(511g) Invited Talk: Biomanufacturing Process Development and Characterization of Extracellular Vesicles Pseudotyped with VSV Glycoprotein for Nucleic Acid Delivery

Extracellular vesicles pseudotyped with vesicular stomatitis virus glycoprotein (named gesicles) demonstrated promising abilities to deliver nucleic acids into different mammalian cell lines and primary cells. The delivery of nucleic acids using gesicles is called “gesifection” and requires mixing nucleic acids, gesicles and a chemical reagent, the hexadimethrine bromide before incubation with cells. Typically, gesicles are manufactured using methods well known to be difficult to scale up, such as transient transfection of HEK293 adherent cells in serum containing media and purification using iodixanol gradients. We describe here a robust gesicle production process in serum-free media using transient transfection of HEK293 suspension cells as well as an easily scalable chromatographic purification process. We have also studied the transcriptome of HEK293 cells to better understand the cellular metabolic pathways involved in the production of these particles. These transcriptomic analyses have revealed three potential genes involved in gesicles formation. We have successfully transiently overexpressed these genes and their impact on the production of gesicles was analyzed.

Moreover, little is known about the structure of gesicles and gesifection-related mechanisms. The other aim of this study is to provide new insight to enable the next optimizations. Here, the formation of a gesifection complex between gesicles, hexadimethrine bromide and plasmids has been demonstrated for the first time. Furthermore, the protein content profile of gesicles have been defined and revealed the presence of three oncogenic proteins as well as a wide diversity of producer cells proteins. Interestingly, the hexadimethrine bromide and the gesicles are both involved in plasmid penetration into cells while only gesicles enable plasmids expression. Finally, the gesifection is a nucleic acid delivery method that uses the endocytosis pathways and not the passive fusion with the plasma membrane. The main entrance pathways use both the macropinocytosis and the clathrin-dependent endocytosis. Also, the intracellular trafficking of plasmids carry out through the microtubules and the dyneins to reach the nucleus.

Finally, all these findings including biomanufacturing advances deepen the understanding of the gesifection technology and pave the way to launch this technology to market.