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Utilization of cancer cell eLF-4E over-expression /over-activation and a prostate specific promoter for the development of a transcriptionally and translationally regulated gene therapy Scott, Christopher Charles

Abstract

Prostate cancer is the second leading cause of cancer related deaths in males. There is no current cure for advanced and metastatic prostate cancer. The purpose of this research was to design a gene therapy system that will allow for the selective killing of prostate cancer cells, while sparing normal cells. We have utilized the over-expression of eukaryotic initiation factor 4E (eIF4E) protein in many different cancer types, including prostate cancer. eIF4E is required for the translation of mRNA containing a 5’untranslated regions (5’UTR) with large degrees of secondary structure, complex 5’UTRs are most commonly found in growth factors and tumour promoting genes. By constructing lentiviral vectors with 5’UTRs inserted upstream of an EGFP reporter gene or herpes simplex virus thymidine kinase (HTK) therapeutic gene we have observed tumour specific expression and killing. Infection of cancer (LNCaP, PC 3M, DU145, and MCF 7 cells) and non-cancer cell lines (BPH 1,267 B1, Plat-E, and Huvec c cells) with lentiviral vectors encoding a CMV-promoter/EGFP-reporter construct resulted in high levels of EGFP expression in all cell lines; however, introduction of a 5’UTR restricted high expression levels to cancer cell lines and Plat-E cells, an embryonic cell line that expresses relatively high levels of eIF4E. Cell lines infected with a 5’UTR regulated HTK showed differential levels of killing with respects to the concentration of gancyclovir (GCV) administered. At least a 100-fold greater dose of GCV was required for equivalent killing effects in non-cancer cell lines infected with lentiviral vectors containing an inserted 5’UTR than with lentiviral vectors not containing a 5’UTR. To further achieve prostate cancer specificity, the CMV promoter was exchanged with the prostate-specific ARR₂PB promoter. Experimental results show that the killing effects of GCV were restricted to prostate cancer cells and not seen in non-prostate cancer cells. Our results indicate that combined translational regulation, by incorporation of an eIF4EUTR recognition sequence upstream of a therapeutic gene, together with transcriptional regulation, through using a prostate-specific promoter, provides a mechanism for the selective killing of prostate cancer cells and the sparing of normal prostate and non-prostate cell lines.

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