engleski

Vector for Tumor Gene Therapy through Inhibition of Angiogenesis: Adenoviruses Bearing NGR Motifs in the HI-Loop of Adenovirus Fiber Protein Bind Aminopeptidase N and Alpha v Beta 3 Integrins

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 an Adenovirus vector type 5 (Ad5) 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 alpha v beta 3 integrin and coxsackie-adenovirus receptor, that all NGR-bearing adenoviruses exhibited moderately increased attachment and 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 alpha v beta 3 integrin, we found that NGR-bearing viruses bound weakly to this 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 alpha v beta 3 integrin as well, was provided by experiments in which RD cells were treated with TGF-beta 1. Such treatment, which up-regulated APN and alpha v beta 3 integrin, significantly increased the retargeting index of adenovirus containing cyclic but not linear NGR. Since both APN and alpha v beta 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; tumor gene therapy; fiber; aminopeptidase N; integrin

Rad je kao predavanje prezentiran i na skupu Target Definition & Vector Design for Molecular Medicine, održanom od 10.-13.11.2005., Cold Spring Harbor, New York, SAD ; objavljen u Knjizi sažetaka / Michael Barry, David T. Curiel, Stephen Russell, Jan Schnitzer (ur.) ; New York : Cold Spring Harbor Laboratory, 2005 ; str. 10-10 ; te kao pozvano predavanje na skupovima: - Treći hrvatski mikrobiološki kongres s međunarodnim sudjelovanjem, održanom od 04.-07.10.2004., Poreč, Hrvatska ; objavljen u Knjizi sažetaka / Mirta Balenović, Velimir Wittner (ur.) ; Zagreb : Hrvatsko mikrobiološko društvo, 2004 ; str. 94-05 ; - 1st Central European Forum for Microbiology (CEFORM) ; objavljen u Knjizi sažetaka, u: Acta Microbiologica et Immunologica Hungarica (ISSN 1217-8950) (Suppl.) str. 92-93.