Hepatitis B virus (HBV) chronically infects 350-400 million people worldwide and causes >1 million deaths yearly. Current therapies prevent new viral genome formation, but do not target pre-existing viral genomic DNA, thus curing only approximately 1/2 of patients. We targeted HBV DNA for cleavage using zinc-finger nucleases (ZFNs), which cleave as dimers. Co-transfection of our ZFN pair with a target plasmid containing the HBV genome resulted in specific cleavage. After 3 days in culture, 26% of the target remained linear, whereas approximately10% was cleaved and misjoined tail-to-tail. Notably, ZFN treatment decreased levels of the hepatitis C virus pregenomic RNA by 29%. 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 ZFN sites. Targeted cleavage and misrepair destroys the XbaI site. After 3 days in culture, approximately 6% of plasmids were XbaI-resistant. Thirteen of 16 clones sequenced contained frameshift mutations that would lead to truncations of the viral core protein. These results demonstrate, for the first time, the possibility of targeting episomal viral DNA genomes using ZFNs.
We describe ongoing work on I2I, a system aimed at fostering opportunistic communication among users viewing or manipulating content on the Web and in productivity applications. Unlike previous work in which the URLs of Web resources are used to group users visiting the same resource, we present a more general framework for clustering work contexts to group users together that accounts for dynamic content and distributional properties of Web accesses which can limit the utility URL based systems.In addition, we describe a method for scaffolding asynchronous communication in the context of an ongoing task that takes into account the ephemeral nature of the location of content on the Web. The techniques we describe also nicely cover local files in progress, in addition to publicly available Web content. We present the results of several evaluations that indicate systems that use the techniques we employ may be more useful than systems that are strictly URL based.
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