Conditionally replicating adenoviruses kill tumor cells via a basic apoptotic machinery-independent mechanism that resembles necrosis-like programmed cell death

Conditionally replicating adenoviruses (CRAds) represent a promising class of novel anticancer agents that are used for virotherapy. The E1ADelta24 mutation-based viruses, Ad5-Delta24 [CRAd(E3-); E3 region deleted] and infectivity-enhanced Ad5-Delta24RGD [CRAd(E3+)] have been shown to potently eradicate tumor cells. The presence of the E3 region in the latter virus is known to improve cell killing that can be attributed to the presence of the oncolysis-enhancing Ad death protein. The more precise mechanism by which CRAds kill tumor cells is unclear, and the role of the host cell apoptotic machinery in this process has been addressed only in a limited way. Here, we examine the role of several major apoptotic pathways in the CRAd-induced killing of non-small-cell lung cancer H460 cells. As expected, CRAd(E3+) was more potent than CRAd(E3-). No evidence for the involvement of the p53-Bax apoptotic pathway was found. Western blot analyses demonstrated strong suppression of p53 expression and unchanged Bax levels during viral replication, and stable overexpression of human papillomavirus type 16-E6 in H460 cells did not affect killing by both CRAds. CRAd activity was also not hampered by stable overexpression of anti-apoptotic Bcl2 or BclXL, and endogenous Bcl2/BclXL protein levels remained constant during the oncolytic cycle. Some evidence for caspase processing was obtained at late time points after infection; however, the inhibition of caspases by the X-linked inhibitor of apoptosis protein overexpression or cotreatment with zVAD-fmk did not inhibit CRAd-dependent cell death. Analyses of several apoptotic features revealed no evidence for nuclear fragmentation or DNA laddering, although phosphatidylserine externalization was detected. We conclude that despite the known apoptosis-modulating abilities of individual Ad proteins, Ad5-Delta24-based CRAds trigger necrosis-like cell death. In addition, we propose that deregulated apoptosis in cancer cells, a possible drug resistance mechanism, provides no barrier for CRAd efficacy.     

Conditionally replicating adenovirus expressing TIMP2 increases survival in a mouse model of disseminated ovarian cancer

Ovarian cancer remains difficult to treat mainly due to presentation of the disease at an advanced stage. Conditionally-replicating adenoviruses (CRAds) are promising anti-cancer agents that selectively kill the tumor cells. The present study evaluated the efficacy of a novel CRAd (Ad5/3-CXCR4-TIMP2) containing the CXCR4 promoter for selective viral replication in cancer cells together with TIMP2 as a therapeutic transgene, targeting the matrix metalloproteases (MMPs) in a murine orthotopic model of disseminated ovarian cancer. An orthotopic model of ovarian cancer was established in athymic nude mice by intraperitonal injection of the human ovarian cancer cell line, SKOV3-Luc, expressing luciferase. Upon confirmation of peritoneal dissemination of the cells by non-invasive imaging, mice were randomly divided into four treatment groups: PBS, Ad-ΔE1-TIMP2, Ad5/3-CXCR4, and Ad5/3-CXCR4-TIMP2. All mice were imaged weekly to monitor tumor growth and were sacrificed upon reaching any of the predefined endpoints, including high tumor burden and significant weight loss along with clinical evidence of pain and distress. Survival analysis was performed using the Log-rank test. The median survival for the PBS cohort was 33 days; for Ad-ΔE1-TIMP2, 39 days; for Ad5/3-CXCR4, 52.5 days; and for Ad5/3-CXCR4-TIMP2, 63 days. The TIMP2-armed CRAd delayed tumor growth and significantly increased survival when compared to the unarmed CRAd. This therapeutic effect was confirmed to be mediated through inhibition of MMP9. Results of the in vivo study support the translational potential of Ad5/3-CXCR4-TIMP2 for treatment of human patients with advanced ovarian cancer.     

Productive replication of human adenovirus type 5 in canine cells

Development of immunocompetent patient-like models that allow direct analysis of human adenovirus-based conditionally replicative adenoviruses (CRAds) would be beneficial for the advancement of these oncolytic agents. To this end, we explored the possibility of cross-species replication of human adenovirus type 5 (Ad5) in canine cells. With a panel of canine tumor cell lines of both epithelial and mesenchymal derivations, we demonstrate that human Ad5 can productively infect canine cells. Since the biological behavior and clinical presentation of certain dog tumors closely resemble those of their human counterparts, our results raise the possibility of exploiting canine models for preclinical analysis of candidate CRAd agents designed for human virotherapy.     

A potent replicative delta-24 adenoviral vector driven by the promoter of human papillomavirus 16 that is highly selective for associated neoplasms

BACKGROUND: Several human epithelial neoplasms are associated with high-risk strains of human papillomavirus (HPV) such as cervical, anorectal, and other carcinomas. For some tumor types the current therapeutic tools are only palliative. Conditionally replicative adenoviruses (CRAds) are promising antineoplastic agents, which also can trigger confined antitumor effects. METHODS: We constructed a series of CRAds driven by the upstream regulatory promoter region (URR) of an Asian-American variant of HPV-16, which contained different mutations at the E1A region (dl1015 and/or Delta24) and wild-type. All vectors were tested in vitro for viral replication and cytotoxicity. Viral DNA replication and E1A expression were also assessed by quantitative PCR. Finally, we confirmed the antitumoral efficacy of this vector in injected and non-injected xenotransplanted cervical tumors in a murine model for tumor regression and survival studies. RESULTS: A vector denominated Ad-URR/E1ADelta24 displayed a potent cytopathic effect associated with high selectivity for HPV+ cell lines. We found that the oncolytic effect of this CRAd was comparable to Ad-wt or Ad-Delta24, but this efficacy was significantly attenuated in HPV- cell lines, an effect that was contributed by the URR promoter. Ad-URR/E1ADelta24 was very effective to control tumor growth, in both, injected and non-injected tumors generated with two different HPV+ cell lines. CONCLUSIONS: CRAd Ad-URR/E1ADelta24 is a highly selective vector for HPV+ cell lines and tumors that preserves the oncolytic efficacy of Ad-wt and Ad-Delta24. Our preclinical data suggest that this vector may be useful and safe for the treatment of tumors induced by HPV, like cervical cancers.     

Expression of p53, or targeting towards EGFR, enhances the oncolytic potency of conditionally replicative adenovirus against neuroblastoma

BACKGROUND: Advanced stage and relapsing neuroblastoma (NB) has a poor prognosis with frequent treatment failures, warranting new treatment options and enhanced local tumor control. Treatment with conditionally replicative adenoviruses (CRAds) has shown effectiveness in various preclinical cancer models, but has not yet been evaluated for local control of NB. Here, we tested the efficacy of the CRAd AdDelta24 and of two AdDelta24 derivatives against NB. Derivative AdDelta24-425S11 infects cells deficient in coxsackie/adenovirus receptor (CAR) via the epidermal growth factor receptor (EGFR). Derivative AdDelta24-p53 expresses the tumor suppressor protein p53 to promote oncolysis. METHODS: Expression of CAR and EGFR, and p53 pathway and DNA damage responses were analyzed in six NB cell lines and two xenografts derived from primary NB using immunohistochemistry, reporter gene transactivation, Western blot and fluorescence-activated cell sorting (FACS) analysis. Efficacy of AdDelta24, AdDelta24-425S11 and AdDelta24-p53 against NB was evaluated in vitro by cell viability analysis and in vivo by monitoring subcutaneous xenograft tumor growth in mice and by histological analysis of treated tumors. RESULTS: Neuroblastoma cell lines were sensitive to oncolysis by AdDelta24, with a higher susceptibility of those with functional p53 and intact DNA damage responses. Compared to AdDelta24, AdDelta24-p53 exhibited enhanced oncolytic potency on all NB cell lines independent of their p53 status and AdDelta24-425S11 was more effective against CAR-low IGR-NB8 cells. Moreover, five daily intratumoral injections of 10(8) plaque-forming units (pfu) of AdDelta24-p53 or AdDelta24-425S11 into subcutaneous IGR-NB8 and IGR-N91 xenografts at an advanced tumor stage yielded significant tumor growth delays (TGD). In contrast, at this dose, AdDelta24 did not cause significant TGD of neuroblastoma xenografts. Injection of AdDelta24-p53 was associated with extensive cell lysis, apoptotic cell death, and fibrous fascicles in the tumors. CONCLUSION: CRAds expressing p53 and targeted towards EGFR appear promising new agents for local control in the treatment of neuroblastoma.     

AdDelta24 and the p53-expressing variant AdDelta24-p53 achieve potent anti-tumor activity in glioma when combined with radiotherapy

BACKGROUND: The conditionally replicating adenovirus (CRAd) AdDelta24-p53 replicates selectively in Rb mutant cells, and encodes the p53 suppressor protein. It has shown improved oncolytic potency compared to the parental control AdDelta24. As exogenous p53 has been shown to enhance radiosensitivity, the combination of AdDelta24-p53 and AdDelta24 with radiotherapy was assessed in vitro and in vivo against the therapy resistant gliomas. METHODS: In glioma cells, multicellular spheroids and animal xenografts the efficacy of combination therapy was assessed. P53 phosphorylation, induction of apoptosis and viral replication were determined following single or combination therapies. RESULTS: In vitro, AdDelta24-p53 was more effective against glioma cells than the control AdDelta24. Addition of irradiation equally increased the efficacy of both AdDelta24-p53 and AdDelta24 resulting in improved oncolysis compared to single agent treatment. Radiotherapy did not significantly change the replication kinetics of AdDelta24-p53 or AdDelta24. No detectable increase in p53 phosphorylation was observed but combination of radiotherapy and AdDelta24-p53 caused an increase in the percentage of apoptotic cells. In vivo, combination therapy with either AdDelta24 or AdDelta24-p53 significantly increased the number of mice demonstrating tumor regression (100%) as well as long-term survival (50%). No differences between viruses were noted. CONCLUSIONS: Exogenous p53 expression does not appear to increase the synergistic interaction of CRAds combined with radiotherapy. These results however do indicate that radiotherapy provides the time frame in which AdDelta24 and AdDelta24-p53 can eradicate established tumors that would otherwise escape treatment, and establishes the need to combine these modalities to form an effective anti-cancer treatment.     

Targeted and shielded adenovectors for cancer therapy

Conditionally replicative adenovirus (CRAd) vectors are novel vectors with utility as virotherapy agents for alternative cancer therapies. These vectors have already established a broad safety record in humans and overcome some of the limitations of non-replicative adenovirus (Ad) vectors. In addition, one potential problem with these vectors, attainment of tumor or tissue selectivity has widely been addressed. However, two confounding problems limiting efficacy of these drug candidates remains. The paucity of the native Ad receptor on tumor tissues, and host humoral response due to pre-existing titers of neutralizing antibodies against the vector itself in humans have been highlighted in the clinical context. The well-characterized CRAd, AdΔ24-RGD, is infectivity enhanced, thus overcoming the lack of coxsackievirus and adenovirus receptor (CAR), and this agent is already rapidly progressing towards clinical translation. However, the perceived host humoral response potentially will limit gains seen from the infectivity enhancement and therefore a strategy to blunt immunity against the vector is required. On the basis of this caveat a novel strategy, termed shielding, has been developed in which the genetic modification of a virion capsid protein would provide uniformly shielded Ad vectors. The identification of the pIX capsid protein as an ideal locale for genetic incorporation of shielding ligands to conceal the Ad vector from pre-existing neutralizing antibodies is a major progression in the development of shielded CRAds. Preliminary data utilizing an Ad vector with HSV-TK fused to the pIX protein indicates that a shield against neutralizing antibodies can be achieved. The utility of various proteins as shielding molecules is currently being addressed. The creation of AdΔ24S-RGD, an infectivity enhanced and shielded Ad vector will provide the next step in the development of clinically and commercially feasible CRAds that can be dosed multiple times for maximum effectiveness in the fight against cancers in humans.This article is a symposium paper from the Annual Meeting of the "International Society for Cell and Gene Therapy of Cancer", held in Shenzhen, China, on 9–11 December 2005.     

Chlorpromazine and apigenin reduce adenovirus replication and decrease replication associated toxicity

BACKGROUND: Adenoviruses can cause severe toxicity in immunocompromised individuals. Although clinical trials have confirmed the potency and safety of selectively oncolytic adenoviruses for treatment of advanced cancers, increasingly effective agents could result in more toxicity and therefore it would be useful if replication could be abrogated if necessary. METHODS: We analyzed the effect of chlorpromazine, an inhibitor of clathrin-dependent endocytosis and apigenin, a cell cycle regulator, on adenovirus replication and toxicity. First, we evaluated the in vitro replication of a tumor targeted Rb-p16 pathway selective oncolytic adenovirus (Ad5/3-Delta24) and a wild-type adenovirus in normal cells, fresh liver samples and in ovarian cancer cell lines. Further, we analyzed the in vitro cell killing efficacy of adenoviruses in the presence and absence of the substances. Moreover, the effect on in vivo efficacy, replication and liver toxicity of the adenoviruses was evaluated. RESULTS: We demonstrate in vitro and in vivo reduction of adenovirus replication and associated toxicity with chlorpromazine and apigenin. Effective doses were well within what would be predicted safe in humans. CONCLUSIONS: Chlorpromazine and apigenin might reduce the replication of adenovirus, which could provide a safety switch in case replication-associated side effects are encountered in patients. In addition, these substances could be useful for the treatment of systemic adenoviral infections in immunosuppressed patients.     

A one-step ligation system for rapid generation of armed, conditionally-replicating adenoviruses

Conditionally-replicating adenoviruses (CRAd) are becoming a promising strategy for cancer therapy. However, the generation of CRAd is complicated and time-consuming, especially for armed CRAd Ad5-D24. A one-step ligation system is now reported for rapid construction of an armed CRAd Ad5-D24 carrying two foreign transgenes. The system has the following features: (1) the CRAd backbone pCRAd5-D24-BS-Lacza contains two unique restriction sites between the fiber and E4 for easy introduction of a transgene expression cassette; and (2) a bi-directional promoter in pshuttle-BS-Dual allows for the expression of two independent transgenes. As proof of principle, Ad5-D24 CRAd carrying eGFP and mCherry was generated and examined. This novel ligation system will be a versatile and efficient tool in cancer viral gene therapy.     

Absent in melanoma 2 enhances anti‐tumour effects of CAIX promotor controlled conditionally replicative adenovirus in renal cancer

Conditionally replicative adenoviruses (CRAds) were promising approach for solid tumour treatment, but its oncolytic efficiency and toxicity are still not satisfactory for further clinical application. Here, we developed the CAIX promotor (CAIX^promotor)‐controlled CRAd armed with a tumour suppressor absent in melanoma 2 (AIM2) to enhance its oncolytic potency. The CAIX^promotor‐AIM2 adenoviruses (Ad‐CAIX^promotor‐AIM2) could efficiently express E1A and AIM2 in renal cancer cells. Compared with Ad‐CAIX^promotor, Ad‐CAIX^promotor‐AIM2 significantly inhibited cell proliferation and enhanced cell apoptosis and cell killing, thus resulting in the oncolytic efficiency in 786‐O cells or OSRC‐2 cells. To explore the therapeutic effect, various Ads were intratumourally injected into OSRC‐2‐xenograft mice. The tumour growth was remarkably inhibited in Ad‐CAIX^promotor‐AIM2‐treated group as demonstrated by reduced tumour volume and weight with a low toxicity. The inflammasome inhibitor YVAD‐CMK resulted in the reduction of anti‐tumour activity by Ad‐CAIX^promotor‐AIM2 in vitro or in vivo, suggesting that inflammasome activation response was required for the enhanced therapeutic efficiency. Furthermore, lung metastasis of renal cancer mice was also suppressed by Ad‐CAIX^promotor‐AIM2 treatment accompanied by the decreased tumour fossil in lung tissues. These results indicated that the tumour‐specific Ad‐CAIX^promotor‐AIM2 could be applied for human renal cancer therapy. The therapeutic strategy of AIM2‐based CRAds could be a potential and promising approach for the therapy of primary solid or metastasis tumours.     

增殖型腺病毒的改良及应用

增殖型腺病毒能在肿瘤细胞中复制并裂解肿瘤细胞,释放出的子代病毒再感染邻近肿瘤细胞直至完全杀灭肿瘤,却不影响正常细胞的功能。同时,增殖型腺病毒还是一种有效的基因治疗载体,可通过病毒自身增殖提高目的基因的拷贝数,从而更高效率地表达外源性治疗基因,增强抗肿瘤效应。本文着重介绍增殖型腺病毒载体改良和应用的最新进展,并对其研究前景进行展望,以期对增殖型腺病毒的发展有所帮助。     

SPECT/CT imaging of hNIS-expression after intravenous delivery of an oncolytic adenovirus and 131I

Oncolytic adenoviruses can be engineered for better tumor selectivity, gene delivery and be armed for imaging and concentrating radionuclides into tumors for synergistic oncolysis. We constructed Ad5/3-hTERT-hNIS where replication is controlled by hTERT-promoter. Ad5/3-hTERT-hNIS expresses hNIS for imaging of transgene expression and for treatment of infected tumors by radioiodine. Ad5/3-hTERT-hNIS efficiently killed prostate cancer cells and induced iodine uptake in vitro and in vivo after intratumoral virus administration. Survival of mice treated with intravenous Ad5/3-hTERT-hNIS significantly prolonged survival over mock or radioiodine only but the combination of virus with radioiodine was not more effective than virus alone. Temporal and spatial changes in hNIS-expression during therapy were detected with SPECT, demonstrating feasibility of evaluation of the combination therapy with hNIS-expressing adenoviruses and radioiodide.     

Vesicular stomatitis virus as an oncolytic agent against pancreatic ductal adenocarcinoma

Vesicular stomatitis virus (VSV) is a promising oncolytic agent against a variety of cancers. However, it has never been tested in any pancreatic cancer model. Pancreatic ductal adenocarcinoma (PDA) is the most common and aggressive form of pancreatic cancer. In this study, the oncolytic potentials of several VSV variants were analyzed in a panel of 13 clinically relevant human PDA cell lines and compared to conditionally replicative adenoviruses (CRAds), Sendai virus and respiratory syncytial virus. VSV variants showed oncolytic abilities superior to those of other viruses, and some cell lines that exhibited resistance to other viruses were successfully killed by VSV. However, PDA cells were highly heterogeneous in their susceptibility to virus-induced oncolysis, and several cell lines were resistant to all tested viruses. Resistant cells showed low levels of very early VSV RNA synthesis, indicating possible defects at initial stages of infection. In addition, unlike permissive PDA cell lines, most of the resistant cell lines were able to both produce and respond to interferon, suggesting that intact type I interferon responses contributed to their resistance phenotype. Four cell lines that varied in their permissiveness to VSV-ΔM51 and CRAd dl1520 were tested in mice, and the in vivo results closely mimicked those in vitro. While our results demonstrate that VSV is a promising oncolytic agent against PDA, further studies are needed to better understand the molecular mechanisms of resistance of some PDAs to oncolytic virotherapy.     

A new type of adenovirus vector that utilizes homologous recombination to achieve tumor-specific replication

We have developed a new class of adenovirus vectors that selectively replicate in tumor cells. The vector design is based on our recent observation that a variety of human tumor cell lines support DNA replication of adenovirus vectors with deletions of the E1A and E1B genes, whereas primary human cells or mouse liver cells in vivo do not. On the basis of this tumor-selective replication, we developed an adenovirus system that utilizes homologous recombination between inverted repeats to mediate precise rearrangements within the viral genome resulting in replication-dependent activation of transgene expression in tumors (Ad.IR vectors). Here, we used this system to achieve tumor-specific expression of adenoviral wild-type E1A in order to enhance viral DNA replication and spread within tumor metastases. In vitro DNA replication and cytotoxicity studies demonstrated that the mechanism of E1A-enhanced replication of Ad.IR-E1A vectors is efficiently and specifically activated in tumor cells, but not in nontransformed human cells. Systemic application of the Ad.IR-E1A vector into animals with liver metastases achieved transgene expression exclusively in tumors. The number of transgene-expressing tumor cells within metastases increased over time, indicating viral spread. Furthermore, the Ad.IR-E1A vector demonstrated antitumor efficacy in subcutaneous and metastatic models. These new Ad.IR-E1A vectors combine elements that allow for tumor-specific transgene expression, efficient viral replication, and spread in liver metastases after systemic vector application.     

Trans-complementing adenoviral vectors for oncolytic therapy of malignant melanoma

BACKGROUND: Despite attempts to develop efficient viral-based gene transfer therapies for the treatment of malignant tumors, only limited progress has been made to improve the efficacy of this approach. As an alternative, the use of replicating oncolytic adenoviruses with and without the expression of therapeutic transgenes is an area of active investigation. METHODS: We used a human melanoma xenograft tumor nude mouse model to test the efficacy of a bivalent vector approach consisting of two trans-complementing replication-incompetent adenoviral vectors that resulted in tumor-restricted oncolysis. We combined an E1-deleted non-replicating adenoviral vector expressing the herpes simplex virus thymidine kinase gene (AV.C2.TK) and Ad5.dl1014, an E4-deleted/E4orf4-only expressing adenovirus, to allow full replication competence when tumor cells were co-infected with both vectors. RESULTS: A375 tumors showed apoptosis at the ultrastructural level after transduction with the trans-complementing vector system that was not seen with injection of either vector alone. Apoptotic DNA fragments could be co-localized to sites of infection with the adenoviral vectors. A significant survival benefit was achieved for the trans-complementing vector treated animals compared to animals treated with either vector alone. Interestingly, the administration of GCV did not further increase animal survival over treatment with the trans-complementing system of viruses alone, and long-term survival was only seen in the trans-complementing vector treatment group. Intraperitoneal administration of a pseudo-wild-type vector Ad.dl327 resulted in significant hepatotoxicity, while intraperitoneal administration of the trans-complementing vectors resulted in only mild liver abnormalities. CONCLUSIONS: The trans-complementing vector approach using a combination of E1- and E4-deleted adenoviral vectors showed similar antitumor efficacy as reported for monovalent replicating vector systems, but may offer additional safety by reducing the risk of dissemination of the replication-competent vectors by requiring the presence of both vectors in a cell to achieve replication competence.     

Analysis by in situ hybridization and autoradiography of sites of replication and storage of single- and double-stranded adenovirus type 5 DNA in lytically infected HeLa cells

The distribution in the different compartments of infected nuclei of double-stranded (ds) and single-stranded (ss) adenovirus type 5 (Ad5) DNA and of the sites of viral DNA replication were examined on thin sections of Low-icryl-embedded material. The DNA is visualized with a biotinylated viral probe and immunogold labeling of biotin, and its replication is monitored by high-resolution autoradiography after short pulses with tritiated thymidine. The first detectable sites of viral DNA, named early replicative sites, contained all the ss and ds viral DNA and viral replicative activity. At a later stage of nuclear transformation, they gave rise to two new structures. The compact fibrillar ssDNA accumulation sites enlarged greatly and became transformed functionally to become a transient site of accumulation of large numbers of ss replicative intermediates. Double-stranded viral DNA and its replicative activity shifted primarily into immediately surrounding fibrillogranular peripheral replicative zones. Ad5 DNA replication continues in the ssDNA accumulation sites but it is intermittent, whereas in the peripheral replicative zones it is continuous. Still later in infection, a single, large, centrally located mass of dense fibrils, the viral genome storage site, developed in each nucleus which proved to be the main site of storage of nonreplicating, nonencapsidated, ds viral genomes. We discuss the possible distribution of the various viral DNA replicative intermediates among these virus-induced intranuclear structures.     

An oncolytic herpes simplex virus type 1 selectively destroys diffuse liver metastases from colon carcinoma.

Viruses used for gene therapy are usually genetically modified to deliver therapeutic transgenes and prevent viral replication. In contrast, replication-competent viruses may be used for cancer therapy because replication of some viruses within cancer cells can result in their destruction (oncolysis). Viral ribonucleotide reductase expression is defective in the HSV1 mutant hrR3. Cellular ribonucleotide reductase, which is scarce in normal liver and abundant in liver metastases, can substitute for its viral counterpart to allow hrR3 replication in infected cells. Two or three log orders more of hrR3 virions are produced from infection of colon carcinoma cells than from infection of normal hepatocytes in viral replication assays. This viral replication is oncolytic. A single intravascular administration of hrR3 into immune-competent mice bearing diffuse liver metastases dramatically reduces tumor burden. hrR3-mediated tumor inhibition is equivalent in immune-competent and immune-incompetent mice, suggesting that viral oncolysis and not the host immune response is the primary mechanism of tumor destruction. HSV1-mediated oncolysis of diffuse liver metastases is effective in mice preimmunized against HSV1. These results indicate that replication-competent HSV1 mutants hold significant promise as cancer therapeutic agents. Yoon, S. S., Nakamura, H., Carroll, N. M., Bode, B. P., Chiocca, E. A., Tanabe, K. K. An oncolytic herpes simplex virus type 1 selectively destroys diffuse liver metastases from colon carcinoma.     

Analysis by in Situ hybridization and autoradiography of sites of replication and storage of single- and double-stranded adenovirus type 5 DNA in lytically infected HeLa cells

The distribution in the different compartments of infected nuclei of double-stranded (ds) and single-stranded (ss) adenovirus type 5 (Ad5) DNA and of the sites of viral DNA replication were examined on thin sections of Lowicryl-embedded material. The DNA is visualized with a biotinylated viral probe and immunogold labeling of biotin, and its replication is monitored by high-resolution autoradiography after short pulses with tritiated thymidine. The first detectable sites of viral DNA, named early replicative sites, contained all the ss and ds viral DNA and viral replicative activity. At a later stage of nuclear transformation, they gave rise to two new structures. The compact fibrillar ssDNA accumulation sites enlarged greatly and became transformed functionally to become a transient site of accumulation of large numbers of ss replicative intermediates. Double-stranded viral DNA and its replicative activity shifted primarily into immediately surrounding fibrillogranular peripheral replicative zones. Ad5 DNA replication continues in the ssDNA accumulation sites but it is intermittent, whereas in the peripheral replicative zones it is continuous. Still later in infection, a single, large, centrally located mass of dense fibrils, the viral genome storage site, developed in each nucleus which proved to be the main site of storage of nonreplicating, nonencapsidated, ds viral genomes. We discuss the possible distribution of the various viral DNA replicative intermediates among these virus-induced intranuclear structures.     

Adenoviruses Using the Cancer Marker EphA2 as a Receptor In Vitro and In Vivo by Genetic Ligand Insertion into Different Capsid Scaffolds

Adenoviral gene therapy and oncolysis would critically benefit from targeted cell entry by genetically modified capsids. This requires both the ablation of native adenovirus tropism and the identification of ligands that remain functional in virus context. Here, we establish cell type-specific entry of HAdV-5-based vectors by genetic ligand insertion into a chimeric fiber with shaft and knob domains of the short HAdV-41 fiber (Ad5T/41sSK). This fiber format was reported to ablate transduction in vitro and biodistribution to the liver in vivo. We show that the YSA peptide, binding to the pan-cancer marker EphA2, can be inserted into three positions of the chimeric fiber, resulting in strong transduction of EphA2-positive but not EphA2-negative cells of human melanoma biopsies and of tumor xenografts after intratumoral injection. Transduction was blocked by soluble YSA peptide and restored for EphA2-negative cells after recombinant EphA2 expression. The YSA peptide could also be inserted into three positions of a CAR binding-ablated HAdV-5 fiber enabling specific transduction; however, the Ad5T/41sSK format was superior in vivo. In conclusion, we establish an adenovirus capsid facilitating functional insertion of targeting peptides and a novel adenovirus using the tumor marker EphA2 as receptor with high potential for cancer gene therapy and viral oncolysis.