Modulation of Telomerase Promoter Tumor Selectivity in the Context of Oncolytic Adenoviruses

Abstract

The telomerase RNA (hTR) and reverse transcriptase (hTERT) promoters are active in most cancer cells, but not in normal cells, and are useful for transcriptional targeting in gene therapy models. Telomerase-specific conditionally replicating adenoviruses (CRAd) are attractive vectors because they should selectively lyse tumor cells. Here, we compare CRAds, in which either the hTR or hTERT promoter controls expression of the adenovirus E1A gene. In replicationdefective reporter adenoviruses, the hTR promoter was up to 57-fold stronger in cancer cells than normal cells and up to 49-fold stronger than hTERT. In normal cells, hTERT promoter activity was essentially absent. Doses of telomerase-specific CRAds between 1.8 and 28 infectious units per cell efficiently killed cancer cells, but normal cells required higher doses. However, CRAd DNA replication and E1A expression were detected in both cancer and normal cells. Overall, tumor specificity of the CRAds was limited compared with nonreplicating vectors. Surprisingly, both CRAds expressed similar E1A levels and functional behavior, despite known differentials between hTR and hTERT promoter activities, suggesting that the promoters are deregulated. Rapid amplification of cDNA ends analysis of hTR-/hTERT-E1A transcripts ruled out cryptic transcription from the vector backbone. Blocking E1A translation partially restored the hTR-/hTERT-E1A mRNA differential, evidencing feedback regulation by E1A.

SPECT/CT imaging of oncolytic adenovirus propagation in tumours in vivo using the Na/I symporter as a reporter gene

“…This region was chosen for its previously-described ability to support transgene expression 31 and for the dispensable nature of gp19K. 13,32 Using this design, both AdIP1 which is otherwise identical to the wild-type adenovirus and AdAM6 in which replication is driven by the hTR promoter 23,29 show replication and functional hNIS expression in vitro. In vivo, the viruses demonstrate functional hNIS expression and importantly, the kinetics of adenoviral replication can be monitored using molecular imaging (Figures 3 and 4).…”

“…This promoter has previously been shown to be strong and tumour specific in non-replicating 23 and replicating adenoviruses. 29 These viruses were used to establish whether the hNIS imaging system could be used to visualize virus propagation in tumours in vivo, in the living animal.…”

An oncolytic adenovirus expressing granulocyte macrophage colony-stimulating factor shows improved specificity and efficacy for treating human solid tumors

“…29 However, the relatively weak antitumor activity obtained in some tumor cells and the nonspecific viral replication obtained in normal cells have restricted the potential utility of such agents. For instance, recent work published by Bilsland et al 11 indicated that high levels of viral DNA and E1a expression are detectable in both cancerous and normal cells following infection of the adenovirus controlled by the wild-type hTERT promoter. Therefore, there is a practical requirement to enhance the specificity of this promoter to improve its usefulness in cancer gene therapy.…”

“…7,8 However, some studies have detected hTERT-specific viral gene expression in normal cells. 9,10 Bilsland et al 11 reported the detection of viral replication, E1a expression, and cytotoxicity of wildtype hTERT promoter-controlled replicating adenovirus in normal cells, suggesting the need for an improved hTERT promoter for tumor-specific oncolytic adenoviruses. Several research groups have attempted to improve the selectivity of telomerase-specific oncolytic adenovirus to tumor cells by introducing other transcriptional-factor-binding sequences such as c-Myc and Sp1-binding sites, or E-boxes, into the core sequence of the hTERT promoter.…”

Targeting of P53-transcriptional dysfunction by conditionally replicating adenovirus is not limited by P53-homologues

“…Additionally, E1A accumulation due to basal promoter activity in normal cells can finally lead to replication onset and loss of selectivity, in particular at high multiplicities of infection. 1,2 Consequently, alternative strategies are required that ensure stringent regulation of viral replication in response to crucial oncogenic pathway aberrations.…”