A Multi Targeting Conditionally Replicating Adenovirus Displays Enhanced Oncolysis while Maintaining Expression of Immunotherapeutic Agents.

Studies have demonstrated that oncolytic adenoviruses based on a 24 base pair deletion in the viral E1A gene (D24) may be promising therapeutics for treating a number of cancer types. In order to increase the therapeutic potential of these oncolytic viruses, a novel conditionally replicating adenovirus targeting multiple receptors upregulated on tumors was generated by incorporating an Ad5/3 fiber with a carboxyl terminus RGD ligand. The virus displayed full cytopathic effect in all tumor lines assayed at low titers with improved cytotoxicity over Ad5-RGD D24, Ad5/3 D24 and an HSV oncolytic virus. The virus was then engineered to deliver immunotherapeutic agents such as GM-CSF while maintaining enhanced heterogenic oncolysis.

Analysis of purified wild type and mutant adenovirus particles by SILAC based quantitative proteomics.

We used SILAC (stable isotope labelling of amino acids in cell culture) and high-throughput quantitative MS mass spectrometry to analyse the protein composition of highly purified WT wild type adenoviruses, mutant adenoviruses lacking an internal protein component (protein V) and recombinant adenoviruses of the type commonly used in gene therapy, including one virus that had been used in a clinical trial. We found that the viral protein abundance and composition were consistent across all types of virus examined except for the virus lacking protein V, which also had reduced amounts of another viral core protein, protein VII. In all the samples analysed we found no evidence of consistent packaging or contamination with cellular proteins. We believe this technique is a powerful method to analyse the protein composition of this important gene therapy vector and genetically engineered or synthetic virus-like particles. The raw data have been deposited at proteomexchange, identifer PXD001120.

Species D human adenovirus type 9 exhibits better virus-spread ability for antitumor efficacy among alternative serotypes.

Species C human adenovirus serotype 5 (HAdV-C5) is widely used as a vector for cancer gene therapy, because it efficiently transduces target cells. A variety of HAdV-C5 vectors have been developed and tested in vitro and in vivo for cancer gene therapy. While clinical trials with HAdV-C5 vectors resulted in effective responses in many cancer patients, administration of HAdV-C5 vectors to solid tumors showed responses in a limited area. A biological barrier in tumor mass is considered to hinder viral spread of HAdV-C5 vectors from infected cells. Therefore, efficient virus-spread from an infected tumor cell to surrounding tumor cells is required for successful cancer gene therapy. In this study, we compared HAdV-C5 to sixteen other HAdV serotypes selected from species A to G for virus-spread ability in vitro. HAdV-D9 showed better virus-spread ability than other serotypes, and its viral progeny were efficiently released from infected cells during viral replication. Although the HAdV-D9 fiber protein contains a binding site for coxsackie B virus and adenovirus receptor (CAR), HAdV-D9 showed expanded tropism for infection due to human CAR (hCAR)-independent attachment to target cells. HAdV-D9 infection effectively killed hCAR-negative cancer cells as well as hCAR-positive cancer cells. These results suggest that HADV-D9, with its better virus-spread ability, could have improved therapeutic efficacy in solid tumors compared to HAdV-C5.

An adenovirus vector incorporating carbohydrate binding domains utilizes glycans for gene transfer.

BACKGROUND: Vectors based on human adenovirus serotype 5 (HAdV-5) continue to show promise as delivery vehicles for cancer gene therapy. Nevertheless, it has become clear that therapeutic benefit is directly linked to tumor-specific vector localization, highlighting the need for tumor-targeted gene delivery. Aberrant glycosylation of cell surface glycoproteins and glycolipids is a central feature of malignant transformation, and tumor-associated glycoforms are recognized as cancer biomarkers. On this basis, we hypothesized that cancer-specific cell-surface glycans could be the basis of a novel paradigm in HAdV-5-based vector targeting. METHODOLOGY/PRINCIPAL FINDINGS: As a first step toward this goal, we constructed a novel HAdV-5 vector encoding a unique chimeric fiber protein that contains the tandem carbohydrate binding domains of the fiber protein of the NADC-1 strain of porcine adenovirus type 4 (PAdV-4). This glycan-targeted vector displays augmented CAR-independent gene transfer in cells with low CAR expression. Further, we show that gene transfer is markedly decreased in cells with genetic glycosylation defects and by inhibitors of glycosylation in normal cells. CONCLUSIONS/SIGNIFICANCE: These data provide the initial proof-of-concept for HAdV-5 vector-mediated gene delivery based on the presence of cell-surface carbohydrates. Further development of this new targeting paradigm could provide targeted gene delivery based on vector recognition of disease-specific glycan biomarkers.

Adenoviral protein V promotes a process of viral assembly through nucleophosmin 1.

Adenoviral infection induces nucleoplasmic redistribution of a nucleolar nucleophosmin 1/NPM1/B23.1. NPM1 is preferentially localized in the nucleoli of normal cells, whereas it is also present at the nuclear matrix in cancer cells. However, the biological roles of NPM1 during infection are unknown. Here, by analyzing a pV-deletion mutant, Ad5-dV/TSB, we demonstrate that pV promotes the NPM1 translocation from the nucleoli to the nucleoplasm in normal cells, and the NPM1 translocation is correlated with adenoviral replication. Lack of pV causes a dramatic reduction of adenoviral replication in normal cells, but not cancer cells, and Ad5-dV/TSB was defective in viral assembly in normal cells. NPM1 knockdown inhibits adenoviral replication, suggesting an involvement of NPM1 in adenoviral biology. Further, we show that NPM1 interacts with empty adenovirus particles which are an intermediate during virion maturation by immunoelectron microscopy. Collectively, these data implicate that pV participates in a process of viral assembly through NPM1.

Therapeutic efficacy of an oncolytic adenovirus containing RGD ligand in minor capsid protein IX and Fiber, Δ24DoubleRGD, in an ovarian cancer model.

Ovarian cancer is the leading cause of gynecological disease death despite advances in medicine. Therefore, novel strategies are required for ovarian cancer therapy. Conditionally replicative adenoviruses (CRAds), genetically modified as anti-cancer therapeutics, are one of the most attractive candidate agents for cancer therapy. However, a paucity of coxsackie B virus and adenovirus receptor (CAR) expression on the surface of ovarian cancer cells has impeded treatment of ovarian cancer using this approach. This study sought to engineer a CRAd with enhanced oncolytic ability in ovarian cancer cells, "Δ24DoubleRGD." Δ24DoubleRGD carries an arginine-glycine-aspartate (RGD) motif incorporated into both fiber and capsid protein IX (pIX) and its oncolytic efficacy was evaluated in ovarian cancer. In vitro analysis of cell viability showed that infection of ovarian cancer cells with Δ24DoubleRGD leads to increased cell killing relative to the control CRAds. Data from this study suggested that not only an increase in number of RGD motifs on the CRAd capsid, but also a change in the repertoir of targeted integrins could lead to enhanced oncolytic potency of Δ24DoubleRGD in ovarian cancer cells in vitro. In an intraperitoneal model of ovarian cancer, mice injected with Δ24DoubleRGD showed, however, a similar survival rate as mice treated with control CRAds.

An adenoviral vector expressing human adenovirus 5 and 3 fiber proteins for targeting heterogeneous cell populations.

Human adenovirus serotype 5 (HAdV-5) attaches to its primary receptor, the coxsackie and adenovirus receptor (CAR) as the first step of infection. However, CAR expression decreases as tumors progress, thereby diminishing the utility of HAdV-5-based vectors for cancer therapy. In contrast, many aggressive tumor cells highly express CD46, a cellular receptor for HAdV-3. We hypothesized that a mosaic HAdV vector, containing two kinds of fiber proteins, would provide extensive transduction in a heterogeneous population of tumor cells with varying expression levels of HAdV receptors. We therefore generated a fiber-mosaic HAdV vector displaying both a chimeric HAdV-3 fiber and the HAdV-5 fiber protein. We verified the structural integrity of purified viral particles and confirmed that the fiber-mosaic HAdV vector has expanded tropism. We conclude that the use of fiber-mosaic HAdV vectors is a promising approach for transducing a heterogeneous cell population with different expression levels of adenovirus receptors.

Evaluation of adenovirus capsid labeling versus transgene expression.

Adenoviral vectors have been utilized for a variety of gene therapy applications. Our group has incorporated bioluminescent, fluorographic reporters, and/or suicide genes within the adenovirus genome for analytical and/or therapeutic purposes. These molecules have also been incorporated as capsid components. Recognizing that incorporations at either locale yield potential advantages and disadvantages, our report evaluates the benefits of transgene incorporation versus capsid incorporation. To this end, we have genetically incorporated firefly luciferase within the early region 3 or at minor capsid protein IX and compared vector functionality by means of reporter readout.

In vitro dynamic visualization analysis of fluorescently labeled minor capsid protein IX and core protein V by simultaneous detection.

Oncolytic adenoviruses represent a promising therapeutic medicine for human cancer therapy, but successful translation into human clinical trials requires careful evaluation of their viral characteristics. While the function of adenovirus proteins has been analyzed in detail, the dynamics of adenovirus infection remain largely unknown due to technological constraints that prevent adequate tracking of adenovirus particles after infection. Fluorescence labeling of adenoviral particles is one new strategy designed to directly analyze the dynamic processes of viral infection in virus-host cell interactions. We hypothesized that the double labeling of an adenovirus with fluorescent proteins would allow us to properly analyze intracellular viruses and the fate of viral proteins in a live analysis of an adenovirus as compared to single labeling. Thus, we generated a fluorescently labeled adenovirus with both a red fluorescent minor capsid protein IX (pIX) [pIX monomeric red fluorescent protein 1 (mRFP1)] and a green fluorescent minor core protein V (pV) [pV enhanced green fluorescent protein (EGFP)], resulting in Ad5-IX-mRFP1-E3-V-EGFP. The fluorescent signals for pIX-mRFP1 and pV-EGFP were detected within 10 min in living cells. However, a growth curve analysis of Ad5-IX-mRFP1-E3-V-EGFP showed an approximately 150-fold reduced production of the viral progeny at 48 h postinfection as compared to adenovirus type 5. Interestingly, pIX-mRFP1 and pV-EGFP were initially localized in the cytoplasm and nucleolus, respectively, at 18 h postinfection. These proteins were observed in the nucleus during the late stage of infection, and relocalization of the proteins was observed in an adenoviral-replication-dependent manner. These results indicate that simultaneous detection of adenoviruses using dual-fluorescent proteins is suitable for real-time analysis, including identification of infected cells and monitoring of viral spread, which will be required for a complete evaluation of oncolytic adenoviruses.

Chimeric adenoviral vectors incorporating a fiber of human adenovirus 3 efficiently mediate gene transfer into prostate cancer cells.

BACKGROUND: We have developed a range of adenoviral (Ad) vectors based on human adenovirus serotype 5 (HAdV-5) displaying the fiber shaft and knob domains of species B viruses (HAdV-3, -11, or -35). These species B Ads utilize different cellular receptors than HAdV-5 for infection. We evaluated whether Ad vectors displaying species B fiber shaft and knob domains (Ad5F3Luc1, Ad5F11Luc1, and Ad5F35Luc1) would efficiently infect cancer cells of distinct origins, including prostate cancer. METHODS: The fiber chimeric Ad vectors were genetically generated and compared with the original Ad vector (Ad5Luc1) for transductional efficiency in a variety of cancer cell lines, including prostate cancer cells and primary prostate epithelial cells (PrEC), using luciferase as a reporter gene. RESULTS: Prostate cancer cell lines infected with Ad5F3Luc1 expressed higher levels of luciferase than Ad5Luc1, as well as the other chimeric Ad vectors. We also analyzed the transductional efficiency via monitoring of luciferase activity in prostate cancer cells when expressed as a fraction of the gene transfer in PrEC cells. In the PC-3 and DU145 cell lines, the gene transfer ratio of cancer cells versus PrEC was once again highest for Ad5F3Luc1. CONCLUSION: Of the investigated chimeric HAdV-5/species B vectors, Ad5F3Luc1 was judged to be the most suitable for targeting prostate cancer cells as it showed the highest transductional efficiency in these cells. It is foreseeable that an Ad vector incorporating the HAdV-3 fiber could potentially be used for prostate cancer gene therapy.

Derivation of a triple mosaic adenovirus for cancer gene therapy.

A safe and efficacious cancer medicine is necessary due to the increasing population of cancer patients whose particular diseases cannot be cured by the currently available treatment. Adenoviral (Ad) vectors represent a promising therapeutic medicine for human cancer therapy. However, several improvements are needed in order for Ad vectors to be effective cancer therapeutics, which include, but are not limited to, improvement of cellular uptake, enhanced cancer cell killing activity, and the capability of vector visualization and tracking once injected into the patients. To this end, we attempted to develop an Ad as a multifunctional platform incorporating targeting, imaging, and therapeutic motifs. In this study, we explored the utility of this proposed platform by generating an Ad vector containing the poly-lysine (pK), the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK), and the monomeric red fluorescent protein (mRFP1) as targeting, tumor cell killing, and imaging motifs, respectively. Our study herein demonstrates the generation of the triple mosaic Ad vector with pK, HSV-1 TK, and mRFP1 at the carboxyl termini of Ad minor capsid protein IX (pIX). In addition, the functionalities of pK, HSV-1 TK, and mRFP1 proteins on the Ad vector were retained as confirmed by corresponding functional assays, indicating the potential multifunctional application of this new Ad vector for cancer gene therapy. The validation of the triple mosaic Ad vectors also argues for the ability of pIX modification as a base for the development of multifunctional Ad vectors.

Genetic incorporation of the protein transduction domain of Tat into Ad5 fiber enhances gene transfer efficacy.

BACKGROUND: Human adenovirus serotype 5 (Ad5) has been widely explored as a gene delivery vector for a variety of diseases. Many target cells, however, express low levels of Ad5 native receptor, the Coxsackie-Adenovirus Receptor (CAR), and thus are resistant to Ad5 infection. The Protein Transduction Domain of the HIV Tat protein, namely PTD tat, has been shown to mediate protein transduction in a wide range of cells. We hypothesize that re-targeting Ad5 vector via the PTD tat motif would improve the efficacy of Ad5-mediated gene delivery. RESULTS: In this study, we genetically incorporated the PTD tat motif into the knob domain of Ad5 fiber, and rescued the resultant viral vector, Ad5.PTD tat. Our data showed the modification did not interfere with Ad5 binding to its native receptor CAR, suggesting Ad5 infection via the CAR pathway is retained. In addition, we found that Ad5.PTD tat exhibited enhanced gene transfer efficacy in all of the cell lines that we have tested, which included both low-CAR and high-CAR decorated cells. Competitive inhibition assays suggested the enhanced infectivity of Ad5.PTD tat was mediated by binding of the positively charged PTD tat peptide to the negatively charged epitopes on the cells' surface. Furthermore, we investigated in vivo gene delivery efficacy of Ad5.PTD tat using subcutaneous tumor models established with U118MG glioma cells, and found that Ad5.PTD tat exhibited enhanced gene transfer efficacy compared to unmodified Ad5 vector as analyzed by a non-invasive fluorescence imaging technique. CONCLUSION: Genetic incorporation of the PTD tat motif into Ad5 fiber allowed Ad5 vectors to infect cells via an alternative PTD tat targeting motif while retaining the native CAR-mediated infection pathway. The enhanced infectivity was demonstrated in both cultured cells and in in vivo tumor models. Taken together, our study identifies a novel tropism expanded Ad5 vector that may be useful for clinical gene therapy applications.

Thermostability/infectivity defect caused by deletion of the core protein V gene in human adenovirus type 5 is rescued by thermo-selectable mutations in the core protein X precursor.

Mastadenoviruses represent one of the four major genera of the Adenoviridae family comprising a variety of mammalian pathogens including human adenovirus (Ad), whose genomes encode a gene for minor core protein V (pV), not found in other genera of Adenoviridae. Deletion of other genus-specific genes (gene IX and E3 genes) from the Ad type 5 (Ad5) genome has been studied experimentally in vitro and the results on biological characterization of the mutants support the phylogenetic evidence of those genes being non-essential for Ad viability. On this basis it seemed logical to suggest that a deletion of gene V from the Ad5 genome could also be tolerated. To test this hypothesis we constructed and rescued the first pV-deletion mutant of human Ad5. As compared to Ad5, this mutant formed small plaques, had dramatically reduced thermostability and lower infectivity. A subsequent thermoselection screen of the pV-deleted Ad5 allowed isolation of a suppressor mutant Ad5-dV/TSB with restored biological characteristics. Since replication and viral assembly of Ad5-dV/TSB could still occur in the absence of pV, we conclude that pV is a non-essential component of the virion. The observed rescue of the biological defects appears to be associated with a cluster of point mutations in the gene encoding the precursor for the other core protein, X/Mu. This finding, thus, suggests possible roles of pV and protein X/Mu precursor in viral assembly. It also provides an interesting insight into genetic events that mediate molecular adaptation of viruses to possible changes in the genetic background in the course of their evolutionary divergence. The possible mechanism of the observed genetic suppression is discussed.

Derivation, maintenance, and induction of the differentiation in vitro of equine embryonic stem cells.

We describe here the isolation and maintenance of pluripotent embryonic stem (ES) cells from equine blastocysts that have been frozen and thawed. Equine ES cells appear to maintain a normal diploid karyotype in culture. These cells express markers that are characteristic of mouse ES cells, namely, alkaline phosphatase, stage-specific-embryonic antigen 1, STAT3, and Oct4. We also describe protocols for the induction of differentiation in vitro to neural precursor cells in the presence of basic fibroblast growth factor (bFGF), epidermal growth factor, and platelet-derived growth factor and to hematopoietic and endothelial cell lineages in the presence of bFGF, stem cell factor, and oncostatin M. Equine ES cells provide a powerful tool for gene targeting and the generation of transgenic clonal offspring.

Accumulation of infectious mutants in stocks during the propagation of fiber-modified recombinant adenoviruses.

In infected cells, replication errors during viral proliferation generate mutations in adenoviruses (Ads), and the mutant Ads proliferate and evolve in the intracellular environment. Genetically fiber-modified recombinant Ads (rAd variants) were generated, by modification of the fiber gene, for therapeutic applications in host cells that lack or express reduced levels of the Coxsackievirus and adenovirus receptor. To assess the genetic modifications of rAd variants that might induce the instability of Ad virions, we examined the frequencies of mutants that accumulated in propagated stocks. Seven of 41 lines of Ad variants generated mutants in the stocks and all mutants were infectious. Moreover, all the mutations occurred in the modified region that had been added at the 3' end of the fiber gene. Our results show that some genetic modifications at the carboxyl terminus of Ad fiber protein lead to the instability of Ad virions.

A database of recombinant viruses and recombinant viral vectors available from the RIKEN DNA bank.

BACKGROUND: Viral vectors are required as gene-delivery systems for gene therapy and basic research. Recombinant adenoviruses (rAds) expressing genes of interest are being developed as research tools and many studies in vitro and in vivo have already been performed with such rAds. METHODS: Shuttle vectors for rAds were constructed with full-length cDNAs and rAds were generated in HEK293 cells by the COS-TPC method. The rAds and shuttle vectors were developed by the Japanese research community and deposited in the RIKEN DNA Bank (RDB; http://www.brc.riken.jp/lab/dna/en/) for distribution to the scientific community. The Recombinant Virus Database (RVD; http://www.brc.riken.jp/lab/dna/rvd/) was established at the RIKEN BioResource Center (BRC) in Japan as the source of information about and distribution of the various resources. RESULTS: The RIKEN BRC is releasing more than 300 recombinant viruses (RVs) and 500 shuttle vectors, as well as all related information, which is included in a newly established database, the RVD. The RVD consists of (i) information about the RVs, the inserted cDNAs and the shuttle vectors; (ii) data about sequence-tagged sites (STSs) that are markers of viral DNAs; and (iii) experimental protocols for the use of RVs. CONCLUSIONS: The new database and available resources should be very useful to scientists who are studying human gene therapy and performing related basic research. It is a web-interfaced flat-file database that can be accessed through the internet. Moreover, all of the resources deposited in the RDB, which is a public facility in Japan, are available to researchers around the world.

Purification of infectious adenovirus in two hours by ultracentrifugation and tangential flow filtration.

Adenoviruses are excellent vectors for gene transfer and are used extensively for high-level expression of the products of transgenes in living cells. The development of simple and rapid methods for the purification of stable infectious recombinant adenoviruses (rAds) remains a challenge. We report here a method for the purification of infectious adenovirus type 5 (Ad5) that involves ultracentrifugation on a cesium chloride gradient at 604,000g for 15 min at 4 degrees C and tangential flow filtration. The entire procedure requires less than two hours and infectious Ad5 can be recovered at levels higher than 64% of the number of plaque-forming units (pfu) in the initial crude preparation of viruses. We have obtained titers of infectious purified Ad5 of 1.35x10(10) pfu/ml and a ratio of particle titer to infectious titer of seven. The method described here allows the rapid purification of rAds for studies of gene function in vivo and in vitro, as well as the rapid purification of Ad5.

Effective gene therapy of biliary tract cancers by a conditionally replicative adenovirus expressing uracil phosphoribosyltransferase: significance of timing of 5-fluorouracil administration.

In order to enhance the efficacy of conditionally replicating adenoviruses (CRAd) in the treatment of cancers of the biliary tract, we studied the efficacy in vitro and in vivo of AxE1CAUP, a CRAd vector that carries a gene for uracil phosphoribosyltransferase (UPRT), which converts 5-fluorouracil (5-FU) directly to 5-fluorouridine monophosphate and greatly enhances the cytotoxicity of 5-FU. AxE1CAUP replicated and induced an increased UPRT expression in biliary cancer cells more efficiently than AxCAUP, a nonreplicative adenovirus carrying the UPRT gene. Whereas AxCAUP and AxE1AdB, a CRAd without the UPRT gene, modestly increased the sensitivity of BC cells to 5-FU, AxE1CAUP markedly increased the sensitivity, especially when the timing of 5-FU administration was appropriately chosen. AxE1CAUP replicated much less efficiently in normal WI-38 fibroblasts without any change in the sensitivity to 5-FU. In nude mice with s.c. biliary cancer xenografts, i.t. AxE1CAUP/5-FU therapy inhibited tumor growth significantly more strongly than AxCAUP/5-FU or AxE1AdB/5-FU therapy. Furthermore, in mice with peritoneally disseminated biliary cancer, i.p. AxE1CAUP efficiently proliferated in the tumors, decreased the tumor burden, and prolonged the survival of the mice when 5-FU was started 10 or 15 days after the vector inoculation, whereas earlier initiation of 5-FU resulted in early eradication of the vector and no survival benefit. The present study shows that the CRAd expressing UPRT was a more potent sensitizer of biliary cancer to 5-FU, than was a nonreplicative UPRT-encoding vector or a CRAd without UPRT gene, even at a lower dose of the vector, and that timing of 5-FU administration was a key factor to maximize the efficacy. This gene therapy with appropriately timed administration of 5-FU should be useful in overcoming the resistance of biliary cancers to 5-FU.

A simple method for the simultaneous detection of E1A and E1B in adenovirus stocks.

Recombinant adenoviral vectors have been developed for use as therapeutic agents and for the introduction of exogenous genes into living cells. However, the occurrence of replication-competent adenoviruses (RCA) in adenovirus stocks produced in 293 cells remains a major problem in terms of the safe use of such vectors. To overcome the problems associated with the occurrence of RCA, we have established a simple method for the simultaneous detection of amplified E1A and E1B from RCA that might contaminate adenoviral stocks. The products amplified by polymerase chain reaction (PCR) were fractionated by regular electrophoresis on agarose gels and visualized by staining with ethidium bromide. This method is rapid and inexpensive for detection of RCA in the preparation of adenoviruses.

E1A, E1B double-restricted adenovirus for oncolytic gene therapy of gallbladder cancer.

New treatments, such as gene therapy, are necessary for advanced gallbladder cancer (GBC), but little has been studied. Recent studies have introduced mutant adenoviruses (Ads) with either defective E1B-55kD or mutated E1A, focusing on tumor-specific replication, and the results have been promising. To enhance the safety of this approach, we constructed AxdAdB-3, a double-restricted Ad with a mutant E1A and E1B-55kD deletion. We studied the effects of this Ad in vitro and in vivo on GBC, as well as its safety for normal human cells. We compared the replication and cytopathic effects of AxdAdB-3 in several lines of GBC and primary normal cells with those of wild-type Ad or of AxE1AdB, an E1B-55kD-deleted Ad. The efficacy in vivo was examined in nude mice with s.c. implanted or i.p. disseminated GBC. AxdAdB-3 replicated in and caused oncolysis of GBC cell lines (TGBC-44TKB and Mz-ChA2) as efficiently as wild-type Ad or AxE1AdB in vitro. By contrast, AxdAdB-3 replicated much less effectively in primary normal cells (e.g., epithelial cells, endothelial cells, and hepatocytes) than in GBC cells and had only mild cytopathic effects, unlike wild-type Ad. Furthermore, cytotoxicity of AxdAdB-3 in normal cells was milder than that of AxE1AdB. AxdAdB-3 significantly (P < 0.01) suppressed the growth of GBC (TGBC-44TKB) xenografts. AxdAdB-3 was also effective in the treatment of mice with peritoneally disseminated GBC (TGBC-44TKB), demonstrating tumor-selective replication and oncolysis that resulted in significantly (P < 0.05) prolonged survival. The present study shows that the E1 double-restricted Ad effectively and selectively replicates in and causes oncolysis of GBC in vitro and in vivo with reduced negative effects on normal cells, suggesting that this approach could be a promising tool for gene therapy of GBC.