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

• 作者: Amar Deep Sharma Asha Balakrishnan Christien Bednarski Michael Ott Michael P Manns Norman Junge Qinggong Yuan Simon Krooss Thu Huong Vu Toni Cathomen Ulrich Baumann
• 来源期刊: 世界肝病学杂志：英文版（电子版）
• 学科: 医学
• 关键词: Gene therapy AAV8 LIVER based METABOLIC DISEASE TARGETED integration ROSA26 PAEDIATRIC LIVER DISEASE
• 年，卷（期）: sjgbxzzywbdzb,（2）
• 页码范围: 277-286
• 页数: 10页
• 分类号: R

摘要

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 FahΔexon5 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.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 FahΔexon5 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 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