Inhibition of Hepatitis C Virus Replication by Intracellular Delivery of Multiple siRNAs by Nanosomes

Abstract

Sustained antiviral responses of chronic hepatitis C virus (HCV) infection have improved recently by the use of direct-acting antiviral agents along with interferon (IFN)-α and ribavirin. However, the emergence of drug-resistant variants is expected to be a major problem. We describe here a novel combinatorial small interfering RNA (siRNA) nanosome-based antiviral approach to clear HCV infection. Multiple siRNAs targeted to the highly conserved 5'-untranslated region (UTR) of the HCV genome were synthesized and encapsulated into lipid nanoparticles called nanosomes. We show that siRNA can be repeatedly delivered to 100% of cells in culture using nanosomes without toxicity. Six siRNAs dramatically reduced HCV replication in both the replicon and infectious cell culture model. Repeated treatments with two siRNAs were better than a single siRNA treatment in minimizing the development of an escape mutant, resulting in rapid inhibition of viral replication. Systemic administration of combinatorial siRNA-nanosomes is well tolerated in BALB/c mice without liver injury or histological toxicity. As a proof-of-principle, we showed that systemic injections of siRNA nanosomes significantly reduced HCV replication in a liver tumor-xenotransplant mouse model of HCV. Our results indicate that systemic delivery of combinatorial siRNA nanosomes can be used to minimize the development of escape mutants and inhibition of HCV infection.

Establishment of Animal Models with Orthotopic Hepatocellular Carcinoma

“…Additionally, various human or animal HCC cell lines are used to study comparative viability, apoptosis, and toxicity in response to test substances [24]. Typically, Hep 3B, Hep G2, Huh 7.5 and SK-Hep1 in human HCC cell lines and N1S1 and McA-RH7777 in rat HCC cell lines are commonly used in HCC models [25][26][27][28][29][30].…”

“…[27] used HCC models with Hep G2 cell lines in their study that demonstrated and characterized the inhibition of both tumor progression, and proliferation of liver cancer in vitro and in vivo. Chandra et al [28] used Huh 7.5 cell lines to design xenograft mouse models. They evaluated the inhibition of hepatitis C virus (HCV) replication by small interfering RNA (siRNA) encapsulated into lipid nanoparticles (nanosomes).…”

Expression of Duplex shRNAs through a Lentiviral Vector against Cellular and Viral Genes Inflicts Sustained Inhibition of Hepatitis C Virus Replication

“…In recent years, siRNA-mediated gene silencing has been extensively employed to study different RNA viruses, especially HCV [10][11][12]. HCV often causes chronic infections and, therefore, a consistent presence of effective doses of siRNA would be DOI: 10.1159/000492220 obligatory for a sustained virological response (SVR), which is feasible either by frequent deliveries of exogenous siRNA or in vivo expression of shRNA [13,14]. However, repetitive administration of exogenous siRNA may not be feasible in the clinical setting; therefore, approaches for in vivo expression of shRNAs have been evaluated [15,16].…”

Composite vector formulation for multiple siRNA delivery as a host targeting antiviral in a cell culture model of hepatitis C virus (HCV) infection

“…Several studies using both in vitro and in vivo models of HCV infection, showed that repeated treatments with two siRNAs were better than a single siRNA treatment at minimizing the development of escape mutants, resulting in a rapid inhibition of viral replication. 11,12 Moreover, targeting various HTAs would be advantageous by controlling different steps of viral infection, from viral entry to cell re-infection. 6,13 Finally, previous studies have demonstrated that, in contrast to the siRNA technology, the overexpression of exogenously introduced shRNA competes with that of endogenous miRNA and thus leads to the saturation of the endogenous miRNA pathway, resulting in serious toxicity in the mouse liver and, in some instances, death.…”

“…Our results are in accordance with a study published by Sokolova et al showing that complexation of PEI with calcium phosphate particles improves siRNA delivery. 34 As combining different siRNAs has been shown to increase antiviral efficacy, 11 we prepared nanoparticles coated with two different siRNAs. HTAs constitute a promising strategy to treat HCV infection with low viral resistance to treatment.…”