A triple regulated oncolytic adenovirus armed with IL-24

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Adenoviruses carry proteins with them, which change cellular mechanisms inside the host cell, in a way that makes it possible for the virus to be reproduced by the host and to induce cell lysis at the end of the viral replication cycle. These alterations of the host cell share some characteristics with cancer cells, namely extended stimulation of the cell cycle and inhibition of apoptosis. Since this cancer like conditions are required, a virus without the ability to create this conditions on it self, would only replicate in cancer cells and could therefore have oncolytic effects.

Oncolytic adenoviruses present a recent approach to achieve virus induced cell lysis in cancer cells but not in other healthy cells. The first presented oncolytic virus ONYX-15 consisted of an Adenovirus that was deleted in its 55kDa gene for E1B. E1B was believed to be involved in degradation of p53. Therefore ONYX-15 would only be replicated by cells with impaired p53-function. Most human cancers include mutations in p53 or its downstream players. Unfortunately ONYX-15 was slightly capable of infecting normal replicating cells and normal non-replicating cells (Xiao et al., 2010).

This may be due to E1B having another function than previously believed. E1B has more recently been shown to be responsible for late viral RNA-export rather than breakdown of p53. Therefore an altered mechanism of RNA-export in cancer cells may be responsible for the selectivity of E1B restricted adenoviruses (O’Shea et al. , 2004). Figure 1 Schematic structure of the genomes of all viruses discussed (Adapted from Xiao et al. , 2010): Promoters or genes, which have not been changed in any of the viruses, are not shown.

(ITR: inverted terminal repeat; hSurP: human Survivin promoter; E1B? 55: deletion of the complete gene for the 55kDa E1B protein; E1A? 24: deletion of 24bp form the E1A gene; CMV: cytomegalovirus promoter; EGFP: inserted gene for enhanced green fluorescent protein; IL-24: inserted gene for Interleukin 24) Deletion of 24bp from the adenoviral gene E1A impairs the inhibitory binding of E1A to pRB, which has been another strategy to make the adenovirus specific for cancer cells.

To reduce toxicity and further restrict the replicating ability to cancer cells, both tissue and tumor specific promoters have been used, including the promoter of survivin. Survivin inhibits apoptosis and is highly expressed in many tumors. Figure 2 Comparison of three oncolytic viruses in vitro (Xiao et al. , 2010): The effect of Ad·sp·E1A(? 24)·E1B(? 55)·IL24 was compared to Ad·sp·E1A(? 24)·E1B(? 55)·EFGP and ONYX15 by measuring cell viability after treatment compared to untreated cells. Panel A shows the viability of four tumour cell lines in the four days after treatment at 10 MOI.

Panel B shows the viability of two additional more cell lines 96h after treatment with MOIs of 0. 1, 1, 5 and 10 respectively. Cell viability was measured by MTT assay. Adenoviruses have also been used to insert therapeutic genes, such as IL-24 into cancers cells. Il-24 does not induce apoptosis in normal cells but is involved in mitochondrial and also exogenous apoptosis in cancer cells. Nonetheless specificity has been an impediment because replication in normal cells is disadvantageous.

By combining cancer specific viruses with therapeutic genes, which are therefore selectively expressed in infected cancer cells, a higher efficacy and specificity can be achieved than in oncolytic viruses and gene therapy alone. Il-24 is not only capable of inducing apoptosis in the cancer cells where it is produced, it is also secreted and may therefore induce exogenous apoptosis in metastases throughout the body. An oncolytic virus can highly amplify inside human tumors and cause a very high production of therapeutic genes as Il-24.

The most recent approach combined all four strategies. This virus called Ad·sp·E1A(? 24)· E1B(? 55)·IL-24 included a survivin promoter replacing the promoter for E1A. In the E1A gene 24bp were deleted and E1B was deleted in its complete 55kDa gene. Finally the therapeutic gene Il-24 was inserted into the adenovirus, accompanied by a cytomegalovirus (CMV) promoter. The oncolytic capabilities were tested on different cancer cell lines in comparison with ONYX-15 and Ad·sp·E1A(? 24)·E1B(? 55)·EGFP.

This third virus, that had the enhanced green fluorescent protein inserted instead of IL-24, served as a control to show what part of the improvement was due to Il-24. The efficacy of the three viruses was assessed by the viability of the target cells compared to untreated controls in 24h intervals form 0h to 96h (figure 2A) and at different concentrations of infectious agents (figure 2B). The viability was measured in a MTT assay, which reveals the activity of an MTT reducing enzyme present in every cell (Xiao et al. , 2010).

MTT is a yellow tetrazole that turns purple after it is reduced (Cory et al. , 1991). Viral concentrations are expressed in MOI (multiplicities of infection) that describes the ratio of the number of viral particles to the number of cells. In all cell lines ONYX-15 caused the least and the IL-24 armed virus caused the most reduction in viability. The difference between the EGFP virus and the IL24 virus was bigger than between ONYX-15 and the EGFP virus. This distinction advanced with both the concentrations and the incubation time.

Overall the results shown in figure 2 indicate a larger decrease in viability by the new adenovirus compared to ONYX-15 and IL24 seems to be largely responsible for this effect. The described EGFP virus and the Il-24 virus have also been tested in a naked mouse model of human lung cancer xenografts, in which they were able to achieve 45% and 60% growth reduction respectively. The in therapeutic aspects still moderate reduction in growth, is assumed to be at least partially caused by not representative experimental conditions (Xiao et al. , 2010). It remains to determine weather this will improve under medical conditions.

It should also be noted that although human cancer xenografts in nude mice are a widely used model for human cancers, they do not include an immune system, which is plays an essential role in the fight against cancer. The combination of multiple regulated oncolytic viruses and gene therapy into one adenovirus seems to greatly improve the oncolytic capabilities in the resulting virus. Ad·sp·E1A(? 24)·E1B (? 55)·IL-24 will hopefully yield similar results in other cancer models and may potentially lead the way to a new category of therapies against cancer. 1065 Words.

References See references for pictures or see https://www.dropbox. com/s/boazfx8wvp84fcr/XEROX. Oncolytic_adenovirus_layout. pdf Xiao, L. L. , Wu, Y. M. , Qian, J. , et al. (2010) The antitumor efficacy of IL-24 mediated by E1A and E1B triple regulated oncolytic adenovirus. Cancer Biol Ther vol. 10 pp. 242-50 O’Shea, C. C. , Johnson, L. , Bagus, B. , et al. (2004).

Late viral RNA export, rather than p53 inactivation, determines ONYX-015 tumor selectivity. Cancer Cell vol. 6 pp. 611-23 Cory, A. H. , Owen, T. C. , Barltrop, J. A. and Cory, J. G. (1991) Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture. Cancer Commun vol. 3 pp. 207-12.

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