诱导性多潜能干细胞（iPSCs）技术的兴起为以基因打靶为核心的自体化基因治疗创造了全新的契机，然而在干细胞中较低的基因打靶效率极大限制了基因治疗在临床转化中的广泛应用。前期的研究中，我们原创性构建出了小鼠核糖体基因区（rDNA）打靶载体pMrn，并实现了对小鼠胚胎干细胞（mESCs）rDNA区打靶。在此基础和经验上，本项目我们将围绕pMrn打靶载体，引入最新的靶向基因组编辑技术- - CRISPR/Cas9，高效介导核糖体基因区的特异性打靶，以此实现FIX表达框对血友病B特异性诱导多潜能干细胞（iPSCs）的基因打靶。再将已经定点靶入了FIX基因的iPSCs定向诱导分化为肝细胞，移植血友病B模型小鼠，测定小鼠病情改善情况。最后通过将定点整合FIX的iPSCs克隆发育成嵌合体模型小鼠，进一步证实rDNA区打靶后的长期有效性及安全性。此研究将为实现血友病B自体化基因治疗的临床前研究奠定良好的基础。

Generation of induced pluripotent stem cells (iPSCs) offers great potential for autologous gene therapy. In order to have iPSCs fully used in gene therapy purposes, stable genetic modification should be introduced. In previous study, we originally constructed a mouse-derived ribosomal locus targeting vector(pMrn), which could be targeted at the rDNA locus in mESCs genome. Base on this background and experience, in this proposal a novel targeting genome-editing technology CRISPR/Cas9 will be introduced for improvement of gene targeting efficiency at rDNA locus. The FIX expression cassette will be targeted into mouse Hemophilia B derived iPSCs by combination with pMrn and CRISPR/Cas9, trying to establish a gene targeting system at rDNA locus of iPSCs with high efficiency. After selection and identification of the integrated iPSC clones, direct differentiation of hepatic cells will be performed, along with the detection of FIX expression. Finally, the differentiated cells will be transplanted into Hemophilia B model mice to ameliorate the bleeding phenotype. Our results will provide a substantial basis for the preclinical research of autologous gene therapy for Hemophilia B patients.