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Efficient targeted integration in proliferating patient hepatocytes for inherited liver diseases

ABSTRACT: Hepatocyte transplantation has shown great potential for treating inherited liver diseases. However, its prolonged clinical therapeutic efficacy is still hindered by serious immune rejection from the host immunological responses to the allogeneic hepatocytes and there is still no an effective approach to generate clinically available autologous hepatocytes for transplantation therapy. Here, we report advanced medium conditions that allow 10,000-fold expansion of primary human hepatocytes from patients suffering inherited liver diseases, providing a new autologous hepatocytes source to this problem. Moreover, we developed a CRISPR–Cas9 genome-targeting system that combines Cas9 ribonucleoproteins and adeno-associated viral vector delivery of a homology-independent large donor (greater than three kilobase) to achieve efficient targeted integration at the AAVS1 safe harbor locus in patient-derived proliferating human hepatocytes (ProliHHs). We further applied this strategy to correct a pathogenic OTC and FAH mutation in ProliHHs and demonstrated improved gene expression, while preserving cell viability for the expansion and purification of edited ProliHHs. Importantly, these edited ProliHHs repopulate into injured mouse liver at a level near to primary human hepatocytes, and they undergo maturation to successfully treat the tyrosinemia mouse model following transplantation in vivo. Thus, this study provides a new autologous hepatocyte therapy techniques that enable large-scale expansion and ex vivo gene correction in patient-derived transplantable ProliHHs, which holds the potential for modeling and treating human liver disease.

ORGANISM(S): Homo sapiens

PROVIDER: GSE233425 | GEO | 2023/09/12

REPOSITORIES: GEO

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Project description:We previously showed that severe liver diseases are characterized by expansion of liver progenitor cells (LPC), which correlates with disease severity. However, the origin and role of LPC in liver physiology and in the hepatic response to injury remains a contentious topic. We have now used genetic lineage tracing of Hnf1Î²-expressing biliary duct cells to assess their contribution to LPC expansion and hepatocyte generation during normal liver homeostasis, and following different types of liver injury. We found that ductular reaction cells in human cirrhotic livers express HNF1Î². However, HNF1Î² expression was not present in newly generated EpCAM-positive hepatocytes. Using a tamoxifen-inducible Hnf1Î²CreER/R26RYFP/LacZ mouse, we show that there is no contribution of the biliary epithelium to hepatocyte turnover during liver homeostasis in healthy mice. Moreover, after loss of liver mass, Hnf1Î²+ LPC did not contribute to hepatocyte regeneration. We also assessed the contribution of Hnf1Î²+ cells following acute and repeated liver injury. All animal models showed expansion of LPC, as assessed by immunostaining and gene expression profile of sorted YFP-positive cells. A contribution of Hnf1Î²+ LPC to hepatocyte generation was not detected in animal models of liver injury with preserved hepatocyte regenerative potential such as acute acetaminophen, carbon tetrachloride injury, or chronic diethoxycarbonyl-1,4-dihydro-collidin (DDC)-diet. However, in mice fed with choline-deficient ethionine-supplemented (CDE)-diet, which causes profound hepatocyte damage and arrest, a small number of hepatocytes were derived from Hnf1Î²+ cells. Conclusion: Hnf1Î²+ cells do not participate in hepatocyte turnover in the healthy liver or during liver regeneration after partial hepatectomy. After liver injury, LPC arise from the biliary duct epithelium, which gives rise to a limited number of hepatocytes only when hepatocyte regeneration is compromised. Transcriptomic profile using MoGeneST-2.0 chip from 3 samples of YFP+ CDE, 3 samples of YFP+ DDC, 2 samples of YFP+ UTR and 3 samples YFP-

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Efficient targeted integration in proliferating patient hepatocytes for inherited liver diseases

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