Hepatitis C virus (HCV) infection is a common and a major worldwide causative of chronic hepatitis, cirrhosis, and hepatocellular carcinoma potentially accounting for up to 0.5 million deaths annually (Lozano et al. 2012). HCV is an enveloped positive sense single stranded RNA virus and is a member of the genus Hepacivirus in the family of Flaviviridae (Giannini and Brechot 2003). The HCV genome has 9600 bases, forming a continuous open reading frame edged by 5′ and 3′ non-translated regions (Bartenschlager and Sparacio 2007). The 5′ non-translated region contains an internal ribosome entry (IRES) that is essential to initiate the translation of the HCV genome (Tsukiyama et al 1992). The IRES–mediated translation produces an approximately 3000 amino acid polyprotein precursor. It subsequently cleaves co- and post translationally into mature viral structural and nonstructural (NS) proteins. The proteolytic processing of the polyprotein is by cellular peptidases (Hijikata et al 1993) and two viral proteases, NS2/3 and NS3 (Grakoui et al 1993; Bartenschlager et al 1993), which breaks it into 10 functional subunits: structural core (C), envelope (E1 and E2) proteins, ion channel p7, and NS proteins NS2, NS3, NS4A, NS5A and NS5B (Pallaoro et al 2001). Subunits C, E1, and E2 form the virus particles in which nucleocapsid is constructed from repeated copies of the core protein, while E1 and E2 form the envelope glycoproteins. P7 may have a function in assembly and release of the viral particles. The NS proteins from NS3 to NS5B form the viral replicase complex. Also, NS5B forms the RNA-dependent RNA polymerase (Han et al 2014).
The variability of viral RNA enables the classification of HCV into six genotypes (1 to 6) and several subtypes epidemiologically associated with risk factors and geographical areas (Simmonds 2013). HCV genetic variation occurs (Zein and Persing 1996) as a result of the high error rate of RNA-dependent RNA polymerases during HCV replication (Bartenschlager and Lohmann 2000). HCV Genotype 1 (G1) is the most predominant in the world (~83.4 million people) followed by G3 (~54.3 million), then G2, G4 and G6 (~15.6 million), and G5 (~1.4 million) (Messina et al 2015). HCV transmission mainly occurs through contaminated blood and blood products transfusion, injection drug use, hemodialysis and organ transplantation; however unprotected sexual intercourse and birth from an infected mother have also been documented as other modes of transmission (Alter 1997; Alter 2002).
Until 2011, the standard treatment was a blend of subcutaneous pegylated interferon (PEG-IFN) in addition to oral ribavirin (RBV), administered for 24 or 48 weeks (McEwan et al 2015), but this strategy failed in more than 40% of the patients and has a high cost and serious side effects. The cure rate depends on the viral genotypes where IFN-alpha and RBV failed to eliminate HCV in 50–60% of patients infected with G1 and G4 and about 20% of patients with G2 and G3 (Pawlotsky 2015). Development of a new, specifically targeted antiviral therapy for HCV was a must to overcome the shortcomings of PEG-IFN/RBV therapy.
There had been no effective vaccine for HCV; and there are many FDA-approved anti-HCV therapies, and many diverse scientific strategies have been developed for HCV therapy, but some are still in different clinical trial phases. So we need more attempts to develop other treatments. Targeting HCV replication by RNA interference is an essential strategy for antiviral therapy. RNA interference is a process of post-transcriptional gene silencing that has been identified in all eukaryotes (Fire et al 1998; Hannon 2002). Since its first report, this mechanism has been found to be useful both as a molecular biology tool for the study of gene function and as a therapeutic agent (Harborth et al 2001; Pecot et al 2011). A key component of the RNAi pathway is the RNA-induced silencing complex (RISC), which is responsible for the cleavage of mRNA in a sequence-specific fashion. The specificity of this reaction is provided by a 21 nt antisense strand incorporated into the RISC complex. This antisense strand originates from the digestion of double stranded RNA (dsRNA) by DICER endonuclease (Elbashir 2001). Synthetic 21 nt siRNAs can be introduced into cells and can selectively suppress a specific gene of interest. A number of reports have shown that RNA interference can efficiently inhibit HCV replication via different methodologies (Kapadia et al 2014; Wilson et al 2003).
In the present study, we aimed to study the effect of RNAi to specifically target core gene of Saudi HCV-4 genotype isolates as new option for developing a rational antiviral strategy. It is expected that cleavage of core mRNA will inhibit virus replication. We report here that RNAi targeted against core effectively inhibited core gene RNA and protein expression in a dose dependant manner in HepG-2/C3A cells irrespective of mode of delivery. The present study demonstrates that the RNAi-mediated silencing of the HCV-4 core gene may be one of the important therapeutic opportunities against this particular genotype.