VLA-4 is a surface protein of immune cells that plays an important role in their extravasation into tissues during an immune response. In multiple sclerosis (MS), pathogenic T cells enter the brain and attack nerve cells by using VLA-4 to bind VCAM-1, a cell adhesion molecule on endothelial cells that line blood vessels. Current MS treatments rely on antibodies that bind VLA-4 and block interaction with VCAM-1, preventing a pathogenic immune response. However, antibodies are expensive to manufacture, and their binding cannot be easily regulated to control drug-induced side effects. Aptamers are single-stranded DNA or RNA molecules that fold into sequence-defined structures capable of binding targets with affinities and specificities comparable to antibodies. Being chemically synthesized, they are much cheaper to manufacture and offer no batch-to-batch differences. Unlike antibodies, their binding in vivo is rapidly reversible, which could alleviate some side effects of disease treatments. However, aptamers have limitations in vivo – degradation by nucleases in serum, and rapid clearance into urine. This project designs and assesses modifications to a novel VLA-4 binding aptamer to improve in vivo function, with the goal of developing an alternative for MS treatment. We designed various modifications to the aptamer backbone to prevent nuclease degradation and conjugated the aptamer to a polymer to increase size and reduce clearance. To assess aptamer functionality, an in vitro model of T cell adhesion is used. VCAM-1 coated plates are used to simulate endothelial cells, and VLA-4+ T cells are incubated in the plates to allow adhesion in the presence of modified versions of the aptamer and serum. VLA-4 inhibition by our aptamer designs is assessed by characterizing the extent of cell adhesion inhibition. Successfully designing a modification that significantly improves the in vivo function of aptamers will have broad implications for their clinical translation to in vivo use.

Stimulator of Interferon Genes (STING) signaling contributes to tumor immunity. However, treatments targeting the STING pathway are limited by route of administration, insufficient STING activation, and off-target toxicity. We introduce poly-STING, a copolymerized, mannosylated variant of the diABZI STING agonist-3 known to activate the cGAS-STING signaling pathway, promoting the release of type-1 interferons and pro-inflammatory cytokines leading to tumor immunogenicity. The STING agonist-3 is a non-nucleotide molecule that activates the STING pathway, but it has poor solubility, which limits its usage in-vivo. The developed poly-STING platform improves the drug's solubility, is designed to target immune cells, and provides enzyme-triggered drug release upon delivery, which has been shown to induce improved therapeutic efficacy compared to the free drug. The Pun and Stayton labs seek to investigate modalities for optimization of the cGAS-STING pathway activation and characterize the mechanism of action. Specifically, my project will evaluate STING activation by observing macrophage repolarization from type M2, as the mannose from the poly-STING binds to the CD206 receptors on M2 macrophages. This activates the STING pathway, repolarizing the macrophage to pro-inflammatory type M1. To test effects in vitro, I will culture bone marrow-derived M2 macrophages with various formulations of poly-STING, and repolarization will be measured through flow cytometry and RT-qPCR to quantify expression of macrophage markers. We expect to find higher M1 activity in macrophages treated with poly-STING as opposed to the free drug. Next, I evaluate the therapeutic efficacy of the STING formulations through an in-vivo tumor reduction study using murine models of breast cancer and melanoma, expecting to find longer survival of mice treated with poly-STING. The culmination of this project will result in a polymer-based STING agonist delivery platform that solves the solubility and bioavailability issues associated with the STING-3 agonist, with enhanced efficacy and decreased toxicity after systemic administration.