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Human Liver Microphysiological System for Assessing Drug-Induced Liver Toxicity In Vitro
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A Sensitive In Vitro Approach to Assess the Hybridization-Dependent Toxic Potential of High Affinity Gapmer

Andreas Dieckmann1, Peter H Hagedorn2, Yvonne Burki1

  • 1Roche Pharma Research and Early Development, Roche Innovation Center Basel, 4070 Basel, Switzerland.

Molecular Therapy. Nucleic Acids
|March 4, 2018
PubMed

Insights

A new in vitro assay accurately predicts liver toxicity for locked nucleic acid antisense oligonucleotides (LNA-ASOs). Maintaining a melting temperature below 55°C significantly reduces LNA-ASO hepatotoxicity, aiding drug development.

Area of Science:

  • Pharmacology
  • Oligonucleotide Therapeutics
  • Toxicology

Background:

  • High-affinity antisense oligonucleotide (ASO) therapeutics, including those with locked nucleic acid (LNA) or constrained ethyl (cEt) substitutions, face challenges due to potential hepatotoxicity.
  • Predicting and mitigating liver toxicity is crucial for the successful development of these ASO drugs.

Purpose of the Study:

  • To develop and validate an in vitro assay for predicting the hepatic liabilities of LNA-modified ASOs (LNA-ASOs).
  • To identify key factors correlating with LNA-ASO-induced hepatotoxicity and establish methods for its reduction.

Main Methods:

  • Utilized transfected mouse fibroblasts as an in vitro model to assess 236 different LNA-ASOs with known in vivo hepatotoxic potential.
  • Correlated in vitro findings with in vivo results, examining RNase H1 activity, off-target RNA downregulation, and LNA-ASO binding affinity.
  • Evaluated the translatability of the assay across different cell types and species.

Main Results:

  • The in vitro assay demonstrated high accuracy in predicting in vivo hepatotoxicity of LNA-ASOs.
  • Hepatotoxicity was linked to RNase H1 activity, off-target effects, and binding affinity.
  • The toxic potential was observed across various cell types and species, suggesting human relevance.
  • Maintaining LNA-ASO melting temperature (Tm) below approximately 55°C substantially reduced hepatotoxicity.

Conclusions:

  • An effective in vitro screening method for assessing hybridization-dependent toxicity of LNA-ASOs has been established.
  • This assay facilitates the prioritization of LNA-ASO candidates during drug discovery and early development.
  • Controlling LNA-ASO Tm is a viable strategy to mitigate liver toxicity and advance therapeutic development.
Keywords:
LNAantisense oligonucleotidesapoptosishepatotoxicityhigh affinityin vitro assaymelting temperatureoff-target RNA cleavage

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