Novel adenovirus-based helper system to support production of recombinant parvovirus

Abstract

Preclinical studies using various cell culture and animal systems highlight the potential of recombinant rodent parvoviruses (recPVs) for cancer therapy. Production of these viruses is, however, not efficient and this hampers the clinical applications of these agents. In this study, we show that the adenovirus genes E2a, E4(orf6) and VA RNA increase the production of recPVs by more than 10-fold and reduce the time of production from 3 to 2 days in HEK293T cells. The helper effects of these genes can be observed with different recPVs, regardless of the nature and size of the inserted transgene. Furthermore, we generated a recombinant Adenovirus 5 carrying the parvovirus VP transcription unit. This helper, named Ad-VP, allows recPVs to be efficiently produced through a protocol based only on cell infection, making possible to use cell lines, such as NB324K, which are good producers of parvoviruses but are hardly transfectable. Hence, we could further improve viral titers and reduce time and costs of production. This Ad-VP helper-based protocol could be scaled up to a bioreactor format for the generation of the large amounts of recPVs needed for future clinical applications.

682: Synergistic combination of valproic acid and oncolytic parvovirus H-1PV as a potential therapy against cervical and pancreatic carcinomas

“…Remarkably, both VPA and NaB were found to enhance NS1 transcriptional activity (Fig 3B and Supporting Information Fig S5A). To confirm these results, we infected HeLa cells with a recombinant H-1PV in which the VP gene was replaced with the EGFP encoding gene (recPV-EGFP; El-Andaloussi et al, 2011). VPA was found to enhance NS1-triggered EGFP but not NS1 expression (Fig 3C).…”

Generation of an Adenovirus-Parvovirus Chimera with Enhanced Oncolytic Potential

“…The Ad component of the chimera could also assist parvovirus replication in cancer cells at levels other than entry and nuclear trafficking. We have recently shown that specific Ad genomic elements stimulate recombinant PV production by more than 100-fold (10). Similarly, Ad enhances the production of the human B19 parvovirus (14).…”

“…Similarly to any other recombinant adenovirus vector, the chimera should be produced at high titers, solving the problem of the difficulty related to the production of parvoviruses. Furthermore, we recently reported that expression of adenovirus genomic elements boosted the production of recombinant parvovirus in different cell lines (10). Therefore, we speculated that the Ad-PV chimera may enhance PV replication in cancer cells through the concomitant expression of Ad helper functions.…”

“…Lysis was stopped by adding 960 l of 40 mM HCl. Quantification of viral DNA was carried out by real-time quantitative PCR (qPCR) with an NS1-specific TaqMan probe (Applied Biosystems, Darmstadt, Germany), as previously described (10). With this method, we calculated that 1 PFU of hH-1-TO and 1 Adeno IU (see below) of Ad-hH-1-TO corresponded to approximately 500 and 100 viral genome-containing particles, respectively.…”

Viral Vectors for Delivering Gene Material into Cells and Their Application in Neurobiology (Review)

“…Plasmid DNA is most commonly transferred into cells, but other macromolecules can also be translated inside the cell such as small interfering RNA, oligonucleotides, RNA, and proteins [74]. Transfection may comprise several stages, but formation of pores in the plasmatic membrane, through which extracellular material can penetrate inside the cell, is an obligatory stage [75,76].…”