Millions of individuals are affected by diseases caused by mutations in mitochondrial-encoded genes which lead to a diverse set of clinical disorders ranging from visual impairment (optic neuropathy) to accelerated aging to hypertrophic cardiomyopathy. Unfortunately, there are no commercially available technologies that can enable investigators to dissect the function of these genes. With several thousand mitochondrial genomes per cell, combined with the impermeability of the mitochondria to nucleic acids, it is not possible to create gene knockout mutations in all copies of the mitochondrial genome or to effectively ablate mitochondrial gene expression using RNA interference technology. Because investigators cannot dissect the function of mitochondrial-encoded genes, it has been difficult to develop effective therapies for diseases caused by mutations in the mitochondrial genome. In this proposal, we outline a new enzyme- mediated method to ablate mitochondrial DNA-encoded RNA expression using Artificial Site-Specific RNA Endonucleases (ASREs), which can be engineered to be transported into every mitochondrion where these ASREs can bind to and destroy specific mitochondrial RNAs. The distinguishing feature of ASREs is the presence of an RNA binding domain (PUF domain) which consists of a series of ~36 amino acid modules that recognize one ribonucleotide that can be arranged in an array to recognize specific nucleotide sequences. By combining ASRE ablation of a target gene (phenocopy of a knockout mutation) with the expression of corresponding human disease allele integrated into the nuclear genome, unique cell culture models of human mitochondrial diseases can be generated. To assess feasibility of this approach, Enzerna Biosciences, in collaboration with the company co-founder and inventor of the technology (Dr. Zefeng Wang), will create ASREs to ablate expression of the mitochondrial Complex I ND1 and ND5 genes using drug inducible expression systems in which the ASREs are integrated into defined transcriptionally active regions of the nuclear genome using homologous recombination based strategies. Success will be indicated by the identification of ND1 or ND5 ASRE targets that mediate >90% reduction in RNA/protein expression accompanied by >80% reduction in Complex I functional activity. Second, we will create cell lines that in which mito-ND1 expression is ablated while a wild type (wt) ND1 or mutant ND1 [ND1 (G3460A)] gene associated with optic neuropathy is simultaneously expressed to replace the ablated mito- ND1 protein. Success will be indicated by rescue of ND1 protein expression in the mitochondria, rescue of functional activity by the expression of the wt allele in the presence of ASRE-mediated mito-ND1 knockdown, and inability of mutant ND1 protein expression to rescue Complex I functional activity. In Phase 2, once proof-of concept is demonstrated, we will generate a bank of cell lines in which the expression of each mitochondrial gene is ablated. In the long term, this bank of mitochondrial gene knockout cell lines will open new opportunities to examine the molecular and cellular mechanisms of mitochondrial disease and provide valuable models for the development of novel therapeutic agents for intervention.
Although millions of individuals in the US and throughout the world are affected by diseases caused by mutations in mitochondrial-encoded genes, there are no technologies that enable investigators to dissect the function of these genes. With over 1000 mitochondria per cell, with each containing at least two genomes per mitochondrion combined with the impermeability of the mitochondria to nucleic acids, there are no commercially available technologies to dissect the function of each gene of the mitochondrial genome. As a result, it has been difficult to develop effective therapies for diseases caused by mutations in the mitochondrial genome. In this proposal, we outline a new enzyme-mediated method to ablate mitochondrial DNA-encoded RNA expression using Artificial Site-Specific RNA Endonucleases (ASREs) which can be engineered to be transported into every mitochondrion and bind to and destroy specific mitochondrial RNAs. Combining ASRE ablation of target genes with the expression of human disease (variant) alleles, unique cell culture models of human mitochondrial diseases, associated with mitochondrial-encoded genes, can be generated, which will open new opportunities to examine the molecular and cellular mechanisms of disease and provide valuable models for the development of novel therapeutic agents.
