) Endothelial cells in tumor vessels express angiogenic markers that are not detectable in normal vessels. We have developed an in vivo selection system in which phage capable of homing to tumors are recovered from a phage display peptide library following intravenous administration. Using this strategy, we have isolated several tumor-homing phage. Among those were phage displaying the tripeptides asparagine-glycine-arginine (NGR), glycine-serine-arginine (GSL), and a double cyclic RGD (RGD-4C). We have shown that each of those peptides binds to three different receptors that are upregulated in tumor angiogenic vasculature. Based on our in vivo studies targeting tumors with NGR-, GSL- and RGD-4C-phage, we will develop a peptide-based molecular adaptor for targeted delivery of genes to angiogenic vasculature. This goal will be achieved by screening phage peptide libraries on immobilized adenovirus and AAV. The libraries will be engineered so that phage display peptides that contain tumor-homing domains in addition to all possible permutations of a 10aa-long insert (i.e., CNGRC-X10, CGSLC-X10, and RGD-4C-X10). We will also study which of the three receptor systems is best suited for gene delivery using the adaptors. Receptor-mediated internalization and gene expression levels will be studied in vitro by confocal microscopy using endothelial cells expressing the receptors. We plan to evaluate the efficiency of gene expression and angiogenesis inhibition upon targeted delivery of three classes of genes to angiogenic vasculature: (i) suicide; (ii) pro-apoptotic; and (iii) genes encoding angiogenesis inhibitors such as endostatin and angiostatin. Transgenic models of pancreatic carcinoma (Rip-Tag) and prostate carcinoma (TRAMP) will be used. Finally, we will examine the therapeutic effects of targeted delivery of suicide and pro-apoptotic genes to angiogenic vasculature using an animal model of oxygen-induced retinal angiogenesis. These studies may lead to development of new gene therapy-based tumor treatment strategies that rely on inhibition of angiogenesis. Vector targeting would represent a major advance in cancer treatment. Given that our peptides also target angiogenic vasculature in the retina, these advances are also likely to extend the potential for neovasculature-specific gene therapy targeting to other diseases involving angiogenesis.
