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.
Highlights
Nucleic acid-based gene interfering technologies, such as antisense oligonucleotides and RNA interference (RNAi), have been shown to be a promising gene targeting approach for use in basic research and clinical therapeutic applications [1]
We have shown that External guide sequences (EGSs) that were derived from a natural tRNA effectively induced human Ribonuclease P (RNase P) to cleave the mRNAs of herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) in vitro [10,12,13]
Our results indicated that an EGS variant, S-C386, is about 50 times more active [Vmax(apparent) / K ] m(apparent) in directing RNase P to cleave the S mRNA sequence in vitro than S-SER, an EGS derived from the natural tRNASer sequence
Summary
Nucleic acid-based gene interfering technologies, such as antisense oligonucleotides and RNA interference (RNAi), have been shown to be a promising gene targeting approach for use in basic research and clinical therapeutic applications [1]. Ribonuclease P (RNase P) has been found in all organisms examined and its enzymatic activity is responsible for the maturation of 5’ termini of all tRNAs which account for about 2% of total cellular RNA [23.–4]. This enzyme is a ribonucleoprotein complex and catalyzes a hydrolysis reaction to remove the leader sequence of precursor tRNA (Figure 1A) [2,3,4]. EGS RNAs derived from natural tRNA sequences can be effective in blocking gene expression in bacteria and in mammalian cells [7,8,9,10]. A reduction of ~75% in HSV and HCMV gene expression was observed in viral infected cells that expressed these functional EGS RNAs
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