Huai Cong, Jia Chenqiang, Sun Ruilin, Xu Peipei, Min Taishan, Wang Qihan, Zheng Chengde, Chen Hongyan, Lu Daru
Key Laboratory of Genetic Engineering, MOE Key Laboratory of Contemporary Anthropology, Room C613, Building for School of Life Sciences, Fudan University, No. 2005 Songhu Rd, Shanghai, 200433, China.
Shanghai Model Organisms Center, No.3577 Jinke Rd., Shanghai, 201203, China.
Hum Genet. 2017 Jul;136(7):875-883. doi: 10.1007/s00439-017-1801-z. Epub 2017 May 15.
Hemophilia B (HB) is an X-linked disorder caused by defects of F9 encoded coagulation factor IX, which is an ideal model for gene therapy. Most existing HB gene therapies are based on viral mediated gene supplementation, which could increase immunoreaction. In this study, CRISPR/Cas9 system was used for gene correction in an F9 mutant HB mouse model in both adult mice (in vivo) and in germline cells (ex vivo). In vivo, naked Cas9-sgRNA plasmid and donor DNA were delivered to HB mice livers to recover the mutation via hydrodynamic tail vein (HTV) injection. 62.5% of the HTV-treated mice showed a detectable gene correction (>1%) in the F9 alleles of hepatocytes, which was sufficient to remit the coagulation deficiency. Ex vivo, three different forms of Cas9 were microinjected into germline cells of HB mice to investigate their efficiency and safety in gene correction. Cas9 protein showed higher gene recovery rates, less embryo toxicity, and lower mosaic repair percentage, making it more suitable for germline gene therapy. Our study strongly supports that CRISPR/Cas9-mediated genome editing is feasible in gene therapy of genetic disorders.
血友病B（HB）是一种由凝血因子IX编码基因F9缺陷引起的X连锁疾病，是基因治疗的理想模型。现有的大多数HB基因治疗都基于病毒介导的基因补充，这可能会增加免疫反应。在本研究中，CRISPR/Cas9系统用于F9突变HB小鼠模型的成年小鼠（体内）和生殖细胞（体外）的基因校正。在体内，将裸露的Cas9-sgRNA质粒和供体DNA通过尾静脉高压注射（HTV）导入HB小鼠肝脏以修复突变。62.5%的HTV处理小鼠在肝细胞的F9等位基因中显示出可检测到的基因校正（>1%），这足以缓解凝血缺陷。在体外，将三种不同形式的Cas9显微注射到HB小鼠的生殖细胞中，以研究它们在基因校正中的效率和安全性。Cas9蛋白显示出更高的基因恢复率、更低的胚胎毒性和更低的嵌合修复百分比，使其更适合生殖系基因治疗。我们的研究有力地支持了CRISPR/Cas9介导的基因组编辑在遗传性疾病基因治疗中是可行的。
