Tumor-Specific Gene Delivery Mediated by a Novel Peptide–Polyethylenimine–DNA Polyplex Targeting Aminopeptidase N/CD13

We have developed a novel polyethylenimine (PEI)–DNA vector formulation that is capable of efficient tumor-specific delivery after intravenous administration to nude mice. To further increase the specificity of delivery, we have attached the peptide CNGRC to the vector, which is specific for aminopeptidase N (CD13). The strategy for coupling this peptide to PEI was based on a novel method involving the strong affinity between phenyl(di)boronic acid (PDBA) and salicylhydroxamic acid (SHA) as well as a polyethylene glycol (PEG) linker to reduce steric hindrance between the vector and the peptide. In vitro assessment of targeting by the CNGRC/PEG/PEI/DNA vector carrying a β-galactosidase (β-Gal)-expressing plasmid showed as much as a 5-fold increase in transduction, relative to the untargeted PEG/PEI/DNA-βgal vector, of CD13-positive lung cancer, fibrosarcoma, bladder cancer, and human umbilical vein endothelial cells. Competition with free peptide resulted in up to a 90% reduction in delivery, indicating that gene delivery was specific for CD13-positive cells. Intravenous administration of the CNGRC/PEG/PEI/DNA-βgal vector to nude mice bearing subcutaneous tumors resulted in as much as a 12-fold increase in β-Gal expression in tumors as compared with expression in either lungs or tumors from animals treated with the original PEI/DNA-βgal vector. In vivo transduction analysis using the CNGRC/PEG/PEI/DNA vector to target the intravenous delivery of a yellow fluorescence protein (YFP)-expressing plasmid to subcutaneous H1299 tumors confirmed delivery of YFP to both tumor cells and tumor endothelial cells. The use of this peptide to further increase tumor-specific delivery mediated by our novel PEI/DNA vector now provides a basis for developing tumor-targeted gene therapies for use in the clinical treatment of cancer.