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

Fumito Wada; Tsuyoshi Yamamoto; Tadayuki Kobayashi; Keisuke Tachibana; Kosuke Ramon Ito; Mayumi Hamasaki; Yukina Kayaba; Chisato Terada; Asako Yamayoshi; Satoshi Obika; Mariko Harada-Shiba

doi:10.1016/j.omtn.2021.10.008

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

Mol Ther Nucleic Acids. 2021 Oct 19:26:957-969. doi: 10.1016/j.omtn.2021.10.008. eCollection 2021 Dec 3.

Authors

Fumito Wada^{1

2

3}, Tsuyoshi Yamamoto^{3

4}, Tadayuki Kobayashi^{1

2

3}, Keisuke Tachibana³, Kosuke Ramon Ito³, Mayumi Hamasaki², Yukina Kayaba⁴, Chisato Terada⁴, Asako Yamayoshi⁴, Satoshi Obika³, Mariko Harada-Shiba^{1

2}

Affiliations

¹ Department of Molecular Innovation in Lipidology, National Cerebral and Cardiovascular Center Research Institute, 6-1 Kishibe-shinmachi, Suita, Osaka 564-8565, Japan.
² Department of Molecular Pathogenesis, National Cerebral and Cardiovascular Center Research Institute, 6-1 Kishibe-shinmachi, Suita, Osaka 564-8565, Japan.
³ Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
⁴ Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8131, Japan.

Abstract

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 Ca²⁺ 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.

Keywords: AmNA; BNA gapmer; CEM transfection; GalNAc; LDL-cholesterol; PCSK9; antisense oligonucleotide; drug delivery; ligand targeting.