Adenoviral vectors do not generally perform well following intravenous delivery because they are rapidly inactivated and cleared by the innate and adaptive immune system. Our group has previously shown that stealthing adenovirus particles with multivalent reactive copolymers based on poly-[N-(2-hydroxypropyl)methacrylamide] (pHPMA) protects virus surface epitopes from interaction with the environment 
and gives an impressive extension in plasma circulation kinetics following intravenous delivery 
. The tropism-ablated polymer coated virus can then be retargeted with various molecules including growth factors [23,28–30]
, monoclonal antibodies 
and peptides 
to infect cells via endosome-internalising receptors of interest.
Endothelial selectins represent an attractive target, given their physiological roles in binding circulating blood cells under shear blood flow conditions, and their pattern of selective expression at sites of inflammation such as in tumours. In vitro a 4-h activation of HUVECs with rhTNF-α is sufficient to induce a substantial (8-fold increase in GMean value) up-regulation in the E-selectin level. This enables ligand-specific transduction of E-selectin-expressing endothelial cells, following infection with the MHES-retargeted virus.
While the linkage of E-selectin antibodies to the surface of adenovirus has previously been reported for targeting activated endothelial cells, previous strategies have employed PEG to mediate attachment 
. Ogawara et al. used bifunctional PEG containing N-hydroxysuccinimide ester (NHS) and vinyl sulfone (VS) groups at each end of the molecule, to react (via NHS esters) with primary amino groups on the surface of the virus preventing fibre/CAR attachment, while remaining VS groups were available to bind targeting antibodies. The use of multivalent pHPMA based copolymers, which are able to bind to the virus surface by multiple linkages 
, provides not only a platform to prevent CAR/intergrin mediated uptake and attachment of targeting antibodies but can also improve shielding against components within the blood 
. Avoidance of neutralising antibodies, coagulation factors and complement is a prerequisite for systemic delivery routes accessing tumours via the vasculature.
The use of antibodies for direct conjugation to polymer coated viruses, remains however problematic. This strategy is subject to variability between different antibody molecules depending on their content of reactive amino groups, leading to a mixture of orientations sometimes with exposed Fc regions, which might resemble immune complexes. Hence, strategies to minimise Fc exposure were deemed important and alternative approaches of orientated ligand-conjugation were investigated based on adaptor proteins.
Following the reported success of using S. aureus
protein A to correctly orientate antibody based ligands 
, we tested the use of protein A to attach MHES to polymer coated Ad. Transduction of TNF-α stimulated HUVECs resulted in a 30-fold increase in transgene expression from MHES-StaphApcAdluc compared with Adluc (data not shown). However the use of an isotype control antibody also substantially increased transduction of the endothelial cells compared to the unmodified virus (data not shown). Even without addition of targeting antibody, the presence of the S. aureus
protein A increased transgene expression 4-fold over Adluc (data not shown). Given the high degree of non-specific binding engendered by protein A in this configuration, attention was focussed on an alternative mechanism of attachment namely Streptococcal aureus
Using in vitro studies we have validated the concept of using protein G-modified virus to allow spontaneous binding of monoclonal antibodies against E-selectin, leading to receptor-mediated infection. Importantly this method unlike protein A did not result in substantial non-specific binding and transduction of target cells (c and d, compare pcAdluc and IgGpcAdluc). This system could clearly be easily adapted for linkage of any Fc-containing retargeting ligand. In this manner we believe that protein G modification will provide a useful tool to screen candidate cell surface receptors for targeting, by displaying correctly oriented antibodies on the surface of the polymer coated virus.
In an ex vivo
model of human umbilical vein cord transduced with Adluc or MHES-StrepGpcAdluc following treatment with PBS or rhTNF-α, the ability of the retargeted virus to infect a polarised layer of endothelial cells was demonstrated. Following stimulation of the cells with rhTNF-α, the non-modified (Ad) virus failed to show transduction, while the E-selectin-targeted virus gave a high frequency of transduced cells. This is in agreement with rhTNF-α-induced over expression of E-selectin in vitro, ex vivo
and in vivo
. The inability of unmodified Ad to infect endothelial cells in the presence of rhTNF-α might reflect down-regulation of viral receptors. While rhTNF-α increases the expression of E-selectin, P-selectin, ICAM-1, and vascular cell adhesion molecule (VCAM-1) on endothelial cells 
, it can also change the distribution of some surface proteins away from sites of inter-endothelial cell contact 
. It can also down-regulate cell surface expression of some proteins such as: platelet endothelial cell adhesion molecule-1 (PECAM-1) [37,38]
as well as αvβ3 integrin 
and CAR 
; the pivotal receptors for Ad binding and internalisation [41,42]
This system was also tested in vivo, where a protein G-modified virus retargeted with a chimeric P-selectin Glycoprotein Ligand-1 (PSGL-1)-Fc fusion protein was injected i.v. to target tumour-associated vasculature in tumour-bearing mice. The retargeted virus showed endothelial cell transduction and a better tumour accumulation with an average of 36 fold higher virus uptake compared with unmodified virus.
The ability of free antibodies to outcompete the retargeting ligand for the protein G-modified virus (b) must be taken into consideration in the design of extended duration experiments, although it appears to have only limited consequences for in vivo targeting studies where virus kinetics are generally short.
These findings highlight the possibility to deliver genes selectively to tumour-associated vasculature providing an alternative to direct tumour cell targeting. Endothelial targeting bypasses the need for extravasation which can limit targeting delivery.
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