In the last granting period, we proposed to create and analyze cellular models of mitochondrial disorders due to pathogenetic mutations in mtDNA-encoded subunits of the respiratory chain, with the ultimate goal of developing a gene therapy approach to treat such disorders. We focused on the mtDNA-encoded ATPase 6 subunit of complex V (ATP synthase), as it is the locus for mutations causing two related disorders, MILS (maternally inherited Leigh syndrome) and NARP (neuropathy, ataxia, and retinitis pigmentosa). We have made significant progress towards this goal. We now propose two specific aims to follow up on this work. First, we will continue our efforts to develop a genetic approach to treat these disorders in cellular models of MILS/NARP. Using a strategy called """"""""allotopic expression"""""""", we """"""""recorded"""""""" the ATPase 6 gene to contain the universal (i.e. nuclear) genetic code, added a mitochondrial targeting sequence to the recorded gene, and transferred this construct to the nucleus. We successfully demonstrated allotopic expression in transient assays and showed that the recoded polypeptide is targeted to mitochondria. We will now devoted our main efforts to improving the efficiency of mitochondrial importation, and to developing stably transfected cell lines expression our constructs. If successful, allotopic expression of the recoded wild-type gene, which has been engineered to be targeted and imported into mitochondria, should ameliorate the effects of the ATPase 6 mutation in mutant cells. Second, we will begin to devise a pharmacological approach to treat these disorders, based on our recent finding that treatment of heteroplasmic cells containing the MILS/NARP mutation with a complex V-specific inhibitor (oligomycin) in medium containing galactose results in a rapid and stable shift in heteroplasmy in favor of wild-type mtDNAs, with a concomitant improvement in mitochondrial function. We will now ask if the same shift can occur in heteroplasmic postmitotic MILS/NARP (myotubes), and also ask if this type of strategy can be generalized to mitochondrial deficiencies in other respiratory complexes. Finally, we will begin to assess the pharmacological ramifications of using oligomycin (and related compounds) in cells and in mice, as a prelude to considering a treatment protocol in MILS patients.
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