Drug designs have become increasingly complex and costly without improved outcomes for the patient. The solution is the implementation of precision medicine paired with comprehensive treatments that overcome the limitations of current therapies.
Extracellular vesicles (EVs) are cell membrane-derived particles that are a promising biologic to lead this change that have already proven to overcome many limitations of conventional molecular therapies. These particles are produced by all cells and have naturally evolved as delivery vehicles for cell-to-cell communication throughout the body, making them excellent candidates for drug delivery. Furthermore, EVs contain both surface bound and internalized functional molecules, useful for applications requiring molecular interaction (i.e., vaccines) or molecular delivery (i.e., therapeutic proteins or genes). While EVs have shown great promise in preclinical models, challenges in scalability and homogeneity have hindered their advancement in clinical translation.
To address these challenges, we developed chemical production methods which induced rapid membrane blebbing for the production of micro- and nano-scale EV-like particles, termed extracellular blebs (EBs). Chemically-induced production of EBs resulted in greater yields at significantly shorter timescales compared to traditional EV production methods. For the first time, production mechanisms and properties of EBs were investigated. These included investigation into proteomic, transcriptome, and molecular presentation properties at the bulk and single-particle level. These studies confirmed that EBs are particles composed of plasma membrane, cytoskeletal fragments, and soluble cytosolic elements, free of organelles. Furthermore, at the single-particle scale, EBs were more homogeneous than both producer cells and naturally produced EVs, providing an advantage for controlled therapeutic outcomes. Based on these properties, in addition to RNA content profiles that supported applications in immunotherapy, EBs were then utilized in a vaccine model.
In a preventative cancer vaccination model followed by a tumor challenge, EBs from bone marrow dendritic cells (BMDCs) were compared against BMDCs for the ability to prevent tumor growth. Molecular conservation studies demonstrated that EBs emulated producer cells nearly identically and maintained more consistent T cell stimulation ability over time. EBs also outperformed BMDCs in vivo, with a 65% complete remission and survival rate compared to cell vaccine groups with 12-25% survival.
Previously used chemically-induced production methods proved to negatively influence molecular contents of EBs. For applications in gene therapy, which require delivery of functional molecules such as RNA, new production methods were necessary. To overcome these challenges, novel chemically-induced and photo-initiated EB production were explored. All methods were compared for influences on proteins, RNA, and DNA, with photo-initiated methods maintaining the highest level of molecular function. Methods were compared for applications in gene therapy by delivery of adeno-associated virus (AAV).
This dissertation provides an exploration on EB production, molecular analysis on contents, and example applications in preventative cancer vaccination and gene therapy.