(205f) Improvements in Nucleic Acid Delivery Using VSV-G Vesicles

An assembly of viral structural proteins that is similar to the coat of natural viruses is known as a virus-like particle (VLP). Due to the lack of nucleic acid molecules these structures are replication deficient. The fusiogenic envelope G glycoprotein of the vesicular stomatitis virus (VSV-G) can form VLPs that are released into culture medium after being expressed by animal cells. These VSV-G particles have the capacity to bind naked plasmid DNA and resulting complexes can transfect various animal cell types in presence of polybrene. Actually, production and purification methods of these particles are not fully optimized: Indeed, VSV-G vesicles are generated by transient transfection of HEK293 cells cultivated in adherence with serum and partially purified by ultracentrifugation. These methods are difficult to scale up making costly DNA transfer technology using VSV-G vesicles. Moreover, due to the risk of viral or prion contamination and problems with variability across product lots, the use of serum is not recommended to synthetize molecules dedicated to therapeutic applications. For these reasons VSV-G vesicles-based transfection technology is not yet commercialized.

Recently, we have developed an optimized protocol to produce high level of VSV-G vesicles from transfected animal cells. Different parameters have been evaluated: promoter used to control the VSV-G expression (CMV, CMV5 and CR5 promoter), transfection reagent (Calcium phosphate, polyethylenimine and lipofectamine), cell type (HEK293 and CHO cells), cell culture conditions (adherent vs suspension cell culture +/- fetal bovine serum) and production mode (batch, fed batch and batch replacement).

In an attempt to improve efficiency of the VSV-G vesicles-based transfection technology, we have also studied on various human cell types: effects of DNA, vesicles and polybrene concentration, DNA size, cell density, complex formation time, volume of transfection reagent and post-transfection incubation time.  

Furthermore, we have investigated the potential of VSV-G vesicles to deliver siRNA because the therapeutic application of these molecules is promising due to efficient and specific gene silencing. Our strategy consisted to load purified VSV-G vesicles with exogenous siRNA by electroporation.

In conclusion, nucleic acid delivery using VSV-G vesicles is a powerful technology which can be used instead of viral vectors to transfect refractory cells.