利用TALEN技术建立高效的iPSCs核糖体基因区打靶体系并应用于DMD的自体化基因治疗研究

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. At present, gene targeting is the most effective biomedical technology for precise genome modification. However, extremely low recombination rate in gene targeting of stem cells limits the clinical transformation of gene therapy study. For this, on the basis of the human ribosomal DNA (hrDNA) locus targeting vector named pHrneo (a stable and safe gene targeting vector originally constructed by our group), a novel technology TALEN will be introduced to significantly improve gene targeting efficiency. The TALEN expression vector and pHrneo carrying MiniDystrophin-GFP fusion gene will be co-transfected into DMD patient-specific iPSCs, trying to establish a gene targeting system at rDNA locus of hiPSCs with high efficiency. After selection and identification of the integrated iPSC clones, direct differentiation of myoblasts and mesenchymal stem cells will be performed, along with the detection of sarcolemmal expression and location of MiniDystrophin. Finally, the differentiated cells will be transplanted into DMD model mice to ameliorate the dystrophic phenotype. Our results will provide a substantial basis for the preclinical research of autologous gene therapy for DMD patients.

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应用。针对于此，本研究拟在我室原创性构建的安全稳定的核糖体基因区(hrDNA)打靶载体（pHrneo）基础上，携带MiniDystrophin-GFP融合基因，同时引入最新的能特异性提高基因打靶效率的TALEN技术，通过核转的方式共同转至杜氏肌营养不良（DMD）病人特异性iPSCs中，并测定打靶效率，尝试以此建立对iPSCs核糖体基因区的高效打靶体系。再将已经定点靶入目的基因的iPSCs定向诱导分化为成肌细胞以及间充质干细胞，检测分化后细胞MiniDystrophin的表达及定位，再移植模型小鼠，测定小鼠运动能力改善情况，病生理学改变等指标。本研究将为DMD实现自体化基因治疗的临床前研究奠定良好的实验基础。

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，本研究在我室原创性构建的安全稳定的核糖体基因区(hrDNA)打靶载体（pHrneo）基础上，携带 MiniDystrophin-GFP 融合基因，同时引入最新的能特异性提高基因打靶效率的 TALEN 技术，通过核转及电转的方式共同转至杜氏肌营养不良（DMD）病人特异性 iPSCs 中。本课题通过优化完善了打靶方案，以此建立对 iPSCs 核糖体基因区的高效打靶体系；同时建立了iPSCs非病毒诱导平台，以规避病毒诱导存在的潜在应用风险。再将已经定点靶入目的基因的 iPSCs 定向诱导分化为成肌细胞，为 DMD 实现自体化基因治疗的临床前研究奠定良好的实验基础。