Creation of animal models of mitochondrial DNA disease for translational research applications

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 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 GM 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 ND 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 RNAs will be transduced using piggyBac PB based vectors into C BL 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 ND protein downregulation in MT TI and ND 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 ND 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 ND germ line chimeras will be intercrossed to wild type females to generate dpc embryos and neonates for derivation of primary fibroblast and cardiac cultures to assess ASRE activity Criteria for success For ASRE MT TI andgt knockdown of MT TI expression with no off target ablation of mtRNA expression global downregulation of mt DNA encoded proteins and andgt decrease in Complex I II and IV activities For ASRE ND andgt knockdown of MT ND expression with no off target ablation of mtRNA expression andgt decrease in ND protein expression and andgt 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 Collectively millions of individuals are affected by mitochondrial DNA disease in have or are at risk for mtDNA disease for which there are no curative treatments There are two roadblocks that have limited the development of curative therapies a the absence of cell line models that accurately recapitulate the human disease state to screen identify drug candidates and b the absence of pre clinical models of mitochondrial DNA disease to assess the in vivo efficacy of drug candidates In this proposal Enzerna seeks to develop mouse stem cell and animal models of mitochondrial DNA disease to provide investigators valuable and innovative translational models to screen for and test curative therapies