Safety and toxicity assessment of antisense oligonucleotides in brain-relevant human cellular models

Antisense oligonucleotides (ASOs) have become an established therapeutic modality for central nervous system (CNS) disorders, enabling selective modulation of gene expression through splice-switching or steric-hindrance mechanisms. Backbone and sugar chemistry modifications improved nuclease resistance, tissue retention, and pharmacodynamic durability, facilitating clinical translation of intrathecally delivered ASOs. This review summarizes current in vitro approaches for assessing safety and toxicity of CNS-targeted ASOs, alongside the evolving regulatory landscape shaping their preclinical evaluation. Human-relevant platforms-induced pluripotent stem cell (iPSC)-derived neurons, astrocytes, microglia, and brain organoids-enable evaluation of target engagement, cytotoxicity, and immune activation in disease-relevant genetic backgrounds, though each carries distinct trade-offs in throughput, maturity, and translational fidelity. Delivery strategies and cytotoxicity assays, including calcium imaging, lactate dehydrogenase release, metabolic activity measurements, apoptosis markers, and transcriptomic and proteomic profiling, allow detection of sequence- and chemistry-dependent liabilities, including off-target and neuroexcitability effects. Emerging evidence demonstrates sequence and chemical modifications predicting acute neurotoxicity, highlighting the value of systematic in vitro screening. As personalized "n-of-1" ASO therapies expand, robust and standardized in vitro safety profiling becomes increasingly critical. Comprehensive evaluation in relevant CNS models will be essential to minimize risk and improve translational predictability for developing safe and effective ASO treatments for neurological diseases.