Triplex Hybridization of siRNA with Bifacial Glycopolymer Nucleic Acid Enables Hepatocyte-Targeted Silencing.

Abstract

Herein, we describe a versatile non-covalent strategy for packaging nucleic acid cargo with targeting modalities, based on triplex hybridization of oligo-uridylate RNA with bifacial polymer nucleic acid (bPoNA). Polyacrylate bPoNA was prepared and side chain-functionalized with N-acetylgalactosamine (GalNAc), which is known to enable delivery to hepatocytes and liver via binding to the asialoglycoprotein receptor (ASGPR). Polymer binding resulted in successful delivery of both native and synthetically modified siRNAs to HepG2 cells in culture, yielding in low nanomolar IC50 silencing of the endogenous ApoB target, in line with observations of expected Dicer processing of the polymer-siRNA targeting complex. Indeed, in vitro Dicer treatment of the polymer complex indicated that triplex hybridization does not impede RNA processing and release from the polymer. The complex itself elicited a quiescent immunostimulation profile relative to free RNA in a cytokine screen, setting the stage for a preliminary in vivo study in a high-calorie-diet mouse model. Gratifyingly, we observed significant ApoB silencing in a preliminary animal study, validating bPoNA as an in vivo carrier platform for systemic siRNA delivery. Thus, this new siRNA carrier platform exhibits generally useful function and is accessible through scalable synthesis. In addition to its utility as a carrier, the triplex-hybridizing synthetic platform could be useful for optimization screens of siRNA sequences using the identical polymer carriers, thus alleviating the need for covalent ligand modification of each RNA substrate.