Date of Award
Spring 3-26-2025
Embargo Period
3-26-2030
Document Type
Dissertation - MUSC Only
Degree Name
Doctor of Philosophy (PhD)
Department
Molecular and Cellular Biochemistry and Pathobiology
Additional Department
Ophthalmology
College
College of Graduate Studies
First Advisor
Bärbel Rohrer
Abstract
Mitochondrial dysfunction, along with widespread accumulation of mitochondrial (mt)DNA damage, contributes to the development of a range of diseases. Thus, treatments targeting one of the underlying mechanisms of mitochondrial dysfunction have the potential to be applicable to a variety of diseases, from neurodegenerative disorders to ocular pathologies to enhancing cancer treatments. While there are a variety of therapies and supplements being investigated to restore mitochondrial function, no cure for mitochondrial dysfunction/disease has been developed. Gene therapy approaches for mtDNA have thus far been limited, largely due to the heteroplasmy of mtDNA mutations observed within and between patients. Despite advancements in single gene therapy for diseases with specific mtDNA mutations and progress in mitochondrial transplantation, no method exists for restoring the entire mtDNA molecule in a clinically translatable manner. In nature, mtDNA can be restored in damaged cells via transfer of intact mtDNA within extracellular vesicles, but this method is not clinically controllable. Here, we present for the first time a strategy to deliver an exogenous, fully intact, and healthy mtDNA template into cells to provide a template for replication of undamaged mtDNA and synthesis of functional mitochondrial proteins. We tested nanoparticle formulations for: 1) their ability to deliver exogenous mtDNA to retinal pigment epithelial (RPE) cells; 2) the long-term (i.e. greater than 4 weeks) retention of the delivered mtDNA; and 3) the functionality of the retained mtDNA, that is, its ability to fulfil the central dogma and produce RNA/protein. The final prototype was created by combining a cell-penetrating peptide (CPP) with purified mtDNA, in conjunction with a mitochondrial targeting reagent. The generated nanoparticle complexes were taken up by cells and successfully localized to mitochondria. In mitochondria-depleted ARPE-19 cells, exogenously delivered mtDNA was retained for at least four weeks following a single mtDNA nanoparticle treatment, with evidence of mitochondrial RNA and protein production. These data demonstrate the feasibility of restoring mtDNA in cells, with the therapeutic potential to correct mtDNA damage independent of the number of gene mutations found within the mtDNA.
Recommended Citation
Wilson, Kyrie, "Development of a Novel Approach to Mitochondrial Gene Therapy: Nanoparticle Mediated Delivery of Intact, Functional Mitochondrial DNA" (2025). MUSC Theses and Dissertations. 1061.
https://medica-musc.researchcommons.org/theses/1061
Rights
Copyright is held by the author. All rights reserved.