Developing Guanidinylated Neomycin-Enzyme Conjugates to Treat Lyososomal Storage Disorders Linked to Parkinson’s Disease
β-glucocerebrosidase (GBA) is a lysosomal enzyme that degrades glucosylceramide, a biomolecule believed to stabilize the toxic protein aggregates that lead to Parkinson's disease. Genetic variations that decrease β-glucoce (GBA) activity have been linked to an increased risk of Parkinson's disease. One strategy to restore lysosomal function is to deliver active recombinant GBA directly to neurons, but lysosomal targeting can present a challenge. Guanidinylated neomycin (GNeo), however, facilitates lysosomal localization when covalently appended to proteins. Our approach is thus to prepare GNeo-GBA conjugates, test their activity
in vitro and confirm lysosomal localization and activity in cultured neurons. Once optimized, the
in vivo effectiveness of GB/GNeo conjugates can be assessed using a mouse model system.
Upper airway mucosal responses to MRSA in CRS
Chronic Rhinosinusitis (CRS) affects 10% of adults and Staphylococcus aureus (SA) colonization is increased in CRS. One hypothesis to delineate the role of SA in pathogenesis of CRS is that SA stimulates production of c-type lectin Reg3g which binds to peptidoglycan layer of Gram+ bacteria along with type 2 inflammatory cytokines (thymic stromal lymphopoietin [TSLP] and IL-33) that causes hyper production of mucins (Muc5AC and Muc5B) resulting in chronic inflammatory state involving aberrantly activated innate immune and T cells.
Glycosaminoglycan-based lung cancer immunotherapy
Lung cancer is the leading cause of cancer death in the U.S. and worldwide. Dendritic cells (DCs) recognize tumor antigen and instruct T cells to initiate antitumor immunity. In a tumor microenvironment, however, dendritic cells are suppressed from properly expressing mature phenotype to carry on this function. Heparan sulfate (HS) glycosaminoglycan, when altered on the DC surface, restores mature DC phenotype and inhibits immature DC trafficking to the draining lymph nodes demonstrating anti-tumor therapeutic potential, however, its effect on T cell associated lung cancer immunity has not been previously investigated. In my studies, I will uncover how DC specific HS alteration will influence the antitumor associated T cell function and downstream signaling pathways. Additionally, genetic and chemical strategies will be developed targeting DC HS.
Design and Synthesis of Fluorogenic Proteoglycan Mimetic Polymers
Our goal is to use these fluorescent polymers bearing heparan sulfate (HS) chains to better understand how heparan sulfate impacts the pathophysiology of Alzheimer's disease. This HS-polymer will be modified with a lysosome-targeting molecule to hopefully clear the amyloid plaques upon binding to the HS chains.
CRISPR-Cas9 Dissection of Heparan Sulfate
Heparan sulfate proteoglycans (HSPGs) are expressed on all animal cells and in the extracellular matrix. Each HSPG consists of a core protein with one or more covalently attached linear heparan sulfate (HS) chains composed of alternating glucosamine and uronic acids that are heterogeneously N- and O-sulfated. These complex cell surface carbohydrates regulate important biological processes including cell proliferation and development through their interaction with many matrix proteins and growth factors. The arrangement and orientation of the sulfated sugar residues of HS specify the location of distinct ligand binding sites on the cell surface, and these modifications can vary temporally during development and spatially across tissues. While most of the enzymes involved in HS biosynthesis have been studied extensively, less information exists regarding the mechanisms that give rise to the variable composition and binding properties of HS. The overall goal of this project is to understand the genetic factors that control the formation of heparan sulfate in mammalian cells. A genome-wide CRISPR/Cas9-mediated screen will be developed to uncover novel genes other than those encoding known HS biosynthetic enzymes. A lentiviral single guide RNA (sgRNA) library will be utilized to knock down gene expression across the entire genome in a human cancer cell lines. Subsequently, a high-throughput screen will be adapted to identify sgRNAs that induce resistance to cytotoxins whose action depends on HSPGs or decrease binding of HS-dependent ligands to the cell surface. Overall, these studies will provide a better understanding of the genetic regulatory factors involved in HS biosynthesis, as well as lead us to methods to manipulate HS and its activities in other cellular processes that go awry in human diseases.