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Adenoviruses Bearing NGR Motifs in the HI-Loop of Adenovirus Fiber Protein Bind Aminopeptidase N and Alpha v Beta 3 Integrins

Genetic retargeting of adenovirus type 5 (Ad) through insertion of sequences in the fiber protein should permit targeted gene delivery. The APN (aminopeptidase N) expressed on the endothelial cells of angiogenic vasculature, represents a target molecule for tumor gene therapy aimed at inhibition of angiogenesis. It has been shown by phage display that the motif NGR (asparagine-glycine-arginine) binds to APN. The aim of this work was to design a vector retargeted on the APN molecule via incorporation of specific NGR-containing ligands into the HI loop of a fiber protein and to examine the effect of linear and cyclic sequences on its targeting properties. We constructed four replication defective adenoviruses bearing cyclic or linear NGR-containing sequences. These insertions did not affect structure or incorporation of the fiber protein in viral particles. We have shown on the rhabdomyosarcoma (RD) cell line, which expresses APN but only very low levels of avbeta 3 integrin and coxsackie-adenovirus receptor, that all NGR-bearing adenoviruses exhibited increased transduction efficacy in comparison to wild type virus. NGR-bearing adenoviruses containing cyclic motifs were more efficient than those containing linear ones. The increased transduction efficacy of NGR-bearing Ads was completely abolished by the APN-specific peptide CNGRC and the integrin-specific peptide CRGDC. By measuring transduction efficacy on human laryngeal carcinoma cells with graded expression of avbeta 3 integrin, we found that NGR-bearing viruses bound weakly to avbeta 3 integrin. Nevertheless, adenoviruses bearing linear motifs were more efficient than those bearing cyclic NGR. Additional evidence that the improved entry of NGR-bearing Ads into RD cells was mediated by binding to APN, and perhaps to avbeta 3 integrin as well, was provided by experiments in which RD cells were treated with TGF-beta . Such treatment, which up-regulated APN and avbeta 3 integrin, significantly increased the retargeting index. Since both APN and avbeta 3 integrin are up-regulated in endothelial cells, NGR-bearing adenoviruses could be suitable vectors for tumor gene therapy aimed at inhibition of angiogenesis.

adenovirus; gene therapy; retargeting

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