# Effect of chemical modification on the exon-skipping activity of heteroduplex oligonucleotides

## TL;DR

This paper shows that using a special type of DNA called heteroduplex oligonucleotides improves the ability of another DNA to regulate gene splicing in cells and in mice.

## Contribution

The study introduces how complementary strand design in HDOs enhances exon-skipping activity of LNA-based oligonucleotides.

## Key findings

- HDO technology increases the exon-skipping activity of LNA-based SSOs in vitro.
- Complementary oligonucleotide design significantly affects intracellular stability and exon skipping.
- HDSSOs show higher exon-skipping activity than single-stranded SSOs in mdx mice.

## Abstract

We applied heteroduplex oligonucleotide (HDO) technology, which uses an oligonucleotide hybridized with a complementary strand, to efficiently deliver locked nucleic acid (LNA)-based splice-switching oligonucleotides (SSOs) to the nucleus. Using an in vitro assay involving cationic lipids, we revealed that HDO technology increased the exon-skipping activity of LNA-based SSOs. To assess the effect of heteroduplex SSOs (HDSSOs) on exon-skipping activity, we designed and evaluated various HDSSOs using a series of complementary oligonucleotides with different sugar chemistries (DNA, RNA, and LNA), linkages (phosphodiester; PO and phosphorothioate; PS linkages), and lengths. HDO with different complementary oligonucleotide designs demonstrated a variety of exon-skipping activities. Next, we investigated the intracellular behavior of HDOs, which seemed to affect their efficient exon-skipping activity. We found that HDO technology increased the uptake of both SSOs and complementary oligonucleotides into the nuclei. Additionally, a series of complementary oligonucleotides showed different intracellular stabilities, and complementary oligonucleotide design appears to be one of the key factors affecting efficient exon skipping. Finally, we examined the exon-skipping activity of HDSSOs in mdx mice and found that HDSSOs exhibited higher exon-skipping activity than single-stranded LNA-based SSOs in these mice under intramuscular injections.

Obika, Yokota, and colleagues have applied heteroduplex oligonucleotide (HDO) technology to achieve efficient splicing regulation induced by antisense oligonucleotides. They elucidated the effects of various complementary strands in HDOs with sugar chemistries, linkages, and lengths on the exon-skipping activity in vitro and in vivo using the model mouse of Duchenne muscular dystrophy.