Role of the putative heparan sulfate glycosaminoglycan-binding site of the adenovirus type 5 fiber shaft on liver detargeting and knob-mediated retargeting

Abstract

Liver tropism hampers systemic administration of adenovirus in gene therapy and virotherapy. In consequence, tumour targeting requires the combination of capsid modifications that abrogate liver transduction and redirect adenoviral vectors to tumour cells. Coxsackievirus and adenovirus receptor (CAR), integrins and heparan sulfate glycosaminoglycans (HSG) are receptors involved in adenovirus type 5 (Ad5) entry into cells. The in vitro and in vivo properties of Ad5 vectors unable to bind CAR, integrins and HSG with and without Arg-Gly-Asp (RGD) inserted at the HI loop of the fiber were studied. As was previously observed with CAR-ablated vectors, CAR and integrin double binding-ablated vectors transduced hepatocytes less efficiently in vitro but not in vivo. On the contrary, the role of HSG on Ad5 infectivity was evident in vitro only when CAR binding was abrogated, but the shaft mutation that ablated HSG binding on the background of a normal capsid was sufficient to abrogate liver transduction in vivo. The insertion of amino acids RGD at the HI loop in a shaft-mutated fiber only partially rescued integrin-mediated infectivity. These results indicate that the shaft mutation precluded HSG binding and affected the structure of the fiber. The insertion of ligands at the hexon or protein IX may be required to benefit from the fiber shaft mutation-detargeting properties.

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

“…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. 2,7,8 On the other hand, the HI-loop of the fiber knob domain has been used to accommodate a broad range of tumor-selective peptides for tumor targeting [9][10][11] Among all peptides incorporated into the HI loop, the CDCRGDCFC motif (known as RGD-4C) has been broadly used and confers on Ad5 a CAR-independent entry mechanism by using aV-b3 and aV-b5 integrins as primary receptors. 12,13 As integrins are a large family of adhesive receptors involved in the spread and adhesion of tumor cells, the RGD-4C motif leads to the enhancement of tumor cell infectivity and oncolytic effect both in vitro and in vivo.…”

“…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.…”

Changing the adenovirus fiber for retaining gene delivery efficacy in the presence of neutralizing antibodies

“…27,28 Further studies are needed to dissect the putative role of fiber shaft in the context of infection in the presence of NAb. [29][30][31] Nevertheless, the empiric assays performed here suggest that anti-fiber knob NAbs have an important role in determining the success of gene transfer in vitro and in vivo.…”

“…Intriguingly, anti-Ad5 NAb blocked gene transfer by capsid-modified viruses to normal human lung explants much more than what was seen with tumor explants. This suggests that fiber-knob-independent (but perhaps fiber shaft dependent) mechanisms that were postulated as important in the context of systemic biodistribution in mice and primates [29][30][31] may also apply to humans. Experiments in syngeneic mouse or hamster models might shed more light on these phenomena.…”

Osteosarcoma cells as carriers to allow antitumor activity of canine oncolytic adenovirus in the presence of neutralizing antibodies

“…The viral genomic structures were verified by restriction analysis and sequenciation. The anti-hexon staining-based method 22 cell was used for functional tittering (transducing units or TU) of viruses using DkCre cells (CAV2, OCCAV and CAVGFP) and HEK293 (AdTL).…”