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

 Caitlin VanLith  ;  Rebekah Guthman  ;  Clara T. Nicolas  ;  Kari Allen  ;  Zeji Du  ;  Dong Jin Joo  ;  Scott L. Nyberg  ;  Joseph B. Lillegard  ;  Raymond D. Hickey 
 HUMAN GENE THERAPY, Vol.29(11) : 1315-1326, 2018 
Journal Title
Issue Date
Animals ; Base Sequence ; CRISPR-Associated Protein 9/metabolism ; Cells, Cultured ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Dependovirus/metabolism ; Disease Models, Animal ; Gene Editing* ; Genetic Therapy* ; Genetic Vectors/metabolism ; Hepatocytes/metabolism* ; Hepatocytes/transplantation ; Hydrolases/genetics ; Lentivirus/genetics ; Liver Failure/pathology ; Liver Failure/therapy ; Mice ; Tyrosinemias/genetics* ; Tyrosinemias/pathology ; Tyrosinemias/therapy*
CRISPR/Cas9 ; gene therapy ; hepatocytes ; hereditary tyrosinemia type 1 ; metabolic liver disease
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.
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1. College of Medicine (의과대학) > Dept. of Surgery (외과학교실) > 1. Journal Papers
Yonsei Authors
Joo, Dong Jin(주동진) ORCID logo https://orcid.org/0000-0001-8405-1531
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