A case study of mechanistic investigation using <i>in vivo</i> and <i>in vitro</i> studies for toxicological change observed in oligonucleotide development

<p>In drug discovery, in vitro safety assessment play an important role not only for screening to select safer compounds, but also mechanistic investigation of toxicity. However, despite its huge contribution, further investigation is required to develop appropriate safety assessment for new modality including oligonucleotide (ON) drugs. ONs that modulate gene expression via a wide range of processes such as RNA interference and non-coding RNA inhibition has potential therapeutic applications for various indications and recently gaining approval with several ON drugs. ON-related toxicities are mainly classified as hybridization-dependent/independent effects, and hybridization-independent effects include blood coagulation, thrombocytopenia, immune stimulation, nephrotoxicity and hepatotoxicity. It is reported that DNA/RNA heteroduplex oligonucleotides (HDOs) conjugated to cholesterol (Chol-HDO) may overcome the limited efficacy of antisense ONs targeting the central nervous system without requiring intrathecal administration; however, a mild decrease in platelets and focal necrosis in the brain were noted in mice after intravenous administration of Chol-HDOs (Nagata T. et al. Nat Biotechnol. 2021 Dec;39(12):1529-1536.). To clarify the mechanism, we focused on in vivo hematological and histopathological changes first. Next, given the evidence from their investigations, in vitro/ex vivo approaches were performed. This presentation will provide that these approaches contributed to revealing the mechanism and serving a strategy for creating safer ON therapeutics.</p>