PROJECT SUMMARY/ABSTRACT Currently, there are no FDA-approved treatments for mitochondrial diseases, which affect approximately 1 in 4000 children in the U.S. To address this unmet medical need, I propose to apply a novel technology, random shRNA selection, developed in my lab. Using a pooled approach, we can perform unbiased screens of 3 million bioactive molecules (shRNAs) in a single tissue-culture dish, selecting for beneficial phenotypes and retrieving “hit” shRNA-encoding sequences by PCR. Our previous studies demonstrate that we can combine our random shRNA-screening approach with gene-expression and bioinformatic analyses to identify conventional chemical-compound therapeutic candidates that recapitulate the phenotypes induced by our hit shRNAs. I focus herein on two of most common mitochondrial diseases, MELAS (Mitochondrial Encephalopathy, Lactic acidosis, and Stroke-like episodes) and LHON (Leber Hereditary Optic Neuropathy), cell lines for which exhibit a slow-growth phenotype in galactose-based media. We have already identified three shRNA clones that reverse this phenotype in a MELAS cell line. Our Specific Aims are to identify candidate drugs and drug targets for MELAS and LHON, respectively, using random shRNA selection. We will complete our MELAS screen and initiate a random shRNA screen in LHON cells. We will then (i) optimize hit shRNAs by random mutagenesis and re-screening; (ii) identify drug and drug-target candidates from gene-expression and bioinformatic analyses of optimized hits; and (iii) confirm drug and drug-target candidates in additional cell-culture models for reversal of the slow-growth phenotype in galactose-based media and for improvement in mitochondrial function. Our approach represents a new and substantive departure from conventional approaches to drug and drug-target discovery and therefore has the potential to identify effective therapeutics for MELAS and LHON that would not be identified in any other way. The envisioned products of this research are: 1) small-RNA therapeutic candidates, 2) conventional chemical-compound therapeutic candidates, 3) drug-target candidates, and 4) elucidation of disease mechanisms, which may inform additional therapeutic initiatives. Drugs and drug targets confirmed in our cell-culture models will be considered for additional testing, including medicinal chemistry, animal testing, and further clinical development. We expect that our results will serve as a proof-of- concept for additional mitochondrial disorders in addition to MELAS and LHON, including Leigh syndrome, NARP, MNGIE, MERRF, and KSS, cells and cell lines for which are readily available here in our Center for Mitochondrial and Epigenomic Medicine. It is possible that drugs and drug targets discovered in this project will be useful for one or more of these disorders, which we can determine using the cells and cell lines available to us. Given the current lack of effective therapies for mitochondrial diseases, and t...