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.

Keywords: SV40 viral vectors; SVac; SuperVero; packaging cells.

Figure 1

Schematic Representation of the SV40 Vector Destination Plasmid pSVac The Gateway gene cassette comprising the ccdB and the chloramphenicol resistance (Cm^R) genes present in pSVac is substituted by the transgene using Gateway recombination yielding an SV40 vector expression plasmid.

Figure 2

Vero Cells Stably Transfected with pHY359 Support the Production of SVLuc Vector Particles (A) Schematic representation of the expression plasmids pHY338 and pHY359. Plasmid pHY338 contains the SV40 genomic T antigen sequence encoding both LTag and STag. Plasmid pHY359 contains the LTag cDNA sequence encoding LTag only. In both plasmids, transgene expression is driven by the EF-1α promoter. Both plasmids contain the puromycin resistance (Puro^R) gene under transcriptional control of the CMV_ie promoter and BGH pA signal. (B) Ratio between the number of SVLuc particles produced in Vero cells transfected with pHY338 and pHY359 plasmid DNA and those produced in COS-1 cells transduced with SVLuc particles. (C) Luminescence of COS-1 cells transduced with SVLuc particles produced in transfected Vero or COS-1 cells. (D) Luminescence of COS-1 cells transduced with SVLuc particles produced in stably transfected Vero cell clones. COS-1 cells were used as positive control.

Figure 3

SuperVero Cells Contain pHY359 DNA and Express SV40 LTag (A) Schematic representation of the cDNA reads from the MP-seq analysis corresponding with the SV40 LTag mRNA expressed in SuperVero cells. (B and C) Detection of the LTag protein in SuperVero cells (B) by western blot and (C) by immunocytochemistry using a mouse monoclonal antibody specific to the amino-terminal region of the LTag and STag. Vero cells are used as a negative control. COS-1 cells are used as a positive control expressing both LTag and STag. (D) Densitometric analysis of the western blot bands shown in (B). The graph represents the ratio between the density units of LTag and α-tubulin, used as an internal loading control.

Figure 4

The SV40 Late Gene Is Only Expressed in SuperVero Cells during the Production of Vector Particles and Not in Human Target Cells Detection of GFP, SV40 LTag, and major capsid protein VP1 in cell lysates from SuperVero and HeLa cells transfected with pSVGFP DNA by western blot analysis using antibodies specific for GFP, LTag, and VP1. An anti-GAPDH antibody was used as a loading control.

Figure 5

SuperVero Cells Consistently Accumulate Large Amounts of Functional SV40 Vector Particles (A) Determination of the number of SVLuc particles produced in COS-1 and SuperVero cells using qPCR. (B) Determination of the number of SVLuc particles after passaging in SuperVero cells. (C) Determination of the potency of SVLuc particles after passaging in SuperVero cells. (D) Determination of the number of SV40 vector particles expressing different transgenes produced in SuperVero cells. Error bars represent SD, n = 3.

Figure 6

SV40 Vector Particles Remain Functional after Storage for 6 Months at 4°C or Lower Temperatures SVLuc particles were stored at +4°C, −20°C, −80°C, and −150°C. (A) The number of vector particles was determined by qPCR. (B) The potency of the vector particles was determined by the TCID₅₀ analysis.

Figure 7

Production of SV40 Vectors in SuperVero and COS-1 Cells The production of SV40 particles in COS-1 cells (right side) results in the emergence of RC SV40 particles because of homologous recombination between chromosomal inserted SV40 sequences and episomally replicating SV40 DNA sequences. The generation of RC variants is prevented in SuperVero cells (left side) harboring chromosomally inserted SV40 LTag DNA sequences without sequence overlap to the episomally replicating SV40 vector DNA sequences.

Figure 8

Primers and Probes Used for the qPCR Analysis to Detect RC and RD SV40 Vector Particles (A) Schematic representation of the SV40 genome showing the position of the primers and probe for the detection of RC SV40 particles. (B) The sequences of primers and probes used in the qPCR analysis.