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

Human Gene Therapy
Caitlin VanLithRaymond D Hickey

Abstract

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...Continue Reading

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Citations

Mar 28, 2019·Journal of Inherited Metabolic Disease·Martijn G S RuttenMaaike H Oosterveer
Jun 9, 2020·Clinical Genetics·Roya RamezankhaniCatherine M Verfaillie
Jul 19, 2020·International Journal of Molecular Sciences·Neha SarodayaSuresh Ramakrishna
Jul 28, 2019·Human Gene Therapy·Nerea ZabaletaGloria Gonzalez-Aseguinolaza
Nov 26, 2020·Molecular Therapy : the Journal of the American Society of Gene Therapy·Garima SharmaChiranjib Chakraborty
Nov 6, 2020·Therapeutic Advances in Gastroenterology·Guang-Peng ZhouZhi-Jun Zhu
Aug 4, 2018·Biochimica Et Biophysica Acta. Molecular Basis of Disease·Sharie J HaugabookElizabeth A Ottinger

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Methods Mentioned

BETA
transfect
enzyme-linked immunosorbent assay
transfection
PCR
flow cytometry

Software Mentioned

GraphPad Prism
GraphPad
Aperio ImageScope

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