Chemical optimization improves oligonucleotide delivery

Forty years after their initial discovery, oligonucleotide therapeutics had begun to show potential to reach clinical maturity. By 2014, several drugs had been approved for disease treatment, and many others were undergoing clinical trials at various stages. However, being able to deliver effective doses of oligonucleotides to a specific set of diseased cells or organs was still challenging.

Small interfering RNAs (siRNAs) had been successfully used to inhibit the expression of disease-causing genes, and the implementation of antibody-mediated and lipid-nanoparticle-mediated delivery had significantly improved their efficiency. However, these treatments often required high doses and repeated intravenous injections to be therapeutically effective, which limited their clinical applicability in cases where intravenous drug administration was not feasible. Therefore, researchers worked to develop chemically modified oligonucleotides to enable efficient delivery to target cells by subcutaneous administration.

How such chemical optimisation could improve siRNA delivery was demonstrated in 2014 by Manoharan and colleagues, who covalently conjugated siRNA to N‑acetylgalactosamine (GalNAc), a ligand of the asialoglycoprotein receptor (ASGPR). This receptor is expressed on the surface of liver cells and is responsible for the uptake of circulating glycoproteins with exposed GalNAc glycans. This design enabled targeted siRNA–GalNAc delivery to the liver owing to the high-affinity binding between receptor and ligand. The first tests were performed in cultured mouse liver cells and showed that receptor binding-affinity correlated with siRNA uptake efficiency. Encouraged by these results, the researchers tested the ability of siRNA–GalNAc conjugates to silence gene expression in vivo. They observed a robust and durable silencing of the targeted gene, transthyretin, in the liver of mice after a single or multiple low-volume subcutaneous administrations. The extent of silencing was higher following subcutaneous administration compared with intravenous administration.

In a study published a year later, the same research group optimised the design of the conjugates to improve therapeutic effectiveness. The researchers tested various attachment sites for the GalNAc ligand on the RNA molecule, and evaluated silencing activity both in vitro and in vivo. They found that placing three monovalent GalNAc units in close proximity to each other on the RNA sense strand resulted in a higher-affinity binding to ASGPR on liver cells, and a more robust silencing in vivo.

This seminal work established siRNA−GalNAc as a promising therapeutic delivery approach to treat diseases involving liver-expressed genes. Despite halting of the development of the first siRNA−GalNAc-based drug (Revusiran) during clinical trials in 2016, the impressive silencing efficiency, good safety profile and encouraging results from more recent clinical trials of drugs for acute hepatic porphyria (Givosiran) and cardiovascular disease with elevated LDL cholesterol (Inclisiran), established GalNAc conjugation as a promising solution for therapeutic siRNA delivery to the liver.