Top of pageAbstract In an attempt to overcome the individual hurdles inherent in non viral gene delivery, we have generated a targeting multi-component gene therapy vector that has advantages over each modality administered as a mono-therapy. Taking advantage of the Simian Virus (SV) 40 Nuclear Localization Signaling (NLS) peptide as well as its DNA Nuclear Targeting Signal (DNTS), we have developed a polyethylenimine (PEI)-based multi-component therapeutic gene delivery vector to serve as a proof of principle in increasing tumor-specific gene delivery. In this novel vector, the high affinity and specificity of Peptide Nucleic Acid (PNA) binding to its complementary sequence was also utilized to bind NLS peptide to plasmid DNA containing the DNTS, PNA binding site and either bgal or the p53 gene. Furthermore, the inclusion of PEG in the vector reduced the steric hindrance between the components and ensured a more stable and efficient delivery of the complex to the tumor cells. This newly developed vector was up to a 40-fold more efficient than our previously reported CNGRC-mediated vector which targets aminopeptidase N/CD13 in a nude mouse model (Human Gene Therapy, 2005, in press) and J591 mAb-mediated vector which targets PSMA in an orthotopic mouse model (Molecular Therapy, 2005, in press), when administered systemically to mice bearing H1299 (non-small cell lung cancer) subcutaneous tumors. The targeting polyplex, CNGRC/PEG/PEI/DNA-DNTS-bgal/NLS, expressing bgal, also showed as much as a 100-fold increase in transduction over our previously reported CD13-targeted vector in vitro and up to 10-fold increase in gene delivery over either CNGRC/PEG/PEI/DNA-DNTS-bgal or CNGRC/PEG/PEI/DNA-bgal/NLS alone, indicating the additive effects of the NLS motifs in gene delivery. Examination of nuclear localization as an indicator of gene expression in H1299 cells showed CNGRC/PEG/PEI/DNA-DNTS-bgal/NLS conjugates in the cell nucleus but not the conjugates without NLS signals. Furthermore, a 40-fold increase in in-vivo gene delivery to tumors over our previously reported CNGRC-targeted vector resulted in a significant (80%) reduction in tumor volumes over a 21-day period when bgal was replaced with p53 as the therapeutic gene in the targeting vector, thereby showing a very promising efficacy for systemic p53 gene therapy in a human lung cancer metastasis model. An increase in gene delivery with the therapeutic targeting vector, CNGRC/PEG/PEI/DNA-DNTS-p53/NLS, was corroborated by a more robust p53 mRNA and gene expression in tumors and stained tumor sections as compared to other organs from the same mice or mice injected with vector without the NLS motifs. Pharmacologic and toxicity studies showed no marked adverse effects on major organs. This |[ldquo]|artificial virus|[rdquo]| we have developed here certainly needs further optimization as a potentially suitable multi-component delivery system in overcoming most of the current hurdles in non viral gene delivery. We are currently in the process of extensive evaluation of this novel vector for use in p53 clinical trials.