Adenovirus Toxicity and Tropism In Vivo: Not as Simple as A,B,C (or D,E,F)
Authors: Andrew H Baker
Journal: Molecular Therapy (2007)

Intravenous delivery of adenoviruses is appealing for several clinical applications, such as the delivery of therapeutic genes to disseminated metastases and the targeting of selected diseased organs and tissues. However, such a mode of delivery presents an important set of scientific and regulatory issues in the translational setting. Difficulties relate to poor knowledge of adenovirus receptor usage in vivo, as well as the efficacy and safety of the proposed gene medicine. Much of this is unpredictable, particularly in vivo. Why is it that we still understand relatively little about the mechanisms that drive adenoviral tropism following this delivery route? Even for adenovirus 5 (Ad5), the most commonly used adenovirus serotype thus far, there is still a substantial lack of knowledge relating to its in vivo infectivity mechanisms when delivered directly into the bloodstream (for a recent review, see Baker et al. 1Baker AH Mcvey JH Waddington SN Di Paolo NC Shayakhmetov DM The influence of blood on in vivo adenovirus bio-distribution and transduction.Mol Ther. 2007; 15: 1410-1416Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). In this issue of Molecular Therapy, Stone and colleagues begin to address this issue by studying the biodistribution, blood levels, blood cell interactions, and toxicity of several serotypes (Ad3, Ad4, Ad5, Ad11p, Ad35, and Ad41) from different subgroups following intravenous injection into CD46-transgenic mice.2Stone D Liu Y Li Z-Y Tuve S Strauss R Lieber A Comparison of adenoviruses from species B, C, E, and F after intravenous delivery.Mol Ther. 2007; 15: 2146-2153Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar Gene therapists have pursued alternative adenoviruses for in vivo gene delivery, including relatively simple fiber-swapping strategies as well as development of vectors based entirely on non-Ad5 serotypes (both human and nonhuman). This is driven principally by the previously observed inadequacies of Ad5-mediated gene transfer, including the high rates of neutralizing antibodies in the human population,3Vogels R Zuijeest D Rijnsoever RV Hartkoorn E Damen I Bethune M-PD et al.Replication-deficient human adenovirus type 35 vectors for gene transfer and vaccination: efficient human cell infection and bypass of preexisting adenovirus immunity.J Virol. 2003; 77: 8263-8271Crossref PubMed Scopus (405) Google Scholar toxicity induced by high viral loads,4Raper SE Chirmule N Lee FS Wivel NA Bagg A Gao G-P et al.Fatal systemic inflammatory response syndrome in an ornithine transcarbamylase deficient patient following adenoviral gene transfer.Mol Genet Metab. 2003; 80: 148-158Abstract Full Text Full Text PDF PubMed Scopus (1179) Google Scholar and the substantial liver and spleen accumulation observed following intravascular injection.5Huard J Lochmuller H Ascadi G Jani A Massie B Karpati G The route of administration is a major determinant of the transduction efficiency of rat tissues by adenoviral recombinants.Gene Ther. 1995; 107: 107-115Google Scholar Potential benefits using many of the alternative serotypes of adenovirus relate specifically to vaccination strategies and cancer gene therapy. The latter ideally requires intravenous vector delivery with “built-in” homing to diseased cells while (ideally) sparing gene transfer to non-target tissue. This is a very difficult task with available vectors without extensive re-engineering to improve target selectivity. However, it is important to optimize (as much as possible) each vector system to achieve target-cell transduction while limiting potential off-target side effects. Perhaps surprisingly, a relatively unexplored application of alternative serotypes is for applications involving the intravascular routing of gene delivery. Although this has been explored in depth for some subgroup B viruses, particularly using fiber-swapping strategies, relatively little is known about many full serotypes, with the exception of Ad35.6Sakurai FK Koizumi N Inoue N Okabe M Yamaguchi T Hayakawa T et al.Adenovirus serotype 35 vector-mediated transduction into human CD46-transgenic mice.Gene Ther. 2006; 13: 1118-1126Crossref PubMed Scopus (33) Google Scholar Are biodistributions and tropism very different from Ad5? Are they less toxic than Ad5? CD46-transgenic mice are used in the new study, because they express the cognate receptor for many subgroup B adenoviruses in a profile similar to that in humans, thus allowing the correct comparisons between serotypes from alternative subgroups of adenovirus, since all envisaged receptors will be expressed. Although this is a relatively simple screening approach aimed at identifying potentially useful new vectors, several observations are worth highlighting. First and foremost, some serotypes gave rise to unexpected high-grade toxicity, with Ad3 and Ad4 both evoking significant death rates (>50%) in recipient mice (Ad11 also showed 25% death). For Ad5, mice are relatively resistant to fatal toxicity and can tolerate large input titers. Moreover, for Ad5 significant species differences have been observed.7Cichon G Boeckh-Herwig S Kuemin D Hoffmann C Schmidt HH Wehnes E et al.Titer determination of Ad5 in blood: a cautionary note.Gene Ther. 2003; 10: 1012-1017Crossref PubMed Scopus (35) Google Scholar Perhaps the same is true for different serotypes, but the nature of this may be entirely unpredictable. This is critically important in a translational setting, because the definitive mechanism(s) of toxicity may or may not differ among serotypes. What defines the striking toxicity of Ad3 and Ad4 in particular is unknown, but it is acute and profound. Second, despite similarities in cell transduction in vitro between serotypes using CD46 (Ad35 and Ad11), upon injection into the bloodstream clear differences in in vivo biodistribution are observed. Notably, accumulation in blood cells and serum is high for Ad11 but very low for Ad35. Unfortunately, definitive clearance curves are not presented, but the analysis at 10 min after infusion does highlight large differences between these viruses. Ad4 and Ad11 were present at the highest levels. Both these viruses also show acute toxicity. Moreover, Ad5, Ad5/11, and Ad5/35 showed identical serum and blood cell levels, strongly suggesting that fiber-independent mechanism(s) underpin these observations. Likewise, although liver accumulation for Ad11 and Ad35 was similar, accumulation in lung and spleen was marked for Ad35 but low for Ad11. What governs these substantial differences in vector targeting is important to understand for both clinical translation and additional vector engineering strategies. What is clear is that, despite these differences in blood levels and organ accumulation, all viruses induce substantial release of proinflammatory cytokines and chemokines. Ad3, 4, 5, 11, 35, and 41 all evoke increased interleukin-6, monocyte chemotactic protein-1, and interferon-γ concentrations. Indeed, for interleukin-6, Ad5 showed the weakest induction at the equivalent dose. It is unclear why Ad5/35 was not included in this part of the analysis. This is unfortunate, because this particular capsid configuration is being developed by the same researchers for cancer gene therapy in humans. Hepatic and splenic co-localization with platelets was evident for all viruses tested, again suggesting generic mechanisms governing such processes. Furthermore, platelet activation and D-dimer formation were induced by all adenoviruses. Overall, therefore, multiple common phenotypes are observed for all viruses, yet some unpredicted differences relating to tropism are also evident. This suggests the need for further work to pinpoint the generic mechanisms that govern common phenotypes and the capsid components that define the largely unpredicted differences observed in tropism (especially Ad11 vs. Ad35). Although more than 50 human serotypes have been isolated, the potential of these, as well as some nonhuman serotypes, remains to be investigated further. There remain significant scope and potential for exploiting adenovirus vectors based on new serotypes; perhaps viruses from the larger subgroup D hold potential. However, their application to in vivo gene delivery via bloodstream injection will not necessarily solely involve the utilization of receptors identified by in vitro techniques—in that in vivo tropism seems generally far more complex. Despite this potential, it is obvious that high doses of any serotype adenovirus via this route should be used with caution because of substantial toxicological consequences. This study, therefore, provides an initial look into some alternative serotypes following intravascular injection. It shows that switching to alternatives from Ad5 will not necessarily reap reward in terms of acute toxicity, cytokine induction, or indeed preferred biodistribution and transgene delivery. Further work is required to both improve knowledge of viral components that mediate toxicity and develop strategies to prevent this. Such strategies may be generically relevant. Moreover, we still understand relatively little about blood cell and serum interactions of adenovirus vectors and the mechanisms that govern tropism in vivo. There is a great deal still to be learned.