PROJECT SUMMARY Mitochondrial diseases are caused by mutations in genes that encode structural mitochondrial proteins or proteins involved in mitochondrial function. Mitochondrial function abnormalities are among the most common genetic neurological disorders, making them an ideal therapeutic target. The growing knowledge of links between aberrant mitochondrial gene transcription and human diseases critically necessitates an effective approach to controlling mitochondrial DNA (mtDNA) transcription. To this end, developing a method to modulate mtDNA transcription sites specifically is vital for understanding and treating mitochondria-related diseases. However, the Inefficient delivery of DNA binding motifs into mitochondria and difficulty activating mitochondria genes with current technologies limit mitochondrial genome editing/gene manipulation. Advanced techniques for regulating mtDNA transcription have relied on the delivery of exogenous transcription factors, such as mitochondrial transcription factor A (TFAM), DNA oligomers, or DNA-base editing nucleases. Furthermore, translating these advanced tools into therapeutics would require substantial advances in their targeted delivery into mitochondria, as most mitochondrial transcription factors (TFs) and base editing tools face significant hurdles during circulation in blood or other biofluids. Therefore, there is an urgent need to develop novel methods to achieve selective and efficient gene activation in the mitochondria. Addressing the above challenges, the main goal of this proposal is to develop a nanoparticle-based synthetic mitochondrial DNA (mtDNA) transcription regulator to investigate mitochondrial dysfunction in neurodegeneration. The modular MitoScript platform will be assembled from: i) ultra-small fluorescent gold nanoclusters (NC) as scaffolds for assembly of biomolecular ligands; ii) synthetic PIP oligomers as mtDNA binding domains (DBDs) for site-specific mitochondrial transcription regulation; iii) mitochondria-penetrating peptides (MPPs) as mitochondrial localization domains; and iv) a mitochondrial transcription factor motif derived from TFAM as an activation domain (AD). By doing so, we aim to construct an artificial/synthetic mitochondrial TF that can: i) efficiently target mitochondria genes with no cytotoxic effects or immunogenic (e.g., viral vectors) carriers; ii) selectively bind to any target DNA sequences in the mitochondria genome, and iii) controllably downregulate and upregulate mitochondria genes that can eventually alter neural cell fates. We propose to objectively test our central hypothesis and achieve our objectives by addressing the following specific aims: AIM #1 − Construct ND6-targeting MitoScript (ND6-MitoScript) to regulate ND6 genes in mitochondria efficiently; AIM #2 − Validate ND6-MitoScript for ND6 gene overexpression and improved survival in PD patient iPSC- derived neurons; Collectively, we anticipate that our proposed studies will provide an innovative, highly effective, and selective method for developing therapeutic interventions for mtDNA-mediated neurological disorders.

PROJECT NARRATIVE Our proposed studies are relevant to public health, as developing a non-viral method to investigate mitochondrial dysfunction in neurodegeneration to facilitate CNS regeneration and prevent the pathophysiology of mitochondrial disease. Therefore, the proposed research is relevant to the part of NIH’s mission that relates to treating human illness and disability by investigating how innovative technology can safely translate to clinics.