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World J Hepatol. 2018 Feb 27;10(2):277-286. doi: 10.4254/wjh.v10.i2.277.

Homologous recombination mediates stable Fah gene integration and phenotypic correction in tyrosinaemia mouse-model.

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Department of Pediatric Gastroenterology and Hepatology, Hannover Medical School, Hannover 30625, Germany.
Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover 30625, Germany.
TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover 30625, Germany.
Medical Center, University of Freiburg, Institute for Cell and Gene Therapy, Freiburg 79108, Germany.



To stably correct tyrosinaemia in proliferating livers of fumarylacetoacetate-hydrolase knockout (Fah-/-) mice by homologous-recombination-mediated targeted addition of the Fah gene.


C57BL/6 Fah∆exon5 mice served as an animal model for human tyrosinaemia type 1 in our study. The vector was created by amplifying human Fah cDNA including the TTR promoter from a lentivirus plasmid as described. The Fah expression cassette was flanked by homologous arms (620 bp and 749 bp long) of the Rosa26 gene locus. Mice were injected with 2.1 × 108 VP of this vector (rAAV8-ROSA26.HAL-TTR.Fah-ROSA26.HAR) via the tail vein. Mice in the control group were injected with 2.1 × 108 VP of a similar vector but missing the homologous arms (rAAV8-TTR.Fah). Primary hepatocytes from Fah-/- recipient mice, treated with our vectors, were isolated and 1 × 106 hepatocytes were transplanted into secondary Fah-/- recipient mice by injection into the spleen. Upon either vector application or hepatocyte transplantation NTBC treatment was stopped in recipient mice.


Here, we report successful HR-mediated genome editing by integration of a Fah gene expression cassette into the "safe harbour locus" Rosa26 by recombinant AAV8. Both groups of mice showed long-term survival, weight gain and FAH positive clusters as determined by immunohistochemistry analysis of liver sections in the absence of NTBC treatment. In the group of C57BL/6 Fah∆exon5 mice, which have been transplanted with hepatocytes from a mouse injected with rAAV8-ROSA26.HAL-TTR.Fah-ROSA26.HAR 156 d before, 6 out of 6 mice showed long-term survival, weight gain and FAH positive clusters without need for NTBC treatment. In contrast only 1 out 5 mice, who received hepatocytes from rAAV8-TTR.Fah treated mice, survived and showed few and smaller FAH positive clusters. These results demonstrate that homologous recombination-mediated Fah gene transfer corrects the phenotype in a mouse model of human tyrosinaemia type 1 (Fah-/- mice) and is long lasting in a proliferating state of the liver as shown by withdrawal of NTBC treatment and serial transplantation of isolated hepatocytes from primary Fah-/- recipient mice into secondary Fah-/- recipient mice. This long term therapeutic efficacy is clearly superior to our control mice treated with episomal rAAV8 gene therapy approach.


HR-mediated rAAV8 gene therapy provides targeted transgene integration and phenotypic correction in Fah-/- mice with superior long-term efficacy compared to episomal rAAV8 therapy in proliferating livers.


AAV8; Gene therapy; Liver based metabolic disease; Paediatric liver disease; ROSA26; Targeted integration

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