Cas9-nickase-mediated genome editing corrects hereditary tyrosinemia in rats
Cas9-nickase-mediated genome editing corrects hereditary tyrosinemia in rats
复制标题
Cas9切口酶介导的基因组编辑可纠正大鼠遗传性酪氨酸血症
DOI：
10.1074/jbc.ra117.000347
复制
发表时间：
2018-05-04
影响因子：
4.8
通讯作者：
Li, Dali
中科院分区：
文献类型：
--
作者：
Shao, Yanjiao;Wang, Liren;Li, Dali
遗传性酪氨酸血症 I 型（HTI）是一种由延胡索酰乙酰乙酸水解酶（FAH）突变引起的代谢遗传性疾病。由于有毒代谢物的积累，HTI会导致严重的肝硬化、肝功能衰竭，甚至肝细胞癌。 HTI 是基因治疗的理想模型，并且已在动物模型中证明了多种策略可以改善 HTI 症状。尽管 CRISPR/Cas9 介导的基因组编辑能够纠正小鼠模型中的 Fah 突变，但 WT Cas9 会诱导许多不需要的突变，这引起了临床应用的安全问题。为了开发一种高保真基因校正的新方法，我们构建了 Fah 突变大鼠模型，以研究 Cas9 切口酶 (Cas9n) 介导的基因组编辑是否可以有效校正 Fah。首先，我们证实 Cas9n 很少在细胞系的靶点和脱靶位点诱导插入缺失。使用 WT Cas9 作为阳性对照，我们通过腺病毒载体将 Cas9n 和修复供体模板/单引导 (sg)RNA 递送至 HTI 大鼠体内。对初始基因组编辑效率的分析表明，只有 WT Cas9 而不是 Cas9n 在肝组织的靶位点引起插入缺失。在接受 Cas9n 或 WT Cas9 介导的基因校正治疗后，HTI 大鼠体重稳定增加并存活。治疗9个月后，表达Fah的肝细胞占据了肝组织的95%以上。此外，CRISPR/Cas9 介导的基因治疗阻止了肝硬化的进展，这种表型无法在 HTI 小鼠模型中重现。这些结果强烈表明 Cas9n 介导的基因组编辑是治疗这种遗传病的一种有价值且安全的基因治疗策略。
Hereditary tyrosinemia type I (HTI) is a metabolic genetic disorder caused by mutation of fumarylacetoacetate hydrolase (FAH). Because of the accumulation of toxic metabolites, HTI causes severe liver cirrhosis, liver failure, and even hepatocellular carcinoma. HTI is an ideal model for gene therapy, and several strategies have been shown to ameliorate HTI symptoms in animal models. Although CRISPR/Cas9-mediated genome editing is able to correct the Fah mutation in mouse models, WT Cas9 induces numerous undesired mutations that have raised safety concerns for clinical applications. To develop a new method for gene correction with high fidelity, we generated a Fah mutant rat model to investigate whether Cas9 nickase (Cas9n)-mediated genome editing can efficiently correct the Fah. First, we confirmed that Cas9n rarely induces indels in both on-target and off-target sites in cell lines. Using WT Cas9 as a positive control, we delivered Cas9n and the repair donor template/single guide (sg)RNA through adenoviral vectors into HTI rats. Analyses of the initial genome editing efficiency indicated that only WT Cas9 but not Cas9n causes indels at the on-target site in the liver tissue. After receiving either Cas9n or WT Cas9-mediated gene correction therapy, HTI rats gained weight steadily and survived. Fah-expressing hepatocytes occupied over 95% of the liver tissue 9 months after the treatment. Moreover, CRISPR/Cas9-mediated gene therapy prevented the progression of liver cirrhosis, a phenotype that could not be recapitulated in the HTI mouse model. These results strongly suggest that Cas9n-mediated genome editing is a valuable and safe gene therapy strategy for this genetic disease.