Project Summary/Abstract The ~sixteen-thousand nucleotides of the mitochondrial genome play an outsized role in human health. This gene-dense, circular genome is contained, replicated, transcribed, and regulated independently from our nuclear genome. Its contents are critical to the function of nearly every cell in our body, so when a nucleotide is mutated or lost, physiological processes in cells break down. Mutations to the mitochondrial genome cause diseases often associated with degeneration of central nervous system, heart, and muscle. One in ~five-thousand people inherits a disease-causing mutation to the mitochondrial genome (e.g. Leber’s hereditary optic neuropathy), but that likely underestimates the total burden of disease, as one in ~two-hundred people carries a suspected pathogenic mutation. We do not understand the ramifications of all mitochondrial mutations, which are often tissue-specific and complex to diagnose. Moreover, the mitochondrial genome accumulates mutations throughout the life of an individual, which are thought to play a role in degenerative diseases like Parkinson’s and even normal cellular aging. Unlike the nuclear genome, where emerging technologies to re-write DNA in living cells have enabled breathtaking experimental insights and will soon enable precision genetic medicine, the mitochondrial genome has remained nearly untouched by scientists. The inner membrane of the mitochondria is impermeable to polynucleotides, so template-based repair or augmentation with new DNA is out of reach. This means we can only manage, and never truly cure an individual with a disease that stems from a mutation to their mitochondrial DNA. It also means that we cannot experimentally introduce precise mutations to the mitochondrial DNA to test the effect of particular changes under controlled conditions. The research contained in this proposal is aimed at overcoming these limitations. We will test means of engineering mitochondria to enable active transplantation of mitochondrial populations into living cells as a therapeutic in diseases of the mitochondrial genome. We will assess therapeutic efficacy of mitochondrial transplantation in models of degenerative diseases such as Leber’s hereditary optic neuropathy. We will also modify the genome of the transplanted mitochondria to introduce or fix mutations, allowing for phenotypic analysis in a controlled experimental framework. Completion of this work will yield new opportunities to treat intractable disease and a new molecular paradigm to investigate a biological system that is critical to all cells.