Use of in vivo phage display to engineer novel adenoviruses for targeted delivery to the cardiac vasculature.

We 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.

Adenovirus 5 fibers mutated at the putative HSPG-binding site show restricted retargeting with targeting peptides in the HI loop.

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.

Multiple vitamin K-dependent coagulation zymogens promote adenovirus-mediated gene delivery to hepatocytes.

Upon local delivery, adenovirus (Ad) serotype 5 viruses use the coxsackie and Ad receptor (CAR) for cell binding and alpha(v) integrins for internalization. When administered systemically, however, their role in liver tropism is limited because CAR-permissive and mutated viruses show similar biodistribution, a finding recently attributed to blood coagulation factor (F) IX or complement protein C4BP binding to the adenovirus fiber and "bridging" to either low-density lipoprotein receptor-related protein or heparan sulfate proteoglycans. Here, we show that hepatocyte transduction in vitro can be enhanced by the vitamin K-dependent factors FX, protein C, and FVII in addition to FIX but not by prothrombin (FII), FXI, and FXII.

Targeting endothelial cells with adenovirus expressing nitric oxide synthase prevents elevation of blood pressure in stroke-prone spontaneously hypertensive rats.

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.

In vivo biopanning: A methodological approach to identifying novel targeting ligands for delivery of biological agents to the vasculature.

In order for gene delivery to be clinically acceptable, a number of crucial developments need to be made to existing vectors. Significant advances have been made in the identification of novel platform vectors that possess modified tropism to the native vector, directing infectivity away from nontarget tissues such as the liver. In order to fully optimize these detargeted platform vectors, they need to be retargeted toward a chosen, defined site, which will be defined according to the disease studied.

Effect of adenovirus serotype 5 fiber and penton modifications on in vivo tropism in rats.

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 and in vivo characterisation of endothelial cell selective adenoviral vectors.

Background: Both viral and non-viral gene transfer vectors transduce vascular endothelial cells (EC) with low efficiency compared with other cell types such as hepatocytes. Generation of EC-selective vectors would enhance the clinical utility of gene therapy for diverse vascular-targeted applications.

Methods: 12mer peptides derived by in vitro phage display with EC binding specificity [MTPFPTSNEANL (MTP) and MSLTTPPAVARP (MSL)] were inserted at position T542 in the exposed HI loop of the adenovirus (Ad) serotype 5 fiber using overlapping oligonucleotides; in combination with a double point mutation (KO1) to ablate virus : cell binding via the coxsackie-adenovirus receptor (CAR).