Files and links (1)
Free to read and download, Open Access
Dissertation
Designing zinc finger nucleases that specifically cleave Hepatitis B viral DNA
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Autumn 2009
DOI: 10.17077/etd.484mzmj5
Abstract
<p>Hepatitis B virus chronically infects 350-400 million people worldwide. It often leads to hepatocellular carcinoma, which causes >1 million deaths yearly. Current therapies prevent new viral genome formation but do not target pre-existing viral genomic DNA, thus curing only ~1/2 of patients. We targeted hepatitis B virus DNA for cleavage using zinc finger nucleases, which cleave as dimers. Co-transfection of our zinc finger nuclease pair with a target plasmid containing the hepatitis B virus genome resulted in specific cleavage. After three days in culture, 26% of the target remained linear, while ~10% was cleaved and mis-joined tail-to-tail.</p>
<p>A portion of cleaved plasmids are repaired in cells, often with deletions and insertions. To track misrepair, we introduced an XbaI restriction site in the spacer between the zinc finger nuclease sites. Targeted cleavage and misrepair destroys the XbaI site. After three days in culture, ~6% of plasmids were XbaI resistant. 13 of 16 clones sequenced contained frameshift mutations that would lead to dramatic truncations of the viral core protein. These results demonstrate for the first time the feasibility of targeting episomal viral DNA genomes in cells using zinc finger nucleases. This strategy is broadly applicable toward inactivating other DNA viruses within cells.</p>
<p>A major concern for the therapeutic use of zinc finger nucleases is off-target cleavage. To measure specificity, we employed in vitro assays and developed a bioinformatics method to find off-target cleavage sites in cultured cells. These sites can then be PCR amplified and tested using a mutation detection assay that we developed.</p>
Details
- Title: Subtitle
- Designing zinc finger nucleases that specifically cleave Hepatitis B viral DNA
- Creators
- Thomas James Cradick - University of Iowa
- Contributors
- Anton P. McCaffrey (Advisor)Aloysius J. Klingelhutz (Committee Member)Paul B. McCray (Committee Member)Curt D. Sigmund (Committee Member)Marc S. Wold (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Molecular and Cellular Biology
- Date degree season
- Autumn 2009
- Publisher
- University of Iowa
- DOI
- 10.17077/etd.484mzmj5
- Number of pages
- xiv, 140 pages
- Copyright
- Copyright © 2009 Thomas James Cradick
- Comment
This thesis has been optimized for improved web viewing. If you require the original version, contact the University Archives at the University of Iowa: https://www.lib.uiowa.edu/sc/contact/.
- Language
- English
- Description bibliographic
- Includes bibliographical references (pages 129-140).
- Academic Unit
- Interdisciplinary Graduate Program in Molecular Medicine
- Record Identifier
- 9983777290102771
Metrics
669 File views/ downloads
355 Record Views