Mechanistic Insights into Hybridization-Based Off-Target Activity of GalNAc-siRNA Conjugates

Nonclinical safety screening of small interfering RNAs (siRNAs) conjugated to a trivalent <i>N</i>-acetylgalactosamine (GalNAc) ligand is typically carried out in rats at exaggerated exposures in a repeat-dose regimen. We have previously shown that at these suprapharmacological doses, hepatotoxicity observed with a subset of GalNAc-siRNAs is largely driven by undesired RNA-induced silencing complex (RISC)-mediated antisense strand seed-based off-target activity, similar to microRNA-like regulation. However, the RISC component requirements for off-target activity of siRNAs have not been evaluated. Here, we evaluate the roles of major RISC components, AGO and TNRC6 (or GW182) proteins, in driving on- and off-target activity of GalNAc-siRNAs in hepatocytes, <i>in vitro</i> and <i>in vivo</i>. We demonstrate that knocking down AGO2, but not AGO1 or AGO4, is protective against GalNAc-siRNA-driven off-target activity and hepatotoxicity. As expected, knocking down AGO2, but not AGO1 or AGO4, reduces the on-target activity of GalNAc-siRNA. Similarly, knocking down TNRC6 paralogs, TNRC6A or TNRC6B, but not TNRC6C, is protective against off-target activity and hepatotoxicity while having minimal impact on the on-target activity of GalNAc-siRNA. These data indicate that while AGO2 is the only RISC component required for the on-target activity of GalNAc-siRNAs, the undesired off-target activity and hepatotoxicity of a subset of GalNAc-siRNAs are mediated via the RISC composed predominantly of AGO2 and TNRC6 paralogs TNRC6A and/or TNRC6B.