Mutations in mitochondrial DNA (mtDNA) cause untreatable disease in ~1:5000 humans. There is a fundamental gap in studying mtDNA diseases due to a lack of faithful animal models. In particular, our complete inability to engineer the mitochondrial genome has prevented us from creating animal models containing the same deletions/mutations present in the human population. The key technical gap in mtDNA editing is our inability to introduce nucleic acids into the mitochondrion. Bridging this gap is extremely important in order to enhance our understanding of disease pathophysiology, as well as provide a platform for the development and testing of therapeutics. Our long-term goal is to understand the progression of mtDNA disease over an organism's lifespan, and develop appropriate therapeutics. The overall objective is to develop faithful mouse models of mtDNA disease which recapitulate the genetics and physiology seen in human patients. The central hypothesis is that engineering mtDNA deletions or mutations in mouse tissues should recapitulate key features of human mtDNA disease. Our rationale is that studying such models has the potential to enhance our understanding of disease progression in humans. This proposal utilizes multiple approaches to engineer mtDNA disease in mice. First, we will engineer a ?competency? system of mitochondria, utilizing evolutionarily conserved bacterial species as a model. By screening through the known competency machineries in bacteria, we will discover and design a protocol for introducing nucleic acids into mammalian mitochondria. In combination with CRISPR/Cas9 technology, this tool will allow the precise engineering of mtDNA for the first time. We envision this approach can be utilized to create animal models containing the same precise genetic mutations found in the human population. Second, we will utilize cybrid technology in order to create mouse cells containing human mtDNA. These cells can then be utilized to create genetic models with the complement of mtDNA from human patients. At the end of the project period, we will have created mice containing engineered mtDNA mutations from human patients. These approaches are innovative in that they attempt to recapitulate the genetics of mtDNA disease as it occurs in patients, thus departing from current models which utilize nuclear-gene knockout alleles to mimic mtDNA mutations. The research is significant in that it will create new animal models which recapitulate the genetics of human disease ? a characteristic lacking in currently available models. We expect the availability of these models to the scientific community will accelerate the development of effective therapeutics for patients.
The proposed research is relevant to public health as the availability of animal models with mitochondrial DNA mutations will increase our understanding of disease progression and pathogenesis, as well as provide a valuable and relevant platform for the testing of therapeutics. Thus, the research relates to the NIH's mission to foster innovative research strategies and their applications as a basis for ultimately protecting and improving health.
