Recent studies have demonstrated the importance of coagulation factor X (FX) in adenovirus (Ad) serotype 5-mediated liver transduction in vivo. FX binds to the adenovirus hexon hypervariable regions (HVRs). Here, we perform a systematic analysis of FX binding to Ad5 HVRs 5 and 7, identifying domains and amino acids critical for this interaction. We constructed a model of the Ad5-FX interaction using crystallographic and cryo-electron microscopic data to identify contact points. Exchanging Ad5 HVR5 or HVR7 from Ad5 to Ad26 (which does not bind FX) diminished FX binding as analyzed by surface plasmon resonance, gene delivery in vitro, and liver transduction in vivo. Exchanging Ad5-HVR5 for Ad26-HVR5 produced deficient virus maturation. Importantly, defined mutagenesis of just 2 amino acids in Ad5-HVR5 circumvented this and was sufficient to block liver gene transfer.In addition, mutation of 4 amino acids in Ad5-HVR7 or a single mutation at position 451 also blocked FX-mediated effects in vitro and in vivo. We therefore define the regions and amino acids on the Ad5 hexon that bind with high affinity to FX thereby better defining adenovirus infectivity pathways. These vectors may be useful for gene therapy applications where evasion of liver transduction is a prerequisite. (Blood. 2009;114:965-971) IntroductionAdenovirus (Ad)-based vectors are used frequently for preclinical gene delivery and therapy and have been used in more than 25% of gene therapy clinical trials conducted to date. Although adenovirus serotype 5 is the most commonly used serotype, the human and nonhuman adenovirus families are large, and many of these are being exploited in diverse clinical applications, such as cancer gene therapy and vaccination. [1][2][3][4] However, the use of Ad vectors as gene delivery tools has raised several safety concerns. The importance of such issues was highlighted in the recent STEP trial in which patients were vaccinated against human immunodeficiency virus using an Ad5 gene delivery vector. The trial was terminated because the vaccine did not function as expected, but actually increased infection rates in those patients with preexisting antibodies to Ad5. Together with other adverse events in humans transduced with Ad5, 5 this highlights the importance of understanding fundamental aspects of Ad biology.In vitro, the interaction of the Ad5 fiber and the coxsackie and adenovirus receptor (CAR) is the major pathway for Ad cell binding. 6,7 Similarly, engagement with integrins by the penton base protein mediates internalization after cell binding. 8 Although other candidate receptors for Ad5 have emerged since the interaction with CAR was identified, 9,10 the role of these receptors in gene transfer after intravascular gene delivery has not been substantiated.It is well established that Ad5 predominantly transduces rodent liver after intravascular injection, 11 however mutations of the Ad5 fiber and/or penton show limited effects on liver gene transfer mediated by Ad5 (reviewed in Nicklin et al 12 ). Thereafter, it w...

Rationale The pathogenesis of PAH remains unclear. The four microRNAs representing the miR-143 and miR-145 stem loops are genomically clustered. Objective To elucidate the transcriptional regulation of the miR-143/145 cluster, and the role of miR-143 in PAH. Methods and Results We identified the promoter region that regulates miR-143/145 miRNA expression in pulmonary artery smooth muscle cells (PASMCs). We mapped PAH-related signalling pathways, including estrogens receptor (ER), liver X factor/retinoic X receptor (LXR/RXR), TGF-β (Smads), and hypoxia (HRE) that regulated levels of all pri-miR stem loop transcription and resulting miRNA expression. We observed that miR-143-3p is selectively upregulated compared to miR-143-5p during PASMC migration. Modulation of miR-143 in PASMCs significantly altered cell migration and apoptosis. In addition, we found high abundance of miR-143-3p in PASMCs-derived exosomes. Using assays with pulmonary arterial endothelial cells (PAECs) we demonstrated a paracrine pro-migratory and pro-angiogenic effect of miR-143-3p enriched exosomes from PASMC. Quantitative PCR and in situ hybridisation showed elevated expression of miR-143 in calf models of PAH as well as in samples from PAH patients. Moreover, in contrast to our previous findings that had not supported a therapeutic role in vivo, we now demonstrate a protective role for miR-143 in experimental PH in vivo in miR-143−/− and antimiR143-3p-treated mice exposed to chronic hypoxia in both preventative and reversal settings. Conclusions miR-143-3p modulated both cellular and exosome-mediated responses in pulmonary vascular cells, while inhibition of miR-143-3p blocked experimental PH. Taken together these findings confirm an important role for the miR-143/145 cluster in PAH pathobiology.

A major limitation for adenoviral transduction in vivo is the profound liver tropism of adenovirus type 5 (Ad5). Recently, we demonstrated that coagulation factor X (FX) binds to Ad5-hexon protein at high affinity to mediate hepatocyte transduction after intravascular delivery. We developed novel genetically FX-binding ablated Ad5 vectors with lower liver transduction. Here, we demonstrate that FX-binding ablated Ad5 predominantly localize to the liver and spleen 1 hour after injection; however, they had highly reduced liver transduction in both control and macrophagedepleted mice compared with Ad5. At high doses in macrophage-depleted mice, FX-binding ablated vectors transduced the spleen more efficiently than Ad5. Immunohistochemical studies demonstrated transgene colocalization with CD11c ؉ , ER-TR7 ؉ , and MAdCAM-1 ؉ cells in the splenic marginal zone. Systemic inflammatory profiles were broadly similar between FX-binding ablated Ad5 and Ad5 at low and intermediate doses, although higher levels of several inflammatory proteins were observed at the highest dose of FX-binding ablated Ad5. Subsequently, we generated a FX-binding ablated virus containing a high affinity Ad35 fiber that mediated a significant improvement in lung/liver ratio in macrophage-depleted CD46 ؉ mice compared with controls. Therefore, this study documents the biodistribution and reports the retargeting capacity of IntroductionOf the 54 different adenoviral serotypes isolated to date, adenovirus serotype 5 (Ad5) has been the most commonly used vector in gene therapy clinical trials. This is, in part, due to clear advantages over alternate strategies including the relatively easy manipulation of its viral genome and feasible scale-up production to high titers (up to 10 13 viral particles (vp)/mL). Nevertheless, Ad5 presents 2 substantial limitations that have required attention to optimize the use of Ad5 in gene therapy. These include the observation that the majority of the human population has pre-existing neutralizing antibodies against Ad5 1-3 and the profound liver tropism observed for Ad5 after intravascular delivery. 4,5 For this reason, fundamental aspects of Ad5 biology need to be further studied to provide safer and target-specific Ad5 gene therapy vectors. The mechanism of Ad5-mediated gene transfer has now been relatively well characterized. In vitro studies have shown that Ad5 and those Ads from subspecies A, C, D, E, and F may use the coxsackievirus and Ad receptor (CAR) as a primary binding receptor. [6][7][8][9] This interaction occurs via the fiber knob domain with subsequent interaction of the Ad5 penton base with cellular integrins (␣v␤3 and ␣v␤5), mediating capsid internalization. 10,11 Although CAR and integrin-binding ablated mutant Ad vectors show a substantial reduction in transduction in vitro, these vectors still predominantly transduce hepatocytes in vivo after intravascular administration. 12,13 Injection of Ad5 into the bloodstream leads to a complex series of interactions that impact on the resulting biodistri...

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