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Efficient liver repopulation of transplanted hepatocyte prevents cirrhosis in a rat model of hereditary tyrosinemia type I.

ABSTRACT: Hereditary tyrosinemia type I (HT1) is caused by a deficiency in the enzyme fumarylacetoacetate hydrolase (Fah). Fah-deficient mice and pigs are phenotypically analogous to human HT1, but do not recapitulate all the chronic features of the human disorder, especially liver fibrosis and cirrhosis. Rats as an important model organism for biomedical research have many advantages over other animal models. Genome engineering in rats is limited till the availability of new gene editing technologies. Using the recently developed CRISPR/Cas9 technique, we generated Fah(-/-) rats. The Fah(-/-) rats faithfully represented major phenotypic and biochemical manifestations of human HT1, including hypertyrosinemia, liver failure, and renal tubular damage. More importantly, the Fah(-/-) rats developed remarkable liver fibrosis and cirrhosis, which have not been observed in Fah mutant mice or pigs. Transplantation of wild-type hepatocytes rescued the Fah(-/-) rats from impending death. Moreover, the highly efficient repopulation of hepatocytes in Fah(-/-) livers prevented the progression of liver fibrosis to cirrhosis and in turn restored liver architecture. These results indicate that Fah(-/-) rats may be used as an animal model of HT1 with liver cirrhosis. Furthermore, Fah(-/-) rats may be used as a tool in studying hepatocyte transplantation and a bioreactor for the expansion of hepatocytes.

SUBMITTER: Zhang L

PROVIDER: S-EPMC4980609 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Efficient liver repopulation of transplanted hepatocyte prevents cirrhosis in a rat model of hereditary tyrosinemia type I.

Zhang Ludi L Shao Yanjiao Y Li Lu L Tian Feng F Cen Jin J Chen Xiaotao X Hu Dan D Zhou Yan Y Xie Weifen W Zheng Yunwen Y Ji Yuan Y Liu Mingyao M Li Dali D Hui Lijian L

Scientific reports 20160811

Hereditary tyrosinemia type I (HT1) is caused by a deficiency in the enzyme fumarylacetoacetate hydrolase (Fah). Fah-deficient mice and pigs are phenotypically analogous to human HT1, but do not recapitulate all the chronic features of the human disorder, especially liver fibrosis and cirrhosis. Rats as an important model organism for biomedical research have many advantages over other animal models. Genome engineering in rats is limited till the availability of new gene editing technologies. Us ...[more]

PMID: 27510266

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Project description:Considerable progress has been made in developing antifibrotic agents and other strategies to treat liver fibrosis; however, significant long-term restoration of functional liver mass has not yet been achieved. Therefore, we investigated whether transplanted hepatic stem/progenitor cells can effectively repopulate the liver with advanced fibrosis/cirrhosis. Stem/progenitor cells derived from fetal livers or mature hepatocytes from DPPIV(+) F344 rats were transplanted into DPPIV(-) rats with thioacetamide (TAA)-induced fibrosis/cirrhosis; rats were sacrificed 1, 2, or 4 months later. Liver tissues were analyzed by histochemistry, hydroxyproline determination, reverse-transcription polymerase chain reaction (RT-PCR), and immunohistochemistry. After chronic TAA administration, DPPIV(-) F344 rats exhibited progressive fibrosis, cirrhosis, and severe hepatocyte damage. Besides stellate cell activation, increased numbers of stem/progenitor cells (Dlk-1(+), AFP(+), CD133(+), Sox-9(+), FoxJ1(+)) were observed. In conjunction with partial hepatectomy (PH), transplanted stem/progenitor cells engrafted, proliferated competitively compared to host hepatocytes, differentiated into hepatocytic and biliary epithelial cells, and generated new liver mass with extensive long-term liver repopulation (40.8 ± 10.3%). Remarkably, more than 20% liver repopulation was achieved in the absence of PH, associated with reduced fibrogenic activity (e.g., expression of alpha smooth muscle actin, platelet-derived growth factor receptor ?, desmin, vimentin, tissue inhibitor of metalloproteinase-1) and fibrosis (reduced collagen). Furthermore, hepatocytes can also replace liver mass with advanced fibrosis/cirrhosis, but to a lesser extent than fetal liver stem/progenitor cells.This study is a proof of principle demonstration that transplanted epithelial stem/progenitor cells can restore injured parenchyma in a liver environment with advanced fibrosis/cirrhosis and exhibit antifibrotic effects.

Project description:Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disorder caused by deficiency of fumarylacetoacetate hydrolase (FAH). It has been previously shown that ex vivo hepatocyte-directed gene therapy using an integrating lentiviral vector to replace the defective Fah gene can cure liver disease in small- and large-animal models of HT1. This study hypothesized that ex vivo hepatocyte-directed gene editing using CRISPR/Cas9 could be used to correct a mouse model of HT1, in which a single point mutation results in loss of FAH function. To achieve high transduction efficiencies of primary hepatocytes, this study utilized a lentiviral vector (LV) to deliver both the Streptococcus pyogenes Cas9 nuclease and target guide RNA (LV-Cas9) and an adeno-associated virus (AAV) vector to deliver a 1.2 kb homology template (AAV-HT). Cells were isolated from Fah-/- mice and cultured in the presence of LV and AAV vectors. Transduction of cells with LV-Cas9 induced significant indels at the target locus, and correction of the point mutation in Fah-/- cells ex vivo using AAV-HT was completely dependent on LV-Cas9. Next, hepatocytes transduced ex vivo by LV-Cas9 and AAV-HT were transplanted into syngeneic Fah-/- mice that had undergone a two-thirds partial hepatectomy or sham hepatectomy. Mice were cycled on/off the protective drug 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) to stimulate expansion of corrected cells. All transplanted mice became weight stable off NTBC. However, a significant improvement was observed in weight stability off NTBC in animals that received partial hepatectomy. After 6 months, mice were euthanized, and thorough biochemical and histological examinations were performed. Biochemical markers of liver injury were significantly improved over non-transplanted controls. Histological examination of mice revealed normal tissue architecture, while immunohistochemistry showed robust repopulation of recipient animals with FAH+ cells. In summary, this is the first report of ex vivo hepatocyte-directed gene repair using CRISPR/Cas9 to demonstrate curative therapy in an animal model of liver disease.

Dataset Information

Efficient liver repopulation of transplanted hepatocyte prevents cirrhosis in a rat model of hereditary tyrosinemia type I.

Publications

Efficient liver repopulation of transplanted hepatocyte prevents cirrhosis in a rat model of hereditary tyrosinemia type I.

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