359. Enhanced Gene Delivery by a Novel Tumor-Specific Vector Containing Peptide and Nucleic Acid Based Nuclear Translocation Signals

Abstract

We have developed a novel formulation between Polyethylenimine (PEI) and DNA that exhibits extremely efficient gene delivery both in vitro and in vivo following an intravenous administration. In addition, this formulation can also serve as a foundation to develop targeted vectors which we have demonstrated by attaching the peptide CNGRC to the vector to further increase the resulting tumor specific delivery by targeting aminopeptidase N (CD13) expression on tumor and tumor endothelial cells. However, as is the case for most non-viral vectors, our formulation is still limited by the inability of delivered genes to efficiently translocate to the nucleus, the site of gene transcription. Previous reports have shown the ability to use nuclear localization signals and other sequence-specific targeting motifs to deliver genes to the nucleus of different cell types in vitro. When we incorporated a peptide based on the SV40 nuclear localization signal (NLS) into our CD13 targeted vector using peptide nucleic acid (PNA) coupling technology, gene expression was increased by as much as 7 times over expression in control samples based on quantitation of bgal in the human cancer cell lines H1299 (lung) and UC-3 (bladder) 24 hours after transduction. After 72 hours, gene expression increased to 16 times levels in control samples. A further increase in gene expression was also obtained by using a plasmid containing the 72bp DNA nuclear targeting sequence (DNTS) of SV40. Interestingly, the intravenous administration of the CD13 targeted PEI/DNA vector to animals bearing H1299 tumors resulted in 6 times greater expression in tumor than in lung. We also demonstrate by confocal microscopy that only the vector bearing the NLS peptide localizes in the nucleus of tumor cells. These results provide proof of principle that nuclear targeting using NLS based peptides and DNA sequences can increase the resulting gene expression obtained with our novel vector formulations, providing improved non–viral vectors for the treatment of cancer.