Development of an in vitro screening assay to predict acute CNS toxicity induced by antisense oligonucleotides and mechanistic exploration of its toxicity

In the development of antisense oligonucleotides (ASOs) indicated for central neurological diseases, local administration to central nervous system (CNS) (e.g., intrathecal injection) is selected as a clinical route of administration. However, some ASOs injected to CNS induce acute CNS toxicity such as ataxic gait, decreased locomotor activity, and convulsions in in vivo non-clinical studies. Therefore, it is desirable to screen the potential toxicities in the early stage of drug discovery. In this study, we tried to develop an in vitro screening tool to predict the risk of acute CNS toxicity induced by ASOs. We also explored the mechanism of the toxicity using in vitro methods. First, we investigated the effects of ASOs on neuronal activity by Ca²⁺ oscillation (CaO) assays using rat primary cerebral cortical neurons. A total of 27 gapmer-type ASOs, which had been evaluated in mouse intracerebroventricular (ICV) single dose toxicity studies, were evaluated as test articles. The results showed that P-rate (oscillation peak numbers per one minute) was inhibited in an ASO in concentration-proportional manner and that IC50 values generally correlated with the intensity of toxicity in the mouse ICV studies. Using “IC50 = 17 μM” as a cutoff value, intolerable acute toxicity observed in the mouse ICV studies could be predicted with a high accuracy (sensitivity: 83 %, specificity: 100 %). Furthermore, multielectrode array (MEA) assays using human iPS cell-derived neurons were conducted for some ASOs to investigate the potential mechanism of the acute CNS toxicity. Two types of similarity analysis (principal component analysis and AI analysis) based on the MEA data including reference small molecules suggested that the toxic ASOs were similar to AMPA glutamate receptor antagonists. Indeed, this mechanism was confirmed in patch clamp assay using HEK293 cells expressing human AMPA receptor subunits, GluR2. In summary, we developed a useful in vitro assay to screen the potential risk of acute CNS toxicity induced by ASOs and revealed that one of the mechanisms of the acute toxicity may be AMPA glutamate receptor antagonism.