Simultaneous CAR- and αV integrin-binding ablation fails to reduce Ad5 liver tropism

Abstract

Targeting adenovirus encoding therapeutic genes to specific cell types has become a major goal in gene therapy. Coxsackievirus and adenovirus receptor (CAR) and alpha(V) integrins have been identified as the primary cell surface components that interact with adenovirus type 5 (Ad5)-based vectors during in vitro transduction. Redirecting Ad5-based vectors requires abrogation of the natural interaction between the viral capsid and its cellular receptors and simultaneous introduction of a new binding specificity into the viral capsid. To abrogate native Ad5 tropism, fiber knob mutations Pro409Glu and Lys417Ala were each incorporated into adenoviral vectors, while the RGD motif was deleted from the penton base. In vitro transduction experiments showed that these capsid mutations eliminated Ad5 interactions with CAR and alpha(V) integrins. Moreover, incorporation in the fiber HI loop of a vitronectin-derived ligand (VN4) specific for the uPAR/CD87 receptor provided the Lys417Ala virus with an alternative entry pathway specific for uPAR-expressing cells, indicating a successful in vitro retargeting of the vector. Unexpectedly, however, simultaneous disruption of Ad5 binding to CAR and alpha(V) integrins had no effect on liver gene transfer following systemic administration in mice. This study highlights the need to understand better the molecular determinants involved in adenovirus uptake by the liver to control the fate of adenoviral vectors in vivo.

Wild-type adenoviruses from groups A–F evoke unique innate immune responses, of which HAd3 and SAd23 are partially complement dependent

“…41,57 For example, CAR-ablated Ads seem to retain their liver targeting capabilities in vivo. 58 Our findings that group B member HAd3 was found at high levels in the lung is consistent with previous findings noted with the CD46 utilizing group B HAd11. 59 However, this contrasts with a recently published study that evaluated biodistribution of HAd3 in CD46 transgenic mice.…”

Improved systemic antitumor therapy with oncolytic adenoviruses by replacing the fiber shaft HSG-binding domain with RGD

“…The significance of differences in relative tumor size between treatment groups was assessed by a two-tailed Student's unpaired t-test. For Kaplan-Meier survival curves, end point was established at X500 mm 3 . The survival curves obtained were compared for the different treatments.…”

“…To achieve liver detargeting, several mutations on capsid proteins, such as the double ablation of CAR and integrin binding, have failed to reduce hepatocyte transduction in vivo owing to the role of blood factors in adenovirus liver transduction. 2,3 Recently, ablation of Factor X binding by the hexon has resulted in successful liver-detargeting, 4,5 although this might be at the expense of lower tumor targeting. 6 Alternatively, the mutation of the heparan sulfate glycosaminoglycan (HSG) putative-binding site KKTK of the Ad5 fiber shaft domain detargets liver transduction, probably because of affectation of the bending or structure of the fiber.…”

Efficient infection of tumor endothelial cells by a capsid-modified adenovirus

“…Retargeting strategies by modification of the viral genome include deleting the binding sites for CAR and a v b 3 /a v b 5 integrins and inserting short peptide ligands with specificity for target cells. [15][16][17][18][19] These approaches have produced somewhat conflicting results with regard to liver transduction. Some papers have reported significant reduction in liver transduction using vectors deleted for both CAR and integrin binding sites, while others demonstrate that similar deletions have no effect in detargeting the liver.…”