Despite recent advancements in cancer treatment, the overall 5-year survival rate for glioblastoma, a very aggressive brain cancer, is only 7.4%. The greatest challenge in brain cancer treatment is the blood-brain barrier (BBB), a physical barrier that protects the central nervous system (CNS) from circulating solutes in the blood but prevents therapeutics from entering the brain space. While surgery is the gold-standard treatment, this procedure is high-risk. Thus, research into nanoscale injectable therapies that can cross the BBB to treat brain tumors is critical as they are non-invasive and can be targeted to specific cells. The Pun Lab is developing nanoparticles to cross the BBB via receptor-mediated transcytosis (RMT) via the transferrin receptor. To further this research, I developed a cellular model of the BBB to assess the ability of different nanoparticle formulations to cross the BBB in vitro. Specifically, I developed a Transwell culture of brain endothelial cells, which are the main regulators of the BBB due to tight junction formation. I investigated additional targeting ligands through analysis of a polymer panel to improve transport through the BBB. Finally, I will validate endosomal escape through the barrier with confocal microscopy. Through this project, I (i) developed a representative in vitro model of the BBB, (ii) explored alternative receptor-binding ligands and (iii) validated the mechanism through which the nanoparticles travel to enhance nanoparticle transport through the BBB. Ultimately, these three aims enable better direction of nanoparticle behavior in vivo and across the BBB. Non-invasive nano-therapeutics are critical to the future treatment of glioblastoma as current treatment options are limited, extremely risky, and lack long term efficacy.

 The CD28 receptor provides co-stimulatory signaling as part of T-cell activation, thereby driving T-cell proliferation, differentiation, cytokine production, and survival required for effective immune responses. Given this important role, CD28 has broad therapeutic implications, serving as a target for cancer immunotherapy, treatment of autoimmune disorders, and production of adoptively transferred T cells. Current approaches for targeting CD28 rely on antibodies, which can be employed in vivo or ex vivo to promote or block CD28 signaling depending on the application. While effective, antibody-based targeting is costly and rigid in design, owing to their biological production and reduced control over binding. Aptamers are small, single-stranded oligonucleotides with sequence-defined architectures that can bind specific targets of interest at high specificity and affinity. Aptamers can be produced at low cost and the inherent properties of oligonucleotides permit flexibility in reversing binding and fine-tuning affinity strength for optimal receptor targeting. This project proposes to develop the first aptamer that targets human CD28 using a combinatorial selection strategy that incorporates protein- and cell-based selections. Aptamer candidates will be identified and characterized to evaluate their binding specificities and kinetics. The selected aptamer will then be used to design a T cell activation assay. A 12-round selection has been completed and binding specificities of individual aptamer candidates will be evaluated. A second selection using a modified approach is currently in progress. If successful, this project has the potential to improve the T-cell activation process in manufacturing adoptive T cell therapies and facilitate the development of novel therapeutics for treating cancer and autoimmune diseases.

The SARS-CoV-2 Delta variant, first detected in India, has contributed significantly to the 78 million global COVID-19 cases throughout the course of the pandemic. As such, effective diagnostic tools remain crucial for controlling widespread infection. DNA aptamers are single-stranded, self-folding oligonucleotides that can bind to specific targets with high specificity and affinity. DNA aptamers are especially useful for diagnostic applications because they are stable, inexpensive, consistent between batches, and allow for additional chemical modifications for diagnostic applications. On the other hand, commonly-used alternatives such as antibodies are difficult to store and are produced through a labor-intensive cellular process that makes them susceptible to batch-to-batch variation. This project selected for DNA aptamers that bound to the S1 domain of the SARS-CoV-2 Delta variant spike protein using the iterative method SELEX (Systematic Evolution of Ligands by Exponential Enrichment). In each round of SELEX, a starting aptamer pool was first exposed to undesirable proteins in a process known as negative selection. Aptamers that bound strongly to these unwanted proteins were removed from the aptamer pool using magnetic Dynabeads. In an analogous process of positive selection, aptamers with high affinity for the Delta S1 spike protein were retained in the aptamer pool, while nonspecific aptamers were discarded. With each passing round of SELEX, the stringency of aptamer binding was increased such that only the highest affinity aptamers remained in the final SELEX rounds. These final rounds were then sequenced through Next-Generation Sequencing (NGS) and the aptamers that displayed the highest enrichment were characterized using a combination of ELISA (enzyme-linked immunosorbent assay) and biolayer interferometry. In the near future, effective aptamers discovered through this process will be applied in antigen testing applications through the use of tools like lateral flow assays.