Genetic aberrations in the mitochondrial genome (mtDNA) can often manifest as clinical pathologies. Along with genetic mutations in nuclear encoded mitochondrial genes, these pathologies form a group of genetic disorders referred to as mitochondrial diseases. Engineered mitochondrially targeted zinc finger nucleases (mtZFNs) have been successfully used to selectively degrade mutation bearing mtDNA both in vivo and in vitro, resulting in a shift in the genetic makeup of affected mitochondria and subsequently to phenotypic rescue. Due to an uneven distribution in the mtDNA mutation load across tissues in patients, as well as a great diversity in pathogenic mutations, it is of interest to develop selective gene therapy techniques that could be delivered to a particular affected tissue and curated for specific mutations. Specifically, many mitochondrial diseases are characterized by dysfunctions of the muscular and/or central nervous systems (CNS). This thesis demonstrated the effectiveness of in vivo mitochondrial gene therapy using mtZFNs in targeting clinically relevant tissues delivered using an adeno-associated viral (AAV) platform to a murine model harboring a pathogenic mtDNA mutation. The work demonstrated effective reduction in mutation load in skeletal muscle, which was accompanied by molecular phenotypic rescue. The gene therapy treatment was shown to be safe when markers of immunity and inflammation were assessed. This work subsequently explored refinements in design methods for mtZFNs using both rational design and directed evolution assays for a novel murine model, with the later method being novel. Lastly, the work demonstrated the phenotypic rescue of a homoplasmic cell line derived from this novel murine model, which was previously an incorrigible genetic defect prior to the advent of mitochondrial base editing. In particular, the combined use of base editing and nuclease treatments showed greater reduction in mutation burden as well as a reduction in the off-target effects associated with base editing. In summary, this work expanded the potential clinical scope of mitochondrial gene therapy, demonstrating effectiveness and safety in vivo, improving the design capabilities for therapeutic nucleases and enabling therapy on homoplasmic cells for the first time.