Home Department: Chemistry and Italian
Mentor: Steven Banik (Chemistry)

“Development of PLA2 Enzymes as Endosomal Escape Therapeutic Agents”

Methods of delivering large biomolecular cargo to the intracellular space (cytoplasm) are limited, preventing successful use of protein drugs to modulate intracellular pathways. Therefore, a better delivery strategy could be revolutionary in targeting previously “undruggable” space and address unmet medical needs. It is particularly difficult to deliver drugs to the cytoplasm because of the lack in key knowledge about the underlying biology of endosomes (membrane-bound vesicles). The mechanisms by which a molecule can escape an endosome or how those endosomes are subsequently repaired are poorly understood. Matteo hopes to better understand enzymatically mediated endosomal escape. He will design, clone, express, and purify phospholipase A2 (PLA2) enzymes used by bacteria to cleave endosomal lipids and enhance intracellular delivery. He will run the purified enzymes in biochemical and cell-based assays - using TEV protease-activated green fluorescent protein (GFP) expressing cells - to detect if they enter the cytosol. To further assess cytosolic penetration, he will use flow cytometry and collect relative GFP turn-on data. He will analyze cytotoxicity and cell lysis to determine the viability of a PLA2 therapeutic delivery strategy. Finally, he will mutagenize PLA2s to investigate the biological mechanism of cell entry. The mechanisms by which a molecule can escape an endosome or how those endosomes are subsequently repaired are not understood. By taking an example from nature, the Banik lab hopes to characterize endosomal escape and develop a tool for mechanistic studies.