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

Area of Science:

• Pharmacology
• Molecular Biology
• Hepatology

Background:

• Nonclinical safety of N-acetylgalactosamine (GalNAc)-conjugated small interfering RNAs (siRNAs) is assessed in rats.
• Suprapharmacological doses of some GalNAc-siRNAs cause hepatotoxicity via RNA-induced silencing complex (RISC)-mediated off-target activity.
• The specific RISC components involved in siRNA on- and off-target activities remain unclear.

Purpose of the Study:

• To investigate the roles of Argonaute (AGO) and TNRC6 proteins in GalNAc-siRNA on- and off-target activities.
• To determine the contribution of specific RISC components to GalNAc-siRNA-induced hepatotoxicity.
• To elucidate the molecular mechanisms underlying off-target effects of GalNAc-siRNAs.

Main Methods:

• In vitro and in vivo studies using hepatocytes.
• Knockdown of AGO (AGO1, AGO2, AGO4) and TNRC6 (TNRC6A, TNRC6B, TNRC6C) paralogs.
• Evaluation of GalNAc-siRNA on-target and off-target activities and associated hepatotoxicity.

Main Results:

• Knockdown of AGO2, but not AGO1 or AGO4, reduced both on-target and off-target GalNAc-siRNA activities and hepatotoxicity.
• Knockdown of TNRC6A or TNRC6B, but not TNRC6C, protected against off-target effects and hepatotoxicity with minimal impact on on-target activity.
• AGO2 is essential for on-target siRNA activity, while AGO2 and TNRC6A/B mediate off-target effects.

Conclusions:

• AGO2 is the sole RISC component required for the on-target activity of GalNAc-siRNAs.
• Off-target activity and hepatotoxicity of certain GalNAc-siRNAs are mediated by RISC complexes containing AGO2 and TNRC6A and/or TNRC6B.
• These findings provide critical insights into the safety profiling of GalNAc-siRNA therapeutics.