TY - JOUR
T1 - A universal system to select gene-modified hepatocytes in vivo
AU - Nygaard, Sean
AU - Barzel, Adi
AU - Haft, Annelise
AU - Major, Angela
AU - Finegold, Milton
AU - Kay, Mark A.
AU - Grompe, Markus
N1 - Funding Information:
We thank L. Wakefield for assisting with the animal husbandry. Funding: This work was supported by NIH grants DK048252 (M.G.) and HL064274 (M.A.K.). Author contributions: S.N. performed the bulk of the experiments and generated figures for the manuscript; A.B. provided the GeneRide construct and the RNA abundance quantitation; A.H. did the animal husbandry and performed the F9 assays; A.M. performed the immunohistochemistry; M.F. analyzed the immunohistochemistry; M.A.K. designed experiments and provided vector constructs; and M.G. oversaw the experiments and wrote the manuscript. Competing interests: M.G. is a shareholder and consultant for Yecuris Corporation, the company that has licensed the Fah-/- mouse technology from Oregon Health & Science University. M.A.K. and A.B. are shareholders for LogicBio Therapeutics that has licensed the GeneRide technology from Stanford University. Data and materials availability: All data and materials are available from the corresponding author upon request. Plasmids containing viral vectors and Fah-/- mutant mice require the completion of a standard academic material transfer agreement.
PY - 2016/6/8
Y1 - 2016/6/8
N2 - 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-OHphenylpyruvate 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.
AB - 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-OHphenylpyruvate 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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U2 - 10.1126/scitranslmed.aad8166
DO - 10.1126/scitranslmed.aad8166
M3 - Article
C2 - 27280686
AN - SCOPUS:84973878623
SN - 1946-6234
VL - 8
JO - Science translational medicine
JF - Science translational medicine
IS - 342
M1 - 342ra79
ER -