Thesis: Ph. D. in Biomedical Engineering, Harvard-MIT Program in Health Sciences and Technology, 2016. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 136-153). / The Hepatitis B virus (HBV) has, at one time or another, infected one third of the world's population, and over 300 million people worldwide are chronically infected, leading often to progressive liver damage, cirrhosis, and the development of hepatocellular carcinoma over decades. While an effective vaccine exists, imperfect vaccine penetrance and perinatal transmission result in the continuous establishment of chronic infection in new patients, for whom there is very little chance for a cure. This thesis sought to move closer toward the development of consistently curative treatments for HBV by developing novel model systems to study HBV infection, profiling the host response to this infection to nominate targets for therapeutic intervention, and repurposing novel genome editing tools to directly attack the long-lived viral form that is resistant to cure. First, we use micro-patterning tools and directed differentiation protocols to develop primary adult human hepatocyte and iPS-derived hepatocyte models of HBV infection, respectively, which enable us to test antiviral therapies with multiple mechanisms of action and identify a significant interferon-driven response to HBV infection. Second, we use global transcriptional profiling of this response in multiple human hepatocyte donors to identify candidate genes and pathways that are putatively important for viral infection, and demonstrate significant variability in this infection response between donors and virus sources. We then confirm the importance of nominated virus-regulated genes by showing that pharmacological inhibition of these targets - such as heat shock proteins - restricts viral infection in both primary hepatocytes and cell lines. Finally, we provide a proof of concept for using the CRISPR/Cas9 system to directly attack HBV. We show that carefully selected guide RNAs can direct CRISPR-based inhibition of HBV both in vitro and in vivo. We importantly show that Cas9 can directly cleave and direct degradation of the long-lived, episomal HBV cccDNA, resulting in >1 log-fold reductions in cccDNA in both constitutively HBV-producing and newly HBV infected cells. Overall, this work takes steps toward both elucidating HBV biology, and accelerating the path toward novel treatments for this disease. / by Vyas Ramanan. / Ph. D. in Biomedical Engineering
| Identifer | oai:union.ndltd.org:MIT/oai:dspace.mit.edu:1721.1/107341 |
| Date | January 2016 |
| Creators | Ramanan, Vyas |
| Contributors | Sangeeta N. Bhatia., Harvard--MIT Program in Health Sciences and Technology., Harvard--MIT Program in Health Sciences and Technology. |
| Publisher | Massachusetts Institute of Technology |
| Source Sets | M.I.T. Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 153 pages, application/pdf |
| Rights | MIT theses are protected by copyright. They may be viewed, downloaded, or printed from this source but further reproduction or distribution in any format is prohibited without written permission., http://dspace.mit.edu/handle/1721.1/7582 |
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