Cancer-specific targeting of a conditionally replicative adenovirus using mRNA translational control.

BACKGROUND: In view of the limited success of available treatment modalities for a wide array of cancer, alternative and complementary therapeutic strategies need to be developed. Virotherapy employing conditionally replicative adenoviruses (CRAds) represents a promising targeted intervention relevant to a wide array of neoplastic diseases. Critical to the realization of an acceptable therapeutic index using virotherapy in clinical trials is the achievement of oncolytic replication in tumor cells, while avoiding non-specific replication in normal tissues. In this report, we exploited cancer-specific control of mRNA translation initiation in order to achieve enhanced replicative specificity of CRAd virotherapy agents. Heretofore, the achievement of replicative specificity of CRAd agents has been accomplished either by viral genome deletions or incorporation of tumor selective promoters. In contrast, control of mRNA translation has not been exploited for the design of tumor specific replicating viruses to date. We show herein, the utility of a novel approach that combines both transcriptional and translational regulation strategies for the key goal of replicative specificity. METHODS: We describe the construction of a CRAd with cancer specific gene transcriptional control using the CXCR4 gene promoter (TSP) and cancer specific mRNA translational control using a 5'-untranslated region (5'-UTR) element from the FGF-2 (Fibroblast Growth Factor-2) mRNA. RESULTS: Both in vitro and in vivo studies demonstrated that our CRAd agent retains anti-tumor potency. Importantly, assessment of replicative specificity using stringent tumor and non-tumor tissue slice systems demonstrated significant improvement in tumor selectivity. CONCLUSIONS: Our study addresses a conceptually new paradigm: dual targeting of transgene expression to cancer cells using both transcriptional and mRNA translational control. Our novel approach addresses the key issue of replicative specificity and can potentially be generalized to a wide array of tumor types, whereby tumor selective patterns of gene expression and mRNA translational control can be exploited.

The human cytomegalovirus virion possesses an activated casein kinase II that allows for the rapid phosphorylation of the inhibitor of NF-kappaB, IkappaBalpha.

We documented that the NF-kappaB signaling pathway was rapidly induced following human cytomegalovirus (HCMV) infection of human fibroblasts and that this induced NF-kappaB activity promoted efficient transactivation of the major immediate-early promoter (MIEP). Previously, we showed that the major HCMV envelope glycoproteins, gB and gH, initiated this NF-kappaB signaling event. However, we also hypothesized that there were additional mechanisms utilized by the virus to rapidly upregulate NF-kappaB. In this light, we specifically hypothesized that the HCMV virion contained IkappaBalpha kinase activity, allowing for direct phosphorylation of IkappaBalpha following virion entry into infected cells. In vitro kinase assays performed on purified HCMV virion extract identified bona fide IkappaBalpha kinase activity in the virion. The enzyme responsible for this kinase activity was identified as casein kinase II (CKII), a cellular serine-threonine protein kinase. CKII activity was necessary for efficient transactivation of the MIEP and IE gene expression. CKII is generally considered to be a constitutively active kinase. We suggest that this molecular characteristic of CKII represents the biologic rationale for the viral capture and utilization of this kinase early after infection. The packaging of CKII into the HCMV virion identifies that diverse molecular mechanisms are utilized by HCMV for rapid NF-kappaB activation. We propose that HCMV possesses multiple pathways to increase NF-kappaB activity to ensure that the correct temporal regulation of NF-kappaB occurs following infection and that sufficient threshold levels of NF-kappaB are reached in the diverse array of cells, including monocytes and endothelial cells, infected in vivo.

Prolonged activation of NF-kappaB by human cytomegalovirus promotes efficient viral replication and late gene expression.

Infection of fibroblasts by human cytomegalovirus (HCMV) rapidly activates the NF-kappaB signaling pathway, which we documented promotes efficient transactivation of the major immediate-early promoter (DeMeritt, I.B., Milford, L.E., Yurochko, A.D. (2004). Activation of the NF-kappaB pathway in human cytomegalovirus-infected cells is necessary for efficient transactivation of the major immediate-early promoter. J. Virol. 78, 4498-4507). Because a second, sustained increase in NF-kappaB activity following the initial phase of NF-kappaB activation was also observed, we investigated the role that this prolonged NF-kappaB activation played in viral replication and late gene expression. We first investigated HCMV replication in cells in which NF-kappaB activation was blocked by pretreatment with NF-kappaB inhibitors: HCMV replication was significantly decreased in these cultures. A decrease in replication was also observed when NF-kappaB was inhibited up to 48 h post-infection, suggesting a previously unidentified role for NF-kappaB in the regulation of the later class of viral genes.