxify specific tcDNA-ASO candidates successfully.

Keywords: Duchenne muscular dystrophy; antisense oligonucleotides; preclinical; sequence-specific toxicity; splice switching; tcDNA-ASOs.