A challenge with treating solid tumors with immunotherapy is their immunologically “cold” tumor microenvironment (TME). Our lab targets the TME through the development of an RNA-LNP platform as an immunotherapeutic strategy. The purpose of my project was to determine the role of key pattern recognition receptors (PRRs) involved in innate immune activation with our RNA-LNP-based innate immune activator strategies. I performed western blots to identify key components of the NFᴋB pathway, along with qRT-PCR to measure relative cytokine expression of type I IFNs such as IFNβ and IL-6. I utilized bone marrow-derived macrophages (BMDMs) from both type I IFN α/β receptor (IFNAR) and MDA5 knockout murine models to assess the requirement for these PRRs. The results showcased components within the NFᴋB pathway to be most crucial for macrophage activation in vitro, as well as the significance of our lab’s innate immune activator that serves as an adjuvant for our vaccine to elicit a more robust activation profile. In addition, the qPCR results from the IFNAR and MDA5 knockout BMDMs demonstrated the requirement for both of these PRR in robust innate immune activation. Ultimately, the results of my project have revealed the significant role of macrophages in innate immune response, the key PRRs involved in innate immune activation, and the necessity for a potent adjuvant which boosts the efficacy of our immunotherapy and will enhance the anti-tumor response in a patient system in the future.

Natural Killer (NK) cells are cytotoxic innate lymphocytes that can both directly kill target cells and produce cytokines that enhance the overall immune response. Their ability to immediately respond to both virally infected and tumor cells makes them a crucial asset of the immune system. Dietary nutrients such as vitamins A, D, and E have well established immunomodulatory effects, where deficiencies hinder the immune response and lead to higher susceptibility to infection or disease. Studies have explored the effects of dietary vitamins, such as vitamin C, which improves tumor infiltration of both CD8+ T cells and macrophages. It has also been shown that vitamin C affects NK cells on a transcriptional level during early differentiation, however, the effects of vitamin C on NK cell effector functions remain unclear. We hypothesized that vitamin C would improve the effector functions of NK cells in a direct and dose-dependent manner. To test this, we isolated primary human NK cells from healthy human donors and tested whether the addition of vitamin C could augment cytokine production and tumor-killing abilities of NK cells. Our study shows that vitamin C did not result in enhanced effector cell functions of naïve or cytokine activated human NK cells. These results suggest that the immunomodulatory role of vitamin C on the immune system is likely extrinsic to human NK cells.        

Ocular diseases can lead to vision impairment and blindness. Extracellular vesicles (EVs) have been shown to possess anti-inflammatory and regenerative properties that can be used to treat ocular diseases. However, effective delivery of therapeutics to the eye remains a challenge due to the structure of the eye. Current delivery methods suffer from low drug bioavailability caused by the corneal epithelium barrier and rapid clearance from blinking. To address these problems, we developed a mucoadhesive hydrogel made of hyaluronic acid (HA) dual functionalized with phenylboronic acid (PBA) and methacryloyl (MA), PBA-MeHA, for ocular delivery of EVs. The PBA interacts with the mucin layer of the cornea to improve adhesion and retention of EVs on the ocular surface. We characterized the hydrogel’s stiffness and mesh size at varying polymer concentrations, and conducted mucoadhesion tests to confirm that the PBA percentage increased hydrogel mucoadhesion. To test EV release, we incorporated EVs into the hydrogel and used a bicinchoninic acid (BCA) assay and nanoparticle tracking analysis (NTA) to measure release over time. We confirmed that the PBA moiety could enhance mucoadhesion of the hyaluronic-based hydrogel, and that EVs can be released in a sustained manner over a prolonged period of time. This PBA-MeHA mucoadhesive hydrogel will be useful for ocular delivery of EVs to treat ocular diseases.

Extracellular vesicles (EVs) are nanoparticles surrounded by a lipid bilayer, which may bud from various membranous surfaces in virtually all cells. EVs can carry cellular products and byproducts, acting as couriers in intercellular communication and disposers of waste, respectively. Central nervous system (CNS)-originating EVs are capable of crossing the blood-brain barrier and have previously been detected in human serum and plasma. These EVs may carry biomarkers, which offer cell-state-specific indicators of health or disease. In neurodegenerative diseases, such as Parkinson’s disease, biomarkers may include pathologic forms of the α-synuclein protein. Through isolation and quantification of such biomarkers, it may be possible to improve diagnosis, track the progression of neurodegenerative diseases, and assess treatment efficacy. Isolation of CNS-originating EVs from serum or plasma requires a moderately invasive blood draw. If a non-invasive modality, such as urine collection, could replace a blood draw, it would offer pain and stress reduction for patients. To that end, my study examines the use of EVs isolated from urine for the measurement of α-synuclein in neuronal EVs (nEVs) as an alternative to the same biomarker measured in serum or plasma. Two workflows yielding successful isolation of CNS-originating EVs from urine included affinity chromatography and a slurry-based isolation, followed by verification and quantification of nEV-specific biomarkers through western blot and an electrochemiluminescence assay. Results indicate promise for a neurodegenerative disease diagnostic assay, with measurable nEV proteins confirmed from urine samples, yet how these biomarkers compare between urine and blood will require confirmation in follow-up studies.

Monoclonal antibodies (mAbs) have been applied as biologic drugs for conditions ranging from autoimmune disease, cancer and infection. Their therapeutic potential arises from their ability to modulate immune pathways, but their utility is often limited by repeat intravenous dosing or expensive, patient-specific gene-therapy workflows. We introduce a capped nanovial platform that encapsulates generic, allogeneic antibody-secreting cells (ASCs) inside spatially functionalizable hydrogel microparticles called nanovials sealed with a semi-permeable cap. In this work, we explore the efficacy of capped nanovials to permit the transport of mAbs at concentrations that are detectable by an immunospot assay and sufficient to drive activation of adjacent immune cells.