A human iPSC-based platform for functional toxicity screening of CNS-targeted RNA therapeutics

Abstract

Human induced pluripotent stem cell (iPSC)-derived neural co-cultures are increasingly recognized as powerful, physiologically relevant systems for modeling central nervous system (CNS) biology in vitro. In the context of RNA-based therapeutics, particularly antisense oligonucleotides (ASOs), CNS toxicity remains a major safety concern during drug development. To address this, we developed a human iPSC-based platform capable of high-throughput functional toxicity screening using calcium imaging as a readout for neuronal network activity.

The platform employs iPSC-derived neurons and astrocytes co-cultured in 384-well plates, enabling scalable assessment of calcium transient dynamics. Key metrics—calcium peak rate (0.15–0.25 Hz), amplitude (ΔF/F ≈ 1.5–2), and oscillation score (80–100%)—were stable under vehicle conditions with <15% inter-well coefficient of variation, confirming assay reproducibility and robustness.

Seventy preclinical ASO candidates were screened, revealing three distinct toxicity profiles based on suppression of spontaneous calcium oscillations. Group 1 ASOs were classified as highly neurotoxic, causing >60% suppression of oscillation scores with no recovery during a 15-min observation period. These candidates correlated with seizures and convulsions observed in vivo, consistent with data from Hagedorn et al. (Nucleic Acid Ther., 2022). Group 2 ASOs exhibited intermediate toxicity, with partial recovery (20–30%) of calcium activity and were associated with milder behavioral phenotypes. Group 3 ASOs maintained normal calcium activity even at high concentrations (up to 100 μM), indicating a lack of neurotoxicity.

Importantly, the in vitro calcium oscillation scores showed an ~80% correlation with CNS toxicity outcomes in rodent models, supporting the translational relevance of the assay. Furthermore, the platform was employed to guide ASO sequence modifications that mitigated neurotoxicity. One optimized ASO demonstrated a significant improvement in calcium oscillation score (~40% compared to baseline) without loss of intended therapeutic activity.

This study underscores the utility of human iPSC-derived neural co-cultures for early-stage, functional neurotoxicity screening of CNS-targeted RNA therapeutics. The platform offers a scalable, human-relevant alternative to animal testing, supporting safer and more efficient ASO drug development.