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

Abstract

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.

Phase 3 Multicenter Study of Revusiran in Patients with Hereditary Transthyretin-Mediated (hATTR) Amyloidosis with Cardiomyopathy (ENDEAVOUR)

“…A clear causative mechanism could not therefore be identified for the mortality imbalance. Short-and longterm rat and non-human primate chronic toxicology studies (of up to 2 years' duration) did not reveal any corresponding toxicities to those observed in the ENDEAVOUR study [25] (data on file). However, the combination of an increase in deaths in the revusiran arm and other notable imbalances in AEs (peripheral neuropathy, hepatic, and renal events) suggests that drug-mediated toxicity may have been a factor in the outcome.…”

“…As such, the exposure to revusiran, given at 500-mg weekly doses in the ENDEAVOUR study, was 28 g of siRNA in the first year, whereas vutrisiran achieves the same degree of TTR reduction at 25 mg every 3 months (100 mg annually), equating to a 280-fold lowered drug exposure. For ESC GalNAc-siRNA conjugates, lowered exposures are expected to lead to more favorable safety results [25]. Consistent with this, several ESC GalNAc-siRNA compounds are in, or have recently completed, Phase 3 studies without similar findings to those seen with revusiran.…”

Manipulating microRNAs for the Treatment of Malignant Pleural Mesothelioma: Past, Present and Future

“…However, even with this selective delivery advantage, the miR-34a-based drug MRX34 targeting hepatocellular carcinoma or liver metastases was terminated due to unexpected immune-related adverse events (88). It is notable that seed sequence-mediated hepatotoxicity has been used to screen siRNA drug candidates prior to clinical development (89), but as the miR-34a mimic used did not cause immune events or hepatocyte damage in mouse models of liver cancer (90) and there are no published results describing these adverse events in more detail, the underlying cause remains unknown. Nevertheless, reaction to the liposomal vehicle, immune stimulation by double-stranded RNA or necrotic cell death may have played a role (88).…”

CHAPTER 14. Strategies for Safe and Targeted Delivery of MicroRNA Therapeutics

“…Right: When tumorsuppressive miRNAs are downregulated in the disease state, miRNA siRNAs. 110 Importantly, while these modifications and shorter passenger strand design have been efficacious for siRNA delivery, attempts to modify miRNA mimics in a similar manner have had mixed results, requiring more careful engineering.…”