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One strategy for improving selectivity of gene therapy is the use of a replication-activated adenovirus vector that mediates transgene expression specifically in tumor cells through homologous recombination of viral genomes. In this study, we compared replication- activated adenovirus containing inverted repeats (Ad.IR-BG) with IR-deficient virus (Ad.BG) for selective gene expression in hepatocellular carcinoma and colon carcinoma metastases in the liver. We found that Ad.IR-BG conferred specific gene expression in both carcinoma cells, with minimal expression in hepatocytes and colon epithelial cells. This occurred through viral DNA recombination in Ad.IR-BG-infected tumor cells but not in normal cells. Hydroxyurea, which blocks DNA replication, inhibited DNA recombination and β-gal expression in Ad.IR-BG-infected but not Ad.BG-infected tumor cells. Finally, systemic injection of Ad.IR-BG into tumor xenografts in nude mice significantly improved selectivity of gene expression in tumors with minimal expression in normal tissues. Viral DNA recombination, which was absent in normal liver, was detected in Ad.IR-BG-infected tumors but not in Ad.BG-infected tissue. These findings demonstrated that replication-activated adenovirus can mediate tumor- specific gene expression through viral DNA recombination, which is otherwise deficient in normal cells. [ABSTRACT FROM AUTHOR]

Clinical applications of tumor gene therapy require tumor-specific delivery or expression of therapeutic genes in order to maximize the oncolytic index and minimize side effects. This study demonstrates activation of transgene expression exclusively in hepatic metastases after systemic application of a modified first-generation (E1A/E1B-deleted) adenovirus vector (AdE1-) in mouse tumor models. The discrimination between tumors and normal liver tissue is based on selective DNA replication of AdE1- vectors in tumor cells. This new AdE1- based vector system uses homologous recombination between inverted repeats to mediate precise rearrangements within the viral genome. As a result of these rearrangements, a promoter is brought into conjunction with a reporter gene creating a functional expression cassette. Genomic rearrangements are dependent upon viral DNA replication, which in turn occurs specifically in tumor cells. In a mouse tumor model with liver metastases derived from human tumor cells, a single systemic administration of replication activated AdE1- vectors achieved transgene expression in every metastasis, whereas no extra-tumoral transgene induction was observed. Here we provide a new concept for tumor-specific gene expression that is also applicable for other conditionally replicating adenovirus vectors. [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs) are crucial players in promoting immune responses. Logically, adoptive DC therapy is a promising approach in cancer immunotherapy. One of the major obstacles in cancer immunotherapy in general is the immunosuppressive tumor microenvironment, which hampers the maturation and activation of DCs. Therefore, human clinical outcomes with DC therapy alone have been disappointing. In this study, we use fully serotype 3 oncolytic adenovirus Ad3-hTERT-CMV-hCD40L, expressing human CD40L, to modulate the tumor microenvironment with subsequently improved function of DCs. We evaluated the synergistic effects of Ad3-hTERT-CMV-hCD40L and DCs in the presence of human peripheral blood mononuclear cells ex vivo and in vivo. Tumors treated with Ad3-hTERT-CMV-hCD40L and DCs featured greater antitumor effect compared with unarmed virus or either treatment alone. 100% of humanized mice survived to the end of the experiment, while mice in all other groups died by day 88. Moreover, adenovirally-delivered CD40L induced activation of DCs, leading to induction of Th1 immune responses. These results support clinical trials with Ad3-hTERT-CMV-hCD40L in patients receiving DC therapy. [ABSTRACT FROM AUTHOR]

Vaccination with dendritic cells (DCs), the most potent professional antigen-presenting cells in the body, is a promising approach in cancer immunotherapy. However, tumors induce immunosuppression in their microenvironment that suppresses and impairs the function of DCs. Therefore, human clinical trials with DC therapy have often been disappointing. To improve the therapeutic efficacy and to overcome the major obstacles of DC therapy, we generated a novel adenovirus, Ad3-hTERT-CMV-hCD40L, which is fully serotype 3 and expresses hCD40L for induction of antitumor immune response. The specific aim is to enhance DCs function. Data from a human cancer patient indicated that this capsid allows effective transduction of distant tumors through the intravenous route. Moreover, patient data suggested that virally produced hCD40L can activate DCsin situ. The virus was efficientin vitroand had potent antitumor activityin vivo.In a syngeneic model, tumors treated with Ad5/3-CMV-mCD40L virus plus DCs elicited greater antitumor effect as compared with either treatment alone. Moreover, virally coded CD40L induced activation of DCs, which in turn, lead to the induction of a Th1 immune response and increased tumor-specific T cells. In conclusion, Ad3-hTERT-CMV-hCD40L is promising for translation into human trials. In particular, this virus could enable successful dendritic cell therapy in cancer patients. [ABSTRACT FROM AUTHOR]

An abstract of the article "Analysis of T-Cell Responses to Helper-Dependent Serotype 5-Based Capsid-Modified Adenovirus (HD.5/35) or First-Generation Serotype 11- Based (Ad11) Vectors," by Nelson C. Di Paolo and colleagues is presented.

An abstract of the article "Identifying Functional Adenovirus-Host Interactions Using Tandem Mass Spectrometry," by Anuj Gaggar, Martin Sadilek, Andre Lieber and Dmitry M. Shayakhmetov is presented.

Twenty-five patients with chemotherapy refractory cancer were treated with a fully serotype 3-based oncolytic adenovirus Ad3-hTERT-E1A. In mice, Ad3 induced higher amounts of cytokines but less liver damage than Ad5 or Ad5/3. In humans, the only grade 3 adverse reactions were self-limiting cytopenias and generally the safety profile resembled Ad5-based oncolytic viruses. Patients that had been previously treated with Ad5 viruses presented longer lasting lymphocytopenia but no median increase in Ad3-specific T-cells in blood, suggesting immunological activity against antigens other than Ad3 hexon. Frequent alterations in antitumor T-cells in blood were seen regardless of previous virus exposure. Neutralizing antibodies against Ad3 increased in all patients, whereas Ad5 neutralizing antibodies remained stable. Treatment with Ad3-hTERT-E1A resulted in re-emergence of Ad5 viruses from previous treatments into blood and vice versa. Signs of possible efficacy were seen in 11/15 (73%) patients evaluable for tumor markers, four of which were treated only intravenously. Particularly promising results were seen in breast cancer patients and especially those receiving concomitant trastuzumab. Taken together, Ad3-hTERT-E1A seems safe for further clinical testing or development of armed versions. It offers an immunologically attractive alternative, with possible pharmacodynamic differences and a different receptor compared to Ad5. [ABSTRACT FROM AUTHOR]

Adenovirus (Ad) vectors are widely used for gene delivery in vitro and in vivo. A solid understanding of the biology of this virus is imperative for the development of novel, effective, and safe vectors. For the group C adenovirus serotypes 2 and 5 that use CAR as a primary attachment receptor, it is known that the penton base RGD motifs interact with cellular integrins and that this interaction promotes virus internalization. However, the RGD motif's impact on the efficiency of postinternalization steps, such as the escape of the virus particle from the endosome, is less defined. Furthermore, the role of penton-integrin interactions remains unknown for new vectors possessing group B Ad fiber knobs that use CD46 as a primary virus attachment receptor. In this study, we used vectors with the RGD motif deleted that contained Ad5 and B-group Ad35 fiber knobs and long fiber shafts and studied the role of RGD-integrin interactions in virus internalization and endosome escape. The deletion of the RGD motif in the penton base did not affect virus attachment, regardless of the type of cellular receptor used for attachment. RGD motif deletion, however, significantly reduced the rate of virus internalization for both the Ad5 and Ad35 fiber knob-containing vectors. This study also demonstrates the role of penton RGD motifs in facilitating the endosome escape step of virus infection and indicates that penton-integrin interactions are involved in internalization of capsid-chimeric CD46-interacting Ads with long fiber shafts. [ABSTRACT FROM AUTHOR]

We previously reported that the HS-4 insulator, derived from the chicken β-globin locus, was able to shield a downstream inducible promoter from viral enhancers or silencers present in the genome of adenovirus vectors. In this study, we constructed two recombinant adenoviruses (Ad) that express an alkaline phosphatase (AP) reporter gene driven by an α-fetoprotein (AFP) enhancer/promoter with and without HS-4 insulator (Ad.HS4.AFP-AP and Ad.AFP-AP). The insulated vector, Ad.HS4.AFP-AP, conferred significantly higher AP expression than Ad.AFP-AP in all AFP-producing hepatocellular carcinoma cell lines (HepG2, Hep3B, and HuH7) examined. AP expression from Ad.HS4.AFP-AP was specific to hepatoma cells and barely detectable in AFP-negative tumor cell lines and normal human cells, including human hepatocytes. Intravenous infusion of viral vectors into mice with liver metastasis derived from Hep3B hepatoma cells resulted in AP expression exclusively localized to tumor cells. The number of tumor cells with detectable AP expression was significantly higher in mice infused with Ad.HS4.AFP-AP than in mice that received the non-insulated vector. This study demonstrates that the HS-4 insulator in the context of an Ad vector can increase the activity of the AFP promoter, while maintaining its tumor-specificity in vitro and in vivo. Considering that the anti-tumor activity of oncolytic vectors often depends on the level of pro-apoptotic or suicide gene expression, insulators might be a useful tool to improve the efficacy and specificity of these vectors. [Copyright &y& Elsevier]

An abstract of the article "Evaluation of Biodistribution and Safety of Adenovirus Vectors Containing B-Group Fibers after Intravenous Injection into Baboons," by Shaoheng Ni, Kathrin Bernt, Zong-Yi Li, Hans-Peter Kiem, and Andre Lieber is presented.

We investigated the mechanisms which confer resistance to viral oncolysis using a capsid-modified, conditionally replicating oncolytic adenovirus (Ad5/35.IR.E1A/TRAIL) and primary ovarian cancer cells. This vector targets tumor cells by binding through the adenovirus serotype 35 fiber to CD46, which is upregulated on most tumor cells in situ. Tumor-specific viral replication and TRAIL gene expression is achieved upon homologous recombination between inverted repeats (IR) in adenovirus genomes (Ad.IR). Ad5/35.IR- E1A/TRAIL was efficient in lysing a variety of established human tumor cell lines in vitro and in eliminating liver metastases in mouse models after intravenous injection. However, in experiments on primary ovarian cancer cell cultures derived from biopsies (N>30), we discovered that in a number of cultures, individual cells remained alive after infection with Ad5/35.IR.E1A/TRAIL, even when high doses of virus were applied. To enrich cultures for cells resistant to viral oncolysis we established a total of 120 cultures derived from single cell clones of a biopsy from a grade IV ovarian cancer patient. Resulting clonal cell cultures were tested for their level of infectibility, their ability to support viral replication and their susceptibility to viral oncolysis by Ad5/35.IR-E1A/TRAIL. We found that about 50% of clonal cultures contained cells resistant to cell lysis, whereby the mechanisms that cause resistance were complex and appeared to include reduced infectivity, reduced ability to support viral DNA replication, and inability to execute apoptosis. Using methylation- specific PCR we found the promoter of a number of cancer-associated genes methylated in resistant cells but not in susceptible cells. Based on this, we tested whether demethylation agents would increase the sensitivity to viral oncolysis. We demonstrated that enhanced viral oncolysis could be detected when Ad5/35.IR.E1A/TRAIL was used in combination with deacetylase inhibitor TSA (trichostatin A) and methyltransferase inhibitor DAC (5-aza-2'-deoxycytidine). We then focused on clones that could be efficiently infected with Ad5/35 vectors and performed genome-wide expression profiling of 15 clones with >90% resistant cells and 15 clones that were 100% susceptible to lysis by Ad5/35.IR-E1A/TRAIL using cDNA arrays. Examples of genes that were upregulated (in array and qRT-PCR analyses) in resistant cells were clusterin, lipocalin2, netrin4, epiregulin, claudin7, defensin beta1, inhibin betaB. We are currently using siRNA specific to these genes to test whether their down-regulation can overcome resistance to oncolysis and results will be reported. Furthermore, we are processing the array data by functional clustering to identify pathways that confer resistance. Our final goal is to “arm” our oncolytic vector with cDNA or siRNA expression cassettes that can sensitize tumor cells to virus and TRAIL mediated cell killing.Molecular Therapy (2006) 13, S18–S18; doi: 10.1016/j.ymthe.2006.08.056 [ABSTRACT FROM AUTHOR]

Intravascular (iv) delivery of adenovirus (Ad) vectors is hindered by acute toxicity that is triggered by trapping of virus in the reticuloendothelial system (RES) within minutes of administration, and subsequent production of pro-inflammatory cytokines/chemokines. While Ad localizes to tissue macrophages and endothelial cells of the liver, spleen and lung within minutes of iv delivery, the mechanism of this reticuloendothelial targeting is unknown. We have identified a novel pathway by which platelets mediate trapping of Ad in the RES. Rapidly after iv administration fiber independent interaction between Ad and circulating platelets can be seen by Southern blot, by transmission electron microscopy (TEM) of isolated platelets and blood cell fractionation of mice receiving radiolabeled Ad. Blood platelet levels drop after Ad administration, while serum levels of the platelet activation marker soluble CD62p rise. Within 5 minutes of administration, Ad and platelets co-localize in the liver sinusoids along with endothelial and Kupffer cells. TEM showed Ad particles inside degranulated platelets and platelet aggregates within the liver sinusoids. Ad particles could also be seen inside Kupffer cells. Immunohistochemistry (IHC) showed widespread co-labelling of platelets (CD41), Kupffer cells (F4/80) and Ad (hexon) throughout the liver. In the spleen, Ad genomes were found within 5 minutes and Ad particles were detected in CD41 and F4/80 rich regions of the marginal zone by IHC. In the lung, Ad genomes were found within 5 minutes. Platelet accumulation was seen by IHC after delivery of Ad vectors, with platelet-endothelial interactions visible in the pulmonary vasculature by TEM. To determine the contribution of Ad-platelet interactions towards toxicity, mice were pre-injected with a platelet depleting antibody prior to virus delivery. At 6 hours post virus delivery, vector genomes in liver, lung and spleen were detected by Southern blot, and circulating serum cytokine levels were determined. Serum levels of the pro-inflammatory cytokines/chemokines IFN-γ, IL-6 and MCP- 1 were lower in platelet depleted mice. The effect of Ad neutralizing antibodies on Ad-platelet interactions was also determined in mice that had been pre-immunized with the same vector prior to iv Ad vector administration. Early after vector administration significantly lower levels of Ad genomes were associated with circulating platelets in pre-immunized mice. While the levels of Ad deposition in the liver were unchanged in pre-immunized mice, there was reduced Ad deposition within the lungs of pre-immunized mice. Taken together these data suggest that rapid interaction between Ad and platelets occurs after iv administration. Platelet-Ad interactions apparently play a role in RES sequestration and degradation of Ad, and subsequent toxicity. We are currently attempting to identify the structural components involved in Ad-platelet interactions in vivo and investigating the contribution of this interaction to sequestration, degradation and toxicity, after Ad administration.Molecular Therapy (2006) 13, S30–S31; doi: 10.1016/j.ymthe.2006.08.089 [ABSTRACT FROM AUTHOR]

An abstract of the article "Trapping of Adenovirus Vectors in Blood," by Daniel Stone, Shaoheng Ni, Zong-Yi Li, Dmitry M. Shayakhmetov and Andre Lieber is presented.

An abstract of the article "Integration Site Analysis of a Helper- Dependent Adenovirus Vector Containing AAV ITRs and 22 kb of the Human Globin LCR," by Hongjie Wang, Tobias Leege, Dmitry Shayakhmetov, Qiliang Li, Thalia Papayannopoulou, George Stamatoyannopolous and Andre Lieber is presented.

An abstract of the article "Evaluation of a Serotype 35 Fiber Containing Adenovirus Vector for Vaccination," by Nelson C. Di Paolo, Shaoheng Ni, Anuj Gaggar, Zong-Yi Li, and Andre Lieber is presented.

An abstract of the article "Localization of Regions in CD46 That Interact with Adenovirus," by Anuj Gaggar, Dmitry M. Shayakhmetov, M. Kathryn Liszewski, John P. Atkinson, and Andre Lieber is presented.