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

Stable E1 transformed cells, like PER.C6, are able to grow at scale and to high cell densities. E1-deleted adenoviruses replicate to high titer in PER.C6 cells whereas subsequent deletion of E2A from the vector results in absence of replication in PER.C6 cells and drastically lowers the expression of adenovirus proteins in such cells. We therefore considered the use of an DeltaE1/DeltaE2 type 5 vector (Ad5) to deliver genes to PER.C6 cells growing in suspension with the aim to achieve high protein yield. To evaluate the utility of this system we constructed DeltaE1/DeltaE2 vector carrying different classes of protein, that is, the gene coding for spike protein derived from the Coronavirus causing severe acute respiratory syndrome (SARS-CoV), a gene coding for the SARS-CoV receptor or the genes coding for an antibody shown to bind and neutralize SARS-CoV (SARS-AB). The DeltaE1/DeltaE2A-vector backbones were rescued on a PER.C6 cell line engineered to constitutively over express the Ad5 E2A protein. Exposure of PER.C6 cells to low amounts (30 vp/cell) of DeltaE1/DeltaE2 vectors resulted in highly efficient (>80%) transduction of PER.C6 cells growing in suspension. The efficient cell transduction resulted in high protein yield (up to 60 picogram/cell/day) in a 4 day batch production protocol. FACS and ELISA assays demonstrated the biological activity of the transiently produced proteins. We therefore conclude that DeltaE1/DeltaE2 vectors in combination with the PER.C6 technology may provide a viable answer to the increasing demand for high quality, high yield recombinant protein.

Induction of the mitogen-activated p70 S6 kinase by adenovirus E1A.

Adenovirus E1A proteins can transform primary cells in culture in conjunction with other oncogenes, such as E1B or activated ras. The modulation of various cell cycle regulators by E1A is thought to be involved in this transformation process. In this paper we show that E1A enhances the expression of the mitogen-inducible p70 S6 kinase (p70s6k), a kinase which is essential for G1 progression. p70s6k mRNA and protein levels are enhanced 3-4-fold in various E1A-expressing cell lines. Similarly, the activity of p70s6k is enhanced in E1A-expressing cells in a manner partially independent of enhanced expression of p70s6k. The induction of p70s6k correlates with the presence of conserved region 1 (CR1) of E1A and with morphological transformation by E1A. These results suggest that induction of p70s6k by E1A might be involved in transformation by E1A.

Feasibility of adenovirus-mediated nonsurgical synovectomy in collagen-induced arthritis-affected rhesus monkeys.

Gene transfer to synovial tissue by adenoviral vectors (Ad) was studied in vitro in cultured human synoviocytes and in vivo in seven primates with arthritis. Hyperplastic synovium was efficiently transduced with Ad.lacZ in vitro and in vivo in rhesus monkeys with collagen-induced arthritis, whereas chondrocytes were not transduced. Intraarticular injection of recombinant Ad harboring the luciferase gene showed the presence of reporter gene products only in Ad-injected joints. In addition, the feasibility of synovectomy by Ad harboring the herpes simplex virus thymidine kinase gene (tk) was studied. In vitro infection of synovium from rheumatoid arthritis patients with Ad.TK, followed by administration of ganciclovir, resulted in death of >90% of the synoviocytes. By mixing Ad.TK-infected with noninfected cells, it appeared that the presence of 10% infected synoviocytes resulted in the killing of more than 85% of the synoviocytes, demonstrating a substantial bystander effect. Intraarticular injection of Ad.TK in the knees of rhesus monkeys with arthritis, followed by treatment with ganciclovir for 14 days, resulted in increased apoptotic cell death in the synovium of Ad.TK-injected as compared with noninjected joints and ablation of the synovial lining layer. The procedure revealed no toxic side effects. These data suggest that nonsurgical synovectomy by tK gene therapy is feasible.

The effect of promoter strength in adenoviral vectors in hyperplastic synovium.

OBJECTIVES: To compare the activity of the CytoMegaloVirus promoter (CMV) and the Major Late promoter (MLP) in synoviocytes in vitro and in vivo. To determine the phenotype of infected cells and the induction of inflammation. To investigate the effects of the cytomegalovirus (CMV) or major late (MLP) promoter on adenovirus-mediated reporter gene transduction of synoviocytes in vitro and in vivo. METHODS: After infection with adenoviral vectors harboring CMV- and MLP-driven luciferase and lacZ genes, gene expression was examined in cultured synoviocytes and in the synovium of rhesus monkeys with collagen-induced arthritis. Immunohistochemical staining for the macrophage-marker CD68 and lacZ expression was performed. Inflammation was scored in the synovial membrane of injected and non-injected joints. RESULTS: CMV-driven reporter gene expression was found to be 6 to 10 times higher than MLP-driven gene expression in both cultured synoviocytes and monkey synovium. Both CD68 positive and CD68 negative cells were lacZ positive. Inflammation in joints injected with CMV-driven adenoviral vectors was not higher than that in MLP-driven adenoviral vectors- or non-injected joints. CONCLUSION: These experiments show that the CMV promoter induces higher gene expression in synoviocytes than the MLP promoter. Both fibroblast-like and macrophage-like synoviocytes can be infected with adenoviral vectors. No deleterious effects of the CMV-promoter driven adenoviral vectors were observed.

Downregulation of MHC class I expression due to interference with p105-NF kappa B1 processing by Ad12E1A.

We have previously demonstrated that expression of major histocompatibility complex (MHC) class I genes is repressed in baby rat kidney cells transformed by early region 1 of oncogenic adenovirus type 12 (Ad12E1). Reduced expression of MHC class I antigens contributes to the escape of Ad12-transformed cells from T-cell-mediated immune surveillance and to tumour induction. In this study, we show that repression of MHC class I expression by Ad12E1A is mediated via the H2TF1 element of the MHC class I promoter. This element binds NF kappa B and KBF1, two factors which play a major role in the regulation of MHC class I expression in vivo. In extracts from Ad12E1-transformed cells, binding of KBF1 and NF kappa B to the H2TF1 element is decreased. This is caused by reduced production of p50-NF kappa B1, the 50 kDa subunit shared by KBF1 and NF kappa B, due to interference with p105-NF kappa B1 processing by Ad12-13S-E1A protein. Overexpression of the p105-NF kappa B1 cDNA, or of a truncated p105-NF kappa B1 cDNA that codes for p50-NF kappa B1, restores MHC class I expression in Ad12E1-transformed cells. These data demonstrate that downregulation of MHC class I expression in Ad12E1-transformed cells is due to interference with processing of p105-NF kappa B1 by the Ad12-13S-E1A protein.

Transposon Tc1 of the nematode Caenorhabditis elegans jumps in human cells.

The transposon Tc1 of the nematode Caenorhabditis elegans is a member of the widespread family of Tc1/mariner transposons. The distribution pattern of virtually identical transposons among insect species that diverged 200 million years ago suggested horizontal transfer of the elements between species. Thishypothesis gained experimental support when it was shown that Tc1 and later also mariner transposons could be made to jump in vitro , with their transposase as the only protein required. Later it was shown that mariner transposons from one fruit fly species can jump in other fruit fly species and in a protozoan and, recently, that a Tc1-like transposon from the nematode jumps in fish cells and that a fish Tc1-like transposon jumps in human cells. Here we show that the Tc1 element from the nematode jumps in human cells. This provides further support for the horizontal spread hypothesis. Furthermore, it suggests that Tc1 can be used as vehicle for DNA integration in human gene therapy.

IkappaB alpha is a target for the mitogen-activated 90 kDa ribosomal S6 kinase.

The activity of transcription factor NFkappaB is regulated by its subcellular localization. In most cell types, NFkappaB is sequestered in the cytoplasm due to binding of the inhibitory protein IkappaB alpha. Stimulation of cells with a wide variety of agents results in degradation of IkappaB alpha which allows translocation of NFkappaB to the nucleus. Degradation of IkappaB alpha is triggered by phosphorylation of two serine residues, i.e. Ser32 and Ser36, by as yet unknown kinases. Here we report that the mitogen-activated 90 kDa ribosomal S6 kinase (p90rsk1) is an IkappaB alpha kinase. p90rsk1 phosphorylates IkappaB alpha at Ser32 and it physically associates with IkappaB alpha in vivo. Moreover, when the function of p90rsk1 is impaired by expression of a dominant-negative mutant, IkappaB alpha degradation in response to mitogenic stimuli, e.g. 12-O-tetradecanoylphorbol 13-acetate (TPA), is inhibited. Finally, NFkappaB cannot be activated by TPA in cell lines that have low levels of p90rsk1. We conclude that p90rsk1 is an essential kinase required for phosphorylation and subsequent degradation of IkappaB alpha in response to mitogens.

Induction of differentiation-regulated transcription factor Oct-6 specifically accompanies major histocompatibility complex class I down-regulation by E1A of oncogenic adenovirus type 12.

The E1A genes from adenovirus (Ad) types 5 and 12 share the capacity to cooperate with a second oncogene to transform primary rodent cells in vitro. However, only Ad12-transformed cells are oncogenic in immunocompetent rodents, an event that requires conserved region 3 (CR3) of E1A to be intact. Ad12-induced tumorigenicity correlates with the E1A-CR3-dependent down-modulation of MHC class I transcription, contributing to escape from CTL-mediated immune surveillance. Expression of MHC class I antigens is also lacking in undifferentiated embryonal carcinoma cells. In these cells, MHC class I expression increases during differentiation in a process possibly involving octamer-binding proteins. We found that both nononcogenic and oncogenic Ad-transformed cells contained the ubiquitously expressed factor Oct-1. In contrast, only oncogenic Ad12-transformed cells that are derived from primary cell cultures expressed an additional octamer-binding factor, which we identified as Oct-6. The induction of Oct-6 expression was at the RNA level and was found to require an intact CR3 domain in Ad12 E1A. Like MHC class I expression, Oct-6 expression was not affected in already established cell lines expressing Ad12 E1A. The presence of Oct-6 in Ad12-transformed cells correlated with an increase in octamer-dependent transcription of a reporter gene, relative to Ad5-transformed cells.(ABSTRACT TRUNCATED AT 250 WORDS)