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Designed zinc finger proteins as novel therapeutics inhibiting the transcription of hepatitis B and duck hepatitis B viruses Open Access

Descriptions

Other title
Subject/Keyword
zinc finger protein
duck hepatitis B
inhibition
cccDNA
hepatitis B
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Zimmerman, Kimberley Anne
Supervisor and department
Tyrrell, Lorne
Examining committee member and department
Schang, Luis (Department of Biochemistry)
Evans, David (Department of Medical Microbiology and Immunology)
Richardson, Chris (Department of Microbiology and Immunology, Dalhousie University)
Foley, Edan (Department of Medical Microbiology and Immunology)
Department
Department of Medical Microbiology and Immunology
Specialization

Date accepted
2010-08-31T19:59:20Z
Graduation date
2010-11
Degree
Doctor of Philosophy
Degree level
Doctoral
Abstract
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.
Language
English
DOI
doi:10.7939/R3NH7K
Rights
License granted by Kimberley Zimmerman (kaz1@ualberta.ca) on 2010-08-31T18:57:54Z (GMT): Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of the above terms. The author reserves all other publication and other rights in association with the copyright in the thesis, and except as herein provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
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