Use of replicating oncolytic adenoviruses in combination therapy for cancer.

Oncolytic virotherapy is the use of genetically engineered viruses that specifically target and destroy tumor cells via their cytolytic replication cycle. Viral-mediated tumor destruction is propagated through infection of nearby tumor cells by the newly released progeny. Each cycle should amplify the number of oncolytic viruses available for infection. Our understanding of the life cycles of cytolytic viruses has allowed manipulation of their genome to selectively kill tumor cells over normal tissue. Because the mechanism of tumor destruction is different, oncolytic virotherapy should work synergistically with current modes of treatment such as chemotherapy and radiation therapy. This article focuses on oncolytic adenoviruses that have been created and tested in preclinical and clinical trials in combination with chemotherapy, radiation therapy, and gene therapy.     

Modified adenoviruses for cancer gene therapy

Adenoviral gene therapy is an exciting novel approach for treating cancers resistant to currently available therapies. However, currently there is little evidence supporting significant clinical benefits with replication-incompetent adenoviruses. Recent data suggest that expression of the primary receptor, the coxsackie-adenovirus receptor (CAR), may be highly variable on tumor cells, resulting in resistance to infection. Consequently, various strategies have been evaluated to modify adenovirus tropism in order to circumvent CAR deficiency, including retargeting complexes or genetic capsid modifications. To improve tumor penetration and local amplification on the antitumor effect, selectively oncolytic agents, i.e., conditionally replicating adenoviruses, have been constructed. Infection of tumor cells results in replication, oncolysis and subsequent release of the virus progeny. Normal tissue is spared due to lack of replication. This review focuses on the various modifications that have been investigated for improving the antitumor effect of adenoviral gene therapy.     

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.     

表达小干扰RNA的条件增殖腺病毒与肿瘤

RNA干扰（RNAi）是一种由双链RNA介导的基因沉默现象。RNAi作为强有力的研究工具正在基因功能组学领域掀起一场真正的革命,也成为当今肿瘤基因治疗研究的热点。条件增殖腺病毒（CRAd）是一类仅在肿瘤细胞内特异增殖新型腺病毒载体,通过CRAd介导的小干扰RNA（siRNA）可以靶向肿瘤细胞,且随着病毒增殖反复表达siRNA,进一步加强siRNA介导的癌基因沉默效果。因此,siRNA-CRAd模式开辟了肿瘤基因治疗的新途径。     

表达小干扰RNA的条件增殖腺病毒与肿瘤

RNA干扰(RNAi)是一种由双链RNA介导的基因沉默现象。RNAi作为强有力的研究工具正在基因功能组学领域掀起一场真正的革命,也成为当今肿瘤基因治疗研究的热点。条件增殖腺病毒(CRAd)是一类仅在肿瘤细胞内特异增殖新型腺病毒载体,通过CRAd介导的小干扰RNA(siRNA)可以靶向肿瘤细胞,且随着病毒增殖反复表达siRNA,进一步加强siRNA介导的癌基因沉默效果。因此, siRNA-CRAd模式开辟了肿瘤基因治疗的新途径。     

Conditionally replicative adenoviruses: a second wind for cancer gene therapy

Cancer gene therapy has offered many hopes but its first use in humans revealed some pitfalls and at least three main problems: lack of efficacy and specificity of current vectors to deliver therapeutic genes, poor diffusion of the therapeutic effects inside the tumor (absence of bystander effect), poor distribution of the vectors injected inside the tissue. To address some of these issues, several teams have developed tumor selective replicating adenoviruses, some of them being already in the clinic. First results are promising but complementary studies are needed to define if these agents will take place in the therapeutic armentorium against cancer.     

肿瘤特异增殖病毒的研究进展

经转录调节修饰的肿瘤特异增殖病毒可特异性地杀伤肿瘤细胞,具有临床治疗肿瘤的潜在价值,是近年来肿瘤研究的热点之一,并取得了很大的进展,现综述肿瘤特异增殖腺病毒的研究和临床应用现状.     

E1A, E1B double-restricted replicative adenovirus at low dose greatly augments tumor-specific suicide gene therapy for gallbladder cancer

Combination therapy with replicative oncolytic viruses is a recent topic in innovative cancer therapy, but few studies have examined the efficacy of oncolytic adenovirus plus replication-deficient adenovirus carrying a suicide gene. We aim to evaluate whether an E1A, E1B double-restricted oncolytic adenovirus, AxdAdB-3, can improve the efficacy for gallbladder cancers (GBCs) of the replication-deficient adenovirus-based herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) therapy directed by the carcinoembryonic antigen (CEA) promoter. Cytopathic effects of AxdAdB-3 plus AxCEAprTK (an adenovirus expressing HSVtk directed by CEA promoter) or AxCAHSVtk (an adenovirus expressing HSVtk directed by a nonspecific CAG promoter) with GCV administration were examined in several GBC lines and normal cells. Efficacy in vivo was tested in severe combined immunodeficiency disease mice with GBC xenografts. Addition of AxdAdB-3 (1 multiplicity of infection, MOI) significantly enhanced the cytopathic effects of AxCEAprTK (10 MOI)/GCV on GBC cells. The augmented effect was attributable to the replication of the AxCEAprTK and also to the enhanced CEA promoter activity, which was presumably transactivated by E1A. In normal cells, AxdAdB-3 (20 MOI) plus AxCEAprTK (200 MOI)/GCV was not cytopathic, whereas AxdAdB-3 (1 MOI) plus AxCAHSVtk (10 MOI)/GCV was significantly toxic. Low-dose AxdAdB-3 (2 x 10(7) PFU, plaque-forming unit) plus AxCEAprTK (2 x 10(8) PFU)/GCV significantly suppressed the growth of GBC xenografts as compared with either AxdAdB-3 (2 x 10(7) PFU)/GCV or AxCEAprTK (2 x 10(9) PFU)/GCV alone. E1A, E1B double-restricted replicating adenovirus at low dose significantly augmented the efficacy of CEA promoter-directed HSVtk/GCV therapy without obvious toxicity to normal cells, suggesting a potential use of this combination for treating GBC and other CEA-producing malignancies.     

Assessment of an altered E1B promoter on the specificity and potency of triple-regulated conditionally replicating adenoviruses: implications for the generation of ideal m-CRAs

Although previous studies modified two components of conditionally replicating adenoviruses (CRAs), which selectively replicate in and kill cancer cells, the most accurate ways to achieve increased cancer specificity (that is, safety) without reducing the anticancer (that is, therapeutic) effects are unknown. Here, we generated two types of survivin-responsive m-CRAs (Surv.m-CRAs), Surv.m-CRA-CMVp and Surv.m-CRA-OCp, which use two and three different mechanisms to target cancer, that is, early region 1A (E1A) regulated by the survivin promoter and mutated E1BΔ55K regulated by the ubiquitously active cytomegalovirus promoter and cancer/tissue-specific osteocalcin promoter, respectively, and carefully examined their safety and anticancer effects. Endogenous osteocalcin mRNA was expressed and further enhanced by vitamin D(3) in all osteosarcoma and prostate cancer cell lines and human osteoblasts, but not in human fibroblasts. The osteocalcin promoter activity was weak even with vitamin D(3) treatment in these osteocalcin-expressing cancers, leading to low E1BΔ55K expression after Surv.m-CRA-OCp infection. Nevertheless, Surv.m-CRA-OCp had significantly increased cancer specificity without reduced anticancer effects in both in vitro and in vivo experiments. The unexpected but favorable fact that strong activity of an altered E1B promoter is unnecessary indicates that the majority of cancer/tissue-specific promoters may be used to generate ideal m-CRAs and will advance the development of m-CRA-based cancer therapies.     

Multidrug-resistant cancer cells facilitate E1-independent adenoviral replication: impact for cancer gene therapy

Resistance to chemotherapy is responsible for a failure of current treatment regimens in cancer patients. We have reported previously that the Y-box protein YB-1 regulates expression of the P-glycoprotein gene mdr1, which plays a major role in the development of a multidrug resistant-tumor phenotype. YB-1 predicts drug resistance and patient outcome in breast cancer. Thus, YB-1 is a promising target for new therapeutic approaches to defeat multidrug resistance. In drug-resistant cancer cells and in adenovirus-infected cells YB-1 is found in the nucleus. Nuclear accumulation of YB-1 in adenovirus-infected cells is a function of the E1 region, and we have shown that YB-1 facilitates adenovirus replication. Here we report that E1A-deleted or mutant adenovirus vectors, such as Ad312 and Ad520, replicate efficiently in multidrug-resistant (MDR) cancer cells and induce an adenovirus cytopathic effect resulting in host cell lysis. Thus, replication-defective adenoviruses are a previously unrecognized vector system for a selective elimination of MDR cancer cells. Our work forms the basis for the development of novel oncolytic adenovirus vectors for the treatment of MDR malignant diseases in the clinical setting.     

Recent developments in the use of adenoviruses and immunotoxins in cancer gene therapy

Despite setbacks in the past and apparent hurdles ahead, gene therapy is advancing toward reality. The past several years have witnessed this new field of biomedicine developing rapidly both in breadth and depth, especially for the treatment of cancer, thanks largely to the better understanding of molecular and genetic basis of oncogenesis and the development of new and improved vectors and technologies for gene delivery and targeting. This article is intended to provide a brief review of recent advances in cancer gene therapy using adenoviruses, both as vectors and as oncolytic agents, and some of the recent progress in the development of immunotoxins for use in cancer gene therapy.     

Selectively replicating adenoviruses targeting deregulated E2F activity are potent,systemic antitumor agents

We have engineered a human adenovirus, ONYX-411, that selectively replicates in human tumor cells, but not normal cells, depending upon the status of their retinoblastoma tumor suppressor protein (pRB) pathway. Early and late viral gene expression as well as DNA replication were significantly reduced in a functional pRB-pathway-dependent manner, resulting in a restricted replication profile similar to that of nonreplicating adenoviruses in normal cells both in vitro and in vivo. In contrast, the viral life cycle and tumor cell killing activity of ONYX-411 was comparable to that of wild-type adenovirus following infection of human tumor cells in vitro as well as after systemic administration in tumor-bearing animals.     

The development of conditionally replicative adenoviruses for cancer therapy.

Replicative viral agents represent a novel approach for treating neoplastic disease. Tumor cell killing by the viral agent is achieved by direct consequence of the viral replication. Relative sparing of nontumor is, however, required to provide a therapeutic index of utility for cancer treatment. To this end, an ideal viral agent would, thus, possess several logical attributes, including stability and efficiency for infection and lateral spread in vivo, a preference for replication in tumor versus nontumor cells, and the capability of avoiding early detection-and eradication-by the immune system. To date, none of the agents has exhibited optimal characteristics with regard to the aforementioned attributes. Adenovirus, however, has lent itself to a process of extensive engineering that is dealing with each and every one of the major requirements and that is realizing its clinical potential. An advanced understanding of the cancer phenotype, as well as achievements in functionally exploiting viral plasticity, predicate the design and realization of conditionally replicative adenoviral agents with improved characteristics for cancer therapy.     

RAD001 (everolimus) improves the efficacy of replicating adenoviruses that target colon cancer

Selectively replicating adenoviruses have the potential to cure cancer but have shown little efficacy in clinical trials. We have tested the ability of the mTOR kinase inhibitor RAD001 (everolimus) to enhance the response of xenografts to an oncolytic adenovirus. The virus has Tcf sites inserted in the early viral promoters and replicates selectively in cells with activation of the Wnt signaling pathway. To enhance tumor cell infection, an integrin targeting peptide (CDCRGDCFC) was inserted into the fiber gene of the virus. RAD001 combines three useful properties: it inhibits tumor cell growth directly, blocks angiogenesis, and suppresses the immune response. RAD001 does not block viral protein expression, DNA replication, or cytopathic effect in tumor cells in vitro. After 6 weeks of daily RAD001 treatment, ongoing viral DNA replication could be detected in tumor xenografts, showing that RAD001 does not inhibit virus replication in vivo. I.v. injection of virus alone produced a small delay in xenograft growth, whereas combination therapy substantially prolonged the survival of the mice. We suggest that collapsing the tumor vasculature after the initial infection traps the virus and facilitates local spread within the tumor. Unlike conventional drugs, which require continued access to the tumor through the vascular system, oncolytic viruses are in principle less sensitive to late reductions in perfusion because they are produced locally within the tumor.     

Gene therapy for prostate cancer by controlling adenovirus E1a and E4 gene expression with PSES enhancer

PSES is a chimeric enhancer containing enhancer elements from prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) genes that are prevalently expressed in androgen-independent prostate cancers. PSES shows strong activity equivalent to cytomegalovirus (CMV) promoter, specifically in PSA/PSMA-positive prostate cancer cells, the major cell types in prostate cancer in the absence of androgen. We developed a recombinant adenovirus (AdE4PSESE1a) by placing adenoviral E1a and E4 genes under the control of the bidirectional enhancer PSES and enhanced green fluorescent protein gene for the purpose of intratumoral virus tracking under the control of CMV promoter. Because of PSES being very weak in nonprostatic cells, including HEK293 and HER911 that are frequently used to produce recombinant adenovirus, AdE4PSESE1a can only be produced in the HER911E4 cell line which expresses both E1 and E4 genes. AdE4PSESE1a showed similar viral replication and tumor cell killing activities to wild-type adenovirus in PSA/PSMA-positive prostate cancer cells. The viral replication and tumor cell killing activities were dramatically attenuated in PSA/PSMA-negative cells. To test whether AdE4PSESE1a could be used to target prostate tumors in vivo, CWR22rv s.c. tumors were induced in nude mice and treated with AdE4PSESE1a via intratumoral and tail vein injection. Compared to tumors treated with control virus, the growth of CWR22rv tumors was dramatically inhibited by AdE4PSESE1a via tail vein injection or intratumoral injection. These data show that adenoviral replication can be tightly controlled in a novel fashion by controlling adenoviral E1a and E4 genes simultaneously with a single enhancer.