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 s...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.展开更多
A recent study published in Nature by Maya M.Arce and colleagues unveils the role of central gene circuits in governing the delicate balance between T cell rest and T cell activation.This work bridges fundamental mole...A recent study published in Nature by Maya M.Arce and colleagues unveils the role of central gene circuits in governing the delicate balance between T cell rest and T cell activation.This work bridges fundamental molecular biology with findings in preclinical models,providing insights into context-specific gene regulation and potential therapeutic targets for immune modulation.展开更多
基金SFB 738the"Deutsche Forschungsgemeinschaft"(Gerok-Grant)for financial support
文摘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.
文摘A recent study published in Nature by Maya M.Arce and colleagues unveils the role of central gene circuits in governing the delicate balance between T cell rest and T cell activation.This work bridges fundamental molecular biology with findings in preclinical models,providing insights into context-specific gene regulation and potential therapeutic targets for immune modulation.
