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

TLDR

Conjugating siRNA to a GalNAc ligand targets hepatocytes via the asialoglycoprotein receptor, enabling efficient delivery in vitro and in vivo while remaining compatible with standard oligonucleotide synthesis. Subcutaneous administration of chemically optimized siRNA–GalNAc conjugates achieved robust, dose‑dependent, long‑lasting liver gene silencing (median ED50 ≈ 1 mg/kg, ≤1 mL volume) with a five‑fold efficacy improvement, sustained effects for over 9 months, no adverse events, and therapeutic potential for liver‑expressed diseases.

Abstract

Conjugation of small interfering RNA (siRNA) to an asialoglycoprotein receptor ligand derived from N-acetylgalactosamine (GalNAc) facilitates targeted delivery of the siRNA to hepatocytes in vitro and in vivo. 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 in vivo 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.