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. 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.

In vitro dendritic cell infection by pseudotyped adenoviral vectors does not correlate with their in vivo immunogenicity.

Expression of antigens in dendritic cells (DC) can stimulate protective immunity against both viral infection and tumor growth, making them important targets for gene therapy. In-vitro-generated DC are commonly used in gene delivery studies with the assumption that the results will correlate with in vivo activity. Adenovirus Type 5 (Ad5) vectors have been widely used with DC, but these cells lack the primary receptor (CAR) used by Ad5 and are poorly infected. We investigated the use of Ad5 vector particles pseudotyped with fibers from other Ad serotypes in DC targeting. Several fiber proteins, including those from Ad16 (Subgroup B) and Ad37 (Subgroup D), conferred dramatically increased in vitro infection. Surprisingly, neither dendritic cell infection nor the immune response to an Ad-delivered antigen was improved when the modified viruses were tested in vivo. These results underscore the importance of using appropriate animal models in gene delivery studies.

Adenoviral serotype 5 vectors pseudotyped with fibers from subgroup D show modified tropism in vitro and in vivo.

Adenovirus (Ad5) serotype 5 vectors are commonly used for gene transfer. Preclinical studies have shown that their application to systemic gene delivery, however, is limited by their highly efficient uptake in the liver, principally mediated by receptor-binding sites on the fiber shaft and knob domain. Using Ad to target other sites in vivo requires vectors that lack hepatic tropism. We therefore sought to exploit Ad family diversity to isolate vectors that possessed poor hepatic tropism. We pseudotyped the fibers from Ad16 (subgroup B; Ad5/16), Ad19p (subgroup D; Ad5/19p), and Ad37 (subgroup D; Ad5/37) onto Ad5 capsids and assessed infectivity profiles in vitro in multiple cell types and in vivo in rats. In rat, mouse, and human hepatocytes, Ad5/19p and Ad5/37 both possessed a striking lack of hepatic cell infectivity compared with Ad5. Both vectors were, however, able to transduce human vascular endothelial and smooth muscle cells with efficiencies equal to or greater than that of nonmodified Ad5. We evaluated liver uptake in 12-week-old male rats after intravenous injection. In contrast to a vector with the wild-type Ad5 fiber, Ad5, both Ad5/19p and Ad5/37 produced significantly less virion accumulation (measured at 1 hr and 5 days) and transgene expression in the liver. Thus, Ad5/19p and Ad5/37 may be useful platforms for the development of targeted Ad vectors.

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). The resulting modified viruses were tested in vitro and in vivo for their ability to direct endothelial-specific gene transfer. RESULTS: Peptide insertion was not deleterious to fiber trimerisation or virion maturation. In vitro gene transfer studies using a panel of cell types demonstrated that both peptide-targeted Ad vectors mediated efficient CAR-independent gene transfer to vascular EC compared with non-modified Ads. Neither peptide supported gene delivery to non-EC. Upon systemic injection into mice and subsequent evaluation of transgene expression we failed to observe a reduction in hepatic Ad accumulation but observed a significant elevation in beta-galactosidase in blood vessels with the MSLTTPPAVARP-targeted Ad vector. CONCLUSIONS: 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.

Development of efficient viral vectors selective for vascular smooth muscle cells.

The vascular smooth muscle cell (SMC) is integral to the pathogenesis of neointimal formation associated with late vein graft failure, in-stent restenosis, and transplant arteriopathy. Viral vectors transduce SMC with low efficiency and hence, there is a need for improvement. We aimed to enhance the efficiency and selectivity of gene delivery to human SMC. Targeting ligands were identified using phage display on primary human saphenous vein SMC with linear and cyclic libraries. Two linear peptides, EYHHYNK (EYH) and GETRAPL (GET), were incorporated into the HI loop of adenovirus (Ad) fibers and the capsid protein of adeno-associated virus-2 (AAV-2). Exposure of human venous SMC to EYH-modified (but not the GET-modified) Ad vector resulted in a significant increase in transgene expression levels at short, clinically relevant exposure times. Similarly, the EYH-modified AAV vector resulted in enhanced gene transfer to human venous SMC but not endothelial cells in a time- and dose-dependent manner. The EYH-modified AAV vector also enhanced (up to 70-fold) gene delivery to primary human arterial SMC. Hence, incorporation of EYH into Ad and AAV capsids resulted in a significant and selective enhancement in transduction of SMC and has implications for improving local gene delivery to the vasculature.

Transductional and transcriptional targeting of cancer cells using genetically engineered viral vectors.

Gene delivery vectors, including adenovirus (Ad) and adeno-associated virus (AAV), are inefficient and non-selective for cancer due to low levels of viral receptors with high levels on other tissues, including liver. We tested Ads and AAVs with the SIGYPLP-targeting peptide inserted into virus capsids for transduction in a panel of cancer cells. Six of twelve lines (C8161, PC-3, G-CCM, MKN-45, LnCAP and A549) were transduced, independently of native viral tropism. Furthermore the candidate cancer gene therapy promoter FLT-1 was active in three of these six cell lines. This offers the potential for dual targeting of selected cancer cells.

Flexibility of the adenovirus fiber is required for efficient receptor interaction.

The adenovirus (Ad) fiber protein mediates Ad binding to the coxsackievirus and Ad receptor (CAR) and is thus a major determinant of viral tropism. The fiber contains three domains: an N-terminal tail that anchors the fiber to the viral capsid, a central shaft region of variable length and flexibility, and a C-terminal knob domain that binds to cell receptors. Ad type 37 (Ad37), a subgroup D virus associated with severe ocular infections, is unable to use CAR efficiently to infect host cells, despite containing a CAR binding site in its fiber knob. We hypothesized that the relatively short, inflexible Ad37 fiber protein restricts interactions with CAR at the cell surface. To test this hypothesis, we analyzed the infectivity and binding of recombinant Ad particles containing modified Ad37 or Ad5 fiber proteins. Ad5 particles equipped with a truncated Ad5 fiber or with a chimeric fiber protein comprised of the Ad5 knob fused to the short, rigid Ad37 shaft domain had significantly reduced infectivity and attachment. In contrast, placing the Ad37 knob onto the long, flexible Ad5 shaft allowed CAR-dependent virus infection and cell attachment, demonstrating the importance of the shaft domain in receptor usage. Increasing fiber rigidity by substituting the predicted flexibility modules in the Ad5 shaft with the corresponding regions of the rigid Ad37 fiber dramatically reduced both virus infection and cell attachment. Cryoelectron microscopy (cryo-EM) single-particle analysis demonstrated the increased rigidity of this chimeric fiber. These studies demonstrate that both length and flexibility of the fiber shaft regulate CAR interaction and provide a molecular explanation for the use of alternative receptors by subgroup D Ad with ocular tropism. We present a molecular model for Ad-CAR interactions at the cell surface that explains the significance of fiber flexibility in cell attachment.

In vivo transduction of photoreceptors or ciliary body by intravitreal injection of pseudotyped adenoviral vectors.

Strategies for retargeting adenoviral (Ad) vectors have been developed, but their in vivo efficacy remains to be demonstrated. Gene delivery to specific ocular cell types represents an approach to treating many diseases that cause irreversible blindness. One of these cell types, the photoreceptor (PR), is not infected by standard Ad5-based vectors. We evaluated gene delivery after intraocular injection of Ads pseudotyped with three different fiber proteins and found three distinct patterns of infection. An intravitreally injected Ad5 vector readily infected the iris, corneal endothelium, and ciliary body, while few cells in the retina expressed transgene product. In contrast, an Ad3-pseudotyped virus selectively transduced ciliary body, of interest for treating diseases such as glaucoma. A vector pseudotyped with the fiber protein of Ad37 transduced PRs as well as ciliary body. This finding has potential application to the treatment of retinal degenerative or neovascular diseases. These studies demonstrate cell type-selective gene delivery in vivo with retargeted Ads, provide information about the cellular tropisms of several Ad serotypes, and should lead to improved strategies for preserving vision.