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

AIM To stably correct tyrosinaemia in proliferating livers of fumarylacetoacetate-hydrolase knockout(Fah-/-) mice by homologous-recombination-mediated targeted addition of the Fah gene.METHODS C57 BL/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 c DNA 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(r AAV8-ROSA26.HAL-TTR.FahROSA26.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(r AAV8-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. RESULTS 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 C57 BL/6 Fah?exon5 mice, which have been transplanted with hepatocytes from a mouse injected with r AAV8-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 r AAV8-TTR.Fah treated mice, survived and showed few and smaller FAH positive clusters. These results demonstrate that homologous recombinationmediated 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 r AAV8 gene therapy approach.CONCLUSION HR-mediated r AAV8 gene therapy provides targeted transgene integration and phenotypic correction in Fah-/-mice with superior long-term efficacy compared to episomal r AAV8 therapy in proliferating livers.