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Hum Gene Ther. 2018 Nov;29(11):1315-1326. doi: 10.1089/hum.2017.252. Epub 2018 Jun 22.

Curative Ex Vivo Hepatocyte-Directed Gene Editing in a Mouse Model of Hereditary Tyrosinemia Type 1.

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1 Department of Surgery, Mayo Clinic , Rochester, Minnesota.
2 Department of Molecular Medicine, Mayo Clinic , Rochester, Minnesota.
3 Department of Surgery, Yonsei University College of Medicine , Seoul, Republic of Korea.
4 Department of William J. von Liebig Center for Transplantation and Clinical Regeneration, Mayo Clinic , Rochester, Minnesota.
5 Midwest Fetal Care Center, Children's Hospital and Clinics of Minnesota , Minneapolis, Minnesota.
6 Pediatric Surgical Associates, Ltd., Minneapolis, Minnesota.


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.


CRISPR/Cas9; gene therapy; hepatocytes; hereditary tyrosinemia type 1; metabolic liver disease

[Available on 2019-11-01]

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