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Abstract: Adenoviral vectors are highly efficient at transferring genes into cells and are broadly used in cancer gene therapy. However, many therapeutic genes are toxic to vector host cells and thus inhibit vector production. The truncated form of E2F-1 (E2Ftr), which lacks the transactivation domain, can significantly induce cancer cell apoptosis, but is also toxic to HEK-293 cells and inhibits adenovirus replication. To overcome this, we have developed binary- and single-vector systems with a modified tetracycline-off inducible promoter to control E2Ftr expression. We compared several vectors and found that the structure of expression cassettes in vectors significantly affects E2Ftr expression. One construct expresses high levels of inducible E2Ftr and efficiently causes apoptotic cancer cell death by activation of caspase-3. The approach developed in this study may be applied in other viral vectors for encoding therapeutic genes that are toxic to their host cells and/or inhibit vector propagation. [Copyright &y& Elsevier]

Abstract: Adenovirus vectors (Adv) are used widely in cancer gene therapy research. However, the clinical application of Adv currently is limited to local, intratumoral administration; systemic administration leads to redundant transgene expression in the liver and subsequent hepatotoxicity. Here we replaced the conventional cytomegalovirus (CMV) promoter of Adv with a tumor-specific telomere reverse transcriptase (TERT) promoter, to restrict expression of the Adv-transduced transgene to tumor tissue alone. We evaluated the therapeutic and side effects after systemic administration of Adv expressing herpes simplex virus thymidine kinase (Ad-HSVtk) in mice bearing Meth-A tumors. Although systemically injected CMV promoter-driven Ad-HSVtk lacked therapeutic effect, mice injected with 2×1011 viral particles containing TERT promoter-driven Ad-HSVtk showed inhibited tumor growth and prolonged survival with minimal side effects. Our results suggest that Adv in which transgene expression is driven by the TERT promoter are a promising prototype of tumor-targeting vectors for effective and safe cancer gene therapy. [Copyright &y& Elsevier]

Purpose: Adenoviral gene therapy could potentially play a role in the treatment of esophageal cancer and Barrett’s esophagus. The adenoviruses can be categorized in different serotypes. The goal of the present study was to investigate the transduction efficacy of different adenoviral serotypes in different models of esophageal cancer and Barrett’s esophagus. Methods: Chimeras of the adenoviral serotype 5 backbone and fibers of serotypes 5, 16, 35, 40, and 50 were constructed with PCR technology. For esophageal cancer, cell lines were used originating from with adenocarcinoma and squamous cell carcinoma, respectively. Differentiating Caco-2 cells were used as an in vitro model for Barrett’s esophagus. GFP was used as a reporter gene and transduction efficacy was assessed by flow cytometry. Results: Overall transduction was rather efficient in the cancer cell lines. Especially serotype 16 and 50 exhibited an improved transduction compared with the other serotypes. In the Caco-2 cell lines, we observed a decreased transduction upon differentiation of the cells. All serotypes had a very limited transduction and no serotype had an additional value in this setting. Conclusions: Some serotypes could have an additional value in the development of gene therapy for esophageal cancer. Especially serotype 16 and 50 exhibited an improved transduction in esophageal cancer cells compared with the native serotype 5. In the setting of Barrett’s esophagus, none of the serotypes had an improved potency as in differentiated intestinal cells all serotypes had a very limited transduction. [Copyright &y& Elsevier]

Abstract: Human prostate and colon gene-1 (PC-1, also known as PrLZ) is an androgen-regulated, prostate tissue and prostate cancer cells specifically expressed novel gene. The increased expression of PC-1 gene appears to promote prostate cancer cells androgen-dependent (AD) and androgen-independent (AI) growth. To clone and investigate the expression and regulation elements of PC-1 gene may provide insight into the function of PC-1 and develop a new promoter that targets therapeutic genes to the AD and AI prostate cancer cells. The goal of the present study is cloning and characterization of the PC-1 promoter. A series of luciferase constructs that contain various fragments of the PC-1 5′-genomic region were transfected into human prostate cancer cells for promoter transactivation analysis. 5′ deletion analysis identified the −1579bp promoter region was required for the maximal proximal promoter activity; two transcriptional suppression and a positive regulatory region were identified; −4939bp promoter fragment of the PC-1 gene retained the characteristic of prostate cancer-specific expression and exhibited higher transcription activity than PSA-6kb promoter in the medium supplemented with steroid-depleted FBS. An androgen response element (ARE) was located in between −345 and −359bp of the PC-1 5′-untranslated region relative to the translation initiation site. Thus, our studies not only provide molecular basis of PC-1 transcription regulation, but also define a new regulatory sequence that may be used to restrict expression of therapeutic genes to prostate cancer in the prostate cancer gene therapy. [Copyright &y& Elsevier]

Abstract: Objectives. : Capsid-modified AAV vectors can mediate enhanced gene transfer to neoplasms characterized by low AAV receptor expression. Here we sought to determine the therapeutic potential of a capsid-modified AAV vector for gene therapy of ovarian carcinoma (OvCa). Methods. : We tested a panel of OvCa cell lines for AAV2-mediated gene transduction and for sensitivity to ganciclovir (GCV) following AAVHSVtk administration. Levels of AAV internalization and attachment receptor were assessed by flow cytometry and immunohistochemistry. The role of receptors in AAV-mediated gene transfer was assessed by competition assays. Finally, we examined the ability of a modified vector with an integrin-binding RGD motif inserted into the AAV capsid to improve gene delivery to OvCa and enhance AAVHSVtk/GCV-mediated killing by cytotoxicity assay. Results. : All OvCa cell lines were poorly transduced with AAV2 vectors and showed variably sensitive to AAVHSVtk/GCV. While OvCa cell lines expressed AAV2 internalization receptors (αv integrins), expression of the AAV2 attachment receptor, HSPG, was variable and not detected on many lines. Analysis of archived clinical specimens showed no detectable HSPG expression on approximately 45% of primary human tumors. Gene transfer to OvCa was increased several fold using the RGD-modified vector. Gene transfer was independent of HSPG and specific to the targeted receptor. Importantly, the RGD-modified capsid markedly increased the ability of the AAVHSVtk to kill OvCa cells in the presence of GCV. Conclusions. : The development of AAV vectors targeted to cell surface receptors other than HSPG will be critical to the advancement of AAV-mediated gene therapy for treating OvCa. [Copyright &y& Elsevier]

Abstract: Objective. : Icodextrin, a novel glucose polymer solution utilized for peritoneal dialysis, has been demonstrated to have prolonged intraperitoneal (IP) instillation volumes in comparison to standard PBS solutions. In an animal model of ovarian cancer, we explored whether a survival advantage exists utilizing icodextrin rather than PBS as a delivery solution for an infectivity enhanced virotherapy approach. Methods. : Initial experiments evaluated whether icodextrin would adversely affect replication of a clinical grade infectivity enhanced conditionally replicative adenovirus (Δ24-RGD). Virus was added to prepared blinded solutions of PBS or icodextrin (20%) and then evaluated in vitro in various human ovarian cancer cell lines (SKOV3.ip1, PA-1, and Hey) and in vivo in a SKOV3.ip1 human ovarian cancer IP murine model. Viral replication was measured by detecting adenovirus E4 gene levels utilizing QRT-PCR. Survival was subsequently evaluated in a separate SKOV3.ip1 ovarian cancer IP murine model. Cohorts of mice were treated in blinded fashion with PBS alone, icodextrin alone, PBS+Δ24-RGD, or icodextrin+Δ24-RGD. Survival data were plotted on Kaplan–Meier curve and statistical calculations performed using the log-rank test. Results. : There was no adverse affect of icodextrin on vector replication in the ovarian cancer cell lines nor murine model tumor samples evaluated. Median survival in the IP treated animal cohorts was 23 days for the PBS group, 40 days for the icodextrin group, 65 days for the PBS+Δ24-RGD group, and 105 days for icodextrin+Δ24-RGD (p =0.023). Of note, 5 of the 10 mice in the icodextrin+Δ24-RGD group were alive at the end of the study period, all without evidence of tumor (120 days). Conclusions. : These experiments suggest that the use of dialysates such as icodextrin may further enhance the therapeutic effects of novel IP virotherapy and other gene therapy strategies for ovarian cancer. Phase I studies utilizing icodextrin-based virotherapy for ovarian cancer are currently in development. [Copyright &y& Elsevier]

Background: Transcription factor PDX-1 is expressed by human pancreatic and breast cancers. Although cytotoxicity of PDX-1-directed RIP-TK/GCV gene therapy to pancreatic cancer cells has been demonstrated, the efficacy of this treatment in breast cancer cells is unknown. The purpose of this study was to determine the expression of PDX-1 and its effect on RIP activation in two human breast cancer cell lines, AU565 and T47D. We also investigated the efficacy of RIP-TK/GCV gene therapy and examined whether exogenous PDX-1 to would enhance its cytotoxic effect. Materials and methods: RT-PCR was used to determine PDX-1 expression. Gene constructs RSVLacZ and RIPLacZ were used for transient transfection and LacZ expression was determined using reporter assays. T47D cells were also transfected with adenoviral vectors. Cells were transfected with RIP-TK and the suboptimal level of GCV was determined for each cell line. Following GCV treatment, cytotoxicity was measured using MTS assays. The effect of exogenous PDX-1 on LacZ expression and RIP-TK cytotoxicity was determined. Results: PDX-1 mRNA was expressed in human breast cancer cells and activated the RIP. Exogenous PDX-1 enhanced LacZ expression in AU565 cells but not in T47D cells. Adenoviral transfection was more efficient in T47D cells than non-viral transfection. RIP-TK treatment was cytotoxic to AU565 and T47D cells and this effect was enhanced by exogenous PDX-1 with both transfection methods. Conclusions: RIP-TK/GCV therapy is cytotoxic to human breast cancer cells and exogenous PDX-1 enhances cytotoxicity. In vivo studies are necessary to determine the tumor specificity and efficacy of this treatment. [Copyright &y& Elsevier]

Abstract: The high mutation rates of retroviruses are a potential problem with retroviral vectors. We studied the mutation rates and spectra of p53 sequences transduced with a retroviral vector in a cancer gene therapy model. When p53-deficient H358 non-small cell lung cancer cells were treated with a retroviral vector carrying normal p53 cDNA, most of transduced cells were killed by apoptosis. However, a small number of clones escaped p53-mediated apoptosis. We examined the p53 cDNA structure in these resistant clones. PCR-based analysis showed that 88/102 clones had detectable mutations in p53, including gross rearrangements, deletions/insertions, and base substitutions. To study the mutation rate of the p53 sequence in all transduced clones, the retroviral vector containing the non-functional p53 gene and the Neo-resistant marker gene was introduced into H358 cells. Only one of 95 isolated clones showed a base substitution. These results indicate that the mutation rate of p53 is not particularly high, but there is a significant risk that cancer cells will resist p53 gene therapy as a result of retroviral replication errors. [Copyright &y& Elsevier]

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a type II transmembrane cytokine molecule of the TNF family. Soluble recombinant TRAIL has been shown to induce apoptosis in a wide variety of cancer cells in vitro and to specifically limit tumor growth without damaging normal cells and tissues in vivo. These results suggest a strong potential of TRAIL as an anticancer therapy. Here we report an artificial TRAIL gene that expresses and secretes trimeric TRAIL into the culture supernatant. This novel TRAIL gene is composed of three functional elements, including a secretion signal, a trimerization domain, and an apoptosis-inducing moiety of TRAIL gene sequence. The expression vectors delivering this TRAIL gene produced secretable forms of trimeric TRAIL proteins. These TRAIL proteins showed greater apoptotic activity than the known TRAIL protein that does not contain an additional trimerization domain. Our data suggest that the gene therapy using our artificial TRAIL gene may be used as an anticancer therapy. [Copyright &y& Elsevier]