Background: Hepatitis C virus infection affects more than 170 million people worldwide and is frequently associated with chronic liver disease and hepatocellular carcinoma. No protective vaccine is yet available and the current standard of care, consisting of pegylated interferon alpha and ribavirin, has limited efficacy. Ribavirin is a key component of any effective anti-HCV regimen. However, accumulation of ribavirin in the red cell compartment not only reduces drug efficacy as a result of diversion to extra-hepatic sites but also produces haemolytic anaemia which can lead to dose reduction or discontinuation of treatment. Lipid or polymer based nanoparticles can be used to deliver therapeutic agents, such as drugs or small interfering RNAs (siRNAs) directly to their site of action. We therefore elected to develop new antiviral strategies based on the targeted delivery of ribavirin to hepatocytes, coupled with the identification of new therapeutic targets. In order to inform the rational use of direct intracellular delivery of ribavirin, we enquired whether variation in expression of the ribavirin transporter may determine drug uptake and permit the identification of individuals who would benefit from these alternative approaches to treatment. Aims: The aims of this study were to: • identify host proteins involved in virus replication • demonstrate reduction of viral replication by modulation of host gene expression • develop and test a nanoparticle based system for the delivery of therapeutic molecules, including siRNAs either alone or in combination with ribavirin. • assess the relationship between ribavirin uptake by primary human hepatocytes and expression of ribavirin receptors Methods: A subgenomic HCV replicon system was established to study the virus-host relationship and identify host proteins supporting viral replication by using stealth siRNA. Viral RNAs were in vitro transcribed and transfected into Huh7 cells and expression assessed using engineered GFP as a reporter gene. siRNAs were co-transfected with viral RNAs using a nucleofector. Modulation of host gene expression was measured by both quantitative RT-PCR and protein blotting. Liposomal nanoparticles containing ApoB-100 duplexes were supplied by Lipoxen. Primary human hepatocytes were isolated by a modified two step collagenase perfusion method and cultured on collagen coated plates. HPLC and real time PCR conditions were used to measure and correlate drug uptake and receptor expression respectively. Equilibrative nucleoside transporter (ENT1) gene was analysed by direct sequencing. Results: A JFH1 (HCV genotype 2a) virus based subgenomic replicon system was successfully established. Using this model system, host proteins VAP-A and STAT3 were shown to positively regulate virus replication while ACTN1 had no effect. Liposomes failed to deliver either siRNA targeted at apoB-100 or ribavirin and this was found to be due to structural instability of the delivery vehicle. In contrast, fluorescently labelled liposomes were stable and could be taken up by human hepatocyte cell lines under optimised conditions. A protocol capable of efficient isolation and culture of hepatocytes from human donor was validated. Data from primary human hepatocytes show that ENT1 expression was highly variable in different sets of primary livers and correlated strongly with ribavirin uptake. Strikingly, Huh7 cells did not take up ribavirin despite expressing wild type ENT1. It was also found that interferon alpha does not modulate ENT1 expression and therefore ribavirin uptake, suggesting it to be a highly unlikely mode of synergism between the two drugs. Conclusion: Modulation of host proteins VAP-A and STAT3 inhibited viral replication, confirming that host genes can be used as a potential target to inhibit viral replication. Liposomes used in this study were, however, found to be ineffective vehicles for the delivery of ribavirin or siRNA, as the majority of drug leaked before cellular uptake. Polymer based nanoparticles are currently being assessed for antiviral drug delivery. Variation in ENT1 expression may account for differences in response rate in patients receiving anti-HCV therapy. Results in the Huh 7 cell line suggest that, while ENT1 is necessary, other factors are also required to mediate ribavirin uptake.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:580279 |
| Date | January 2012 |
| Creators | Baloch, Baby Kanwal |
| Publisher | University of Nottingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://eprints.nottingham.ac.uk/14295/ |
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