The Hepatitis B virus (HBV) chronically infects 350 million individuals worldwide, leading to mortality by end-stage liver disease, liver cirrhosis, and hepatocellular carcinoma. The vaccine to prevent HBV infection is highly effective but is not extensively available in endemic areas, resulting in high infection rates. Nucleoside analogue treatment of HBV has allowed for higher rates of viral clearance in infected individuals, but most patients must remain on therapy long term and viral resistance to the drugs is growing.
The HBV viral genome is an episome in the nucleus of infected hepatocytes. It is called covalently closed circular (ccc) DNA and is highly stable, has a long half-life, and is the template for all viral transcription and progeny production. Nucleoside analogues do not directly target cccDNA, therefore many patients experience rebound when antiviral therapy is stopped. I have designed novel DNA binding proteins called zinc finger proteins (ZFPs) to specifically bind to the cccDNA in infected cells and inhibit viral transcription. Seven ZFPs targeting the model duck HBV (DHBV) and ten ZFPs targeting HBV were developed. Kinetic analyses of the purified ZFPs were performed, characterizing their specificity and binding properties. Using the DHBV tissue culture model system, I have demonstrated that the DHBV-specific ZFPs can specifically inhibit transcription from the viral template, resulting in reduced viral RNA, protein products and progeny virions. The DHBV-specific ZFPs were tested in primary duck hepatocytes (PDH) and in vivo in the Pekin duck model. ZFPs failed to express in PDH transduced by baculovirus vectors when DHBV was present in the cells. In vivo gene delivery of the ZFPs was carried out by portal vein injection of chitosan-based nanospheres. Unfortunately, non-specific reductions in viral levels masked any direct effect by the ZFPs. Testing of the HBV-specific ZFPs in tissue culture was hindered by a lack of transfectable cell culture model. A number of different transfection methods were tested to express the HBV-specific ZFPs, all without success. Further work is being carried out using baculovirus vectors to deliver the HBV-specific ZFPs to HBV-harbouring cell lines and HBV-infected scid-Alb/uPA chimeric mice with human liver cells. / Virology
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:AEU.10048/1367 |
| Date | 11 1900 |
| Creators | Zimmerman, Kimberley Anne |
| Contributors | Tyrrell, Lorne, Evans, David (Department of Medical Microbiology and Immunology), Foley, Edan (Department of Medical Microbiology and Immunology), Schang, Luis (Department of Biochemistry), Richardson, Chris (Department of Microbiology and Immunology, Dalhousie University) |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 105722898 bytes, application/pdf |
| Relation | Zimmerman, K.A., Fischer, K.F., Joyce, M.A., and Tyrrell, D.J.L. (2008) Zinc finger proteins designed to specifically target duck hepatitis B virus covalently closed circular DNA inhibit viral transcription in tissue culture. Journal of Virology. 82: 8013 8021., Zimmerman, K.A., Fischer, K.F., Joyce, M.A., and Tyrrell, D.J.L. (2007) Hepatitis B Virus-binding Polypeptides and Methods of Use Thereof. U.S. Patent Application 60/972,644. |
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