Selection of well-tolerated GalNAc-conjugated siRNAs by screening for RNAi-mediated off-target effects in rodent toxicity studies

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 include toxicities 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.与三价N-乙酰半乳糖胺（N-acetylgalactosamine, GalNAc）配体偶联的小干扰核糖核酸（small interfering RNAs, siRNAs）目前正针对多种适应症开展临床探索性研究。典型的开发候选化合物筛选流程会对活性最强的化合物开展药理放大剂量下的大鼠毒性评价。表现出大鼠肝毒性的GalNAc-siRNA候选化合物不会进入后续临床开发阶段。肝毒性的潜在机制包括寡核苷酸及其代谢物在细胞内蓄积引发的毒性、RNA干扰（RNA interference, RNAi）介导的基于杂交的脱靶效应，以及内源性RNAi通路的扰动。本研究表明，在超治疗暴露剂量下观察到的啮齿类动物肝毒性主要可归因于RNAi介导的脱靶效应，而非化学修饰或RNAi通路的扰动。此外，通过采用热不稳定化学修饰调控siRNA的种子区配对，可缓解此类脱靶效应，这能显著改善GalNAc-siRNA在大鼠体内的安全性特征，并有望降低跨物种肝毒性siRNA的发生概率。