Multivalent <i>N</i>‑Acetylgalactosamine-Conjugated siRNA Localizes in Hepatocytes and Elicits Robust RNAi-Mediated Gene Silencing

Abstract

Conjugation of small interfering RNA (siRNA) to an asialoglycoprotein receptor ligand derived from <i>N</i>-acetylgalactosamine (GalNAc) facilitates targeted delivery of the siRNA to hepatocytes <i>in vitro</i> and <i>in vivo</i>. The ligands derived from GalNAc are compatible with solid-phase oligonucleotide synthesis and deprotection conditions, with synthesis yields comparable to those of standard oligonucleotides. Subcutaneous (SC) administration of siRNA–GalNAc conjugates resulted in robust RNAi-mediated gene silencing in liver. Refinement of the siRNA chemistry achieved a 5-fold improvement in efficacy over the parent design <i>in vivo</i> with a median effective dose (ED₅₀) of 1 mg/kg following a single dose. This enabled the SC administration of siRNA–GalNAc conjugates at therapeutically relevant doses and, importantly, at dose volumes of ≤1 mL. Chronic weekly dosing resulted in sustained dose-dependent gene silencing for over 9 months with no adverse effects in rodents. The optimally chemically modified siRNA–GalNAc conjugates are hepatotropic and long-acting and have the potential to treat a wide range of diseases involving liver-expressed genes