Inducible Overexpression of a Toxic Protein by an Adenovirus Vector with a Tetracycline-Regulatable Expression Cassette

Abstract

We have constructed two new adenovirus expression cassettes that expand both the range of genes which can be expressed with adenovirus vectors (AdV) and the range of cells in which high-level expression can be attained. By inclusion of a tetracycline-regulated promoter in the transfer vector pAdTR5, it is now possible to generate recombinant adenoviruses expressing proteins that are either cytotoxic or that interfere with adenovirus replication. We have used this strategy to generate a recombinant adenovirus encoding a deletion in the R1 subunit [R1(Δ2-357)] of the herpes simplex virus type 2 ribonucleotide reductase. Cell lines expressing the tetracycline-regulated transactivator (tTA) from an integrated vector or following infection with an AdV expressing tTA are able to produce ΔR1 protein at a level approaching 10% total cell protein (TCP) when infected with Ad5TR5ΔR1 before they subsequently die. To our knowledge, this is the first report of the overexpression of a toxic gene product with AdV. We have also constructed a new constitutive adenovirus expression cassette based on an optimized cytomegalovirus immediate-early promoter-enhancer that allows the expression of recombinant proteins at a level greater than 20% TCP in nonpermissive cell lines. Together, these new expression cassettes significantly improve the utility of the adenovirus system for high-level expression of recombinant proteins in animal cells and will undoubtedly find useful applications in gene therapy.

Protective MCMV immunity by vaccination of the salivary glandviaWharton's duct: replication‐deficient recombinant adenovirus expressing individual MCMV genes elicits protection similar to that of MCMV

“…While we have demonstrated that direct injection (9) or retrograde perfusion of the salivary gland resulted in the induction of a protective immune response using an attenuated (tcMCMV) viral system, to be generally applicable as a site for vaccination, it was necessary to demonstrate that direct protein expression in the salivary gland such as that delivered via a noninfectious, conventional subunit vaccine, could also elicit effective and functionally protective immunity. Recombinant adenoviruses are a versatile tool for gene delivery and expression, since they are able to infect a broad range of cell types, and infection is not dependent on active host cell division (31,32). The most commonly used adenoviral vectors are typically devoid of E1 and E3 genes, which not only render the virus incapable of replication but also make room for the insertion of up to 7.5 kb of foreign DNA (33,34).…”

Serum‐free transient protein production system based on adenoviral vector and PER.C6 technology: High yield and preserved bioactivity

“…Several reports have previously described the use of a replication deficient adenovirus in combination with adenovirus production cells (HEK293) with the aim to produce recombinant protein (Andersen and Krummen, 2002;Cô té et al, 1998;Garnier et al, 1994;Lamarche et al, 1990;Massie et al, 1995Massie et al, , 1998. In these studies, the E1-deleted vector was allowed to replicate in HEK293 cells, in the process yielding high amounts (4-20% total cell protein) of the protein of interest.…”

High‐Level Recombinant Protein Production in CHO Cells Using an Adenoviral Vector and the Cumate Gene‐Switch

“…High-level recombinant protein production using AdVs was shown to be possible by using cells other than CHO cells (29,31,41,42). The cells used were either permissive for the replication of AdVs, such as 293 cells, or were non-permissive, such as HeLa, A549, or KB cells.…”

“…This plasmid contains also the 9.4-15.5 map unit segment of Ad5 genome for homologous recombination with Ad5-∆PS (see below). A plasmid (pMPG-BFP/CMV-cTA/tk-neo) expressing the cTA and the blue fluorescent protein (BFP) regulated by the CMV5 promoter (29,31) was constructed by replacing the IgG heavy and light chains of pMPG/chB43_TK/neo (32) with the cTA coding sequence excised from pAdCMV5-cTA (28) and the gene for BFP (29).…”

“…An E1/E3-deleted AdV encoding GFP (Qbiogene) regulated by the CR5 promoter (AdCR5-GFP) was generated by first replacing the Tet-regulated promoter (TR5) from pAdTR5-GFP (31) by the CR5 promoter isolated from pAdCR5-LacZ (28). The resulting plasmid (pAdCR5-GFP) was then used to produce AdCR5-GFP by homologous recombination after transfection of 293A cells with a mixture of linearized pAdCR5-GFP and E1/E3-deleted Ad5 DNA as described previously (31). AdCR5-GFP was then grown by sequential passages on BMAdE1 cells, except for the last passage, which was done on 293A cells, using standard procedure (35).…”