利用碱基编辑工具DdCBE制备线粒体基因ND1和ND5突变Leigh综合征模型猪

Leigh综合征（LS）是一类由于线粒体呼吸链功能缺陷、以中枢神经系统病变为主的遗传性线粒体疾病，目前尚无有效治疗手段，需要合适的动物模型加以研究。LS涉及核基因和线粒体DNA（mtDNA）突变，由于直接对mtDNA进行修饰存在技术障碍，mtDNA突变模型难以建立，现有LS模型以核基因修饰为主。新近出现的DdCBE技术，可以对mtDNA进行修饰，被用于疾病小鼠模型的制备。但由于小鼠线粒体代谢通路与人类差异较大，小鼠模型不能很好模拟人类LS疾病表型。而猪的线粒体代谢通路与人类更接近，经核基因修饰制备的LS模型猪证实，其表型与病人非常接近，但尚无mtDNA修饰的LS模型猪。我们在前期研究中，成功构建了基于DdCBE的mtDNA编辑技术体系，并在细胞水平验证了其有效性。因此，本项目拟针对LS病人两个线粒体基因ND1和ND5的致病突变，采用DdCBE工具直接编辑猪ND1和ND5，培育能够精确模拟人类LS疾病表型的猪，为LS等线粒体疾病的机制研究和治疗手段开发提供更合适的大动物模型。

Leigh syndrome (LS), as a subtype of inherited mitochondrial diseases, is mainly characterized by necrotic lesions in central nervous system due to defective mitochondrial respiratory chain. To date, there is no cure for LS patients, which raises an urgent need for suitable animal models for LS research. LS involves mutations in nuclear genes or mitochondrial DNA (mtDNA), however, it is difficult to establish animal models of LS with mtDNA mutations because of technical challenges to modify mtDNA directly, resulting the majority of existing models are nuclear genes modified. Fortunately, the newly developed genome engineering tool, DdCBE, can be used to perform base editing of mtDNA, and has been employed to generate mouse model of LS with mtDNA mutation. However, the mouse model fails to recapitulate clinical manifestations of LS, due to significant differences in mitochondrial metabolism pathways between mouse and human. In contrast, the mitochondrial metabolism pathways of pigs are considered more similar to those of human, as a pig model with a nuclear gene modified successfully recapitulates the phenotypes of LS patients, however, there is no pig model of LS with mtDNA modified. In preliminary experiments, we have successfully established DdCBE-based methods for mtDNA modification and verified the effectiveness of mtDNA editing in cells. Therefore, we intend to generate pig models of LS in this project, utilizing DdCBE tool to target porcine mitochondrial ND1 and ND5, which are two genes harboring pathogenic mutations in LS patients. These pig models are expected to reproduce pathological phenotypes of LS accurately, providing more suitable large animal models for research of mechanisms and treatments of LS and other mitochondrial diseases.