Tunable Barley Stripe Mosaic Virus for PD-L1 Degradation

• Jillian Seno, Chemical Engineering, University of Delaware

• Kevin Solomon, Chemical and Biomolecular Engineering, University of Delaware

Targeted protein degradation (TPD) serves as a promising alternative to traditional protein-targeting therapies by degrading target proteins through intracellular protein degradation machinery.  In other therapeutic strategies, cancerous biomarkers, such as programmed death-ligand 1 (PD-L1), go through unwanted endosomal recycling. TPD addresses this issue by promoting lysosomal-mediated degradation instead, which decreases PD-L1 cellular abundance. Nanoparticle usage in TPD has been of interest, but limited data exists on the effects of their size and shape on degradation efficiency. Based on current research, nanorods with longer aspect ratios appear to be optimal for cellular uptake, making them a prospective TPD modality. Thus, this study aims to examine the optimal length of barley stripe mosaic virus (BSMV) nanorods for cellular uptake and degradation efficiency. Target BSMV nanorod lengths of 200 nm, 150 nm, 100 nm, and 50 nm were chosen based on endocytic vesicle sizes. Varied nanorod lengths were synthesized in vitro using purified capsid proteins and modulated RNA templates. RNA templates were obtained by linearizing and transcribing plasmid vectors using T7 RNA polymerase from the MEGAscript T7 high yield transcription kit. Capsid proteins were engineered to express a chitin binding domain, which binds to a chitin resin in the IMPACT system. Through this system, capsid proteins are purified in a single chromatographic step and are fully removed from the residual intein tag, ensuring complete self-assembly of nanorods. RNA templates of desired lengths were successfully obtained and verified with RNA gel electrophoresis. Upon completion of in vitro assembly, transmission electron microscopy was used to examine nanorod assembly. While nanorods of a 50 nm length were obtained, nanorods of desired lengths of 200 nm and 150 nm were unable to assemble to the full length. Further research on the most optimal method for nanorod assembly is required in order to test endocytosis efficiency.