Engineered Extracellular Vesicles for biogenesis and immunomodulation studies

Small Extracellular Vesicles (sEV), as naturally occurring vesicles, have a low intrinsic immunogenic profile, thus showing great therapeutic potential. Over the past few years, several engineering strategies have been devised to manipulate tumor-derived sEVs in order to induce cellular and innate immunity. In our study we use a mutant Human Immunodeficiency Virus (HIV)-1 Nef protein that presents a N-terminal palmitoylation (NefG3C), which increase the specificity of the protein for sEV association and a mutated NefG3C with two additional mutations (Nefmut) to render the protein biologically inactive. Both Nef mutants were fused at C-terminus with Green Fluorescent Protein (GFP) (NefG3C/Nefmut-GFP). To follow the biogenesis of these engineered sEV we used a novel methodology developed in our laboratory to metabolically label exosomes by incubating cells with a red fluorescent fatty acid BODIPY 558/568 C12 (C12). The lipid is readily taken up by cells and transformed into phospholipids that will ultimately form the exosome lipid bilayer. By transfecting HEK293 cells with the NefG3C-GFP or Nefmut-GFP vectors and pulsing them with C12 we could purify exosomes containing NefG3C/mut-GFP and/or C12 (C12 exo). Results show that the number of cell secreted sEV greatly differs for the two constructs probably due to a different association with sEV. Fluorescent sEV were also characterized for typical exosomes markers and analyzed in iodixanol density gradients. NefG3C/Nefmut-GFP/C12 exo could be separated in two distinct peaks whereas C12 exo displayed only one fluorescent peak. Further analysis will show if these two different populations of sEV display different behaviour in terms of efficiency of transfer to recipient cells and ultimately stimulation of the immune system.