Abstract
Mutations in the mitochondrial DNA are the cause of several debilitating neuromuscular disorders, for which there currently are no effective treatments or cures. Gene therapies are a promising method to treat, and potentially even cure, these diseases. The focus of this study is to characterize the efficacy of a novel DNA-editing enzyme called a meganuclease. Precision Biosciences has patented a method in which they can modify the meganuclease to recognize novel target sequences. This gene editing platform is named ARCUS. In the first aim, I used a mitochondrial-targeted meganuclease (mitoARCUS) specific to the mouse m.5024C>T point mutation to eliminate mutant mtDNA in Mouse Embryonic Fibroblast (MEF) cell culture. The therapy effectively eliminated mutant mtDNA and improved cellular respiration. In the second aim, I used Adeno-Associated Virus (AAV) serotype 9, which has broad tropism including the liver and skeletal muscles, to eliminate mutant mtDNA in these tissues. I found that AAV9-mitoARCUS was effective at eliminating nearly all mutant mtDNA in liver in both juvenile and adult mice. However, younger mice had a more robust elimination of mutant mtDNA in skeletal muscle. In the third aim, I used AAV-PHP.eB to transduce the brain with mitoARCUS. Initially, I found that high titers of treatment caused detrimental effects in mutant and wildtype mice. It appeared that the mitoARCUS lost its specificity in high titers. Based on these results, I diluted the treatment to find the optimal dose of the gene therapy, where mutant mtDNA could be significantly eliminated without causing mtDNA depletion. Together, these findings showed that mitoARCUS is a promising new treatment option for heteroplasmic mitochondrial diseases.