Stem Cell & Neurotherapies, Faculty of Medical and Human Sciences, University of Manchester, 3.721 Stopford Building, Manchester M13 9PT, UK.
Pharm Res. 2011 Nov;28(11):2863-70. doi: 10.1007/s11095-011-0527-1. Epub 2011 Jul 9.
Development of a human mitochondrial gene delivery vector is a critical step in the ability to treat diseases arising from mutations in mitochondrial DNA. Although we have previously cloned the mouse mitochondrial genome in its entirety and developed it as a mitochondrial gene therapy vector, the human mitochondrial genome has been dubbed unclonable in E. coli, due to regions of instability in the D-loop and tRNA(Thr) gene.
We tested multi- and single-copy vector systems for cloning human mitochondrial DNA in E. coli and Saccharomyces cerevisiae, including transformation-associated recombination.
Human mitochondrial DNA is unclonable in E. coli and cannot be retained in multi- or single-copy vectors under any conditions. It was, however, possible to clone and stably maintain the entire human mitochondrial genome in yeast as long as a single-copy centromeric plasmid was used. D-loop and tRNA(Thr) were both stable and unmutated.
This is the first report of cloning the entire human mitochondrial genome and the first step in developing a gene delivery vehicle for human mitochondrial gene therapy.
开发人类线粒体基因传递载体是治疗线粒体 DNA 突变引起的疾病的关键步骤。尽管我们之前已经完整地克隆了小鼠线粒体基因组，并将其开发为线粒体基因治疗载体，但由于 D 环和 tRNA(Thr)基因的不稳定性区域，人类线粒体基因组在大肠杆菌中被称为不可克隆。
我们测试了多种和单拷贝载体系统，以在大肠杆菌和酿酒酵母中克隆人线粒体 DNA，包括转化相关重组。
人线粒体 DNA 在大肠杆菌中不可克隆，并且在任何条件下都不能保留在多拷贝或单拷贝载体中。然而，只要使用单拷贝着丝粒质粒，就有可能在酵母中克隆并稳定维持整个人类线粒体基因组。D 环和 tRNA(Thr)都是稳定且未突变的。
这是首次报道克隆完整的人类线粒体基因组，也是开发人类线粒体基因治疗基因传递载体的第一步。
