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In this study, we report a miRNA122a based targeted gene therapy for hepatocellular cancer stem cells (CSCs). First, we assessed the levels of miRNA122a in normal human hepatocytes, a panel of hepatocellular carcinoma (HCC) cell lines and hepatocellular CSCs observing its significant downregulation in HCC and CSCs. The miRNA122a binding site was then incorporated at the 3'-UTR of reporter genes gaussia luciferase (GLuc) and eGFP which resulted in significant hepatocyte detargeting. Using this strategy for the delivery of gene directed enzyme prodrug therapy (GDEPT) utilizing the cytosine deaminase/5-fluorocytosine (CD/5-FC) system, we showed significant killing in cells with low or no miRNA122a while those cells, such as hepatocytes with high miRNA122a were largely spared. Next, we showed that CSC enriched tumorspheres exhibit a significant downregulation of miRNA122a expression providing a rational to exploit its binding site for targeted gene delivery. Using plasmids harboring reporters GLuc and eGFP with or without miR122a binding sites, we showed high reporter expression in the CSCs and little reported expression in the non-enriched cultures. Finally, we demonstrate the efficacy of miRNA122a based post-transcriptionally targeted GDEPT for hepatocellular CSCs.
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