620. A Longer Human Telomerase Reverse Transcriptase Promoter-Based Conditionally Replicative Adenovirus Shows Oncolysis with Less Toxicity for Ovarian Cancer Treatment
Authors: Yosuke Kawakami, Koichi Takayama, Masato Yamamoto, Dirk M. Nettelbeck, Minghui Wang, Mack N. Barnes, Gene P. Siegal, Ronald D. Alvarez, David T. Curiel, Jerry L. Blackwell
Journal: Molecular Therapy (2004)

Conditionally replicative adenovirus (CRAd) is one of the approaches to allow selective viral replication which lead to infection of neighboring cancer cells with progeny virus. It is reported that the human telomerase reverse transcriptase (hTERT) promoter, which is active in many cancers but mostly inactive in normal organs including the liver, maintains its useful profile in a CRAd construct for ovarian cancers. It is relevant to determine whether low levels of transgene expression driven by the hTERT promoter could occur in normal cells including normally dividing ones, since a leaky promoter that results in low levels of expression of E1 could results in severe toxicity. In many instances, cell-specific promoters have worked well in vitro but have demonstrated leakiness or a lack of specificity in vivo. To improve the specificity of the hTERT promoter to ovarian cancer cells, we therefore constructed a CRAd agents by configuring a longer hTERT promoter (1.5kb, hTERT L) as well as a conventional shorter one (0.4kb, hTERT S) driving an E1 expression cassette in place of the wild type Ad5E1 region and evaluated its oncolytic effect and specificity both in vitro and in vivo. First, transcriptional and translational activity of hTERT in ovarian cancer cell lines were examined by RT-PCR. Then, we employed recombinant Ad vectors containing luciferase reporter genes under the control of the hTERT L or hTERT S promoter (AdhTERT L Luc, AdhTERT S Luc) to evaluate the transductional activity of this promoter in ovarian cancer cell lines in vitro. Ad vectors with these reporter genes under the control of cytomegalovirus (CMV) promoter were used as control vectors (AdCMVLuc). We next examined the biodistribution of these vectors administered intravenously within mice. Finally, we evaluated the oncolytic effect of AdhTERT L E1 and AdhTERT S E1 in ovarian cancer cell lines by an MTS assay and crystal violet assay. RT-PCR analyses showed significant transcriptional and translational activity of the hTERT gene in ovarian cancer cells. hTERT L and hTERT S promoter activity was relatively high in ovarian cancer cells, whereas low activity of them was observed in the hTERT negative cells (normal human keratinocyte, NHEK) with luciferase expression vectors. The specificity (RLU in ovarian cancer cells/RLU in NHEK) of hTERT L was higher than hTERT L. AdhTERT L E1 showed a significant oncolytic effect in all the ovarian cancer cell lines tested as well as AdhTERT S E1, whereas little effect was seen in NHEK cells. Biodistribution of AdhTERT L Luc showed ten times lower luciferase activity in the liver than AdhTERT S Luc, indicating that AdhTERT L vector is more specific to the ovarian cancer cells and less hepatotoxic. These results suggest that hTERT L promoter activity is relatively efficient and more specific in ovarian cancer cells than that of the conventional hTERT S, making it an attractive candidate as a tissue specific promoter for ovarian cancer cells. This specificity-enhanced hTERT L CRAd may be useful for the gene therapy of ovarian cancer.