AAV8-CRISPR/Cas9联合NHEJ技术介导的血友病A小鼠高效表达完整功能BDD-hF8的研究

血友病是一类单基因遗传病，基因治疗有望为其带来治愈希望。但目前血友病基因治疗手段存在F8的长基因编码序列与转基因载体包装容量不匹配、载体免疫原性、F8异位靶位高效持续表达等问题。本课题拟以腺相关病毒AAV8为载体，利用CRISPR/Cas9联合非同源末端接合（NHEJ）完成基因组目标定位、剪切及DNA修复的技术，构建密码子优化的删除B区的人凝血因子8（BDD-hF8）或其变构体，并精确敲入血友病A小鼠肝脏清蛋白基因Albumin位点，使完整功能F8蛋白与清蛋白在体内共同持续表达。并通过血友病A小鼠体内基因编辑技术的构建、优化hF8表达水平，降低AAV-8载体量，降低非需要基因的植入率及免疫原性，实现体内安全高效的完整功能F8基因持续表达，以期修复血友病A缺陷基因、恢复正常凝血功能。本项目研究结果将为基因编辑治疗血友病A新策略提供可靠的理论依据。

Hemophilia is a monogenic hereditary disease, and gene therapy is expected to bring hope for its cure. However, there are many problems in gene therapy for hemophilia, such as the mismatch between the long gene coding sequence of F8 and the packaging capacity of the transgene vector, the immunogenicity of the vector and the efficient and sustained expression of F8 in heterotopic target. In this study, we will construct the codon-optimized BDD-hF8 or its variants in AAV vector by using CRISPR/Cas9 combined with NHEJ to complete genome targeting, splicing and DNA repair technology. F8 will be further accurately knocked in the hepatic albumin gene loci of hemophilia A mice to make the complete functional F8 protein and albumin co-expressing in vivo. In this application, the new strategy of gene editing technology in hemophilia A mice, will optimize the expression level of human F8, will reduce the quantity of AAV-8 vector, will reduce the rate of non-essential gene implantation and immunogenicity, and will enhance the safety and efficiency of continuous expression of complete functional F8 gene in vivo, and will finally repair the defective gene of hemophilia A and restore normal coagulation function. The results of this project will provide a reliable theoretical basis for the new strategy of gene editing for the treatment of hemophilia A.