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.

Production and Use of Gesicles for Nucleic Acid Delivery.

Over-expression of the vesicular stomatitis virus glycoprotein (VSVG) in mammalian cells can induce the formation of VSVG-pseudotyped vesicles (named "gesicles") from membrane budding. Its use as a nucleic acid delivery tool is still poorly documented. Naked-plasmid DNA can be delivered in animal cells with gesicles in presence of hexadimethrine bromide (polybrene). However, little is known about gesicle manufacturing process and conditions to obtain successful nucleic acid delivery. In this study, gesicles production process using polyethylenimine (PEI)-transfected HEK293 cells was developed by defining the VSVG-plasmid concentration, the DNA:PEI mass ratio, and the time of gesicle harvest. Furthermore, parameters described in the literature relevant for nucleic acid delivery such as (i) component concentrations in assembly mixture, (ii) component addition order, (iii) incubation time, and (iv) polybrene concentration were tested by assessing the transfection capacity of the gesicles complexed with a green fluorescent protein (GFP)-coding plasmid. Interestingly, freezing/thawing cycles and storage at + 4 °C, - 20 °C, and - 80 °C did not reduce gesicles' ability to transfer plasmid DNA. Transfection efficiency of 55% and 22% was obtained for HeLa cells and for hard-to-transfect cells such as human myoblasts, respectively. For the first time, gesicles were used for delivery of a large plasmid (18-kb) with 42% of efficiency and for enhanced green fluorescent protein (eGFP) gene silencing with siRNA (up to 60%). In conclusion, gesicles represent attractive bioreagents with great potential to deliver nucleic acids in mammalian cells.

Evaluation of novel HIV vaccine candidates using recombinant vesicular stomatitis virus vector produced in serum-free Vero cell cultures.

Acquired Immune Deficiency Syndrome (AIDS) in humans is a result of the destruction of the immune system caused by Human Immunodeficiency Virus (HIV) infection. This serious epidemic is still progressing world-wide. Despite advances in treatment, a safe and effective preventive HIV vaccine is desired to combat this disease, and to save millions of lives. However, such a vaccine is not available yet although extensive amounts of resources in research and development have been invested over three decades. In light of the recently approved Ebola virus disease vaccine based on a recombinant vesicular stomatitis virus (rVSV-ZEBOV), we present the results of our work on three novel VSV-vectored HIV vaccine candidates. We describe the design, rescue, production and purification method and evaluate their immunogenicity in mice prior to preclinical studies that will be performed in non-human primates. The production of each of the three candidate vaccines (rVSV-B6-NL4.3Env/SIVtm, rVSV-B6-NL4.3Env/Ebtm and rVSV-B6-A74Env(PN6)/SIVtm) was evaluated in small scale in Vero cells and it was found that production kinetics on Vero cells vary depending on the HIV gp surface protein used. Purified virus preparations complied with the WHO restrictions for the residual DNA and host cell protein contents. Finally, when administered to mice, all three rVSV-HIV vaccine candidates induced an HIV gp140-specific antibody response.

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.

New developments in lentiviral vector design, production and purification.

INTRODUCTION: Lentiviruses are a very potent class of viral vectors for which there is presently a rapidly growing interest for a number of gene therapy. However, their construction, production and purification need to be performed according to state-of-the-art techniques in order to obtain sufficient quantities of high purity material of any usefulness and safety. AREAS COVERED: The recent advances in the field of recombinant lentivirus vector design, production and purification will be reviewed with an eye toward its utilization for gene therapy. Such a review should be helpful for the potential user of this technology. EXPERT OPINION: The principal hurdles toward the use of recombinant lentivirus as a gene therapy vector are the low titer at which it is produced as well as the difficulty to purify it at an acceptable level without degrading it. The recent advances in the bioproduction of this vector suggest these issues are about to be resolved, making the retrovirus gene therapy a mature technology.