Date of Award

6-2022

Document Type

Thesis

Publisher

Santa Clara : Santa Clara University, 2022.

Degree Name

Master of Science (MS)

Department

Bioengineering

First Advisor

Bill Lu

Abstract

Brain cancer treatments have long been restricted by the blood-brain barrier (BBB). Semi-permeable in the most selective way, this barrier may be crossed by one of three methods: membrane integrity disruption, passive transport, or active transport. The third option offers the opportunity for many medically relevant cargoes, such as proteins and nucleic acids, to be shuttled into the brain. The concept of a therapeutic “Trojan horse” builds on this capability by employing engineered nanovesicles—in this work, engineered exosomes—whose surfaces are studded with ligands functioning as the keys to crossing the BBB. Exosomes are a powerful methodology for this approach because they have evolved to be the body’s natural messengers, carrying and delivering bioactive cargo from one cell to another. This work was built on years of research in the field of engineering the exosome’s natural capacities—cargo loading, in vivo survivability, targeting, cellular uptake—in order to execute specific therapeutic functions. Namely, the engineerability of three proteins—rabies virus glycoprotein (RVG), transferrin (TN), and neural cell adhesion molecule (NCAM)—were investigated because they have individually been reported to mediate either active transport across the BBB or targeting of neurons. When put together on an engineered exosome’s surface, these two functionalities are vital pieces of the brain cancer treatment development puzzle. Herein, the tools of confocal microscopy, liquid chromatography-mass spectroscopy (LC-MS), and next-generation sequencing (NGS) provided insight into the efficacy of engineering exosomes with two different combinations of these proteins of interest. An aspect of preclinical safety was evaluated by further NGS analysis, and an initial uptake assay was performed. Notably, as a proof of concept, the sample sizes were low; therefore, conclusions should be considered as flexible to the findings of future research.

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