Nygaard Sean, Barzel Adi, Haft Annelise, Major Angela, Finegold Milton, Kay Mark A, Grompe Markus
Oregon Stem Cell Center, Oregon Health & Science University, Portland, OR 97239, USA.
Departments of Pediatrics and Genetics, Stanford Medical School, Stanford, CA 94305, USA.
Sci Transl Med. 2016 Jun 8;8(342):342ra79. doi: 10.1126/scitranslmed.aad8166.
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.
许多遗传性和获得性肝脏疾病都适合进行基因和/或细胞治疗。然而，细胞植入和稳定基因修饰的效率很低，往往达不到治疗效果。特别是，同源重组的靶向基因修饰是罕见事件。如果经过基因修饰的肝细胞能够被选择性扩增，这些障碍是可以克服的。我们描述了一种普遍适用的系统，用于在任何遗传背景下对基因修饰的肝细胞进行体内选择和扩增。在这个系统中，治疗性转基因与短发夹RNA（shRNA）共表达，该shRNA赋予修饰后的肝细胞对药物诱导毒性的抗性。一种针对酪氨酸分解代谢酶4-羟基苯丙酮酸双加氧酶的shRNA保护肝细胞免受4-[(2-羧乙基)-羟基磷酰基]-3-氧代丁酸酯的影响，后者是富马酰乙酰乙酸水解酶的小分子抑制剂。为了选择特定的基因靶向事件，将保护性shRNA嵌入微小RNA中，并插入到设计用于位点特异性整合到高活性白蛋白基因座的重组腺相关病毒载体中。在选择了基因靶向细胞后，转基因表达增加了10到1000倍，在小鼠体内达到了人因子9蛋白超生理水平（50,000 ng/ml）。这种耐药系统可用于在任何情况下在肝细胞中实现与治疗相关的转基因水平。
