Cre-LoxP Mediated Strong Enhancement of pBIRC5 Promoter Driven Suicide of Cancer Cells with CD/UPRT and Fluorocytosine

ABSTRACT

Suicide gene therapy (gene-directed enzyme prodrug therapy, GDEPT), based on tumor-specific promoter driven expression of genes encoding an enzyme capable of intracellularly converting a extracellularly administered nontoxic prodrug into a toxin that selectively kills cancer cells, is a promising approach for cancer treatment. An important prerequisite for the practical use of the approach is cancer-specific suicide gene expression. Since many tumor specific promoters used for intratumoral expression of suicide genes are relatively weak, it was suggested to use tumor specific expression of Cre recombinase to enhance the expression of the therapeutic transgene driven by a strong promoter, artificially suppressed with a floxed insertion, but reactivated by the excision of this insertion with Cre.

In this report, we demonstrate that the expression level of the chimeric suicide gene (FCU1), encoding a cytosine deaminase (CD)/uracil phosphoribosyltransferase (UPRT) fusion protein, under control of the cancer-specific human pBIRC5-1.5 promoter, as well as its cytotoxicity in the presence of 5-FC in various cell lines, is essentially lower than under control of the strong ubiquitous pCMV promoter. However, the use of the binary system including Cre enzyme expressed under control of the pBIRC5-1.5 promoter and the FCU1 driven by pCMV connected to the gene via a floxed transcriptional stop signal allows to increase the cell specific expression level and cytotoxicity of CD/UPRT up to the values comparable to those achieved with the pCMV promoter immediately adjacent to FCU1. Thus, this combination may be useful for human gene therapy applications.

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