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A.S. Lewin, X. Qi, J. Guy; Reduction in Expression of a Gene Encoded by mtDNA: A Potential Model System for LHON . Invest. Ophthalmol. Vis. Sci. 2003;44(13):634.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Last year we showed that ribozymes designed to reduce the expression of a critical subunit gene of complex I (NDUFA1) encoded by nuclear DNA killed cultured cells and, when injected into the eyes of mice, led to the pathologic hallmarks of Leber Hereditary Optic Neuropathy (LHON)--loss of ganglion cells, axons and myelin. We now propose to test whether a ribozyme can be used to reduce expression of a mitochondrial gene. Methods: We constructed a hammerhead ribozyme against cytochrome oxidase subunit II (COX II), a mitochondrially encoded gene. While mutations in COX have rarely been associated with LHON, mutations in mtDNA encoding complex I (NADH:ubiquinone oxidoreductase) subunit genes cause most cases of LHON. We chose COX II as a ribozyme target rather than a complex I subunit gene, since reductions in COX activity confer resistance to cyanide poisoning, thus increasing cell survival in KCN media. To import the COX II ribozyme into mitochondria we cloned the ribozyme downstream of the 5S rRNA gene. The hybrid gene was then re-cloned in AAV vector pTR-UF5. In this vector, expression of the 5S-ribozyme fusion was controlled only by the pol III promoter contained in the 5S gene. We infected human osteosarcoma cells and measured cell survival in 160 nM KCN, COX enzyme activity by the oxidation of reduced cytochrome c, expression of COX II by immunochemistry and immunobloting relative to mock-infected cells. Results: After four days of KCN selection, there was a four-fold increase in surviving cell numbers in COX II ribozyme inoculated cells compared to mock infection (p=0.02). When measured directly, there was a 70% reduction in cytochrome oxidase activity in RzCOX II transfected cells relative to mock infection (p=0.05). Using antibodies directed at COX II, we detected at least a 50% reduction in protein by immunobloting and found some cells totally devoid of COX II immunofluorescent labeling, although most retained some level of COX II immunofluorescence. Conclusions: Our results showing that mitochondrial gene expression can be substantially suppressed with a ribozyme opens the door for testing of the effects of reduced expression of other subunit genes encoded by mtDNA (ND4, ND1 or ND6) to elucidate the mechanism for the optic neuropathy of Leber.
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