Thiomorpholino oligonucleotides as a robust class of next generation platforms for alternate mRNA splicing.

Abstract

Recent advances in drug development have seen numerous successful clinical translations using synthetic antisense oligonucleotides (ASOs). However, major obstacles, such as challenging large-scale production, toxicity, localization of oligonucleotides in specific cellular compartments or tissues, and the high cost of treatment, need to be addressed. Thiomorpholino oligonucleotides (TMOs) are a recently developed novel nucleic acid analog that may potentially address these issues. TMOs are composed of a morpholino nucleoside joined by thiophosphoramidate internucleotide linkages. Unlike phosphorodiamidate morpholino oligomers (PMOs) that are currently used in various splice-switching ASO drugs, TMOs can be synthesized using solid-phase oligonucleotide synthesis methodologies. In this study, we synthesized various TMOs and evaluated their efficacy to induce exon skipping in a Duchenne muscular dystrophy (DMD) in vitro model using <i>H2K mdx</i> mouse myotubes. Our experiments demonstrated that TMOs can efficiently internalize and induce excellent exon 23 skipping potency compared with a conventional PMO control and other widely used nucleotide analogs, such as 2'-O-methyl and 2'-O-methoxyethyl ASOs. Notably, TMOs performed well at low concentrations (5-20 nM). Therefore, the dosages can be minimized, which may improve the drug safety profile. Based on the present study, we propose that TMOs represent a new, promising class of nucleic acid analogs for future oligonucleotide therapeutic development.