engleski

Vector for tumor gene therapy: Human adenovirus type 5 retargeted on aminopeptidase N

Recombinant adenovirus type 5 (Ad5) is distinguished from other available systems for gene transfer by its unique ability to accomplish in situ gene delivery to differentiated target cells. One of it’ s limitations in the use is widely distribution of its primary cellular receptor. To permit targeted gene delivery, novel strategies need to be developed. One of them is genetic retargeting of Ad5 vector. Angiogenesis is the formation of new blood vessels and among other physiological processes is involved in tumor progression. The APN (aminopeptidase N) is expressed on the endothelial cells of angiogenic but not normal vasculature. It has been shown by phage display that NGR (asparagine-glycine-arginine) motif binds to APN. Thus, an adenovirus vector retargeted on APN could be useful for gene therapy of tumors through inhibition of angiogenesis of tumor endothelial cells. The aim of this project was to design a vector retargeted on APN molecule via incorporation of specific NGR ligands into the fiber protein. In addition we wanted to test the influence of NGR amino acid environment on binding specificity and affinity. We constructed four defective adenoviruses bearing an NGR motif in a HI loop of a fiber protein. Ad5TVTM5 and Ad5TVTM6 contain linear sequences that are naturally present in fibronectin type III repeat. Ad5ARAP (containing the sequence originally isolated by phage display) and Ad5NGR4C contain cyclic NGR sequences, i.e. they posses two pairs of cysteine residues introduced into the sequence to stabilise it via formation of disulfide bonds. All the constructions were performed by cloning of a duplex made of oligonucleotides and by manipulation of the full lenght adenovirus genome as a stable plasmid in E. coli, using the bacterial homologous recombination machinery. All constructions were further confirmed by partial sequencing of the fiber gene. The infectivity indexes on 293 cells (the ratio of physical particles to infectious particles) of CsCl purified viruses were similar showing that incorporation of ligands in HI loop did not change virus infectivity. Subsequent analysis of viruses by Western blotting, using polyclonal antibodies against fiber, showed that the lenghts of the fibers corresponded to those expected for fibers with insertions in HI loop. To determine whether incorporation of NGR-peptides could alter the tropism of adenoviruses we transduced RD cells (embryonal rhabdomyosarcoma) expressing high levels of APN and integrins, and lacking the primary adenovirus receptor CAR, with all viruses normalized on number of viral particles. By counting of  -galactosidase positive cells under the light microscope and by measurement of  -galactosidase activity, we observed 3 to 4-fold increased transduction of cells infected with adenoviruses bearing cyclic NGR sequences while cells infected with viruses bearing linear NGR sequences showed even smaller transduction efficacy. In conclusion, the findings described here show the feasibility of incorporation of targeting sequences into the HI loop of the fiber knob domain. We were able to generate human adenoviruses containing cyclic versions of the NGR and they showed altered tropism i.e. increased trunsduction of APN positive cell line. However, by incorporation of linear forms of NGR we did not obtain similar effect. It remains to be determined whether this retargeted adenoviruses can be used for in vivo targeting on aminopeptidase N.

adenovirus; gene retargeting

Rad je kao poster prezentiran i na skupovima: - Eighth international summer school on biophysics Supramolecular structure and function / Greta Pifat Mrzljak (ur.) ; održanom od 14.-26.09.2003., Rovinj, Hrvatska ; Knjiga sažetaka ; Zagreb : Institut "Ruđer Bošković", 2003. ; str. 150-150 ; - 2. Znanstveni simpozij s međunarodnim sudjelovanjem 45 godina molekularne biologije u Hrvatskoj i 50 godina dvostruke uzvojnice, održanom od 20.-21.11.2003., Zagreb, Hrvatska ; Knjiga sažetaka / Andreja Ambriović Ristov, Anamaria Brozović (ur.) : Zagreb : Institut Ruđer Bošković, 2003. ; str. 49-49.