Enhancing the intracellular delivery of antisense oligonucleotides (ASO) : a comparative study of aptamer, vitamin E, and cholesterol ASO conjugates

Abstract

Antisense oligonucleotides (ASOs) have emerged as powerful tools for gene modulation; however, their clinical application is often hindered by inefficient intracellular delivery. Recent advances suggest that conjugation to biomolecules such as aptamers, lipids, or vitamins may enhance uptake and efficacy. This study investigates the potential of aptamer, vitamin E, and cholesterol-conjugated ASOs to improve delivery and functional activity in cancer cell models. The ASO PNAT524 was conjugated to two DNA aptamers—AS1411 and S2.2—via thiol and triethylene glycol (TEG) linkers, respectively. In parallel, PNAT524 was modified with vitamin E and cholesterol moieties. The conjugates were evaluated for cellular uptake, exon-skipping activity, and cytotoxicity in cancer cell lines. Fluorescence microscopy was used to determine subcellular localization. Aptamer conjugation (AS1411, S2.2) did not significantly enhance exon-skipping efficiency compared to unconjugated PNAT524, consistent with previous findings. In contrast, vitamin E and cholesterol conjugates demonstrated potent, dose-dependent exon-skipping activity and cytotoxic effects. Among all formulations, the cholesterol-conjugated ASO (524-Chol) showed the highest efficacy, with superior splice-modulating and cytotoxic outcomes. Fluorescence microscopy confirmed nuclear and cytoplasmic localization of lipid-conjugated ASOs. These findings indicate that aptamer conjugation provides minimal benefit for ASO delivery, while cholesterol and vitamin E conjugation significantly enhance intracellular delivery and therapeutic activity. The 524-Chol conjugate holds strong potential for adaptation in ASOs targeting EGFR and other oncogenes, representing a promising avenue for ASO-based cancer therapeutics.