Mitochondria are the power plants of the cell and are also the only cellular organelle that possess their own DNA in mammals. In humans, mitochondrial DNA (mtDNA) codes for 13 important proteins, which all assemble into the oxidative phosphorylation relay. Mutations in mtDNA occur as a consequence of constant exposure to reactive oxygen species produced by the mitochondrial energy generation process as well as mistakes in mtDNA replication. These mutations accumulate over time due to inefficient repair mechanisms and compromise respiratory chain function. Inherited and acquired mutations in mtDNA result in impaired energy generation and are the cause for several pathologies such as Leber’s hereditary optic neuropathy (LHON), Myoclonic Epilepsy with Ragged Red Fibers (MERRF), Kearns-Sayre syndrome and Leigh syndrome.

Using the allotopic approach, we have identified specific targeting elements/sequences that can improve expression of these essential genes from the nuclear DNA and their transport to the correct location in mitochondria. The Fellow selected will get the opportunity to design a library of constructs to attempt to rescue specific mtDNA mutations in model patient cell lines. The ability of re-engineered genes to rescue function will be evaluated through various techniques, such protein gels, qPCR and activity assays, with the potential of extending the studies to animal models. Time permitting, the Fellow will validate these constructs for expression from a safe harbor locus using the CRISPR/Cas9 and dual integrase mediated cassette exchange (DICE). Candidates with an interest in mitochondrial biology and with exposure to cell culture, molecular biology and protein biochemistry are encouraged to apply.