A basic study for mitochondria-targeting liposome vector using human mitochondrial fusiogenic protein.

线粒体是细胞中的细胞器，具有不同于核基因组的适当DNA。以线粒体DNA为基础的线粒体疾病的根治需要发展一种不同于传统的以核为靶点的基因治疗方法--将物质导入线粒体的新方法。本研究旨在以线粒体融合因子为有效成分，构建以线粒体为靶点的新型脂质体载体。这样的载体不仅可以将正常的线粒体DNA导入线粒体，还可以将调节线粒体功能的药物和/或调节线粒体转录本的外源核酶引入线粒体。这种载体的开发将成为未来许多线粒体功能障碍疾病的新的药物传递系统。我们鉴定了线粒体融合蛋白，人类FZO同源物，并分析了膜融合活性所必需的蛋白的结构域。我们还制备了该结构域的重组蛋白，用该蛋白重组了脂质体，并在体外证明了脂质体的膜融合活性。另一方面，我们检测了具有线粒体融合活性的OPA1蛋白，发现OPA1亚型在人类细胞中存在差异的亚区块化。该因子是线粒体融合装置的辅助因子之一，对提高脂质体载体的效率具有重要意义。下一步需要检查通过显微注射进入细胞的线粒体DNA嵌入脂质体是否真的针对体内的线粒体。本研究是建立线粒体疾病基因治疗基础技术的第一步。

The mitochondria are organelle in the cell and have the proper DNA different from the nuclear genome. The development of the new way of introducing a material into the mitochondria, which is different from the usual gene therapy to the nuclear target, is necessary for the fundamental treatment of the mitochondrial disease based on the mitochondrial DNA. In this research, it aims at the fundamental development of new liposome vector that peculiarly targets the mitochondria, using the mitochondrial fusiogenc factor as an effective component. Such a vector will make it possible that not only normal mitochondrial DNA but also the drug adjusting the mitochondrial function and/or the exogenous ribozyme regulating the mitochondrial transcripts are introduced into the mitochondria. The development of such a vector would become applied for a new drug delivery system for many diseases with dysfunction of the mitochondria in the future.We identified a mitochondrial fusiogenic protein, a human Fzo homolog, and analyzed the domain of the protein necessary for membrane fusiogenic activity. We also produced the recombinant protein of the domain, reconstituted the proteoliposome using the protein, and proved the membrane fusiogenic activity of the liposome in vitro. On the other hand, we examined the OPA1 protein which has the mitochondrial fusiogenic activity, and showed the differential sublocalization of OPA1 isoform in the human cell. This factor is one of the assistant factors of the mitochondrial fusiogenic device, which will be important for improvement for the efficiency of the liposome vector. Next step requires an examination whether the mitochondrial DNA-embedded liposome, introduced in the cell by microinjection, actually targets the mitochondria in vivo. This research is the first step to establish the fundamental technology of the gene therapy of mitochondrial diseases.