Researcher(s)
- Dean Stas, Chemical Engineering, University of Delaware
Faculty Mentor(s)
- April Kloxin, Chemical Engineering, University of Delaware
- Catherine Fromen, Chemical Engineering, University of Delaware
Abstract
Immune cell therapies have revolutionized treatment for otherwise difficult-to-treat cancers and autoimmune conditions. Lentiviral vectors have emerged as an effective, but expensive, tool for engineering these cell therapies. To increase virus engineering efficiency and reduce costs, special environments using tangential flow filtration and centrifugation to colocalize virus and target cells have been employed. However, these environments display variable effectiveness with lentivirus from different manufacturers and across different cell types. We seek to expand our understanding of these differences by examining the influence of the cell environment on the internalization of lentivirus through receptor-mediated endocytosis. Currently, no procedure exists to assess the endocytosis rate of lentiviral vectors used in cell therapy manufacturing. In this work, we present a flow-cytometry based assay tailored towards examining the rate of lentiviral endocytosis across various environmental conditions.
We first sought to verify assay functionality using an analog of lentiviral binding and endocytosis. Immortalized T-cells were stained with fluorescent antibodies targeting the viral entry receptor. To optimize receptor-antibody binding, various staining and washing solution compositions were investigated, revealing that calcium supplementation improved complex stability. Antibody was then internalized by the cell for different periods of time. Flow cytometry was used to measure the extent of endocytosis via fluorescence intensity. Significant increases in fluorescence intensity were observed over the 0-, 30- and 60-minute endocytosis times.
We then moved to adapting the assay for use with lentivirus. The lentivirus was stained with a fluorescent dye, purified through gradient centrifugation, and imaged to verify staining integrity. Future work will use this fluorescently-labeled lentivirus within the successfully developed assay to elucidate differences in lentiviral endocytosis for cells treated in various transduction environments.