Setting the Stage for Replacement of Mitochondrial Genes

Project Summary We propose to develop a monomeric DNA editing platform to eliminated mutant mtDNA (mitoARCUS). In collaboration with Precision Biosciences, we will develop and characterize a new class of mitochondrial editing enzymes that are smaller and simpler than TALENs, while still highly specific for the mutant mtDNA. This platform is based on the I-CreI homing endonuclease, which was modified to work as a monomer, becoming more amenable to viral packaging and delivery in a clinical setting. We plan to characterize mitoArcus for three pathogenic mtDNA mutations, including: a) Human 4.9kbp Common deletion; b) Human MELAS tRNALeu(UUR) m.3243G, d) the mouse tRNAAla m.5024T. Once the mitoArcus enzymes are characterized in established cell lines, we will test their ability to change mtDNA heteroplasmy and restore function to clinically-relevant models. These will include human motor neuron organoids ex vivo and and the mouse retina in vivo. Finally, we will test a newly described base editing tool (DdCBE) in an in vivo model. This new base editor architecture (DdCBE), specifically edit thymidines on mtDNA. This was possible, because this architecture is not based on the CRISPR, thereby not requiring a gRNA component that cannot be easily imported into mitochondria. In collaboration with David Liu's lab at Harvard/Broad, we will test whether mtDNA base editing works in a mouse model harboring a pathogenic mtDNA mutation in the tRNAAla gene.