Mitochondria are essential organelles for mammalian cells. They produce energy (ATP) and TCA cycle metabolites for biosynthetic processes, regulate intracellular Ca2+ flux and Fe-S cluster synthesis, and initiate apoptosis. To assemble a mitochondrion, proteins and RNAs encoded by both the mitochondrial (mtDNA) and nuclear (gDNA) genomes are required. In humans, only 13 of >1,000 proteins that comprise a mitochondrion are encoded within maternally inherited ~16.6kb mtDNA, but these 13 proteins are essential components of the electron transport chain that enables cellular respiration. Mutations in mtDNA affecting the translation, assembly, or function of these 13 proteins results in > 200 named mitochondrial disease syndromes that affect high energy organs such as the brain, muscle, or heart and often result in early death. Unfortunately, there are no effective therapies or supportive measures for mtDNA diseases. The main hope is to eventually correct or compensate for deleterious mtDNA mutations. However, an almost complete field block exists for altering mtDNA, in contrast to comparatively ready access for altering gDNA sequences. Numerous labs are trying to develop mitochondrial reverse genetics, in which altering mtDNAs generates phenotypes for study. However, current approaches are inefficient, poorly controlled stochastic processes often with ethical concerns over the cell source materials. Several labs have managed to isolate, modify, and re-introduce altered mtDNA back into mitochondria in vitro and shown transcriptional activity, strongly suggesting assembly into nucleic acid-protein aggregates called nucleoids. However, there is no way to reintroduce these mtDNA engineered mitochondria back into cells for functional, system-wide studies. Here, we propose to enable mitochondrial reverse genetics and provide an initial approach for correcting devastating mtDNA mutations. In a longstanding collaboration, the Chiou and Teitell labs invented a photothermal nanoblade that can transfer native or engineered mitochondria into mammalian cells and rescue defects in cellular respiration. However, the skill required, slow speed, and bulk system size of our current nanoblade leads to many failed experiments and precludes wide adoption of this approach. To overcome these inhibitory issues, we propose 3 specific study aims.
In Aim 1, we will generate a high throughput, compact, microfluidic platform for massively parallel mitochondrial delivery that we call BLAST.
In Aim 2, we will deploy BLAST to generate or correct specific mtDNA mutations that cause 3 human disease syndromes with native mitochondrial transfers. And in Aim 3, we will alter mtDNA and utilize BLAST to generate hybrid cell lines by transfer of in vitro modified mitochondria back into cells for thorough evaluation of functional activity, including system-wide carbon tracing studies that have been impossible to perform. Combined, our engineering and molecular biology cross-disciplinary approaches will enable the targeted alteration of mtDNA for both fundamental, basic studies and the beginnings of future translational applications in mitochondrial medicine.
This proposal is relevant to public health because it will open a blocked translational field by enabling the purposeful sequence alterations of maternally inherited mitochondrial DNA (mtDNA) to overcome or compensate for mutations that cause > 200 named human disease syndromes. In addition, success in this proposal will also enable so-called mitochondrial reverse genetics, which would allow new knowledge for how specific sequence changes in mtDNA affect cell functions including respiration, metabolism, survival, and proliferation. Our preliminary studies support a wealth of new, unexpected findings from these studies as we have already discovered that retrograde signaling from mitochondria with repaired mtDNA can correct a defect in the translation or stability of a metabolic enzyme that is encoded within the nucleus of a cell, to fix integrated cellular circuits that control cell metabolism.
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