Hepatitis C Virus (HCV) affects more than 170 million people worldwide, or 3% of the world population (Perz et al., 2006), with 4 million new cases and more than 300,000 deaths per year (Bukh, 2012). Clinical conditions of the disease range from an asymptomatic carrier state to persistent infections. Of those individuals infected, 70% will develop chronic HCV infections, and 20% of chronic infections will progress to cirrhosis and terminal hepatocellular carcinoma (Bradley, 2000; Lauer and Walker, 2001; Seeff, 2002; Hoofnagle, 1997). There are currently no vaccines available to the public to prevent HCV due to the high genetic variability of the virus and its ability to escape host immune defenses (Di Lorenzo et al., 2011).
The current standard of care (SOC) treatments may include a combination of pegylated interferon-α and ribavirin (Christie and Chapman, 1999) and direct acting antivirals such as Sofosbuvir and Simeprevir (Belousova et al., 2015). The drug combination has unfavorable side effects and may ultimately lead to drug resistance and relapse. One of the main reasons may be attributed to the generation of quasispecies genome meclofenoxate common for HCV infections, a phenomenon that results in infection by a swarm of microvariants derived from a predominant “master sequence” within an individual host (Bukh et al., 1995). Quasispecies are more prominent in the setting of persistent infections and may be responsible for drug treatment failures (Farci et al., 2000; Domingo and Gomez, 2007). Quasispecies result from the high error rate of the non-proofreading HCV RNA-dependent RNA polymerase (RdRp) leading to continuous production of mutated virus sequences which is one mechanism the virus employs to escape immune system defense (Carmichael, 2002). This warrants a continued intensive search for alternative antiviral approaches to combating HCV.
HCV is a plus-strand RNA virus of the Hepacivirus genus, having a 9600 nt long genome encoding a single ORF flanked by highly conserved 5′ and 3′ untranslated regions (UTRs) (Takamizawa et al., 1991). The ORF encodes a single polyprotein that is modified post-translationally by both cellular and viral proteases to produce 3 structural (C, E1, E2) and 7 non-structural (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins (Fauvelle et al., 2013). The 5′ UTR of the viral RNA contains an internal ribosome entry site (IRES) that is highly conserved among most known HCV quasispecies (Brown et al., 1992). The 5′ UTR of HCV facilitates viral replication and mediates cap-independent viral protein translation by acting as a scaffold and recruiting multiple protein factors during the initiation of translation upon early infection (Rosenberg, 2001; Kieft et al., 1999; Friebe and Bartenschlager, 2002). Because the IRES serves a crucial function for viral infection and propagation and is therefore highly conserved, it represents an ideal target for anti-HCV approaches employing nucleic acid homologies such as trans-splicing group I introns (Ryu et al., 2003).
Trans-splicing group I introns derived from the cis-splicing group I intron of Tetrahymena thermophila mediate RNA splicing through two successive transesterification steps (Cech, 1991). First, the intron recognizes a specific uracil on the target RNA during complementary base pairing with the surrounding sequence. The target RNA is then cleaved at that uracil, and the intron-attached 3′ exon is cleaved from the group I intron and appended onto the cleaved target RNA to create a product RNA. If that product is capable of translation it will express a new protein encoded by the sequence of the 3′ exon (Long et al., 2003). Group I introns have been used successfully in a number of anti-viral applications including targeting of Dengue Fever virus (Carter et al., 2010), HCV (Ryu et al., 2003), and HIV (Kohler et al., 1999) genomes, and in posttranscriptional gene manipulations including the restoration of wild-type p53 activity in three cancerous cell lines (Shin et al., 2004) and the repair of sickle β-globin mRNAs in erythrocyte precursors (Lan et al., 1998).