Evaluation of polyethylene glycol modification of first-generation and helper-dependent adenoviral vectors to reduce innate immune responses

Abstract

Adenoviruses are robust gene delivery vectors in vivo, but are limited by their propensity to provoke strong innate and adaptive responses. Previous work has demonstrated that polyethylene glycol (PEG) modification of adenovirus can protect the vectors from preexisting and adaptive immune responses by reducing protein-protein interactions. To test whether PEGylation can reduce innate immune responses to adenovirus by reducing their interactions with immune cells, first-generation (FG-Ad) and helper-dependent (HD-Ad) Ad5 vectors were PEGylated with SPA-PEG and tested in vitro and in vivo. We demonstrate that increasing PEGylation ablated in vitro transduction, but surprisingly had no negative effect on the level or distribution of in vivo gene delivery. This poor in vitro transduction could be rescued in part by physically forcing the PEGylated vectors onto cells, suggesting that physiological forces in vivo may enable transduction via heparin sulfate proteoglycan and integrin interactions. While transduction remained the same as for unmodified vectors, the PEGylated vectors reduced innate IL-6 responses by 70 and 50% in vivo for FG-Ad and HD-Ad. These reduced innate responses paralleled similar reductions in vector uptake by macrophages in vitro and Kupffer cells in vivo. These data suggest that PEGylation of Ad vectors can reduce innate immune responses without reducing transduction in vivo. These data also suggest that nonspecific vector uptake by macrophages and Kupffer cells may be critically involved in the initial activation of innate immune responses.

An ex vivo loop system models the toxicity and efficacy of PEGylated and unmodified adenovirus serotype 5 in whole human blood

“…This is in contrast to what has been observed in mice where PEGylation had considerable capacity to reduce immune responses and to enhance transgene expression. [24][25][26]28 We observed that the donors' ability to neutralize Ad5 had a considerable impact on the immune response both for unmodified and PEGylated Ad5. Interestingly, the presence of NAbs in plasma did not directly correlate with the neutralizing ability in whole blood, indicating that other mechanisms may be involved as well or that non-neutralizing antibodies, as determined in plasma samples, may be neutralizing in whole blood.…”

“…24,26,28 However, our data suggest that 20K-PEGylated Ad5, which showed the greatest reduction in immune responses and achieved the lowest level of cell association, in combination with compstatin, could represent a feasible avenue for further development of systemic adenoviral therapy. We want to emphasize that this study has again revealed the importance of using relevant models when studying immune responses against adenoviral vectors intended for use in humans.…”

“…24,26,34 Less is known about the protective effects of Ad5 PEGylation in human blood. However, Hofherr et al have illustrated that 5K-PEGylated Ad5 reduces binding to human platelets and erythrocytes in vitro.…”

“…24,25 Furthermore, there is less uptake in mouse Kupffer cells when PEGylated viruses are used. 26 On the basis of previous in vitro experiments it is believed that high molecular weight PEG molecules achieve more effective detargeting of Ad5 than low molecular weight PEG molecules. 27,28 In mice it has recently been shown that viruses coated with 20K-PEG molecules undergo less uptake in the liver compared with viruses coated with PEGs of lower molecular weight.…”

Gutless adenovirus: last-generation adenovirus for gene therapy

“…However, innate response, as these recently reported, may be reduced by PEG modification, probably due to lower vector uptake by Kupffer cells in vivo. 77,78 Surprisingly, gutless vectors have also been proved to be very efficient in vaccination, due to their longer duration of expression, their lower antiviral reactivity and their higher levels of transgene protein in dendritic cells compared to the same amount of first-generation Ad vectors. 79 In vivo administration of gutless adenovirus Gutless adenoviruses have been administered in vivo to different tissues in rodents, dogs and also to non-human primates.…”