(8g) A Tale of Two Targets – Increasing Tumor-Specific Gene Expression By Targeting Overexpressed ?5?1 Integrin and Upregulated Transcriptional Activity of NF-?B

Non-viral gene delivery vehicles targeted to cancer surface receptors in vivo often non-specifically deliver to off target tissues expressing the same receptors, resulting in undesirable side effects. To address this and increase the specificity of gene expression in cancer tissues, we designed a modular delivery system multi-targeted to the overexpressed cancer surface receptor α₅β₁ integrin and the upregulated transcriptional activity of NF-κB. We therefore condensed a luciferase gene under the control of a NF-κB responsive element (pNF-κB-Luc) with polyethyleneimine and encapsulated it in PR_b peptide functionalized stealth liposomes that specifically target cancer cells that overexpress the α₅β₁ integrin. The multi-targeted system achieved increased gene expression in DLD-1 colon cancer cells compared to BJ-fibroblast healthy cells and was also able to differentiate between cancer cells and healthy cells better than either of the individually targeted systems. In addition, we constructed a novel cancer therapeutic plasmid by cloning a highly potent diphtheria toxin fragment A (DTA) expressing gene under the control of an NF-κB responsive element (pNF-κB-DTA). We obtained a dose dependent reduction of cellular protein expression and a dose dependent increase in cytotoxicity in cancer cells transfected with the pNF-κB-DTA plasmid. When pNF-κB-DTA was delivered in PR_b functionalized stealth liposomes to colon cancer cells and healthy cells in vitro, cancer cells were specifically eradicated by more than 60% while healthy cells were minimally affected. The modular nature of our design allows targeting novel pairs of extracellular receptors and upregulated transcription factors for increased specific delivery to a variety of applications.