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Sequestration of adenovirus serotype 5 (Ad5) in liver restricts its use for gene delivery to other target sites in vivo. To date, no studies have systematically assessed the impact of genetic capsid modifications on in vivo tropism in rats, an important preclinical model for many disease types. We evaluated a panel of Ad5 vectors with capsid mutations or pseudotyped with the short fiber from serotype 41 (Ad41s) for infectivity in Wistar Kyoto rats in vitro and systemically in vivo. In vitro studies demonstrated that both coxsackie and adenovirus receptor (CAR) and heparan sulfate proteoglycan (HSPG) binding were predominant predictors of Ad5 tropism. In vivo, neither CAR nor integrin mutations alone affected liver transduction. The HSPG-binding mutation alone moderately reduced rat liver transgene levels by 2-fold (P < 0.05). This was further substantially decreased by additional mutation of CAR binding (95-fold). Combining CAR and integrin mutations reduced transgene levels by >99% (509-fold, P < 0.01), an effect not observed in parallel experiments in mice and highly variable when studied further in an additional two strains of rat. Ad41s mediated very low liver transduction (58-fold lower than AdCTL). Moreover, CAR-binding mutants (KO1-containing) or pseudotyping 41s eliminated hemagglutination of rat and human red blood cells in vitro. This highlights some important potential species and strain differences dictating Ad5 tropism in vivo and identifies vectors that are substantially detargeted from rat liver in vivo.

Adenoviral vectors are commonly used for liver-directed gene therapy following systemic administration owing to their strong propensity for hepatocyte transduction. However, many disease applications would benefit from the delivery of adenoviruses to alternate tissues via this route. Research has thus focused on stripping the virus of native hepatic tropism in conjunction with modifying virus capsid proteins to incorporate novel tropism. Recently, the KO1S* adenovirus serotype 5 fiber mutant, devoid of both coxsackie and adenovirus receptor binding in the fiber knob domain and mutated at the putative heparan sulphate proteoglycan binding site in the fiber shaft, was shown to possess strikingly poor hepatic tropism in mice, rats, and non-human primates. Thus, it is an ideal candidate for retargeting strategies. We therefore assessed the ability of peptide-modified KO1S* fibers to retarget adenovirus. Peptide insertions were well tolerated and virions produced to high titers. However, expected retargeting at the level of transduction was not observed, despite cell-binding studies showing enhanced vector targeting at the cell surface. Cy3 labeling studies showed retarded trafficking of S*-containing fibers. Taken together, our data demonstrates that KO1S* mutant fibers are ineffective for cell retargeting strategies.

Local adenoviral (Ad)-mediated gene transfer to the carotid artery of the stroke-prone spontaneously hypertensive rat (SHRSP) is successful in improving endothelial function. Here we explored the potential of systemic delivery of Ad encoding endothelial nitric oxide synthase (AdeNOS) to prevent elevation of blood pressure in the SHRSP using both nontargeted and vector targeting approaches. Systemic administration of nontargeted AdeNOS failed to modify the rise in blood pressure in SHRSP when administered during the 12th week of age (n = 5, P = 0.088, F = 3.0), an effect likely to result from sequestration of Ad by the liver. Rerouting Ad transduction using a bispecific antibody (anti-ACE/anti-Ad capsid, Fab9B9) that blocks Ad binding to the coxsackie and adenovirus receptor and simultaneously retargets AdeNOS to the angiotensin-converting enzyme resulted in efficient eNOS overexpression in the lung vasculature and a sustained hypotensive effect (n = 5, P = 0.007, F = 7.9). This study highlights the importance of vector targeting to achieve therapeutic gain and represents the first such study in cardiovascular gene therapy.

We have genetically engineered two novel Ads that transduce human EC selectively in vitro, one of which leads to altered Ad biodistribution in vivo. The successful generation of genetically engineered tropism for EC has broad implications for cardiovascular gene therapy. Further modifications to the Ad capsid will be required to improve in vivo biodistribution profiles.

a b s t r a c tWe performed in vivo phage display in the stroke prone spontaneously hypertensive rat, a cardiovascular disease model, and the normotensive Wistar Kyoto rat to identify cardiac targeting peptides, and then assessed each in the context of viral gene delivery. We identified both common and strain-selective peptides, potentially indicating ubiquitous markers and those found selectively in dysfunctional microvasculature of the heart. We show the utility of the peptide, DDTRHWG, for targeted gene delivery in human cells and rats in vivo when cloned into the fiber protein of subgroup D adenovirus 19p. This study therefore identifies cardiac targeting peptides by in vivo phage display and the potential of a candidate peptide for vector targeting strategies.

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