A Sensitive In Vitro Approach to Assess the Hybridization-Dependent Toxic Potential of High Affinity Gapmer Oligonucleotides

The successful development of high-affinity gapmer antisense oligonucleotide (ASO) therapeutics containing locked nucleic acid (LNA) or constrained ethyl (cEt) substitutions has been hampered by the risk of hepatotoxicity. Here, we present an in vitro approach using transfected mouse fibroblasts to predict the potential hepatic liabilities of LNA-modified ASOs (LNA-ASOs), validated by assessing 236 different LNA-ASOs with known hepatotoxic potential. This in vitro assay accurately reflects in vivo findings and relates hepatotoxicity to RNase H1 activity, off-target RNA downregulation, and LNA-ASO-binding affinity. We further demonstrate that the hybridization-dependent toxic potential of LNA-ASOs is also evident in different cell types from different species, which indicates probable translatability of the in vitro results to humans. Additionally, we show that the melting temperature (T(m)) of LNA-ASOs maintained below a threshold level of about 55°C greatly diminished the hepatotoxic potential. In summary, we have established a sensitive in vitro screening approach for assessing the hybridization-dependent toxic potential of LNA-ASOs, enabling prioritization of candidate molecules in drug discovery and early development.