Hepatitis B virus (HBV) is hyperendemic to southern Africa and parts of Asia where it is a
major cause of serious liver disease. Licensed antivirals for chronically infected individuals
are only partially effective and approximately one million deaths occur annually as a result ofpersistent infection with the virus. Although RNA interference (RNAi) based gene silencingof HBV has been successfully demonstrated, difficulties with delivery of anti-HBV RNAieffectors remains an obstacle to their clinical use. Recombinant adenoviruses (Ads), amongst the most efficient hepatotropic gene vectors following systemic administration, have been successfully used to deliver expressed anti-HBV RNAi sequences. However, a drawback of Ad vectors is diminished efficacy and toxicity that results from stimulation of innate andadaptive immunity.To attenuate these effects we used polyethylene glycol (PEG) to modify first generation recombinant Ad (FG Ad) vectors that express an anti-HBV short hairpin (shRNA) sequence. Efficient hepatocyte transduction occurred and expressed shRNAs were processed to generate intended HBV-targeting guides. Inhibition of HBV replication was achieved after intravenous administration of PEGylated or native recombinant first generation Ads (FG Ads) to HBV transgenic mice. Circulating HBV viral particle equivalents (VPEs) remained low for 3 weeks and began to increase after 5 weeks. A second dose of PEGylated anti-HBV Ad caused a less sustained decrease in circulating VPEs, but no silencing after a second dose was observed in animals treated with unmodified vector. Release of inflammatory cytokines was elevated in animals receiving unmodified vectors and only a modest increase in monocyte chemotactic protein-1 (MCP-1) was observed in mice that received a second dose of PEG
Abstract Ads. Also, polymer-conjugated vectors induced a weaker adaptive immune response and were less hepatotoxic than their unmodified counterparts.
To address concerns about the transient nature of transgene expression by FG Ads resulting from immunostimulation, third generation helper-dependent (HD Ad) were utilised to delivered anti-HBV RNAi effectors. Seven days after intravenous administration of infectious HD Ads to HBV transgenic mice, 80-90% of hepatocytes were transduced and markers of HBV replication were decreased by approximately 95% which was sustained for 8 weeks. HD Ad-induced release of proinflammatory cytokines was minimal in preparations that were enriched with infectious particles. PEGylated HD Ad vectors caused similar anti- HBV effects and may be useful to evade interaction with vector-sequestrating receptors and further attenuate immunostimulation. Collectively these observations indicate that PEG modification of Ads and the use of HD Ads may have utility for delivery of therapeutic HBVsilencing sequences. Future work will focus on improving strategies to avoid immune detection and utilisation of HD Ad vectors for other HBV targeting sequences.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/13857 |
| Date | 18 February 2014 |
| Creators | Crowther, Carol |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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