A novel two-step transcriptional activation system for gene therapy directed toward epithelial cells

Abstract

The two-step transcriptional activation (TSTA) mechanism in gene therapy amplifies cell type-specific promoter activity, allowing for increased levels of gene expression in target tissues. In this system, the specific promoter drives expression of a strong transcriptional activator that binds to DNA target sequences located upstream from a second promoter controlling the expression of the therapeutic gene. The majority of previous studies have exploited a fusion between the DNA binding domain of the yeast transcriptional activator Gal4 fused to the VP16 activation domain of herpes simplex virus 1 as the transcriptional activator. In this report, an alternative to this system is described based on a fusion protein containing the DNA binding domain of the bovine papillomavirus 1 transcriptional activator E2 fused to VP16 that induces target gene expression following binding to a minimal bovine papillomavirus 4 promoter containing upstream E2 binding sites and only 3 bp of promoter sequence upstream from the TATA box. VP16-E2 is superior to Gal4-VP16 as the transcriptional activator in a TSTA system driven by either of the two potentially cancer-specific promoters telomerase RNA and telomerase reverse transcriptase in several cell lines. Results also suggest that this new system has an advantage in epithelial cells and is therefore ideal for potential targeting of carcinomas. By incorporating the TRAIL gene as a transgene in the VP16-E2 TSTA system, selective killing of telomerase-positive cells occurs. We propose that our new system should be considered in future TSTA, particularly when targeting epithelial-derived cells.

The use of a human papillomavirus 18 promoter for tissue-specific expression in cervical carcinoma cells

“…Extra E2BSs could be added to the promoter to increase activity [21]. Furthermore, if the HPV promoter is not sufficiently strong, a twostep transcriptional activation system can be utilized to substantially increase promoter activity [57]. In conclusion, the HPV18 LCR800 P 105 promoter could be an excellent starting point to begin the next stage of this gene therapy project.…”

The human and canine TERT promoters function equivalently in human and canine cells

“…This plasmid was a gift from Dr Mart Ustav (Estonian Biocenter). The p6xE2‐3bp‐Luc reporter vector was created as described in Vance et al 31 The plasmid pTERT‐VP16E2 was cloned as described in Arendt et al 15 …”

“…The relatively weak activity of the TERT promoter, and other cancer selective promoters, compared with ubiquitous mammalian promoters has encouraged the development of two‐step transcriptional activation (TSTA) systems that increase the expression of the transgene in target cells, while not jeopardizing the specificity of the gene expression in non‐telomerase expressing cells 6,7,15 …”

Molecular-Genetic Imaging of Cancer

“…The UAS was also replaced with E2 binding sites followed by a minimal bovine papillomavirus 4 promoter containing three base pairs of the promoter sequence upstream from the TATA box (Arendt, Nasir, & Morgan, 2009). VP16-E2 proved to be superior to Gal4-VP16 as the transcriptional activator in a TSTA system driven by either of the two potentially cancer-specific promoters, telomerase RNA and hTERT, in several cell lines (Arendt et al, 2009). Another widely used transactivator is p65, a subunit of human NF-κB (Schmitz & Baeuerle, 1991).…”