Date of Degree
PhD (Doctor of Philosophy)
Molecular and Cell Biology
Anton P. McCaffrey
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.
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.
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.
Copyright 2009 Thomas James Cradick