Browsing by Author "Quirk, Corrine"

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    Expansion, in vivo–ex vivo cycling, and genetic manipulation of primary human hepatocytes
    (National Academy of Sciences, 2020-01-08) Michailidis, Eleftherios; Vercauteren, Koen; Mancio-Silva, Liliana; Andrus, Linda; Jahan, Cyprien; Ricardo-Lax, Inna; Zou, Chenhui; Kabbani, Mohammad; Park, Paul; Quirk, Corrine; Pyrgaki, Christina; Razooky, Brandon; Verhoye, Lieven; Zoluthkin, Irene; Lu, Wei-Yu; Forbes, Stuart J.; Chiriboga, Luis; Theise, Neil D.; Herzog, Roland W.; Suemizu, Hiroshi; Schneider, William M.; Shlomai, Amir; Meuleman, Philip; Bhatia, Sangeeta N.; Rice, Charles M.; de Jong, Ype P.; Pediatrics, School of Medicine
    Primary human hepatocytes (PHHs) are an essential tool for modeling drug metabolism and liver disease. However, variable plating efficiencies, short lifespan in culture, and resistance to genetic manipulation have limited their use. Here, we show that the pyrrolizidine alkaloid retrorsine improves PHH repopulation of chimeric mice on average 10-fold and rescues the ability of even poorly plateable donor hepatocytes to provide cells for subsequent ex vivo cultures. These mouse-passaged (mp) PHH cultures overcome the marked donor-to-donor variability of cryopreserved PHH and remain functional for months as demonstrated by metabolic assays and infection with hepatitis B virus and Plasmodium falciparum. mpPHH can be efficiently genetically modified in culture, mobilized, and then recultured as spheroids or retransplanted to create highly humanized mice that carry a genetically altered hepatocyte graft. Together, these advances provide flexible tools for the study of human liver disease and evaluation of hepatocyte-targeted gene therapy approaches.
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    Experimental Variables that Affect Human Hepatocyte AAV Transduction in Liver Chimeric Mice
    (Elsevier, 2020-09-11) Zou, Chenhui; Vercauteren, Koen O. A.; Michailidis, Eleftherios; Kabbani, Mohammad; Zoluthkin, Irene; Quirk, Corrine; Chiriboga, Luis; Yazicioglu, Mustafa; Anguela, Xavier M.; Meuleman, Philip; High, Katherine A.; Herzog, Roland W.; Jong, Ype P. de; Pediatrics, School of Medicine
    Adeno-associated virus (AAV) vector serotypes vary in their ability to transduce hepatocytes from different species. Chimeric mouse models harboring human hepatocytes have shown translational promise for liver-directed gene therapies. However, many variables that influence human hepatocyte transduction and transgene expression in such models remain poorly defined. Here, we aimed to test whether three experimental conditions influence AAV transgene expression in immunodeficient, fumaryl-acetoactetate-hydrolase-deficient (Fah−/−) chimeric mice repopulated with primary human hepatocytes. We examined the effects of the murine liver injury cycle, human donor variability, and vector doses on hepatocyte transduction with various AAV serotypes expressing a green fluorescent protein (GFP). We determined that the timing of AAV vector challenge in the liver injury cycle resulted in up to 7-fold differences in the percentage of GFP expressing human hepatocytes. The GFP+ hepatocyte frequency varied 7-fold between human donors without, however, changing the relative transduction efficiency between serotypes for an individual donor. There was also a clear relationship between AAV vector doses and human hepatocyte transduction and transgene expression. We conclude that several experimental variables substantially affect human hepatocyte transduction in the Fah−/− chimera model, attention to which may improve reproducibility between findings from different laboratories.