Advanced Transitional Cell Carcinoma Treatments Via Expression-targeted Gene Delivery And Minicell Technology
The objective of this project is to develop novel treatments, using expression-targeted gene therapy and minicell technology, to replace current methodologies used in the clinic for the treatment of carcinomas, especially transitional cell carcinoma of the bladder. The expression-targeted gene therapy procedure involves cancer-specific DNA elements (promoters) to drive the expression of engineered suicide genes to induce apoptosis in cancer cells. Minicells, a kind of bacterial derivative , prevent tumor recurrence and growth through targeted toxicity and an induced immune response that is similar to that induced by Bacille Calmette-Guerin (BCG), but without the risk of infection due to lack of chromosomes. The osteopontin promoter (popn) was selected via currently accepted methods by comparing endogenous gene expression between normal and cancerous cells. The opn gene is expressed in far greater amounts in cancer cells, so it was reasoned that the opn promoter would be more active in cancer cells as well. Reporter constructs using popn were transfected into both cancerous and normal cell types, with maximum Popn-driven reporter intensity in the cancer cells showing up as strong (102.7%) compared to Pcmv-driven positive controls. Popn-driven reporter intensity was reduced by ~90% in the non-cancer cells. Further enhancements to targeting and expression were obtained through the incorporation of single-nucleotide polymorphisms (SNPs) in the promoter sequence. Further investigations to confirm a correlation between endogenous opn mRNA levels and Popn-driven reporter expression produced a surprising lack of correlation (R2=0.24). However, taking into account opn mRNA splicing variants, a strong negative correlation was determined between mRNA levels of the variant opn-a and Popn-driven transgene activity (R2=0.95). Three novel cancer-specific promoter pran, pbrms1 and pmcm5 were identified through a new screening logic. The activities of those promoters were verified to be much higher in the tested cancer cell lines than the current gold standard used to target gene expression to cancer cells: the promoter of human telomerase reverse transcriptase (phTERT). A constitutively active, apoptosis-inducing analog of caspase 3, referred to as Reverse Caspase3 (RevCasp3), was engineered via gene recombination and cloned into expression-targeted plasmid constructs. These constructs showed excellent activity in inducing apoptosis within the cancer cells tested. Moreover, Pran-RevCasp3 constructs were shown to have significant, cancer-specific killing action within both human and murine cell in vitro. The therapeutic effects of minicell constructs known as VAX-IP were tested within our orthotopic, murine model of transitional cell carcinoma of the bladder. In trials focused on the prevention of tumor growth and tumor implantation, bell-shaped curves were produced by data reflecting the relation between drug dose and tumor burden. The median and average bladder weights, used as a surrogate for tumor burden, decreased with increasing doses of VAX-IP minicells administered via intravesical, transurethral delivery. Activity was lost at high doses of VAX-IP minicells. Compared with the sham-treated group, 1x108 VAX-IP minicells, delivered at 24 hours post-surgery with repeated administrations every 7 days for a total of four treatments, yielded a significant survival advantage to the treated animals (P=0.03).