Gene therapy for prostate cancer: toxicological profile of four HSV-tk transducing adenoviral vectors regulated by different promoters

Abstract

Adenoviral vector delivery of the Herpes simplex virus thymidine kinase (HSVtk) gene in combination with the prodrug ganciclovir (GCV) has been tested in phase I clinical trials for prostate cancer and found to exhibit a satisfactory toxicity profile. We have developed additional adenoviral vectors with differing promoters to optimize the expression profile and in the present study evaluate the potential systemic toxicity of these vectors. Four recombinant adenoviral vectors that express the HSV-tk gene were generated using three different promoters: CMV (leftward orientation); RSV (both rightward and leftward orientation); and the mouse caveolin-1 (cav-1) promoter (leftward orientation). Efficacy was determined in vitro by cytotoxicity assays in a mouse prostate cancer cell line, RM-9, and in vivo by treating orthotopic tumors. Potential toxicity was evaluated from liver histology and apoptotic cell counts and enzyme levels in the serum following intravenous adenoviral vector injection. Although there were differences in HSV-tk expression at the protein level among the four vectors there were no significant differences in in-vitro cytotoxicity studies with GCV or in vivo in tumor growth suppression of an orthotopic mouse prostate cancer model in GCV treated mice. Intravenous delivery of high doses of all adenoviral vectors lead to abnormalities in liver function as measured by specific serum markers and histological evaluation of liver tissue and increased levels of apoptosis in the liver. These abnormalities were most prevalent with the vector containing the CMV promoter and the rightward oriented RSV promoter. They were least prevalent in the vector regulated by the cav-1 promoter. Upregulation of specific chemokines, MIP-2 and MIP-1b was correlated with apoptotic counts. Our results demonstrate that comprehensive toxicological analysis of adenoviral vectors provides internally consistent information that can differentiate vectors with comparable efficacy based on toxicity. In these studies vectors with the cav-1 promoter-driven and leftward RSV-driven HSV-tk gene demonstrated minimal toxicities with cytotoxic effectiveness comparable to more toxic vectors. Our studies further suggest that promoter selection can influence the toxic effects of an adenoviral gene therapy vector.

Promising therapeutic efficacy of a novel reduced expression in immortalized cells/dickkopf‐3 expressing adenoviral vector for hepatocellular carcinoma

“…The REIC/Dkk‐3 gene was originally identified at Okayama University (Okayama, Japan) as a tumor suppressor gene whose expression is downregulated in various cancers . We previously developed an adenoviral vector expressing the human REIC/Dkk‐3 gene (Ad‐REIC) and reported that treatment with Ad‐REIC induced cancer‐selective apoptosis, via ER stress as a result of the unfolded protein response, in various cancer cell lines . In order to achieve improved effective therapeutic outcomes, we have developed a new adenoviral vector (Ad‐SGE‐REIC), which was constructed with tandemly arrayed enhancers (hTERT, SV40, and CMV) after the poly A sequence, to enhance the expression of the targeted REIC gene .…”

Cooperative effects of adenoviral vector-mediated interleukin 12 gene therapy with radiotherapy in a preclinical model of metastatic prostate cancer

“…24 A replication competent adenoviral vector with two cytotoxic genes, HSV-tk and cytosine deaminase, has also been safely administered intraprostatically to 15 patients who also received concurrent systemic prodrug treatment, valganciclovir and 5-fluorocytosine, together with conformal XRT to the prostate. 25 Although intratumorally or intralesionally injected adenoviral vectors have the potential to leak into the systemic circulation, infecting parenchymal organs like liver and spleen, [26][27][28] the limited toxicity in the HSV-tk gene therapy clinical trials suggests that in situ delivery may provide a safe approach.…”

Targeted Expression of Suicide Gene by Tissue-Specific Promoter and MicroRNA Regulation for Cancer Gene Therapy

“…In order to efficiently control the transgene expression only in target cells, various studies have used different tissue specific promoters [15], [16], [17], [18], [19], [20]. One such promoter is epithelial glycoprotein-2/EpCAM/17-1A (EGP2) promoter which selectively kills the EpCAM over expressing cells in many cancers by restricted expression of TK (thymidine kinase) followed by Ganciclovir (GCV) treatment [21], [22].…”