Optimized Mitochondrial Targeting of Proteins Encoded by Modified mRNAs Rescues Cells Harboring Mutations in mtATP6.

Mitochondrial disease may be caused by mutations in the protein-coding genes of the mitochondrial genome. A promising strategy for treating such diseases is allotopic expression-the translation of wild-type copies of these proteins in the cytosol, with subsequent translocation into the mitochondria, resulting in rescue of mitochondrial function. In this paper, we develop an automated, quantitative, and unbiased screening platform to evaluate protein localization and mitochondrial morphology. This platform was used to compare 31 mitochondrial targeting sequences and 15 3' UTRs in their ability to localize up to 9 allotopically expressed proteins to the mitochondria and their subsequent impact on mitochondrial morphology. Taking these two factors together, we synthesized chemically modified mRNAs that encode for an optimized allotopic expression construct for mtATP6. These mRNAs were able to functionally rescue a cell line harboring the 8993T > G point mutation in the mtATP6 gene.

Patient-derived lymphoblastoid cell lines harboring mitochondrial DNA mutations as tool for small molecule drug discovery.

OBJECTIVE: Mitochondrial diseases are a group of devastating disorders for which there is no transformative cure. The majority of therapies for mitochondrial disease-approved, previously tested, or currently in development-are small molecules. The implementation of better cell-based models of mitochondrial disease can accelerate and improve the accuracy of small molecule drug discovery. The objective of this study is to evaluate the use of patient-derived lymphoblastoid cell lines for small molecule research in mitochondrial disease. RESULTS: Five lymphoblastoid cell lines derived from mitochondrial disease patients harboring point mutations in mtND1, mtND4, or mtATP6 were characterized in two high throughput assays assessing mitochondrial function. In a pilot "clinical trial in a dish" experiment, the efficacy of idebenone-an approved therapy for mitochondrial disease-on the lymphoblastoid cell lines was tested. Idebenone increased the basal respiration of all lymphoblastoid cell lines except those harboring the 8993T>G point mutation in mtATP6. Our results posit lymphoblastoid cell lines as a strong model for mitochondrial disease research with small molecules and have implications for the clinical efficacy of idebenone.