title: Evaluation of the effects of chemically different linkers on hepatic accumulations, cell tropism and gene silencing ability of cholesterol-conjugated antisense oligonucleotides.
abstract: Cholesterol conjugation of oligonucleotides is an attractive way to deliver the oligonucleotides specifically to the liver. However cholesterol-conjugated antisense oligonucleotides (ASOs) mainly accumulate in non-parenchymal cells (NPCs) such as Kupffer cells. In this study, to increase the hepatic accumulation of cholesterol-conjugated ASOs, we prepared a variety of linkers for cholesterol conjugation to anti-Pcsk9 ASOs and examined their effects on pharmacological parameters. Hepatic accumulation of ASO was dramatically increased with cholesterol conjugation. The increase in hepatic accumulation depended largely on the linker chemistry of each cholesterol-conjugated ASO. In addition to hepatic accumulation, the cell tropism of each cholesterol-conjugated ASO tended to depend on their linker. Although a linker bearing a disulfide bond accumulated mainly in NPCs, hexamethylene succinimide linker accumulated mainly in hepatocytes. To estimate the benefits of releasing ASO from the conjugated cholesterol in hepatocyte, we designed another linker based on hexamethylene succinimide, which has a phosphodiester bond between the linker and the ASO. The cholesterol-conjugated ASO bearing such a phosphodiester bond showed a significantly improved Pcsk9 mRNA inhibitory effect compared to its counterpart, cholesterol-conjugated ASO with a phosphorothioate bond, while the hepatic accumulation of both cholesterol-conjugated ASOs was comparable, indicating the effectiveness of removing the conjugated cholesterol for ASO activity. In toxicity analysis, some of the linkers induced lethal toxicities when they were injected at high concentrations (>600μM). These toxicities were attributed to decreased platelet levels in the blood, suggesting an interaction between cholesterol-conjugated ASO and platelets. Our findings may provide a guideline for the design of molecule-conjugated ASOs.
authors: Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan; Department of Molecular Innovation in Lipidology, National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka 565-8565, Japan.
authors: Shunsuke
authors: S
authors: Wada
authors: Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan; Department of Molecular Innovation in Lipidology, National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka 565-8565, Japan.
authors: Hidenori
authors: H
authors: Yasuhara
authors: Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan; Department of Molecular Innovation in Lipidology, National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka 565-8565, Japan.
authors: Fumito
authors: F
authors: Wada
authors: Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan; Department of Molecular Innovation in Lipidology, National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka 565-8565, Japan.
authors: Motoki
authors: M
authors: Sawamura
authors: Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan.
authors: Reiko
authors: R
authors: Waki
authors: Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan; Department of Molecular Innovation in Lipidology, National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka 565-8565, Japan.
authors: Tsuyoshi
authors: T
authors: Yamamoto
authors: Department of Molecular Innovation in Lipidology, National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka 565-8565, Japan.
authors: Mariko
authors: M
authors: Harada-Shiba
authors: Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan. Electronic address: obika@phs.osaka.u.ac.jp.
authors: Satoshi
authors: S
authors: Obika
mesh_terms: D000818:Animals; D002784:Cholesterol / Q000031:analogs & derivatives*; D020868:Gene Silencing; D008099:Liver / Q000378:metabolism*; D008297:Male; D008810:Mice, Inbred C57BL; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage / Q000737:chemistry* / Q000235:genetics* / Q000493:pharmacokinetics; D000071449:Proprotein Convertase 9 / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*
chemical_list: D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D002784:Cholesterol; C472126:Pcsk9 protein, mouse; D000071449:Proprotein Convertase 9
keywords: Antisense oligonucleotide; Cholesterol conjugation; Linkage