Introduction: Adenoviral gene therapy vectors offer a safe, alternative platform for developing anti-cancer therapeutics. The most recent “next generation” vectors have improved tropism through “de-targeted” infection from natural receptors and “re-targeted” infection through virus-displayed peptides. Methods: We apply a novel adenoviral vector system, pFex, to generate virus with altered tropism through Fiber-displayed peptides. These virus were generated with an HI Loop altered Fiber-gene recombined into replicating adenovirus through uni-directional Cre recombinase-mediated cassette exchange. The adenoviral vector was mutated by deleting fiber amino acids T489AYT492 for ablation of Fiber-Coxsackie-Adenovirus Receptor (CAR) mediated infection (de-targeting). Then the de-targeted viruses were retargeted by displaying an integrin targeting motif (RGD) or Prostate Specific Membrane Antigen (PSMA) targeting peptides in the Fiber HI-loop. These viruses have been evaluated for re-targeting ability by infecting various cell lines using different concentration (MOI = 1 to 10). The viruses then were removed and cells were continually cultured for 48 hours. The number of infected cells was quantified by fluorescent microscopy and flow cytometry. Results: Our results have shown that de-targeted viruses were unable to infect cells, and the peptide re-targeted viruses were capable of re-directing infection through non-CAR mediated pathways. These data demonstrated that cell binding and internalization are mediated by re-targeted peptides. Conclusions: In summary, this novel pFex vector system is highly efficient and capable of generating testable Fiber-modified adenovirus within one week. Fiber-CAR de-targeted and specific peptide re-targeted viruses are capable of infecting cells. Ablation of Fiber-CAR binding, the natural attachment of virus with cells, can be rescued by using re-targeting peptides.
98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA