线粒体捐赠胚中mtDNA单倍型遗传分离的机制研究

Mitochondrial donation technique can effectively reduce the transmission of pathogenic mtDNA in mitochondrial genetic disease. But up to now, the stability of donated mtDNA in offspring can not be guaranteed in the traditional mitochondrial donation technique. The mechanism may be related to the mitochondrial drift. The study showed that even low levels of pathogenic mtDNA carryover could lead to mitochondrial haplotype reversal. Animal study found that the haplotype genetic distance of mtDNA might be related to the genetic drift of mtDNA haplotype. Until now, there is no related researches on human beings. This project intend to completely eliminate the karyoplast-derived mtDNA carryover with the safe and efficient first polar body 1 genome transfer (PB1T) which was created and established by our team. At the same time, the mitochondrial donor embryos with the same mtDNA heterogeneity and different haplotype genetic distances were constructed and the embryonic stem cell lines were induced to establish, in order to explore the effects of mtDNA haplotype genetic distance on the karyotype stability of mtDNA and the inheritance and epigenetics of mitochondrial donor offspring. It is expected to prevent the occurrence of mitochondrial karyotype drift and to guarantee the safety of mitochondrial donor offspring by eliminating the heterogeneity of mtDNA and combining with reasonable mitochondrial genotype matching.

线粒体捐赠技术可有效减少线粒体遗传病中致病mtDNA的传递，但传统的线粒体捐赠技术中，捐赠的mtDNA在子代的稳定性仍不能保障，其机制可能与线粒体漂移有关。研究发现，传统线粒体捐赠重构胚中，即使携带极少量的致病mtDNA残留都有可能发生线粒体单倍型逆转。动物研究发现mtDNA的单倍型遗传距离可能与mtDNA单倍型的遗传漂移有关，遗传距离越大则线粒体捐赠子代mtDNA的分离的可能性越大。目前尚无人类的相关研究报道。本项目拟通过由本团队发明并建立的安全高效的第一极体基因组移植线粒体捐赠技术彻底消除核源性mtDNA残留，并构建mtDNA异质性相同、单倍型遗传距离不同的线粒体捐赠重构胚并诱导建立胚胎干细胞系，探索mtDNA单倍型遗传距离对mtDNA核型稳定性及对子代遗传和表观遗传的影响，期望通过消除mtDNA异质性、结合合理的线粒体基因型配型阻止线粒体核型漂移的发生，保障线粒体捐赠技术的安全。

卵子发生过程中减数分裂产生的极体已经被证实可以替代卵胞浆中核物质进行线粒体置换，从而有效减少甚至阻断致病线粒体DNA向子代传递。然而，极体作为卵母细胞外独立的遗传物质，临床上如何建立安全高效的极体移植方法，以保证第一极体移植的效率呢？线粒体单倍型配型被认为可以有效规避线粒体置换技术，尤其是原核移植和纺锤体移植后的发生的核供体mtDNA遗传漂移现象。但是，目前大多数动物模型研究集中在线粒体功能近似的正常小鼠，且不同研究之间的争议性较大。因此本项目围绕建立高效的人极体基因组移植线粒体捐赠技术体系；同时利用具有相似单倍型的mtDNA相关I型糖尿病模型鼠和C57BL6/J小鼠，研究MRT重构子代mtDNA异质性分离的特征和机制，以及异源mtDNA对子代长期安全性的影响。本项目建立了高效的人第一极体移植的临床平台。同时发现利润MRT中对mtDNA进行单配型“配型”有助于避免核供体mtDNA在子代和组织中的分离。然而，MRT过程中降低核供体mtDNA携带，是保障MRT安全性的前提。异源性mtDNA的存在并未影响MRT重构子代的线粒体功能和疾病表型，而正常mtDNA（C57BL/6J）的引入改善了重构子代的线粒体功能和I型糖尿病疾病表型。因此，进一步证实第一极体移植仍旧是最有希望阻断致病mtDNA传递的方法。