title: Inhibition of Hepatitis C Virus in Mice by a Small Interfering RNA Targeting a Highly Conserved Sequence in Viral IRES Pseudoknot.
abstract: The hepatitis C virus (HCV) internal ribosome entry site (IRES) that directs cap-independent viral translation is a primary target for small interfering RNA (siRNA)-based HCV antiviral therapy. However, identification of potent siRNAs against HCV IRES by bioinformatics-based siRNA design is a challenging task given the complexity of HCV IRES secondary and tertiary structures and association with multiple proteins, which can also dynamically change the structure of this cis-acting RNA element. In this work, we utilized siRNA tiling approach whereby siRNAs were tiled with overlapping sequences that were shifted by one or two nucleotides over the HCV IRES stem-loop structures III and IV spanning nucleotides (nts) 277-343. Based on their antiviral activity, we mapped a druggable region (nts 313-343) where the targets of potent siRNAs were enriched. siIE22, which showed the greatest anti-HCV potency, targeted a highly conserved sequence across diverse HCV genotypes, locating within the IRES subdomain IIIf involved in pseudoknot formation. Stepwise target shifting toward the 5' or 3' direction by 1 or 2 nucleotides reduced the antiviral potency of siIE22, demonstrating the importance of siRNA accessibility to this highly structured and sequence-conserved region of HCV IRES for RNA interference. Nanoparticle-mediated systemic delivery of the stability-improved siIE22 derivative gs_PS1 siIE22, which contains a single phosphorothioate linkage on the guide strand, reduced the serum HCV genome titer by more than 4 log10 in a xenograft mouse model for HCV replication without generation of resistant variants. Our results provide a strategy for identifying potent siRNA species against a highly structured RNA target and offer a potential pan-HCV genotypic siRNA therapy that might be beneficial for patients resistant to current treatment regimens.
authors.affiliation: Department of Biotechnology, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-749, Korea.
authors.forename: Jae-Su
authors.initials: JS
authors.lastname: Moon
authors.affiliation: Department of Biotechnology, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-749, Korea.
authors.forename: Seung-Hoon
authors.initials: SH
authors.lastname: Lee
authors.affiliation: Department of Biotechnology, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-749, Korea.
authors.forename: Eun-Jung
authors.initials: EJ
authors.lastname: Kim
authors.affiliation: Department of Biotechnology, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-749, Korea.
authors.forename: Hee
authors.initials: H
authors.lastname: Cho
authors.affiliation: Department of Biotechnology, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-749, Korea.
authors.forename: Wooseong
authors.initials: W
authors.lastname: Lee
authors.affiliation: Department of Biotechnology, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-749, Korea.
authors.forename: Geon-Woo
authors.initials: GW
authors.lastname: Kim
authors.affiliation: Department of Biotechnology, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-749, Korea.
authors.forename: Hyun-Ji
authors.initials: HJ
authors.lastname: Park
authors.affiliation: Department of Biotechnology, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-749, Korea.
authors.forename: Seung-Woo
authors.initials: SW
authors.lastname: Cho
authors.affiliation: College of Pharmacy, Dongguk University, Goyang 410-820, Korea.
authors.forename: Choongho
authors.initials: C
authors.lastname: Lee
authors.affiliation: Department of Biotechnology, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-749, Korea.
authors.forename: Jong-Won
authors.initials: JW
authors.lastname: Oh
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000737:chemistry*; D002470:Cell Survival; D019295:Computational Biology; D004195:Disease Models, Animal; D005838:Genotype; D057809:HEK293 Cells; D016174:Hepacivirus / Q000187:drug effects* / Q000502:physiology; D006526:Hepatitis C / Q000188:drug therapy*; D006801:Humans; D000067556:Internal Ribosome Entry Sites / Q000235:genetics*; D051379:Mice; D053758:Nanoparticles / Q000737:chemistry; D009711:Nucleotides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000737:chemistry*; D012367:RNA, Viral / Q000737:chemistry; D014162:Transfection; D014779:Virus Replication
chemical_list: D000998:Antiviral Agents; D000067556:Internal Ribosome Entry Sites; D009711:Nucleotides; D034741:RNA, Small Interfering; D012367:RNA, Viral