title: Engineered external guide sequences are highly effective in inhibiting gene expression and replication of hepatitis B virus in cultured cells.
abstract: External guide sequences (EGSs) are RNA molecules that consist of a sequence complementary to a target mRNA and recruit intracellular ribonuclease P (RNase P), a tRNA processing enzyme, for specific degradation of the target mRNA. We have previously used an in vitro selection procedure to generate EGS variants that efficiently induce human RNase P to cleave a target mRNA in vitro. In this study, we constructed EGSs from a variant to target the overlapping region of the S mRNA, pre-S/L mRNA, and pregenomic RNA (pgRNA) of hepatitis B virus (HBV), which are essential for viral replication and infection. The EGS variant was about 50-fold more efficient in inducing human RNase P to cleave the mRNA in vitro than the EGS derived from a natural tRNA. Following Salmonella-mediated gene delivery, the EGSs were expressed in cultured HBV-carrying cells. A reduction of about 97% and 75% in the level of HBV RNAs and proteins and an inhibition of about 6,000- and 130-fold in the levels of capsid-associated HBV DNA were observed in cells treated with Salmonella vectors carrying the expression cassette for the variant and the tRNA-derived EGS, respectively. Our study provides direct evidence that the EGS variant is more effective in blocking HBV gene expression and DNA replication than the tRNA-derived EGS. Furthermore, these results demonstrate the feasibility of developing Salmonella-mediated gene delivery of highly active EGS RNA variants as a novel approach for gene-targeting applications such as anti-HBV therapy.
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei, China.
authors.forename: Zhigang
authors.initials: Z
authors.lastname: Zhang
authors.forename: Gia-Phong
authors.initials: GP
authors.lastname: Vu
authors.forename: Hao
authors.initials: H
authors.lastname: Gong
authors.forename: Chuan
authors.initials: C
authors.lastname: Xia
authors.forename: Yuan-Chuan
authors.initials: YC
authors.lastname: Chen
authors.forename: Fenyong
authors.initials: F
authors.lastname: Liu
authors.forename: Jianguo
authors.initials: J
authors.lastname: Wu
authors.forename: Sangwei
authors.initials: S
authors.lastname: Lu
mesh_terms: D002478:Cells, Cultured; D015967:Gene Expression Regulation, Viral / Q000187:drug effects* / Q000235:genetics; D018390:Gene Targeting / Q000379:methods*; D018014:Gene Transfer Techniques; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D006515:Hepatitis B virus / Q000235:genetics / Q000502:physiology*; D006801:Humans; D012475:Salmonella; D014779:Virus Replication / Q000187:drug effects* / Q000235:genetics; D058727:RNA, Small Untranslated
chemical_list: D058727:RNA, Small Untranslated