| Despite substantial progress in the development of gene therapies and traditional treatments in recent years, the prognosis for many patients with neoplastic diseases remain poor. Cancer gene therapy based on adenoviruses has been extensively studied in pre-clinical and clinical trials. In particular, CRAds has gained increased attention for a number of reasons. Because the promoters in these vectors are selective for cancer cells, these oncolytic viruses have the ability to replicate and to spread to adjacent tumor cells. Furthermore, it has been shown that infection with CRAds generates anti-tumoral immune responses which can complement chemo- and radiotherapies. Importantly, given the proper therapeutic transgenes, CRAds are capable of achieving destruction of primary and distant tumors.In vitro studies showed that a core region of hTERT containing two E boxes and several Spl sites is sufficient for the major tumor-selective promoter activity. Many strategies including CRAds have been developed using the hTERT core promoter, containing two E boxes and several Sp1 sites, to selectively target tumor cells. One approach being evaluated uses CNHK300, a replicative adenovirus that targets telomerase positive cancer cells. A similar replication-competent adenovirus, AdEHT2, in which hTERT promoter was used to control the expression of the adenoviral E4 gene, was capable of tumor selective replication and oncolysis. Analogous results were also obtained in other replicating adenoviruses, such as Adv-TERTp-Ela, hTERT-Ad, and Ad/GT-Bax, which appear to be promising treatment agents for cancer.The applicability of cancer therapies is not only determined by their efficiency in eliminating tumor cells; specificity is an equally important prerequisite. Apoptin has such properties which could potentially achieve these objectives. Various research groups have reported that more than 70 analyzed tumor cell lines were proven to be susceptible to Apoptin whereas it does not affecting variety of normal, non-transformed cells such as human endothelial cells, hepatocytes, hematopoietic stem cells, keratinocytes, or smooth muscle cells. On the other hand, Apoptin become activated in SV40-transformed normal human fibroblasts or UV-irradiated cells with hereditary cancer-prone syndromes. Although. Guelen et al provided data of toxicity of Apoptin towards non-cancerous cells, this study proved cell death only in a fetal cell type and not in other non-transformed cell types. However, the safety of apoptin is underlined by the fact that continuous expression of Apoptin under the H2-Kb promoter in transgenic mice does not interfere with lymphocyte development and proliferation. While the mechanism by which Apoptin is able to distinguish between tumor and normal cells remains unclear but seems to correlate with its cellular localization. Recently, it was shown that Apoptin-induced apoptosis essentially depends on abnormal phosphatidylinositol 3-kinase (PI3-kinase)/Akt activation, resulting in the activation of the cyclin-dependent kinase CDK2. Maddika et al indicated that inhibitors of PI3-kinase or Akt not only inhibited CDK2 activation but also protected cells from Apoptin-induced cell death, and Akt-mediated activation of CDK2 was caused by direct phosphorylation as well as by the phosphorylation-induced degradation of its inhibitor p27 (Kip1). They also identified CDK2 as the principal kinase that phosphorylates apoptin and is crucially required for apoptin-induced cell death. Besides the tumor-selective destruction properties, Apoptin has several important features indicating its application as a novel antitumor agent. One of these characteristics is the ability of Apoptin to induce tumor-specific apoptosis independently of p53. Thus, apoptin is similarly effective in killing tumor cells that are p53-deficient or either express wildtype or mutant p53. Although the role of anti-apoptotic molecules such as Bcl-2 in Apoptin-induced apoptosis is still a matter of debate, another important feature of Apoptin is that in certain tumor cell lines it mediated cell death is independent of the Bcl-2 status and is even stimulated by Bcl-2 or insensitive to Bcr-Abl and Bcl-xl. The main controversy as to the role of Bcl-2 may focus on the involvement of Nur77 that can bind to Bcl-2 and change its properties from an anti-apoptotic to a proapoptotic molecule. Accordingly, the different expression levels of Nur77 in various cell types might explain the opposite effects of Bcl-2 on Apoptin induced apoptosis. Based on these concepts, it therefore reasonable to anticipate that Apoptin can be used to complement radiotherapeutic and chemotherapeutic approaches.More than a quarter (27%) of human gene transfer protocols registered with the Recombinant DNA Advisory Committee (RAC) use adenovirus vectors. The most extensively used first generation human adenovirus (hAd) vectors are replication-incompetent viruses deleted in early region 1 (E1A and E1B) genes. El genes that are expressed rapidly upon adenovirus penetration into host cells, are responsible for inducing expression of further viral genes, orchestrating modifications of cellular gene expression and protein activity to favor viral replication. Two genes of this group, E1A and E1B, act in inactivating tumor suppressor Rb and p53 genes that are frequently mutated in cancer cells. Following the generation of E1 deleted hAd vectors, others hAd vectors (e.g., E1 & E3, E2, E4, E2 & E4, or E1, E2 & E4-deleted vectors) were constructed. The products of the E3 gene have been described as nonessential to viral infection and play a role in modulation of the host immune response against virus-infected cells. E2 and E4 genes are involved in multiple processes, such as transcriptional regulation, DNA recombination and virus assembly. Various deleted strategies such as ONYX-015 (d11520) have been evaluated to be effective in in vitro and animal models and led to clinical trials. Until a fatality case and other reports of inflammation related to adenovirus vector, the use of adenovirus-mediated gene transfer in humans was thought to be fairly benign. Because of the short circulatory half life of naked adenoviruses and the neutralizing antibodies existed in most adults, attempts to increase antitumor efficacy through the administration of high doses of adenovirus vectors can lead to liver toxicity and immune response. For these reasons, Ad has seen limited clinical use as a systemically administered gene therapy vector. To overcome the potential increased toxicity and reduced vector efficacy during the application of adenovirus vectors, it is important that identifying means to evade innate and pre-existing immunity is a major necessity. Current strategies include use of alternative adenovirus serotypes, modification or chimerism of capsid hexon proteins, generation of hybrid vectors that combine viral and non-viral elements, coating virus with PEG or similar polymers, targeting adenovirus to specific organs, tissues or cell types and so on.In the present study, we describe the generation of a recombinant adenovirus, Ad-KVT, in which replication was driven by hTERT promoter, that selectively replicates and specifically induces apoptosis in tumor cells. When administered to hepatoma cells in vitro, the anti-tumor effects were evident within 24 h; a single Ad-KVT treatment at 100 MOI completely inhibited the growth of HepG-2 cells 4 d later, whereas treatment at 10 MOI or 1 MOI was less effective. Although, the growth inhibition rate of Ad-KVT was not significant higher than the growth inhibition rate of Ad-VT, the time that Ad-KVT suppress the HepG-2 cells was less than Ad-VT needed. Furthermore, Annexin V analysis, DAPI staining and AO/EB staining showed that the recombinant adenovirus can induce apoptosis of the infected HepG-2 cells. These findings indicate that the Ad-KVT induces growth suppression in HepG-2 cells through apoptosis pathway.We also observed anti-tumor activity in vivo, which confirmed and extended the results of the in vitro studies. Although the infection of Ad-KVT did not lead to complete elimination of the tumors, effective inhibition was observed in tumor models. It is plausible that the application of the hTERT promoter allows the adenovirus replication, viral dispersion and transgene expression in any tumor tissues in the animal, regardless of receiving intratumoral injections or not. Thus, our data indicate that there is great potential for improving the safety and efficacy of adenovirus vectors for wide application for treatment of neoplastic diseases.
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