Junge Norman, Yuan Qinggong, Vu Thu Huong, Krooss Simon, Bednarski Christien, Balakrishnan Asha, Cathomen Toni, Manns Michael P, Baumann Ulrich, Sharma Amar Deep, Ott Michael
Department of Pediatric Gastroenterology and Hepatology, Hannover Medical School, Hannover 30625, Germany.
Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover 30625, Germany.
World J Hepatol. 2018 Feb 27;10(2):277-286. doi: 10.4254/wjh.v10.i2.277.
To stably correct tyrosinaemia in proliferating livers of fumarylacetoacetate-hydrolase knockout ( mice by homologous-recombination-mediated targeted addition of the gene.
C57BL/6 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 expression cassette was flanked by homologous arms (620 bp and 749 bp long) of the gene locus. Mice were injected with 2.1 × 10 VP of this vector () the tail vein. Mice in the control group were injected with 2.1 × 10 VP of a similar vector but missing the homologous arms (). Primary hepatocytes from recipient mice, treated with our vectors, were isolated and 1 × 10 hepatocytes were transplanted into secondary 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 gene expression cassette into the "safe harbour locus" by recombinant . 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 mice, which have been transplanted with hepatocytes from a mouse injected with 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 treated mice, survived and showed few and smaller FAH positive clusters. These results demonstrate that homologous recombination-mediated gene transfer corrects the phenotype in a mouse model of human tyrosinaemia type 1 ( 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 recipient mice into secondary recipient mice. This long term therapeutic efficacy is clearly superior to our control mice treated with episomal gene therapy approach.
HR-mediated gene therapy provides targeted transgene integration and phenotypic correction in mice with superior long-term efficacy compared to episomal therapy in proliferating livers.
通过同源重组介导的基因靶向添加，稳定纠正富马酰乙酰乙酸水解酶基因敲除小鼠增殖肝脏中的酪氨酸血症。
在我们的研究中，C57BL/6小鼠作为人类1型酪氨酸血症的动物模型。如所述，通过从慢病毒质粒中扩增包含甲状腺转运蛋白启动子的人Fah cDNA来构建载体。Fah表达盒两侧是Fah基因座的同源臂（分别为620 bp和749 bp长）。通过尾静脉向小鼠注射2.1×10个病毒颗粒的该载体（）。对照组小鼠注射2.1×10个病毒颗粒的类似载体，但缺少同源臂（）。从接受我们载体处理的受体小鼠中分离出原代肝细胞，并通过注射到脾脏中将1×10个肝细胞移植到二级受体小鼠中。在应用载体或进行肝细胞移植后，受体小鼠停止NTBC治疗。
在此，我们报告通过重组将Fah基因表达盒整合到“安全港位点”，成功实现了同源重组介导的基因组编辑。两组小鼠在未进行NTBC治疗的情况下，通过肝脏切片的免疫组织化学分析确定，均表现出长期存活、体重增加和Fah阳性簇。在C57BL/6小鼠组中，这些小鼠在156天前接受了注射载体的小鼠的肝细胞移植，6只小鼠中有6只表现出长期存活并体重增加，且有Fah阳性簇，无需NTBC治疗。相比之下，接受处理过的小鼠的肝细胞的5只小鼠中只有1只存活，且Fah阳性簇较少且较小。这些结果表明，同源重组介导的Fah基因转移纠正了人类1型酪氨酸血症小鼠模型（小鼠）的表型，并且在肝脏的增殖状态下是持久的，这通过停止NTBC治疗以及将原代受体小鼠分离的肝细胞连续移植到二级受体小鼠中得以证明。这种长期治疗效果明显优于用游离型Fah基因治疗方法处理的对照小鼠。
与游离型Fah治疗增殖肝脏相比，同源重组介导的Fah基因治疗在小鼠中提供了靶向转基因整合和表型纠正，具有卓越的长期疗效。
