Gla‐domain mediated targeting of externalized phosphatidylserine for intracellular delivery

• Published in: The FASEB journal 2023-08, Vol.37 (8), p.e23113-n/a
• Main Authors: Hardy, Jonathan, Bauzon, Maxine, Chan, Charles Kwok Fai, Makela, Ashley V., Kanada, Masamitsu, Schneider, Doug, Blankenberg, Francis, Contag, Christopher H., Hermiston, Terry
• Format: Article
• Language: English
• Subjects: Animals; cancer; Cell Membrane - metabolism; extracellular vesicles; GLA domain; infection; Neoplasms - metabolism; Phagocytosis; phosphatidylserine; Phosphatidylserines - metabolism; Phospholipids - metabolism; stem cells
• ISSN: 0892-6638; 1530-6860
• DOI: 10.1096/fj.202201250RRR

Phosphatidylserine (PS) is a negatively charged phospholipid normally localized to the inner leaflet of the plasma membrane of cells but is externalized onto the cell surface during apoptosis as well as in malignant and infected cells. Consequently, PS may comprise an important molecular target in diagnostics, imaging, and targeted delivery of therapeutic agents. While an array of PS‐binding molecules exist, their utility has been limited by their inability to internalize diagnostic or therapeutic payloads. We describe the generation, isolation, characterization, and utility of a PS‐binding motif comprised of a carboxylated glutamic acid (GLA) residue domain that both recognizes and binds cell surface‐exposed PS, and then unlike other PS‐binding molecules is internalized into these cells. Internalization is independent of the traditional endosomal–lysosomal pathway, directly entering the cytosol of the target cell rapidly. We demonstrate that this PS recognition extends to stem cells and that GLA‐domain‐conjugated probes can be detected upon intravenous administration in animal models of infectious disease and cancer. GLA domain binding and internalization offer new opportunities for specifically targeting cells with surface‐exposed PS for imaging and delivery of therapeutics. The Gla‐domain can be isolated to selectively recognize and internalize into cells expressing externalized phosphatidylserine. This represents a unique platform for targeted intracellular delivery of molecules to an array of phosphatidylserine expressing cells that include cancer, stem cells and infected cells.