Retargeting of adenovirus vectors. Viral ligands and cellular receptors
Abstract: Gene therapy is one of the newest technologies attempted to treat human cancer. Progress in the field has however been hampered by the lack of efficient and safe vectors. Ideal vectors should specifically transduce desired cells in vivo. This thesis is devoted to the development and evaluation of retargeted adenovirus type 5 (Ad5) vectors. Different types of complex cell binding ligands have been incorporated into various Ad5 capsid proteins to study ligands and anchoring proteins suitable for Ad5 retargeting. Retargeted Ad5 has been evaluated in terms of e.g. specificity, infectivity and capsid protein incorporation. In addition, immunoglobulin binding Ad5 has been generated and used to evaluate potential tumor receptors. Complex ligands containing disulfide bonds can generally not be used to retarget Ad since they are insoluble and do not bind to their corresponding receptor when expressed in the reducing cell cytosol where the virus proteins are synthesized. Affibody moleculesTM and hyperstable single chain antibodies (scFv) could however be incorporated into Ad capsids with retained binding specificity and can predictably be used as scaffolds for generation of tumor specific Ad5 vectors. The incorporation of new viral ligands into truncated knobless fibers resulted in retargeted Ad5 but the infectivity, growth and fiber content of these viruses were decreased. The decreased fiber content was, at least partially, caused by impaired translation of the fiber protein. Several efforts were made to increase the fiber content of retargeted Ad with truncated, affibody liganded fibers without success. Incorporation of a hyperstable scFv into pIX resulted in normal pIX content on virus capsids with retained binding specificity of the scFv and caused less decrease in virus infectivity than incorporation into de-knobbed fibers. It was found that Ad can use EGF-R, CA242, Her2/neu and possibly PSMA as new cellular receptors. However, no gene transfer via CA242 or EGF-R could be detected in two colorectal tumor cell lines even though the receptors are expressed and the virus was shown to bind to the cell surface. In addition, gene transfer mediated by all the tested cellular receptors was shown to be dependent on an intact integrin-binding RGD motif in the penton base.In conclusion, cytoplasmic solubility and ligand reactivity of fiber-ligand fusions seems to be the best prediction criterions for recovery of genetically retargeted Ad. Affibody molecules and hyperstable single chain antibodies can be used for genetic retargeting of Ad. The fiber content, growth and infectivity is impaired in viruses with truncated knobless fibers. The minor capsid protein pIX is an interesting alternative to fibers for incorporation of new viral ligands since a hyperstable scFv can be genetically added to the protein with less decrease in infectivity of the virus than when the ligand is put on truncated deknobbed fibers. Ad5 can use alternative receptors for virus cellular entry but it is possible that blocks to Ad5 transduction exist in tumor cells downstream of virus binding to cellular receptors. One of these factors is the integrin-binding RGD motif of the penton base.
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