638. Expression-Targeted Cancer Gene Therapy Using pran to Drive Caspase 3 Expression

Abstract

Finding specific yet strong DNA control elements is the key component of securing the safety and efficacy of expression-targeted gene therapy treatments. One of barriers which limits the therapeutic success of the technology is that cancer-specific promoters, even through possessing excellent selectivity, are most often not strong enough. As a result, there remains a great need for identifying and developing cancer-specific promoters with targetable and robust activity.Based on a top-down algorithm, a panel of promoter candidates with both excellent specificity and strength was identified. Among those candidates, we found that the promoter of RAS-related Nuclear protein (pran) yielded excellent gene expression levels in cancer cell lines of breast, prostate, and ovarian origin, but was significantly less active in normal cells. The transfection efficacy of plasmids encoding the green fluorescent protein (GFP), driven by the pran was up to 99.8% that of reporter plasmids driven by pcmv-driven positive controls. When compared to GFP-encoding plasmids driven by the human telomerase reverse transcriptase promoter (which is under investigation in clinical trials), pran yielded reporter intensities between 5- and 15-fold greater. In terms of targeting efficiency, GFP expression was highly restricted in normal cells, with no differences observed between pran and a promoterless control vector.The pran was next used to drive the expression of a bioactive exon – a constitutively active form of caspase 3, which induces apoptosis in expressing cells. Morphologic observations and viability assays both confirmed toxic effects of the delivered plasmids in cancer cells, with little, if any, effect in normal cells. Apoptosis was confirmed by caspase 3 detection assays. Cell viability was reduced by up to 60% after one treatment. Multiple treatments, however, are feasible with non-viral gene delivery vectors, and are under investigation in an in vivo model of transitional cell carcinoma.