Substantial evidence exists to support the hypothesis that oxidatively-damaged mitochondrial genomes contribute to the pathophysiology of aging and neurodegeneration. Damaged mitochondrial genes and mitochondrial oxidative stress contribute to or are primarily causal in bioenergetic failure of tissues, premature cell death of non-mitotic muscle cells and neurons and premature cell senescence through telomere dysfunction in dividing tissues. This STTR project further develops novel technologies of mitochondrial genome transfection ("mitofection") and mitochondrial genome removal ("mitoclean") towards commercialization. Three Aims will be addressed. In Aim 1 mitofection technology will be optimized with respect to transfection parameters. In Aim 2, the feasibility of combined RNAi-based gene silencing and mitofection to replace pathogenic mtDNA with wild-type mtDNA and correct abnormal metabolic phenotype will be demonstrated. In Aim 3, RNAi-based gene silencing will be used to produce mtDNA-free (rho0) lines of human primary neurons and fibroblasts that will serve as unique platforms for therapeutic development. This group has already demonstrated the feasibility of both major technologies. The outcome of this proposal will be a unique ability to manipulate the entire mitochondrial genome in mitochondria of cells. From this will develop methodologies for mitochondrial gene replacement therapies to reverse deleterious effects of aging and to prevent neurodegeneration. The results of this Phase I STTR will yield results that can readily be expanded into a Phase II proposal. Both "Mitofection" and "Mitoclean" are technologies that are scientifically compelling and applicable to multiple tissues, neural and non-neural. The interaction between Gencia and University of Virginia CSND is already well established and productive. [unreadable] [unreadable] [unreadable]