We have identified desmoglein 2 (DSG2) as the primary high-affinity receptor used by adenovirus (Ad) serotypes Ad3, Ad7, Ad11, and Ad14. These serotypes represent important human pathogens causing respiratory tract infections. In epithelial cells, adenovirus binding to DSG2 triggers events reminiscent of epithelial-to-mesenchymal transition, leading to transient opening of intercellular junctions. This improves access to receptors, e.g. CD46 and Her2/neu, that are trapped in intercellular junctions. In addition to complete virions, dodecahedral particles (PtDd), formed by viral penton and fiber in excess during viral replication, can trigger DSG2-mediated opening of intercellular junctions as shown by studies with recombinant Ad3 PtDd. Our findings shed light on adenovirus biology and pathogenesis and have implications for cancer therapy.

Adenoviruses (Ad) are efficient vehicles for gene delivery in vitro and in vivo. Therefore, they are a promising tool in gene therapy, particularly in the treatment of cancer and cardiovascular diseases. However, preclinical and clinical studies undertaken during the last decade have revealed a series of problems that limit both the safety and efficacy of Ad vectors, specifically after intravenous application. Major obstacles to clinical use include innate toxicity and Ad sequestration by nontarget tissues. The factors and mechanisms underlying these processes are poorly understood. The majority of intravenously injected Ad particles are sequestered by the liver, which in turn causes an inflammatory response characterized by acute transaminitis and vascular damage. Here, we describe a novel pathway that is used by Ad for infection of hepatocytes and Kupffer cells upon intravenous virus application in mice. We found that blood factors play a major role in targeting Ad vectors to hepatic cells. We demonstrated that coagulation factor IX and complement component C4-binding protein can bind the Ad fiber knob domain and provide a bridge for virus uptake through cell surface heparan sulfate proteoglycans and low-density lipoprotein receptor-related protein. An Ad vector, Ad5mut, which contained mutations in the fiber knob domain ablating blood factor binding, demonstrated significantly reduced infection of liver cells and liver toxicity in vivo. This study contributes to a better understanding of adenovirus-host interactions for intravenously applied vectors. It also provides a rationale for novel strategies to target adenovirus vector to specific tissues and to reduce virus-associated toxicity after systemic application.

After intravenous administration, adenovirus (Ad) vectors are predominantly sequestered by the liver. Delineating the mechanisms for Ad accumulation in the liver is crucial for a better understanding of Ad clearance and Ad-associated innate toxicity. To help address these issues, in this study, we used Ad vectors with different fiber shaft lengths and either coxsackievirus-Ad receptor (CAR)-interacting Ad serotype 9 (Ad9) or non-CAR-interacting Ad35 fiber knob domains. We analyzed the kinetics of Ad vector accumulation in the liver, uptake into hepatocytes and Kupffer cells, and induction of cytokine expression and release in response to systemic vector application. Immediately after intravenous injection, all Ad vectors accumulated equally efficiently in the liver; however, only genomes of long-shafted Ads were maintained in the liver tissue over time. We found that Kupffer cell uptake of long-shafted Ads was mediated by the fiber knob domain and was CAR independent. The short-shafted Ads were unable to efficiently interact with hepatocellular receptors and were not taken up by Kupffer cells. Moreover, our studies indicated that Kupffer cells were not the major reservoir for the observed accumulation of Ads (used in this study) in the liver within the first 30 min after virus infusion. The lower level of liver cell transduction by short-shafted Ads correlated with a significantly reduced inflammatory anti-Ad response as well as liver damage induced by the systemic administration of these vectors. This study contributes to a better understanding of the biology of systemically applied Ad and will help in designing safer vectors that can efficiently transduce target tissues.

Intravenous (i.v.) delivery of recombinant adenovirus serotype 5 (Ad5) vectors for gene therapy is hindered by safety and efficacy problems. We have discovered a new pathway involved in unspecific Ad5 sequestration and degradation. After i.v. administration, Ad5 rapidly binds to circulating platelets, which causes their activation/aggregation and subsequent entrapment in liver sinusoids. Virus-platelet aggregates are taken up by Kupffer cells and degraded. Ad sequestration in organs can be reduced by platelet depletion prior to vector injection. Identification of this new sequestration mechanism and construction of vectors that avoid it could improve levels of target cell transduction at lower vector doses.For more than a decade, adenovirus serotype 5 (Ad5)-based vectors have been used as intravenous (i.v.) gene transfer vectors. Unfortunately, as a non-blood-borne pathogen, Ad5 has not evolved mechanisms to survive in blood and is rapidly cleared from the circulation, with only a fraction reaching the target tissue (1, 28). Most i.v.-delivered Ad5 is sequestered in the liver, and animal studies indicate that Kupffer cells (KCs) play a major role in this trapping (20,25,38,46,51). Recent studies have shown that liver sequestration is not mediated by the Ad5 receptor, CAR, but involves either a direct (44) or a blood factor (coagulation factors IX and X and complement protein C4BP)-mediated (34,35,40) interaction between the Ad fiber and cellular heparansulfate proteoglycans. While these mechanisms of Ad uptake are less efficient if the Ad5 fiber is replaced with a shorter fiber, such as subspecies B serotype Ad35 (4, 41), the persistence of vectors with the Ad35 fiber in blood does not significantly differ from that of Ad5 (4), indicating that there are other pathways of virus clearance and liver sequestration.Ad rapidly binds to and activates circulating platelets in vivo. To investigate the early kinetics of Ad clearance from blood, we used a previously described quantitative PCR method (15) to analyze the levels of Ad5 in blood cell and serum fractions (Fig. 1A) after tail vein delivery of 10 11 Ad5-cytomegalovirus-green fluorescent protein (43) viral particles (VP) to hCD46Ge transgenic mice (27). All experiments involving animals were conducted in accordance with the institutional guidelines set forth by the University of Washington, and all viruses were free of replication-competent Ad and endotoxin. Assuming that a 25-g mouse holds 1 ml of blood, the blood cell and serum fractions contained 4.18 and 0.85% of the input dose, respectively, at 5 min postdelivery. To identify the cell type(s) associated with Ad5, we injected [ 3 H]thymidine-labeled Ad5 and after 10 min isolated platelets and fractionated blood samples. Over 95% of 3 H-labeled Ad5 was associated with isolated platelet and mixed erythrocyte/platelet fractions (data not shown). Isolated erythrocyte and platelet fractions were fixed in 1/2-strength Karnovsky's fixative for transmission electron microscopy (TEM) analysis. TEM revealed Ad particles...