Research ArticleGene Therapy

A universal system to select gene-modified hepatocytes in vivo

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Science Translational Medicine  08 Jun 2016:
Vol. 8, Issue 342, pp. 342ra79
DOI: 10.1126/scitranslmed.aad8166

Gene therapy gets selective

In gene therapy, most quip that the top three challenges are delivery, delivery, and delivery, but selectively expanding the pool of gene-edited cells is a major challenge, too, to ensure that genes reach therapeutic levels. Nygaard et al. came up with a clever platform technology that selects for gene-edited cells in vivo without the hassle, time, and special facilities required for in vitro expansion and selection via cell culture. Alongside the therapeutic transgene, the authors inserted into hepatocytes a short hairpin RNA targeting an enzyme that, when knocked down, made the cells resistant to a drug called CEHPOBA. Healthy animals received liver-specific vectors to express a model gene, human factor 9, and then were given CEHPOBA or saline for several weeks. The animals receiving saline control saw no change in gene expression in hepatocytes, whereas animals receiving the drug CEHPOBA saw an order of magnitude increase in factor 9, indicating that the gene-corrected cells were pharmacologically selected in a living animal. This powerful approach can be used for genetic diseases like hemophilia B and metabolic liver diseases or extended to any tissue that proliferates after injury, including the bone marrow and skin.


Many genetic and acquired liver disorders are amenable to gene and/or cell therapy. However, the efficiencies of cell engraftment and stable genetic modification are low and often subtherapeutic. In particular, targeted gene modifications from homologous recombination are rare events. These obstacles could be overcome if hepatocytes that have undergone genetic modification were to be selectively amplified or expanded. We describe a universally applicable system for in vivo selection and expansion of gene-modified hepatocytes in any genetic background. In this system, the therapeutic transgene is coexpressed with a short hairpin RNA (shRNA) that confers modified hepatocytes with resistance to drug-induced toxicity. An shRNA against the tyrosine catabolic enzyme 4-OH-phenylpyruvate dioxygenase protected hepatocytes from 4-[(2-carboxyethyl)-hydroxyphosphinyl]-3-oxobutyrate, a small-molecule inhibitor of fumarylacetoacetate hydrolase. To select for specific gene targeting events, the protective shRNA was embedded in a microRNA and inserted into a recombinant adeno-associated viral vector designed to integrate site-specifically into the highly active albumin locus. After selection of the gene-targeted cells, transgene expression increased 10- to 1000-fold, reaching supraphysiological levels of human factor 9 protein (50,000 ng/ml) in mice. This drug resistance system can be used to achieve therapeutically relevant transgene levels in hepatocytes in any setting.

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