The HDAC inhibitor FK228 enhances adenoviral transgene expression by a transduction-independent mechanism but does not increase adenovirus replication.

The histone deacetylase inhibitor FK228 has previously been shown to enhance adenoviral transgene expression when cells are pre-incubated with the drug. Upregulation of the coxsackie adenovirus receptor (CAR), leading to increased viral transduction, has been proposed as the main mechanism. In the present study, we found that the highest increase in transgene expression was achieved when non-toxic concentrations of FK228 were added immediately after transduction, demonstrating that the main effect by which FK228 enhances transgene expression is transduction-independent. FK228 had positive effects both on Ad5 and Ad5/f35 vectors with a variety of transgenes and promoters, indicating that FK228 works mainly by increasing transgene expression at the transcriptional level. In some cases, the effects were dramatic, as demonstrated by an increase in CD40L expression by FK228 from 0.3% to 62% when the murine prostate cancer cell line TRAMP-C2 was transduced with Ad[CD40L]. One unexpected finding was that FK228 decreased the transgene expression of an adenoviral vector with the prostate cell-specific PPT promoter in the human prostate adenocarcinoma cell lines LNCaP and PC-346C. This is probably a consequence of alteration of the adenocarcinoma cell lines towards a neuroendocrine differentiation after FK228 treatment. The observations in this study indicate that FK228 enhances adenoviral therapy by a transduction-independent mechanism. Furthermore, since histone deacetylase inhibitors may affect the differentiation of cells, it is important to keep in mind that the activity and specificity of tissue- and tumor-specific promoters may also be affected.

Adenovirus-derived vectors for prostate cancer gene therapy.

Prostate cancer is a leading cause of death among men in Western countries. Whereas the survival rate approaches 100% for patients with localized cancer, the results of treatment in patients with metastasized prostate cancer at diagnosis are much less successful. The patients are usually presented with a variety of treatment options, but therapeutic interventions in prostate cancer are associated with frequent adverse side effects. Gene therapy and oncolytic virus therapy may constitute new strategies. Already a wide variety of preclinical studies has demonstrated the therapeutic potential of such approaches, with oncolytic prostate-specific adenoviruses as the most prominent vector. The state of the art and future prospects of gene therapy in prostate cancer are reviewed, with a focus on adenoviral vectors. We summarize advances in adenovirus technology for prostate cancer treatment and highlight areas where further developments are necessary.

Clinical adenoviral gene therapy for prostate cancer.

Prostate cancer is at present the most common malignancy in men in the Western world. When localized to the prostate, this disease can be treated by curative therapy such as surgery and radiotherapy. However, a substantial number of patients experience a recurrence, resulting in spreading of tumor cells to other parts of the body. In this advanced stage of the disease only palliative treatment is available. Therefore, there is a clear clinical need for new treatment modalities that can, on the one hand, enhance the cure rate of primary therapy for localized prostate cancer and, on the other hand, improve the treatment of metastasized disease. Gene therapy is now being explored in the clinic as a treatment option for the various stages of prostate cancer. Current clinical experiences are based predominantly on trials with adenoviral vectors. As the first of a trilogy of reviews on the state of the art and future prospects of gene therapy in prostate cancer, this review focuses on the clinical experiences and progress of adenovirus-mediated gene therapy for this disease.

A novel TARP-promoter-based adenovirus against hormone-dependent and hormone-refractory prostate cancer.

TARP (T cell receptor gamma-chain alternate reading frame protein) is a protein that in males is uniquely expressed in prostate epithelial cells and prostate cancer cells. We have previously shown that the transcriptional activity of a chimeric sequence comprising the TARP promoter (TARPp) and the PSA enhancer (PSAe) is strictly controlled by testosterone and highly restricted to cells of prostate origin. Here we report that a chimeric sequence comprising TARPp and the PSMA enhancer (PSMAe) is highly active in testosterone-deprived prostate cancer cells, while a regulatory sequence comprising PSAe, PSMAe, and TARPp (PPT) has high prostate-specific activity both in the presence and in the absence of testosterone. Therefore, the PPT sequence may, in a gene therapy setting, be beneficial to prostate cancer patients that have been treated with androgen withdrawal. A recombinant adenovirus vector with the PPT sequence, shielded from interfering adenoviral sequences by the mouse H19 insulator, yields high and prostate-specific transgene expression both in cell cultures and when prostate cancer, PC-346C, tumors were grown orthotopically in nude mice. Intravenous virus administration reveals both higher activity and higher selectivity for the insulator-shielded PPT sequence than for the immediate-early CMV promoter. Therefore, we believe that an adenovirus with therapeutic gene expression controlled by an insulator-shielded PPT sequence is a promising candidate for gene therapy of prostate cancer.

Characterization of the androgen-regulated prostate-specific T cell receptor gamma-chain alternate reading frame protein (TARP) promoter.

TARP (T cell receptor gamma-chain alternate reading frame protein) is uniquely expressed in males in prostate epithelial cells and prostate cancer cells. Here we demonstrate that TARP expression is regulated by testosterone at the transcriptional level through specific binding of androgen receptor to an androgen response element in the proximal TARP promoter. We further demonstrate that the promoter specifically initiates reporter gene expression in TARP-positive prostate cancer cell lines. To develop a regulatory sequence for prostate-specific gene expression, we constructed a chimeric sequence consisting of the TARP promoter and the prostate-specific antigen (PSA) enhancer. We found that in the prostatic adenocarcinoma cell line LNCaP, the transcriptional activity of the regulatory sequence consisting of a TARP promoter and PSA enhancer is 20 times higher than the activity of a regulatory sequence consisting of the PSA promoter and PSA enhancer. Thus, our studies define a regulatory sequence that may be used to restrict expression of therapeutic genes to prostate cancer cells and may therefore play a role in prostate cancer gene therapy.

Ex vivo stimulation of cytomegalovirus (CMV)-specific T cells using CMV pp65-modified dendritic cells as stimulators.

Cytomegalovirus (CMV) infection is a dangerous complication in immunosuppressed individuals such as allogeneic stem cell transplant patients. CMV disease can be prevented by the early post-transplant transfer of donor-derived, CMV-directed, T cells. Fast and cost efficient methods to generate CMV-specific T cells are, therefore, warranted. The current study utilized peptide-pulsed and adenovirus-transduced dendritic cells (DC) to generate CMV-restricted T cells. After one stimulation with CMV pp65495-503 peptide-pulsed DC and three re-stimulations with peptide-pulsed monocytes, virtually all T cells were CD8+, expressed the relevant T cell receptor and exhibited high peptide-specific lytic activity. After only one stimulation, pp65495-503-restricted T cells could be sorted to a purity of higher than 95% and expanded up to 1000-fold in 2 weeks. This technique may prove useful for the rapid generation of large quantities of specific cytolytic T lymphocytes (CTL) for cell therapy. DC transduced with an adenoviral vector encoding the full-length pp65 protein (Adpp65) were able to simultaneously expand CTL against multiple epitopes of pp65. In addition, they activated CMV-specific CD4+ T-helper cells. This approach would stimulate multiple-epitope populations of pp65-specific T cells and could be made available to patients of any human leucocyte antigen (HLA) haplotype. DC transduced with adenoviral vectors to express full-length antigens may prove to be potent vaccines against viral pathogens and cancer.