Deletion of the adenoviral E1b-19kD gene enhances tumor cell killing of a replicating adenoviral vector

Replicating adenoviral vectors are a promising new modality for cancer treatment and clinical trials with such vectors are ongoing. Targeting these vectors to cancer cells has been the focus of research. However, even if perfect targeting were to be achieved, a vector still must effectively kill cancer cells and spread throughout the bulk of the tumor. The adenoviral E1b-19kD protein is a potent inhibitor of apoptosis and may therefore compromise the therapeutic efficacy of an adenoviral vector. In this study we have investigated if an E1b-19kD gene deletion could improve the ability of a replicating adenoviral vector to spread through and kill cancer cells. In several lung cancer cell lines an E1b-19kD-deleted virus (Ad337) induced substantially more apoptosis than did a wild-type virus (Ad309), and tumor cell survival was significantly reduced in three of four cell lines. In addition, the apoptotic effects of cisplatin or paclitaxel were augmented by Ad337, but inhibited by wild-type virus. The number of infectious virus particles in the supernatant of infected cells was increased with Ad337 compared with wild-type virus, indicating enhanced early viral release. Ad337, in contrast to Ad309, induced significantly larger plaques after infection of A549 cells. This well-described large plaque phenotype of an E1b-19kD mutant virus is likely the result of early viral release and enhanced cell-to-cell viral spread. Loss of E1b-19kD function caused only minor cell line-specific increase or decrease in viral yield. We conclude that deletion of the E1b-19kD gene may enhance the tumoricidal effects of a replicating adenoviral vector.     

Hypoxia reduces adenoviral replication in cancer cells by downregulation of viral protein expression

Successful cancer therapy using replicating viral vectors relies on the spread of virus from infected to uninfected cells. To date, there has been limited clinical success in the use of replicating adenoviruses. In animal models, established xenograft tumors are rarely eliminated despite the persistence of high viral titers within the tumor. Hypoxia is a prevalent characteristic of solid tumors, whereas adenovirus naturally infects tissues exposed to ambient oxygen concentrations. Here, we report that hypoxia (1% oxygen) reduces adenoviral replication in H1299 and A549 lung cancer cells, BxPC-3 pancreatic cancer cells, LNCaP prostate cancer cells and HCT116 colon cancer cells. However, hypoxia does not reduce cell viability or restrict S-phase entry. Importantly, the production of E1a and fiber proteins under hypoxic conditions was substantially decreased at 24 and 48 h compared to room air controls. In contrast, Northern analysis showed similar levels of E1a mRNA in room air and hypoxic conditions. In conclusion, a level of hypoxia similar to that found within solid tumors reduces the replication of adenoviral vectors by reduction of viral protein expression without a reduction in mRNA levels. To further improve oncolytic therapy using a replicating adenovirus, it is important to understand the mechanism through which hypoxia and the virus interact to control expression of viral and cellular proteins, and consequently to develop means to overcome decreased viral production in hypoxic conditions.     

Gene therapy for killing p53-negative cancer cells: use of replicating versus nonreplicating agents

Research has focused on the use of viral vectors to attack p53-negative cancer cells. Such agents may be nonreplicating, whereas others are replicating. This paper uses mathematical models to study the conditions under which therapy can lead to tumor remission. It is found that the optimal characteristics of the vector can be quite different depending on whether the virus replicates or not. If it does not replicate, the rate of virus-induced tumor cell killing should be maximized. If the virus does replicate, the rate of virus-induced cell killing should be kept small. If the virus is too lytic in cancer cells, viral spread is compromised, resulting in persistence of both virus and tumor. This has important implications for choosing the correct techniques to evaluate replicating viruses in culture. A low multiplicity of infection must be used for evaluation, because this mimicks the spread of the virus through an established tumor. If a high multiplicity of infection is used, the virus that appears most efficient in this evaluation can be least efficient at eradicating the cancer in vivo. Theoretical results are discussed in the context of experimental data.     

Wild-type adenovirus decreases tumor xenograft growth, but despite viral persistence complete tumor responses are rarely achieved--deletion of the viral E1b-19-kD gene increases the viral oncolytic effect.

Strategies to target viral replication to tumor cells hold great promise for the treatment of cancer, but even with replicating adenoviruses complete tumor responses are rarely achieved. To evaluate replicating adenoviral vectors, we have used A549 human lung cancer nude mouse xenografts as a model system. Intratumoral injection of wild-type adenovirus (Ad309) significantly reduced tumor growth from day 14 (p = 0.04) onward; however, tumor volumes reached a plateau at day 50. At 100 days, high levels of titratable virus were present within persistent viable tumors. In contrast to viral injection into established tumors, when tumor cells were infected in vitro with wild-type virus and then mixed with uninfected tumor cells, 1% of infected cells was sufficient to prevent tumor establishment. An E1b-19kD-deleted viral mutant (Ad337) was more efficient than Ad309 in this cell-mixing model. Just 1 cell in 1000 infected with Ad337 prevented tumor growth. However, although better than wild-type virus, Ad337 was unable to eradicate established flank tumors. These data suggest that although replicating adenoviruses exhibit significant oncolytic activity, barriers within the established tumor, such as connective tissue and tumor matrix, may limit the spread of virus. Strategies to enhance viral spread through established tumors are therefore likely to greatly improve the therapeutic efficacy of replicating adenoviruses.     

Intratumoral expression of a fusogenic membrane glycoprotein enhances the efficacy of replicating adenovirus therapy

We describe here a novel strategy to enhance the in vivo efficacy of replicating adenovirus therapy, using coinjection of plasmid DNA encoding a fusogenic viral glycoprotein. The combination of fusogenic membrane glycoprotein (FMG)-induced tumor cell fusion and infection with replicating adenovirus effectively treats even large established tumors at doses of plasmid DNA and virus that alone are ineffective. Adenoviral infection appears to increase the transduction of the tumor cells to a modest degree thereby boosting the FMG-mediated component of the therapy. Simultaneously, syncytial formation enhances the therapeutic effects of viral infection by increasing spread of adenoviral particles through the tumor cell population and by increasing titer of virus released from the tumor cells. This effect is due probably to release of intracellular viral particles upon tumor cell death and also to increased levels of E1A protein within syncytia, whose increased metabolic rate is associated with enhanced levels of protein expression. Cotransduction of tumor cells with replicating adenovirus and FMG-expressing vectors could either be combined within single replicating vectors or could be used in strategies using separate administration of two components, both at lower doses than required for either therapy alone.     

Viral oncoapoptosis of human tumor cells

Many cancer cells refractory to radiation treatment and chemotherapy proliferate because of loss of intrinsic programmed cell death (apoptosis) regulation. Consequently, the resolution of these cancers are many times outside the management capabilities of conventional therapeutics. We now report that replication-defective delta27 herpes simplex virus (rd delta27) triggers apoptosis in three representative transformed human cell lines. Susceptibility to virus-induced cell death is dependent on the abundance and distribution of modified p53 protein in the tumor cells indicating specific targeting of the treatment. Primary human and mouse fibroblast cells that produce modified p53 are resistant to rd delta27 killing but not to apoptosis induced by nonviral environmental factors. These results suggest that induction of apoptosis by nonreplicating virus is a feasible genetic therapy approach for killing human cancer cells. Our findings may have important implications in designing novel virus-based anticancer strategies in appropriate animal model systems.     

Single-shot, multicycle suicide gene therapy by replication-competent retrovirus vectors achieves long-term survival benefit in experimental glioma.

Achieving therapeutically efficacious levels of gene transfer in tumors has been a major obstacle for cancer gene therapy using replication-defective virus vectors. Recently, replicating viruses have emerged as attractive tools for cancer therapy, but generally achieve only transitory tumor regression. In contrast to other replicating virus systems, transduction by replication-competent retrovirus (RCR) vectors is efficient, tumor-selective, and persistent. Correlating with its efficient replicative spread, RCR vector expressing the yeast cytosine deaminase suicide gene exhibited remarkably enhanced cytotoxicity in vitro after administration of the prodrug 5-fluorocytosine. In vivo, RCR vectors replicated throughout preestablished primary gliomas without spread to adjacent normal brain, resulting in profound tumor inhibition after a single injection of virus and single cycle of prodrug administration. Furthermore, stable integration of the replicating vector resulted in persistent infection that achieved complete transduction of ectopic glioma foci that had migrated away from the primary tumor site. Thus, efficient and stable integration of suicide genes represents a unique property of the RCR vector that achieved multiple cycles of synchronous cell killing upon repeated prodrug administration, resulting in chronic suppression of tumor growth and prolonged survival.     

Tumor suppression and therapy sensitization of localized and metastatic breast cancer by adenovirus p53

We have examined the effects of a replication-defective adenovirus encoding p53 (RPR/INGN 201 [Ad5CMV-p53]; Adp53), alone or in combination with the breast cancer therapeutic doxorubicin (Adriamycin), to suppress growth and induce apoptosis in breast cancer cells in vitro. We have also examined the in vivo effect of intratumoral administration of Adp53, alone or in combination with doxorubicin, to suppress the growth of established subcutaneous MDA-MB-435 breast cancer tumors. Finally, using the MDA-MB-435 orthotopic model of metastatic breast cancer, we have examined the effect of systemic administration of Adp53, alone or in combination with doxorubicin, to reduce the incidence of metastases. We find that whereas in vitro treatment of cells with Adp53 reduces [(3)H]thymidine incorporation by about 90% at 48 hr, cell viability at 6 days is reduced by only some 50% relative to controls. Although apoptosis is detectable in Adp53-treated cultures, these results suggest that a large fraction of Adp53-treated cells merely undergo reversible cell cycle arrest. Combined treatment with Adp53 and doxorubicin results in a greater than additive loss of viability in vitro and increased apoptosis. In vivo, locally administered Adp53 suppresses growth of established subcutaneous tumors in nude mice and suppression is enhanced by doxorubicin. In the metastatic breast cancer model, systemic administration of Adp53 plus doxorubicin leads to a significant reduction in the incidence of metastases relative to Adp53 or doxorubicin alone. Taken together, these data indicate an additive to synergistic effect of Adp53 and doxorubicin for the treatment of primary and metastatic breast cancer.     

A model system for the design of armed replicating adenoviruses using p53 as a candidate transgene

Cancer gene therapy endeavors to overcome the low therapeutic index of currently available therapeutic modalities via the efficient and safe delivery of genetic material into tumor cells. However, despite promising preclinical results, replication-deficient viral vectors have demonstrated a limited efficacy in the clinical setting. To increase vector efficiency, replication-competent viruses have been proposed. Clinical trials have shown the safety of locally injected, conditionally replicative adenoviruses (Ads) but have underscored the need for improved potency. To further increase the therapeutic effect of replicating viral vectors, armed therapeutic viruses (ATVs) have recently been used for high-efficiency transgene expression. However, interference with cellular signaling and viral production by constitutive transgene expression may be counterproductive for ATV replication, thereby hindering the therapeutic outcome. Consequently, studies are equivocal with regard to the potential benefits of ATVs. To address this issue, we hypothesized that induction of replication of an Ad expressing p53 may be a useful strategy in the context of ATV because p53 does not interfere with Ad replication and may even increase its cytolytic effect. We show that in our in vitro ATV model system, E1 transcomplementation of a replication-deficient Ad encoding p53 resulted in dramatic augmentation of cell killing and circumvented resistance to apoptosis. Correlation was found between the degrees of cell killing and apoptosis induction, rather than with viral burst. Furthermore, both Ad5 E1B 55kDa and E4 orf6 genes were required to enhance the cell killing. In conclusion, our p53-ATV model system demonstrates the potential utility of therapeutic transgene expression by a replicating Ad after a rational selection of a candidate transgene.     

Characterization of infectivity-enhanced conditionally replicating adenovectors for prostate cancer radiovirotherapy

Abstract Prostate cancer (PCa) is the second most commonly diagnosed and sixth leading cause of cancer death in American men and one for which no curative therapy exists after metastasis. To meet this need for novel therapies, our laboratory has previously generated conditionally replicating adenovirus (CRAd) vectors expressing the sodium iodide symporter (hNIS). This virus transduced PCa cells and induced functional NIS expression, allowing for noninvasive tumor imaging and combination therapy with radioiodide, referred to as radiovirotherapy. We have now generated two new modified vectors to further improve efficacy. Ad5/3PB-ADP-hNIS and Ad5/3PB-hNIS include a hybrid Ad5/3 fiber knob to improve transduction efficiency, and express NIS from the endogenous major late promoter to restrict NIS expression to target cells. Additionally, Ad5/3PB-ADP-hNIS includes the adenovirus death protein (ADP), which hastens the release of viral particles after assembly. These two vectors specifically induce radioisotope uptake, cytopathic effect, and viral replication in androgen receptor-expressing PCa cell lines with Ad5/3PB-ADP-hNIS showing earlier (131)I uptake and cytolysis at low multiplicity of infection. SPECT-CT imaging of xenograft tumors infected with Ad5/3PB-hNIS showed steady uptake, whereas infection with Ad5/3PB-ADP-hNIS led to increasing uptake, indicating viral spread. Radiovirotherapy of xenograft LNCaP tumors with Ad5/3PB-ADP-hNIS showed the most significant survival extension versus control tumors (p=0.001), but the benefit of radiovirotherapy was not statistically significant compared with virotherapy alone in this model. These results show the potential of Ad5/3PB-ADP-hNIS as a vector for treatment of prostate cancer.     

VSV-G pseudotyped, MuLV-based, semi-replication-competent retrovirus for cancer treatment

Low levels of gene delivery in vivo using replication-defective retroviral vectors have severely limited their application for clinical protocols. To overcome this problem, we describe here a semi-replication-competent retrovirus (s-RCR) in which the gag-pol and envelope (VSV-G, vesicular stomatitis virus G protein) genes were split into two vectors. This system offers potential advantages over both replication-defective vectors, in terms of efficiency of in vivo spread through a tumor, and all-in-one replication-competent vectors in terms of the payload of therapeutic genes that can be carried. We achieved a viral titer of s-RCR viruses approximately 70-fold higher than VSV-G pseudotyped, replication-defective vectors. In addition, s-RCR vectors induced tumor killing by the cytotoxicity of VSV-G during viral spread. Inclusion of the herpes simplex virus thymidine kinase (HSVtk30) gene into vectors significantly improved tumor killing activity followed by ganciclovir (GCV) treatment in vitro under conditions of low-level viral replication. However, at high levels of viral spread, VSV-G-mediated cytotoxicity predominated. Xenografts of human fibrosarcoma HT1080 cells, preinfected by semi-replicative green fluorescent protein vectors (semi-GFP), were completely non-tumorigenic in nude mice. Implantation of cells preinfected by semi-replicative TK30 vectors (semi-TK30) mixed with parental HT1080 cells at a ratio of 1:1 efficiently prevented tumor growth in mice treated by GCV. Direct intratumoral injection of HT1080 tumors growing in nude mice, or B16 murine melanoma in immunocompetent mice, with semi-TK30 viruses significantly prolonged survival. Injection of autologous cells (B16) producing semi-TK30 vector into B16 tumors prolonged survival only in mice treated with GCV but not with phosphate-buffered saline (PBS). In contrast, when xenogeneic cells (293T) producing semi-TK30 vectors were injected into B16 tumors, an optimal survival advantage was obtained in mice treated with PBS rather than GCV. These data indicate that complex interactions exist between direct cytotoxicity of VSV-G and HSVtk expression when placed in the context of additional immune parameters, which combine to determine the efficacy of the therapy. Taken together, our data suggest that s-RCR vectors have some potential advantages for development to deliver genes into tumors for cancer treatment but that a combination of factors will impact on the decision as to whether the s-RCR strategy is worth developing to full clinical trials.     

重组人p53腺病毒提高胃癌细胞对顺铂敏感性的实验研究

目的：研究重组人p53腺病毒增加胃癌细胞对顺铂敏感性的作用。方法：重组人p53腺病毒感染胃癌细胞BGC-823，Western blot法检测p53蛋白在胃癌细胞中高表达；MTT法测定重组人p53腺病毒单独及联合顺铂用药的不同浓度处理细胞的生长抑制率，流式细胞仪检测细胞周期分布和凋亡率。结果：重组人p53腺病毒感染BGC-823 48h，p53蛋白在BGC-823中高表达，并产生G2／M期阻滞和细胞凋亡。重组人p53腺病毒联合顺铂用药增加顺铂的敏感性，有剂量时间的依赖性。结论：腺病毒介导p53基因感染BGC-823细胞诱导凋亡并增加胃癌细胞对顺铂的敏感性，为p53基因治疗与胃癌化疗临床结合提供了可靠的实验依据。     

Synergy between expression of fusogenic membrane proteins, chemotherapy and facultative virotherapy in colorectal cancer

Using Chou-Talalay median effect analysis, we demonstrated in permanent and short-term cultures of colorectal cancer cells that the expression of measles virus fusogenic membrane glycoproteins (FMGs) in combination with chemotherapy often causes over most of the cytotoxic dose range synergistic cell killing. In this combined treatment, we observed strongly enhanced annexin V binding and caspase-3/7 activity when compared to single-agent treatment. Furthermore, we showed increased expression of heat-shock protein (Hsp)70 and Hsp90alpha, but not of Hsp60. In a subcutaneous HT-29 colorectal xenograft model, we demonstrated that the administration of a replication-defective adenoviral or herpes simplex virus (HSV) amplicon vector (Ad.H/F or HSV.H/F) encoding tumor-restricted FMG in combination with FOLFOX significantly enhanced treatment outcome when compared to treatment with each compound individually. To increase the fraction of tumor cells expressing the FMG, we trans-complemented the Ad.H/F and HSV.H/F vector with the respective oncolytic replication-restricted adenovirus Ad.COXDeltaMK or HSV-1 G47Delta vector. At the end of the observation period (day 100), eight out of 10 animals that received G47Delta, HSV.H/F and FOLFOX were alive and tumor free. Administration of the analogous adenovirus-based regimen resulted in four out of 10 long-term survivors. We demonstrated that the expression of FMG in combination with chemotherapy can significantly enhance treatment outcome, which is further enhanced by combination with trans-complementing oncolytic vectors.     

Highly efficient and carcinoma-specific adenoviral replication restricted by the EGP-2 promoter.

Although some successes have been reported using adenoviral vectors for the treatment of cancer, adenoviral cancer gene therapy is still hampered by the lack of sufficient tumor cell killing. To increase the efficiency, adenoviruses have been modified to replicate specifically in tumor tissues by using tumor specific promoters controlling genes essential for adenoviral replication. However, many conditionally replicating adenoviral vectors replicate in one tumor type only, which limits their application. The epithelial glycoprotein-2 (EGP-2) promoter is active in a broad variety of carcinomas, the most common type of cancer. We utilized this promoter to restrict adenoviral replication. In this report we demonstrate that the potency of the replication-competent adenovirus AdEGP-2-E1 to specifically lyse EGP-2 positive cells is comparable to wild-type adenovirus (AdWT). In addition, we show that in vivo AdEGP-2-E1 replicates as efficient as AdWT in EGP-2 positive tumor cells. On the contrary, in EGP-2 negative cell lines as well as in primary human liver samples, the replication was attenuated up to 4-log in comparison to wild-type virus. This report clearly shows the potency of the EGP-2 promoter to mediate highly efficient and specific adenoviral replication for carcinoma gene therapy.