Author ORCID Identifier
Year of Publication
Doctor of Philosophy (PhD)
Dr. Daniel Pack
Gene therapy is currently being studied as a treatment for a variety of indications, including cancer, infectious disease, and cardiovascular diseases, among others. While many of the early treatments in the field involved the use of viral delivery methods, various safety, ethical, and financial concerns limit the potential uses of this methodology. As such, more recent research has focused on developing non-viral delivery platforms to alleviate some of the issues inherent in viral delivery. Recently, the release of the COVID-19 vaccines from Pfizer and Moderna represents a promising use of non-viral delivery as both utilized a lipid-based delivery vector.
Despite the ease of production and adaptability of recent non-viral platforms, these vectors suffer when compared to their viral counterparts in terms of delivery efficiency. This is due to the various biological barriers facing any gene delivery method. While viruses have had millennia to evolve and adapt to these barriers, non-viral vectors still face issues with circulation times, gene encapsulation, cell-surface interactions, internalization, endosomal escape, intracellular trafficking, and nuclear release of the gene payload. Previous research has revealed that the surface charge of certain delivery vectors can affect various of the aforementioned barriers. To further improve upon the knowledge of charge effects, the following thesis outlines the effects of adding folate receptor-targeting to negative polyplexes.
Chapter 1 provides background information on various gene delivery vehicles and folic acid. Chapter 2 begins with the generation and characterization of a folate-tagged polymer for use in generating ternary polyplexes for gene delivery. Early studies utilizing this targeting method show folate tag concentration affects overall gene delivery efficiency, with a lower tag ratio outperforming the highest folate concentration. Chapter 3 then moves on to investigate the effect of folate receptor-targeting on internalization pathway. In a folate receptor-positive cell line, the folate-tagged polyplexes are shown to divert particles down the expected caveolar endocytic pathway, while folate receptor-negative cells show non-specific uptake of particles. Chapter 4 explains an investigation into the intracellular trafficking effects of folate-tagged polyplexes. Both folate receptor-positive and -negative cell lines show strong reliance on an active transport pathway within the cell to reach the nucleus. Finally, chapter 5 includes a prospective on future directions for both this continued project as well as non-viral vectors as a platform.
The goal of this work was to expand upon the benefits of negatively charged ternary polyplexes by utilizing a targeting methodology to improve overall delivery efficiency of the resulting particles. The evolution and continued adaptation of non-viral vectors will be key to developing a true competitor to viral gene delivery. Future improvements upon this platform will include incorporation of immune-shielding methodologies or the alteration of the ligand linkage to generate a potentially improved targeting effect.
Digital Object Identifier (DOI)
Akers, Caleb, "Investigation of Folate-Poly(Glutamic Acid)/Polyethylenimine/DNA Complexes for in vitro Gene Delivery" (2023). Theses and Dissertations--Pharmacy. 155.