New Transformation Technology for Gene Editing of Human Mitochondrial DNA Associated With Mitochondrial Diseases

PROJECT SUMMARY Human mitochondrial DNA (mtDNA) encodes 37 genes in a small genome of 16,569 bp, which accounts for 0.005% of the total human genome size. Despite the small size of this genetic information, mtDNA is indispensable to human cells. As mitochondria are critical for cellular respiration and ATP production, mitochondria are often referred to as “the Powerhouses of the

cell”. Because of their importance, mutations in mtDNA can cause severe diseases and disorders in humans. These include but are not limited to muscular/neurodegenerative and developmental disorders such as Kearns-Sayre syndrome (KSS), Leber’s Hereditary Optic Neuropathy (LHON) and MELAS disorders. Mutations in mtDNA are also suggested to

correlate with a predisposition for common diseases like diabetes, Alzheimer’s disease, Parkinson’s disease and even aging. Although the causal relationships between certain mutations in mtDNA and their corresponding diseases were reported decades ago, cures have not been realized due to the difficulty in accessing the sub-cellular structures in vivo. Also,

CRISPR technology has not been applicable to mitochondria until recently and corresponding gene therapy of mtDNA still remains challenging in humans. The major challenge of mitochondrial gene editing is the lack of DNA transformation approaches for human mitochondria. No selectable marker gene has been developed that enables

mitochondrial transformation in human cells. As part of our efforts for mitochondrial transformation in crop plants, we have used a novel selectable marker gene to develop a method that has shown good efficacy in rice and yeast. In this proposed project, we will apply this method to human mitochondria. A successful demonstration will be a major advancement

for the application of our organelle gene editing technology to human mitochondrial DNA, which will facilitate future gene therapy treatments for the repair of mtDNA mutations in patients suffering from severe mitochondrial diseases.