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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Achieving high efficiency, targeted gene delivery with adenoviral vectors is a long-standing goal in the field of clinical gene therapy. To achieve this, platform vectors must combine efficient retargeting strategies with detargeting modifications to ablate native receptor binding (i.e. CAR/integrins/heparan sulfate proteoglycans) and “bridging” interactions. “Bridging” interactions refer to coagulation factor binding, namely coagulation factor X (FX), which bridges hepatocyte transduction in vivo through engagement with surface expressed heparan sulfate proteoglycans (HSPGs). These interactions can contribute to the off-target sequestration of Ad5 in the liver and its characteristic dose-limiting hepatotoxicity, thereby significantly limiting the in vivo targeting efficiency and clinical potential of Ad5-based therapeutics. To date, various approaches to retargeting adenoviruses (Ad) have been described. These include genetic modification strategies to incorporate peptide ligands (within fiber knob domain, fiber shaft, penton base, pIX or hexon), pseudotyping of capsid proteins to include whole fiber substitutions or fiber knob chimeras, pseudotyping with non-human Ad species or with capsid proteins derived from other viral families, hexon hypervariable region (HVR) substitutions and adapter-based conjugation/crosslinking of scFv, growth factors or monoclonal antibodies directed against surface-expressed target antigens. In order to maximize retargeting, strategies which permit detargeting from undesirable interactions between the Ad capsid and components of the circulatory system (e.g. coagulation factors, erythrocytes, pre-existing neutralizing antibodies), can be employed simultaneously. Detargeting can be achieved by genetic ablation of native receptor-binding determinants, ablation of “bridging interactions” such as those which occur between the hexon of Ad5 and coagulation factor X (FX), or alternatively, through the use of polymer-coated “stealth” vectors which avoid these interactions. Simultaneous retargeting and detargeting can be achieved by combining multiple genetic and/or chemical modifications.

Human adenoviruses from multiple species bind to coagulation factor X (FX), yet the importance of this interaction in adenovirus dissemination is unknown. Upon contact with blood, vectors based on adenovirus serotype 5 (Ad5) binds to FX via the hexon protein with nanomolar affinity, leading to selective uptake of the complex into the liver and spleen. The Ad5:FX complex putatively targets heparan sulfate proteoglycans (HSPGs). The aim of this study was to elucidate the specific requirements for Ad5:FX-mediated cellular uptake in this high-affinity pathway, specifically the HSPG receptor requirements as well as the role of penton base-mediated integrin engagement in subsequent internalisation. Removal of HS sidechains by enzymatic digestion or competition with highly-sulfated heparins/heparan sulfates significantly decreased FX-mediated Ad5 cell binding in vitro and ex vivo. Removal of N-linked and, in particular, O-linked sulfate groups significantly attenuated the inhibitory capabilities of heparin, while the chemical inhibition of endogenous HSPG sulfation dose-dependently reduced FX-mediated Ad5 cellular uptake. Unlike native heparin, modified heparins lacking O- or N-linked sulfate groups were unable to inhibit Ad5 accumulation in the liver 1h after intravascular administration of adenovirus. Similar results were observed in vitro using Ad5 vectors possessing mutations ablating CAR- and/or αv integrin binding, demonstrating that attachment of the Ad5:FX complex to the cell surface involves HSPG sulfation. Interestingly, Ad5 vectors ablated for αv integrin binding showed markedly delayed cell entry, highlighting the need for an efficient post-attachment internalisation signal for optimal Ad5 uptake and transport following surface binding mediated through FX. This study therefore integrates the established model of αv integrin-dependent adenoviral infection with the high-affinity FX-mediated pathway. This has important implications for mechanisms that define organ targeting following contact of human adenoviruses with blood.

Excessive vascular smooth muscle cell (SMC) proliferation, migration and extracellular matrix (ECM) synthesis are key events in the development of intimal hyperplasia, a pathophysiological response to acute or chronic sources of vascular damage that can lead to occlusive narrowing of the vessel lumen. Atherosclerosis, the primary cause of coronary artery disease, is characterised by chronic vascular inflammation and dyslipidemia, while revascularisation surgeries such as coronary stenting and bypass grafting represent acute forms of vascular injury. Gene knockouts of transforming growth factor-beta (TGFβ), its receptors and downstream signalling proteins have demonstrated the importance of this pleiotropic cytokine during vasculogenesis and in the maintenance of vascular homeostasis. Dysregulated TGFβ signalling is a hallmark of many vascular diseases, and has been associated with the induction of pathological vascular cell phenotypes, fibrosis and ECM remodelling. Here we present an overview of TGFβ signalling in SMCs, highlighting the ways in which this multifaceted cytokine regulates SMC behaviour and phenotype in cardiovascular diseases driven by intimal hyperplasia.

The development of adenoviral vectors for intravascular delivery will require improvements to their in vivo safety and efficacy. The hypervariable regions of the Ad5 hexon are a target for neutralizing antibodies, but also interact with FX, facilitating hepatocyte transduction. Ad48, a species D adenovirus, does not bind FX and has low seroprevalence. Therefore, it has been suggested that Ad5HVR48(1-7), a hexon-chimeric vector featuring the seven HVRs from Ad48, should display advantageous properties for gene therapy, by evading pre-existing Ad5 immunity and blocking FX interactions. We investigated the in vivo biodistribution of Ad5, Ad5HVR48(1-7) and Ad48 following iv delivery. Ad5HVR48(1-7) displayed reduced hepatocyte transduction and accumulation in Kupffer cells, but triggered a robust pro-inflammatory response, even at relatively low doses of vector. We detected elevated serum transaminases (48h) and increased numbers of peri-portal CD11b+/Gr-1+ cells in the livers of Ad5HVR48(1-7)-treated animals following intravascular, but not intramuscular, delivery. In contrast, Ad48 did not elevate transaminases or result in the accumulation of CD11b+/Gr-1+ cells. Collectively, these findings suggest that substantial hexon modifications can lead to unexpected properties which cannot be predicted from parental viruses. Therefore, refined mutations may be preferential for the successful development of targeted vector systems which require intravascular administration.