Inhibition of core gene of HCV 3a genotype using synthetic and vector derived siRNAs

Abstract

BackgroundHepatitis C virus (HCV) is a major causative agent of liver associated diseases throughout the world, with genotype 3a responsible for most of the cases in Pakistan. Due to the limited efficiency of current therapy, RNA interference (RNAi) a novel regulatory and powerful silencing approach for molecular therapeutics through a sequence-specific RNA degradation process represents an alternative option.ResultsThe current study was purposed to assess and explore the possibility of RNAi to silence the HCV-3a Core gene expression, which play complex role in regulation of cell growth and host genes expression essential for infectivity and disease progression. To identify the potent siRNA target sites, 5 small interfering RNAs (siRNAs) against Core gene were designed and in vitro transcribed after consensus sequence analysis of different HCV-3a isolates. Antiviral effects of siRNAs showed upto 80% inhibition of Core gene expression by different siRNAs into Huh-7 cells as compared with Mock transfected and control siRNAs treated cells. For long lasting effect of siRNAs, vector based short hairpin siRNAs (shRNAs) were designed and tested against HCV-3a Core which resulted in a similar pattern of inhibition on RNA and protein expression of HCV Core as synthetic siRNAs. Furthermore, the efficacy of cell culture tested siRNA and shRNA, were evaluated for inhibition of HCV replication in HCV infected serum inoculated Huh-7 cells and a significant decrease in HCV viral copy number was observed.ConclusionsOur results support the possibility of using consensus siRNA and shRNA-based molecular therapy as a promising strategy in effective inhibition of HCV-3a genotype.

Down-regulation of IRES containing 5'UTR of HCV genotype 3a using siRNAs

“…Taken advantage of the newly developed cell culture system of HCV infection in liver cells [27,28,41], the effect of siRNAs against whole virus was evaluated by treating cells with siRNA. Huh-7 serum infected cells were treated with 5'UTR siRNAs and subsequently incubated for 3 days.…”

Core Gene Expression and Association of Genotypes with Viral Load in Hepatitis C Virus (HCV) - Infected Patients in Punjab, Pakistan

“…The nucleotide sequence homology analysis indicates the HCV isolates in this study (FR851292) belong to HCV core gene like Japan (D14309) and USA (EU099417). Previously this type of HCV was already isolated from this region [15]. This gives the idea that for endemic HCV this ELISA based kit method is much better because of local recombinant protein [16].…”

Inhibition of expression of hepatitis C virus 1b genotype core and NS4B genes in HepG2 cells using artificial microRNAs

“…As replication occurs in the cytoplasm of liver cells without integration into the host genome DNA, HCV has emerged as a target of RNAi (40). Certain in vivo and in vitro experiments have demonstrated that exogenously administered short-interfering RNAs are able to exert replication inhibition against a variety of viruses (41)(42)(43)(44). In the present study, two sets of amiRNA expression vectors were designed and constructed against HCV1b genotype core and NS4B mRNAs, and it was investigated whether these amiRNAs were capable of efficiently inhibiting the expression of the HCV core and NS4B genes.…”