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 C57BL/6 Fah(Delta 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 x 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 x 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 x 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 C57BL/6 Fah(Delta 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. CONCLUSION 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.