CRISPR/cas9系统介导的血友病乙小鼠基因纠正治疗及其安全性和纠正效率的探究

Hemophilia B (HB) is an X-linkage disorder caused by defects of coagulation factor IX and gene therapy is the only way to cure HB, theoretically. Previous studies on HB gene therapy are mostly based on viral mediated gene-supplement system, which cannot really correct the defective gene in patients and increase the risk of cancer and excessive immunoreaction. CRISPR/cas9 gene editing technology is a new tool for gene therapy which gives a possibility to fundamentally correct the mutant gene in HB. However, there are also some limitations, such as off-target recognition and unwanted gene mutation caused by non-homologous end joining (NHEJ) DNA repair. The applicant of this study is engaged in HB gene therapy research for more than ten years, and has conducted several projects on the establishment, optimization and application of gene editing systems in recent years. Several disease mice model including hemophilia B mice has been established by CRISPR/cas9 or TALEN. In this study, a CRISPR/cas9 mediated FIX corrective gene therapy system is going to be founded. Using CRISPR/cas9 to correct FIX mutation in the embryonic fibroblast (Mef) and induced pluripotent stem cells (iPS) of HB mice, the corrected cells will be induced to iHepSC and be transplanted back to HB mice to treat the defect of coagulation. By applying optimized cas9 enzyme (cas9 RNP, cas9 nickase), offering different types of DNA donor and repressing the expression of NHEJ in gene editing, this study is plan to minimize the side-effect of CRISPR/cas9 mediate gene therapy, and reduce the possibility of off-target and random insertion of donor DNA. In addition, this study is to seek out an appropriate method for cell transplantation to bleeding diseased model, providing a novel view for corrective gene therapy to hemophilia B.

血友病乙是凝血因子IX缺陷导致的性连锁遗传病，理论上基因治疗是唯一根治途径。既往血友病乙基因治疗多采用病毒系统介导的基因补充策略，它不能真正纠正缺陷基因，并存在致癌和高免疫原性隐患。CRISPR/cas9基因编辑技术有望从根本上对血友病乙进行基因纠正治疗，但也存在脱靶和非同源末端连接(NHEJ)导致基因突变等局限。申请者从事血友病乙基因治疗研究十余年，近年来在多种基因编辑系统的建立、应用、优化和安全性上系统探索，建立了血友病乙小鼠等多种疾病鼠模型。本申请拟通过CRISPR系统对血友病乙小鼠成纤维细胞和iPS细胞体外基因纠正、肝细胞诱导分化及细胞回输个体实施治疗。在此基础上，通过cas9蛋白复合物、缺刻酶cas9n、抑制NHEJ途径、单链DNA同源模板等策略降低脱靶效应、避免随机整合，提高同源重组基因纠正效率；同时探索适合出血性疾病细胞移植的有效方法，为血友病乙基因纠正治疗研究提供新依据。

CRISPR/Cas9的高基因编辑效率和诱导重组的能力使其成为了基因治疗的有力工具。因此，我们开展了CRISPR/Cas9介导的血友病乙基因纠正治疗的研究。我们首先构建并筛选得到了F9基因8 bp缺失突变的重症血友病乙小鼠模型，并以其为治疗对象进行肝脏原位基因纠正及细胞离体基因纠正。.在肝脏原位基因纠正中，采用尾静脉高压注射法将CRISPR/Cas9及同源纠正模板导入模型小鼠肝细胞进行治疗，从基因水平、表达水平和凝血表型上对治疗效果进行检测。检测发现，多数治疗小鼠实现了1%以上的肝细胞基因纠正。纠正小鼠血浆中FⅨ表达量显著上升，为对照组的3.39倍；纠正小鼠的凝血障碍也得到缓解，凝血活性最高恢复到正常值的27%。此外，靶向三个不同位点的重组治疗均有一定疗效，单组最高治疗有效率达70%。.在细胞离体基因纠正中，我们在受精卵水平对比了两种Cas9导入形式——Cas9 mRNA及Cas9-sgRNA蛋白复合物进行生殖细胞基因治疗的有效性和安全性。研究发现：RNP注射治疗的有效性更高，其介导的DNA修复效率为mRNA注射组的1.62倍，治疗成功率为1.31倍。同时，RNP注射也显示出更低的细胞毒性和嵌合体形成比例，更适用于生殖细胞的基因纠正。最后，我们从疾病型小鼠的成纤维细胞出发进行多能干细胞的诱导、基因修复和肝向分化，初探体细胞离体基因纠正方案。本研究中的，我们从基因型到表型上证明了CRISPR/Cas9介导的纠正型基因治疗的有效性。.为了实现DNA的精确切割，亟需以结构信息为指导进行Cas9酶的优化，而目前已解析的Cas9复合物晶体结构为非活性状态下、DNA链部分缺失的结构模型。因此，我们进行了活性状态下Cas9-sgRNA-DNA三元蛋白复合物的结构解析。利用冷冻电镜技术获得了含完整DNA双链的三元复合物图像，通过单颗粒三维重构方法获得了分辨率为4.6 Å的复合物三维密度图，进一步构建了活性复合物的原子模型，解析出非互补链DNA的空间走向。在原子模型的基础上，我们还对Cas9蛋白RuvC结构域与DNA非互补链结合的关键氨基酸残基进行了预测和功能验证，找到了关键位点K974；并根据氨基酸空间分布和水分子模拟提出RuvC结构域切割非互补DNA的化学机制。此修正的三元复合物原子模型将为CRISPR/Cas9系统的优化改造提供重要科学指导。