Generation of a Vero-Based Packaging Cell Line to Produce SV40 Gene Delivery Vectors for Use in Clinical Gene Therapy Studies

Abstract

Replication-defective (RD) recombinant simian virus 40 (SV40)-based gene delivery vectors hold a great potential for clinical applications because of their presumed non-immunogenicity and capacity to induce immune tolerance to the transgene products in humans. However, the clinical use of SV40 vectors has been hampered by the lack of a packaging cell line that produces replication-competent (RC) free SV40 particles in the vector production process. To solve this problem, we have adapted the current SV40 vector genome used for the production of vector particles and generated a novel Vero-based packaging cell line named SuperVero that exclusively expresses the SV40 large T antigen. SuperVero cells produce similar numbers of SV40 vector particles compared to the currently used packaging cell lines, albeit in the absence of contaminating RC SV40 particles. Our unique SV40 vector platform named SVac paves the way to clinically test a whole new generation of SV40-based therapeutics for a broad range of important diseases.

How Simian Virus 40 Hijacks the Intracellular Protein Trafficking Pathway to Its Own Benefit … and Ours

“…Clinical studies revealed that because of the immune memory in humans, the in vivo efficacy of AAV vectors is very low. SV40 vectors are the only gene delivery vectors that can be used for inducing immune tolerance to transgene proteins in humans and for this reason the oldest viral gene delivery vector will be key to the successful development of effective interventions for today’s major diseases ( 95 ).…”

Cre Recombinase Mediates the Removal of Bacterial Backbone to Efficiently Generate rSV40

“…To this end, an SV40 vector destination plasmid pSV ac was used, in which the vector genome is flanked by Lox-P sites to allow specific removal of the bacterial backbone by Cre recombinase expressed in SV40 vector packaging cells. 36 We here show that expression of Cre recombinase in COS-1 and SuperVero cells results in an efficient removal of the bacterial backbone.…”

“…COS-1 cells were transfected with the following: (1) the rSV Luc 36 plasmid with Lox-P sites flanking the rSV genome; (2) the rSV LucΔLox-P plasmid without Lox-P site co-transfected with a Cre-expressing plasmid; and, (3) as a negative control, a GFP-expressing plasmid. At 5 days after transfection, the medium was harvested and the titer of rSV Luc was determined using qPCR.…”

“…Recently, a novel Vero-based packaging cell line to produce rSV40 called SuperVero was generated. 36 In this novel packaging cell line, only the LTag gene is integrated into the chromosomal DNA. Because sequence overlap between chromosomally inserted SV40 LTag-coding sequences and episomally replicating SV40 vector sequences is lacking, the emergence of replication-competent particles during the vector production process is highly unlikely.…”

“…Because sequence overlap between chromosomally inserted SV40 LTag-coding sequences and episomally replicating SV40 vector sequences is lacking, the emergence of replication-competent particles during the vector production process is highly unlikely. 36 To further improve the production of rSV40, we therefore generated a stable SuperVero cell line with inducible Cre expression to be used for the production of rSV40 vectors. Our data show that efficient production of rSV40 is possible with this cell line upon the induction of Cre expression using doxycycline ( Figure 5 C).…”