Selection of GalNAc-conjugated siRNAs with limited off-target-driven rat hepatotoxicity

Nat Commun. 2018 Feb 19;9(1):723. doi: 10.1038/s41467-018-02989-4.


Small interfering RNAs (siRNAs) conjugated to a trivalent N-acetylgalactosamine (GalNAc) ligand are being evaluated in investigational clinical studies for a variety of indications. The typical development candidate selection process includes evaluation of the most active compounds for toxicity in rats at pharmacologically exaggerated doses. The subset of GalNAc-siRNAs that show rat hepatotoxicity is not advanced to clinical development. Potential mechanisms of hepatotoxicity can be associated with the intracellular accumulation of oligonucleotides and their metabolites, RNA interference (RNAi)-mediated hybridization-based off-target effects, and/or perturbation of endogenous RNAi pathways. Here we show that rodent hepatotoxicity observed at supratherapeutic exposures can be largely attributed to RNAi-mediated off-target effects, but not chemical modifications or the perturbation of RNAi pathways. Furthermore, these off-target effects can be mitigated by modulating seed-pairing using a thermally destabilizing chemical modification, which significantly improves the safety profile of a GalNAc-siRNA in rat and may minimize the occurrence of hepatotoxic siRNAs across species.

MeSH terms

  • Acetylgalactosamine / chemistry*
  • Acetylgalactosamine / toxicity
  • Animals
  • Liver / drug effects*
  • Liver / metabolism
  • Male
  • RNA Interference
  • RNA, Small Interfering / chemistry
  • RNA, Small Interfering / genetics*
  • RNA, Small Interfering / metabolism
  • RNA, Small Interfering / toxicity*
  • Rats
  • Rats, Sprague-Dawley


  • RNA, Small Interfering
  • Acetylgalactosamine