Mitochondria are the principal generators of cellular ATP by oxidative phosphorylation which links electron transport via Complexes I through IV to production of ATP via ATP synthase. Over 250 pathogenic human mitochondrial DNA (mtDNA) mutations, associated with diseases ranging from cardiomyopathy to juvenile blindness, have been identified. Collectively, millions are affected by mtDNA disease for which there are no curative treatments. The absence of (a) cell line models that accurately mimic the human mtDNA disease state for drug discovery, and (b) pre-clinical disease models to assess in vivo efficacy of drug candidates have hampered the development of curative therapies. In an SBIR-funded project (GM117965), Enzerna is using Artificial Site- Specific RNA Endonucleases (ASREs), engineered to be transported into mitochondria to mediate inducible ablation of specific mt-RNAs, to create cell line models of mtDNA disease to screen for therapeutic candidates. Here, Enzerna will seek to address the second unmet need for mtDNA disease drug development ? the creation of mtDNA disease models to assess the in vivo efficacy of potential therapeutics. Given that most pathogenic mtDNA mutations act recessively, we will develop ?transgenic? ASRE knockdown stem (ES) cell models that can be used as in vitro (after directed differentiation) or in vivo models for drug discovery. ASREs targeted against the tRNA gene, MT-TI (tRNA isoleucine) and ND5 genes, both associated with cardiomyopathy and defects in cardiac mitochondrial function, will be transduced into mouse ES cells. Cumate?inducible ASREs targeted against MT-TI or ND 5 RNAs will be transduced using piggyBac(PB)-based vectors into C57BL6 ES cell lines and selected for puromycin resistance. Pools of puroR ES cells and ES cell-derived cardiomyocytes will be induced with cumate and screened for: (a) ASRE localization to mitochondria; (b) RNA expression of targeted locus and other mt-RNAs to assess specificity; (c) Western blotting to assess global downregulation of mtDNA- encoded proteins and ND5 protein downregulation in MT-TI and ND5 transduced cells, respectively; and (d) Complex I, II and IV activities. We will seek to demonstrate that ASREs are active in mice generated from transduced ES cells. Pools of ASRE(MT-TI) and ASRE(ND5) transduced ES cells will be injected into host blastocysts to generate germ line chimeric animals; four independent transgenic lines will be established for future studies. In parallel, ASRE(MT-TI) and ASRE(ND5) germ line chimeras will be intercrossed to wild type females to generate 13.5 dpc embryos and neonates for derivation of primary fibroblast and cardiac cultures to assess ASRE activity. Criteria for success: For ASRE(MT-TI), >75% knockdown of MT-TI expression with no off- target ablation of mtRNA expression, global downregulation of mt-DNA encoded proteins, and >50% decrease in Complex I, II, and IV activities. For ASRE(ND5), >75% knockdown of MT-ND5 expression with no off-target ablation of mtRNA expression, >50% decrease in ND5 protein expression, and >50% decrease in Complex I activity. With the development of two models that carry mtDNA knockdown mutations, we (in collaboration with Dr. Brown) will expand ongoing studies to develop therapeutics for mtDNA-associated cardiomyopathies in Phase II. Furthermore, the establishment of a general platform for ASRE-mediated knockdown in ES cells will enable Enzerna to build a bank of inducible and tunable ?knockdown? models for each mtDNA gene, which would provide the field with valuable and innovative translational models to screen for and test curative therapies.