Characterization of the Cyclooxygenase-2 Promoter in an Adenoviral Vector and Its Application for the Mitigation of Toxicity in Suicide Gene Therapy of Gastrointestinal Cancers

Abstract

The application of adenoviral molecular chemotherapy for systemic malignant disease using herpes simplex virus thymidine kinase has been limited by ectopic transgene expression in the liver due to the vector hepatotropism. The aim of this study was to mitigate this hepatotoxicity using the promoter of cyclooxygenase-2, inactive in liver but active in many gastrointestinal cancers. To analyze the specificity of transgene expression driven by cyclooxygenase-2 (cox-2) promoters, promoters of two different lengths were incorporated into adenoviral vectors to drive luciferase expression. The specific cytocidal effect and in vivo toxicity were analyzed with thymidine kinase expression vectors. The specificity of the cox-2 promoter was well preserved in the adenoviral vector. In vivo, the cox-2 promoter (-1432/+59) showed very little activity in the liver but attained high activity, comparable to that of the cytomegalovirus promoter, in cyclooxygenase-2-positive subcutaneous tumors. The cox-2 promoter-driven thymidine kinase-expressing vectors showed a cytocidal effect specifically in cyclooxygenase-2-positive cells. When mice were treated with the thymidine kinase-expressing vector and ganciclovir, the cox-2 promoter successfully mitigated the fatal hepatotoxicity, which was observed with the cytomegalovirus promoter-driven vector. The cox-2 promoter successfully mitigated the adverse effects of adenoviral suicide gene therapy by minimizing transgene expression in the liver.

Transcriptional targeting of tumors with a novel tumor-specific survivin promoter

“…Transcriptional targeting methods have been studied that utilize TSPs to restrict transgene expression to tumor cells. 6,7 A wide range of promoters have been evaluated for transcriptional targeting. These results leave no doubt that transcriptional targeting can enhance the tumor specificity and therapeutic index.…”

“…Two control recombinant adenoviruses, AdGL3BCMV and AdGL3BCox-2M, are E1-and E3-deleted and express luciferase, which is induced by enhancer/CMV or the 942 bp Cox-2M promoter, 6 respectively. The viruses were propagated in the adenovirus-packaging cell line, 293, and purified by double CsCl density gradient ultracentrifugation, followed by dialysis against PBS with 10% glycerol.…”

“…This combination of ''tumor on'' and ''liver off '' profile can result in increasing the therapeutic index and limiting the toxicity of vectors and transgenes in vivo. Many promoters have already been evaluated for transcriptional targeting in cancer gene therapy, 1,2 such as the a-fetoprotein (AFP) promoter for hepatic carcinoma, 3 the tyrosinase gene promoter for melanoma, 4 the prostate-specific antigen (PSA) promoter for prostate cancer, 5 and recently, the cyclooxygenase-2 (Cox-2) promoter for gastrointestinal cancer, 6 the midkine (MK) promoter for Wilms' tumor or neuroblastoma, 7 and the CXCR4 promoter for breast cancer and melanoma. 8 Presently, the data appear to show that transcriptional targeting can enhance the tumor specificity.…”

Hypoxia-mediated tumour targeting

“…17,20,30 Several tissue-specific promoters have been investigated for their ability to mitigate the liver toxicity associated with TK-mediated 'suicide' gene therapy. 31,32 In the present study, we reasoned that the OBHRE promoter would be suitable for this purpose due to its low basal activity in normal tissue. We compared the in vivo toxicity of the Ad.HRETK and Ad.CMVTK vectors.…”

“…35 Several different tissue-specific promoters have been used to direct the expression of genes essential for viral replication, leading to the generation of conditionally replicating adenoviruses (CRADs) targeted to tumour cells. [36][37][38] The low basal activity combined with the high tumour activity of the OBHRE promoter suggests that it would be ideally suited for use in the context of a CRAD.…”