Date of Award
Summer 7-14-2025
Embargo Period
7-14-2029
Document Type
Dissertation - MUSC Only
Degree Name
Doctor of Philosophy (PhD)
Department
Oral Health Sciences
Additional Department
Cell and Molecular Biology and Pathobiology
College
College of Dental Medicine
Additional College
College of Graduate Studies
First Advisor
Andrew Jakymiw
Abstract
The development of efficient, stable nucleic acid delivery vehicles remains a critical challenge in gene therapy, as many systems fail to achieve targeted uptake, protect cargo from degradation, or release it effectively inside cells. This dissertation addresses these limitations by investigating RD3AD, a novel peptide carrier designed to enhance nucleic acid delivery through natural cellular entry pathways. We demonstrate that RD3AD significantly improves siRNA uptake, stability, and gene silencing by exploiting filopodia-mediated transport. RD3AD-siRNA complexes displayed highly ordered binding along filopodia, facilitating efficient transport into cells. In 2D and 3D models of HPV+ oropharyngeal squamous cell carcinoma, RD3AD delivery of siRNAs targeting the oncogenic E6 and E7 genes achieved robust silencing, p53 restoration, and disruption of tumor spheroid architecture, addressing the need for less toxic, targeted treatments for HPV-driven cancers.
Beyond siRNA, RD3AD was evaluated for delivering miRNA, mRNA, plasmid DNA (pDNA), and mitochondrial DNA (mtDNA) to meet the demand for versatile nucleic acid carriers. While it effectively bound and protected small nucleic acids, release of mRNA and pDNA remained suboptimal, underscoring the need for further peptide optimization. In mitochondrial therapeutics, where no clinically viable method exists to restore the full mtDNA molecule, RD3AD enabled intracellular delivery of intact, functional mtDNA, restoring mitochondrial gene expression and protein synthesis. This addresses a critical gap in the treatment of mtDNA-associated diseases. Collectively, this work establishes RD3AD as a non-toxic, stable, and adaptable nucleic acid carrier with broad therapeutic potential. Future studies will optimize trafficking, cargo release, and in vivo validation to advance RD3AD toward clinical translation.
Recommended Citation
Holjencin, Charles E., "An Investigation into Filopodia-mediated Cellular Entry and Diversification of Payload Capacity of a Cell-penetrating Peptide System for Expanded Gene Therapy Capabilities" (2025). MUSC Theses and Dissertations. 1081.
https://medica-musc.researchcommons.org/theses/1081
Rights
Copyright is held by the author. All rights reserved.