Drug discovery and development scheme for liver-targeting bridged nucleic acid antisense oligonucleotides

Antisense oligonucleotides (ASOs) containing bridged nucleic acids (BNAs) have been proven to be very powerful. However, ensuring a reliable discovery and translational development scheme for this class of ASOs with wider therapeutic windows remains a fundamental challenge. We here demonstrate the robustness of our scheme in the context of the selection of ASOs having two different BNA chemistries (2,′4′-BNA/locked nucleic acid [LNA] and amido-bridged nucleic acid [AmNA]) targeting human proprotein convertase subtilisin/kexin type 9 (PCSK9). The scheme features a two-step process, including (1) a unique and sensitive in vitro screening approach, called Ca(2+) enrichment of medium (CEM) transfection, and (2) a ligand-targeted drug delivery approach to better reach target tissues, averting unintended accumulation of ASOs. Using CEM screening, we identified a candidate ASO that shows >70% cholesterol-lowering action in monkeys. An N-acetylgalactosamine (GalNAc) ligand then was appended to the candidate ASO to further broaden the therapeutic margin by altering the molecule’s pharmacokinetics. The GalNAc conjugate, HsPCSK9-1811-LNA, was found to be at least ten times more potent in non-human primates (compared with the unconjugated counterpart), with reduced nephrotoxicity in rats. Overall, we successfully showed that our drug development scheme is better suited for selecting clinically relevant BNA-based ASOs, especially for the treatment of liver-associated diseases.