Lentivirus: Fibrin based gene delivery, live cell arrays for high throughput screening and adherens junctions controlled entry
Padmashali, Roshan M.
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With the primary focus on lentiviruses, this dissertation is divided into three prominent sections: 1) Fibrin based non-viral & viral gene delivery We introduce the use of fibrin hydrogels as a novel gene delivery substrate both for plasmid DNA and lentivirus delivery. Apart from obtaining high gene delivery efficiency, other improved aspects brought forth in these studies include sustained delivery, low toxicity, cell controlled fibrinolysis and gene delivery. We encountered localization challenges with lentivirus delivered with fibrin gels towards which we applied modifications to the viral envelope for enzyme mediated crosslinking to the fibrin fibers. We further characterized this system for fibrin binding as well as signal/noise improvements. 2) Lentivirus based live cell screens for high throughput analysis of Mesenchymal stem cell(MSC) differentiation. The lack of a high throughput system that monitors gene regulation during MSC differentiation prompted us to develop this system of live cell arrays with lineage identifying reporter constructs. Unique and lineage specific responses were obtained using these fluorescence reporters constructs providing a significantly larger number of data points and responses that could be simultaneously compared across three MSC lineages. We further applied our system to a high throughput screen for identifying key pathways controlling MSC differentiation and confirmed our results with conventional functional stains. 3) Understanding lentivirus entry in epithelial cells and role of adherens junctions. The last section of the thesis deals with the control of lentivirus entry in epithelial cells via the formation of adherens junctions. Various strategies are employed to weaken adherens junctions and the subsequent effect on viral entry is quantified. The findings of this study hold importance in improving gene delivery efficiency in both in-vitro as well as in-vivo epithelial tissue models.