Date of Graduation


Document Type



The primary objective of this study was to develop a system to deliver antisense oligonucleotides (ONs) via the epidermal growth factor (EGF) receptor. Since many tumor cells derived from epithelial tumors overexpress the EGF receptor they can be preferentially targeted. In this study, a delivery system comprising of a conjugate of EGF covalently linked to poly-L-lysine (an ON binding moiety) was synthesized, characterized and tested for in vitro efficacy. Studies were carried out in A-549 cells showed that EGF-PL promoted up to a 12-fold increase in uptake of the oligonucleotide. Uptake was monitored via a 5{dollar}\\sp\\prime{dollar} BODIPY FL label on the ON. We found that effective ON uptake mediated by the conjugate requires functional domains capable of both ON binding and cell surface recognition. Inclusion of fusogenic peptides, polymyxin B and influenza HA2 peptide, were found to further enhance uptake. We examined the efficacy of this system in inhibiting the ras oncogene in T24 cell-line using phosphorothioate antisense ON sequences. We also tested a new delivery approach utilizing conjugates of antibodies to an external domain of the EGF receptor (EGFR), and PL linked via a disulfide bond. Treatment samples incubated with free ON1 showed up to 35% inhibition in growth as compared to control levels; whereas cells incubated with the ON1:AntiEGFR-PL showed a further enhancement in growth inhibition, up to a 72%, as compared to control levels. A similar trend was seen with the free ON2 which showed up to 58% growth inhibition compared to controls with further enhancement with the ON2:AntiEGFR-PL delivery system i.e. 74% as compared to control levels. The AntiEGFR-PL conjugate alone or the Random ON (10 {dollar}\\mu{dollar}M) did not have any significant effect on cell proliferation. The reduction in the c-Ha-ras oncoprotein was examined using Western blot analysis. This study indicates that the Anti-EGFR-PL and EGF-PL delivery systems are effective in improving antisense efficacy.