利用基因编辑技术靶向清除人卵子中突变线粒体的研究

Mitochondrial genetic disorders is a maternally inherited disease caused by mutations in the mitochondrial genome (mtDNA) and its clinical phenotype is influenced by the threshold of mitochondrial DNA mutation. Currently, there have been no effective methods to block offspring's inheritance. We confirmed that mito-TALENs targeted and eliminated the mutant mitochondria in iPSCs of mitochondrial patients to restore cellular metabolism, it can also target and remove the mutated human mtDNA which fused to pig eggs. We intend to elucidate the molecular mechanisms of mito-TALENs that targeting and scavenging mutant mtDNA in human eggs, reduce the proportion of mutated mtDNA in embryos and eventually, achieve the goal of intervention and treating mitochondrial gene diseases at the embryonic level. We aimed to screen out mito-TALENs with high-efficiency by using mitochondrial patient iPSCs. Then, the selected mito-TALENs mRNA will be injected into mitochondrial patients donated eggs with sperm to clarify the developmental characteristics of early embryos after gene correction. Embryonic stem cell lines will be established by using those embryos after its reach to the blastocyst stage. Through stem cells` mitochondrial genome sequencing, in vitro differentiation and cell metabolism assays to assess the effectiveness and safety of mito-TALENs in treating mitochondrial gene disorders, thus providing a theoretical basis for earlier intervention with treatment in mitochondrial genetic diseases at the embryonic level.

线粒体基因病是一类由线粒体基因组(mtDNA)突变引起的母系遗传病，其临床表型受mtDNA突变阈值效应影响,当前缺乏有效阻断子代遗传的方法。我们证实mito-TALENs可靶向清除线粒体病人iPSCs中突变线粒体，使细胞代谢功能恢复正常，还可靶向清除融合病人细胞的猪卵子中突变的人mtDNA。我们拟明确mito-TALENs靶向清除人卵子中的突变mtDNA，降低突变mtDNA在胚胎中比例的分子机制，实现线粒体基因病在胚胎水平干预治疗的目标。本项目将利用线粒体病人iPSCs筛选高效的mito-TALENs；将mito-TALENs的mRNA和精子注入线粒体病人捐献的卵子中，明确基因编辑线粒体后胚胎早期发育特征；利用囊胚建立胚胎干细胞系，通过干细胞线粒体基因组测序、体外分化及细胞代谢功能实验评估mito-TALENs对线粒体基因病治疗的有效性和安全性，为线粒体基因病在胚胎水平干预治疗提供理论依据

线粒体基因病是一类由线粒体基因组(mtDNA)突变引起的母系遗传病，其临床表型受mtDNA突变阈值效应影响,当前缺乏有效阻断子代遗传的方法。我们研究证实mito-TALENs可靶向清除线粒体病人iPSCs中突变线粒体，使细胞代谢功能恢复正常，还可靶向清除融合病人细胞的猪卵子中突变的人mtDNA。我们通过知情同意利用线粒体基因病患者捐赠的卵子，针对m.8993T>G 的mito-TALENs的mRNA和精子显微注射到相应的线粒体基因病患者的卵子胞浆内，观察受精情况以及胚胎发育情况，评估mito-TALENs技术对卵子突变线粒体靶向清除效率和发育潜能的影响。通过对5个胚胎突变线粒体残留检测结果显示，mito-TALENs可以有效的把突变mtDNA的比例降到触发人类线粒体基因病的阈值以下，5个检测的胚胎有2个胚胎突变线粒体残留率为0，清除效率达到100%。. 我们使用来自MELAS患者的诱导多能干细胞建立类器官模型，发现高水平的Notch信号是与MELAS神经培养相关的神经发生延迟和神经突生长缺陷有关，还发现分泌酶抑制剂DAPT可以逆转这些神经发育缺陷。我们首次开发出“两步法”诱导生成人造胚胎的技术，首先利用人的扩展多能性干细胞（hEPS）体外定向诱导为类滋养层（TE-like）细胞后，将hEPS细胞和TE-like细胞混合培养在微孔板中，这些细胞很快彼此形成连接，产生信息交流，在特定的培养基下形成类囊胚结构（blastoids），该研究成果2021年在Cell Discovery上发表。. 本项目发表署名基金号的SCI论文11篇，其中影响因子大于30分的1篇，大于10分的2篇，申请发明专利2项，授权发明专利1项。项目负责人2021年获得广东省杰出青年基金资助，培养在读博士生4名，博士后3名，硕士生8名，出站博士后2名，毕业博士1名，毕业硕士生2名，圆满完成各项指标任务。

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