Improved Manufacturing Methods of Extracellular Vesicles Pseudotyped with the Vesicular Stomatitis Virus Glycoprotein.

Extracellular vesicles (EV), which expose the vesicular stomatitis virus glycoprotein (VSVG) on their surface, are used for delivery of nucleic acids and proteins in human cell lines. These particles are biomanufactured using methods that are difficult to scale up. Here, we describe the development of the first EV-VSVG production process in serum-free media using polyethylenimine (PEI)-based transient transfection of HEK293 suspension cells, as well as the first EV-VSVG purification process to utilize both ultracentrifugation and chromatography. Three parameters were investigated for EV-VSVG production: cell density, DNA concentration, and DNA:PEI ratio. The best production titer was obtained with 3 × 106 cells/mL, a plasmid concentration of 2 µg/mL, and a DNA:PEI ratio of 1:4. The production kinetics of VSVG was performed and showed that the highest amount of VSVG was obtained 3 days after transfection. Addition of cell culture supplements during the transfection resulted in an increase in VSVG production, with a maximum yield obtained with 2 mM of sodium butyrate added 18 h after transfection. Moreover, the absence of EV-VSVG during cell transfection with a GFP-coding plasmid revealed to be ineffective, with no fluorescent cells. An efficient EV-VSVG purification procedure consisting of a two-step concentration by low-speed centrifugation and sucrose cushion ultracentrifugation followed by a heparin affinity chromatography purification was also developed. Purified bioactive EV-VSVG preparations were characterized and revealed that EV-VSVG are spherical particles of 176.4 ± 88.32 nm with 91.4% of protein similarity to exosomes.

Non-viral nucleic acid delivery methods.

INTRODUCTION: Delivery of nucleic acid-based molecules in human cells is a highly studied approach for the treatment of several disorders including monogenic diseases and cancers. Non-viral vectors for DNA and RNA transfer, although in general less efficient than virus-based systems, are particularly well adapted mostly due to the absence of biosafety concerns. Non-viral methods could be classified in two main groups: physical and vector-assisted delivery systems. Both groups comprise several different methods, none of them universally applicable. The choice of the optimal method depends on the predefined objectives and the features of targeted micro-environment. Areas covered: In this review, the authors discuss non-viral techniques and present recent therapeutic achievements in ex vivo and in vivo nucleic acid delivery by most commonly used techniques while emphasizing the role of 'biological particles', namely peptide transduction domains, virus like particles, gesicles and exosomes. Expert opinion: The number of available non-viral transfection techniques used for human therapy increased rapidly, followed by still moderate success in efficacy. The prospects are to be found in design of multifunctional hybrid systems that reflect the viral efficiency. In this respect, biological particles are very promising.