RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells

Abstract

RNA interference represents an exciting new technology that could have therapeutic applications for the treatment of viral infections. Hepatitis C virus (HCV) is a major cause of chronic liver disease and affects >270 million individuals worldwide. The HCV genome is a single-stranded RNA that functions as both a messenger RNA and replication template, making it an attractive target for the study of RNA interference. Double-stranded small interfering RNA (siRNA) molecules designed to target the HCV genome were introduced through electroporation into a human hepatoma cell line (Huh-7) that contained an HCV subgenomic replicon. Two siRNAs dramatically reduced virus-specific protein expression and RNA synthesis to levels that were 90% less than those seen in cells treated with negative control siRNAs. These same siRNAs protected naive Huh-7 cells from challenge with HCV replicon RNA. Treatment of cells with synthetic siRNA was effective >72 h, but the duration of RNA interference could be extended beyond 3 weeks through stable expression of complementary strands of the interfering RNA by using a bicistronic expression vector. These results suggest that a gene-therapeutic approach with siRNA could ultimately be used to treat HCV.

Stable RNA interference (RNAi) as an option for anti-bcr-abl therapy

“…Accordingly, both foreign genes encoded by infectious organisms and fusion genes resulting from chromosomal translocation have been analyzed by RNAi. [7][8][9][10][11][12][13][14][15][16][17][18] For example, short-term mRNA and protein reduction of the bcr-abl oncogene necessary for leukemic transformation in chronic myeloid and bcrabl+ acute lymphoblastic leukemia (CML, Ph+ALL), as well as inhibition of survival and proliferation of bcrabl+ cell lines have recently been described. [14][15][16] However, the application of RNAi for reverse genetics in mammalian cells is hampered by both the difficulty to deliver RNAi triggers to target cells and by the transient nature of RNAi in mammals.…”

Suppression of Hepatitis C Virus Replicon by RNA Interference Directed against the NS3 and NS5B Regions of the Viral Genome

“…was reported that siRNAs and/or shRNAs against 5'-untranslated region (5'UTR) (44), and nonstructural regions 3 (NS3) (19,49) and NS5B (29) of the HCV genome could effectively suppress the replication of HCV replicons without induction of IFN. In their approaches, however, siRNAs or plasmid vectors expressing shRNAs should be introduced to the target cell by using a transfection reagent or electroporation, that cannot be applied to human use.…”

RNA interference-mediated prevention and therapy for hepatocellular carcinoma

“…Wilson and Richardson (2005) have shown that HCV replicon escape mutants can emerge after multiple siRNA treatments. An 87% reduction of HCV replicon replication and gene expression was previously shown (Wilson et al, 2003) when replicon expressing cells were transfected with an siRNA targeting the NS5B gene. Mutations, speculated to have occurred in the HCV RNA, were thought to be selected for after siRNA treatment.…”

“…This effect was dose dependent and specific in that nucleotide changes within the target siRNA sequence abolished the effect. Wilson et al (2003) showed that long term suppression of HCV RNA replication could be established. Using episomal plasmids expressing pol III driven sense and antisense transcripts against NS5B, inhibition of HCV RNA replication was maintained for up to 3 weeks.…”