线粒体视神经病多为遗传突变所致，视网膜神经节细胞（RGCs）进行性丧失是其普遍特征，迄今尚无有效治疗手段。基因编辑和干细胞技术的快速发展，使得非病毒载体介导的干细胞基因打靶有望成为一种新型治疗方式。申请者前期建立了首个线粒体基因SLC25A46突变导致的遗传性运动和感觉神经病6B型（HMSN VIB）疾病小鼠模型，眼部表型包括RGCs渐进性丢失、视神经退行性病变等。因此本项目拟在课题组长期致力于干细胞核糖体基因区（rDNA）非病毒基因打靶的基础上，创新性地以HMSN VIB小鼠为研究对象，首先建立其iPSCs，再通过CRISPR/Cas9 nickase与rDNA区打靶载体将Slc25a46治疗基因定点整合至iPSCs的rDNA区；并将其定向分化为RGCs相关细胞和MSCs，最后经玻璃体腔共移植至模型鼠，评估视网膜功能改善情况。本研究将为推进线粒体视神经病的临床自体化基因治疗奠定良好基础。

Mitochondrial optic neuropathy is mostly caused by genetic mutations, with a common feature of progressive loss of retinal ganglion cells (RGCs). So far, there is no effective treatment. With the rapid development of genome editing and stem cell technologies, gene targeting in stem cells mediated by non-viral vectors is expected to become a new therapeutic method. In previous study, our group had established the first mouse model of HMSN VIB caused by biallelic mutations of the mitochondrial related gene SLC25A46. The ocular phenotypes of the mouse include early progressive loss of RGCs and degenerative optic nerves. Therefore, on the basis of our long-term commitment to the gene targeting in ribosome gene region (rDNA) of stem cells by non-viral vectors. This study will innovatively take HMSN VIB mouse model as the research object and establish an iPSCs line from the mouse dermal fibrolasts. Slc25a46 therapeutic gene will then be targeted into the rDNA region of iPSCs through the combination of optimized CRISPR/Cas9 nickase and rDNA targeting vectors. The targeted iPSCs would be differentiated into RGCs-related cells, which will be intravitreally co-injected with iPSCs-derived MSCs into the model mice, and finally evaluate the improvement of retinal function. This study will provide a foundation for the clinical autologous gene therapy of mitochondrial optic neuropathy.