In the last five years, mutations of the mitochondrial DNA (mDNA) were identified in several neuro-ophthalmological diseases. However, the detailed molecular events associated with the pathology of these untreatable disorders are poorly understood. The first goal of the present application is to study the molecular pathogenesis of five different mDNA mutations recently identified in patients with mitochondrial disease. Four of these pathogenic mutations are point mutations in mitochondrial tRNA genes (tRNA for Leu, Asn, Pro, and Glu) while the fifth is a 260 bp duplication of the mtDNA regulatory region. These studies will be performed in trans-mitochondrial cybrids cell lines, generated by the fusion of an established mtDNA-less cells (termed p o cells) with enucleated patient's cells. The trans-mitochondrial cybrid system has several advantages over primary cell lines for the study of mtDNA mutations, the most important being their immortal phenotype. Trans-mitochondrial cybrids containing 100% mutated, 100% wild-type, and different percentages of both mtDNAs, will be studied for organelle protein synthesis, mitochondrial transcription, mtDNA replication, RNA processing, and tRNA aminoacylation. Characterized trans-mitochondrial cell lines will also be used as model systems to test the feasibility of expression of mitochondrial genes in the nucleus, the second major goal of this application. To express mitochondrial genes in the nucleus, they have to be extensively modified by site-directed mutagenesis. This is necessary in order to adjust the mitochondrial genetic code to the universal genetic code. Moreover, re-coded genes will need a mitochondrial targeting leader peptide to reach their functional location. Re-engineered genes will be transfected into the nucleus of cultured cells, and their expression, as well as the sub- cellular targeting and functional assembly of their protein products monitored. The knowledge accumulated by the studies described above should help set the stage for future gene replacement therapies for mitochondrial disorders.
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