S. Liu1, J. Wu1, G. Zhou1, J. Yu1, R. Sanchez1, F. Brunicardi1 1University Of California – Los Angeles,Department Of Surgery, David Geffen School Of Medicine,Los Angeles, CA, USA
Introduction: Two limitations of gene therapy are tissue specificity and tissue expression of the transgene. We hypothesize that synthetic promoters have greatly enhanced activities over endogenous promoters to drive gene expression and can be used to enhance the effect of gene therapy without loss of target specificity. We have demonstrated that the synthetic human insulin promoter (SHIP) has far greater activity than HIP or rat insulin promoter (RIP) in driving gene expression with subsequent enhanced effect of gene therapy in neuroendocrine tumors which overexpress pancreatic and duodenal homeobox1 (PDX1) transcription factor. Bidirectional two-step transcriptional amplification (TSTA) has been shown to enhance gene expression. In this study we sought to determine that TSTA enhances the expression and therapeutic effect of SHIP-diphtheria toxin A (SHIPTSTA-DTA) in PDX1 expressing cells, such as insulinoma.
Methods: SHIP-eGFP vs SHIPTSTA-eGFP, SHIP- firefly luciferase (FLuc) vs SHIPTSTA-FLuc and SHIP-DTA vs SHIPTSTA-DTA vectors were constructed using subcloning techniques. Activity and specificity of vectors were determined by bioluminescence and eGFP assays in mouse insulinoma (βTC6) cells, human primary pancreatic (HPP) cells, pancreatic ductal epithelial (HPDE) cells and PDX1-HPDE cells, since PDX1 is the primary activator of SHIP, with or without PDX-1 shRNA co-transfection. Cytotoxicity was determined by MTS assay and glucose-stimulated insulin secretion (GSIS) was determined by ELISA. Statistical analysis was performed via paired T test; p<0.05 = significant.
Results: SHIPTSTA-FLuc activity in βTC6 cells was 8 fold higher than that of SHIP-FLuc (p<0.05)(Fig. A). SHIP-FLuc was equal to CMV-driven expression, however was specific for cells expressing PDX1. PDX-1 shRNA co-transfection resulted in decreased SHIPTSTA-FLuc activity. Similarly, increased cell numbers of eGFP and expression intensity of eGFP were observed in SHIPTSTA-eGFP transfected βTC6 cells vs controls. No FLuc activity was detected in both SHIPTSTA-FLuc and SHIP-FLuc transfected HPP and HPDE cells. SHIPTSTA-DTA resulted in enhanced cytotoxicity and enhanced suppression of proliferation and GSIS from βTC6 cells compared to SHIP-DTA (p<0.05)(Fig. B).
Conclusions: SHIPTSTA markedly enhanced SHIP activity without loss of tissue specificity; SHIPTSTA-DTA in PDX1+ insulinoma cells enhanced cytotoxicity and suppressed proliferation and insulin secretion. The data support the hypothesis that synthetic promoters of the target gene have greatly enhanced expression efficiency over endogenous promoters and can be used to enhance the effect of gene therapy without loss of target specificity.
