Stuart A. Nicklin

Adenovirus Serotype 5 Hexon Mediates Liver Gene Transfer

Adenoviruses are used extensively as gene transfer agents, both experimentally and clinically. However, targeting of liver cells by adenoviruses compromises their potential efficacy. In cell culture, the adenovirus serotype 5 fiber protein engages the coxsackievirus and adenovirus receptor (CAR) to bind cells. Paradoxically, following intravascular delivery, CAR is not used for liver transduction, implicating alternate pathways. Recently, we demonstrated that coagulation factor (F)X directly binds adenovirus leading to liver infection. Here, we show that FX binds to the Ad5 hexon, not fiber, via an interaction between the FX Gla domain and hypervariable regions of the hexon surface. Binding occurs in multiple human adenovirus serotypes. Liver infection by the FX-Ad5 complex is mediated through a heparin-binding exosite in the FX serine protease domain. This study reveals an unanticipated function for hexon in mediating liver gene transfer in vivo.

Genome-wide association study of blood pressure extremes identifies variant near UMOD associated with hypertension

Hypertension is a heritable and major contributor to the global burden of disease. The sum of rare and common genetic variants robustly identified so far explain only 1%–2% of the population variation in BP and hypertension. This suggests the existence of more undiscovered common variants. We conducted a genome-wide association study in 1,621 hypertensive cases and 1,699 controls and follow-up validation analyses in 19,845 cases and 16,541 controls using an extreme case-control design. We identified a locus on chromosome 16 in the 5′ region of Uromodulin (UMOD; rs13333226, combined P value of 3.6×10−11). The minor G allele is associated with a lower risk of hypertension (OR [95%CI]: 0.87 [0.84–0.91]), reduced urinary uromodulin excretion, better renal function; and each copy of the G allele is associated with a 7.7% reduction in risk of CVD events after adjusting for age, sex, BMI, and smoking status (H.R. = 0.923, 95% CI 0.860–0.991; p = 0.027). In a subset of 13,446 individuals with estimated glomerular filtration rate (eGFR) measurements, we show that rs13333226 is independently associated with hypertension (unadjusted for eGFR: 0.89 [0.83–0.96], p = 0.004; after eGFR adjustment: 0.89 [0.83–0.96], p = 0.003). In clinical functional studies, we also consistently show the minor G allele is associated with lower urinary uromodulin excretion. The exclusive expression of uromodulin in the thick portion of the ascending limb of Henle suggests a putative role of this variant in hypertension through an effect on sodium homeostasis. The newly discovered UMOD locus for hypertension has the potential to give new insights into the role of uromodulin in BP regulation and to identify novel drugable targets for reducing cardiovascular risk.

Combined transductional and transcriptional targeting improves the specificity of transgene expression in vivo.

The promise of gene therapy for health care will not be realized until gene delivery systems are capable of achieving efficient, cell-specific gene delivery in vivo. Here we describe an adenoviral system for achieving cell-specific transgene expression in pulmonary endothelium. The combination of transductional targeting to a pulmonary endothelial marker (angiotensin-converting enzyme, ACE) and an endothelial-specific promoter (for vascular endothelial growth factor receptor type 1, flt-1) resulted in a synergistic, 300,000-fold improvement in the selectivity of transgene expression for lung versus the usual site of vector sequestration, the liver. This combined approach should be useful for the design of other gene delivery systems.

Targeted Gene Delivery to Vascular Tissue In Vivo by Tropism-Modified Adeno-Associated Virus Vectors

Background—Gene therapy offers an unprecedented opportunity to treat diverse pathologies. Adeno-associated virus (AAV) is a promising gene delivery vector for cardiovascular disease. However, AAV transduces the liver after systemic administration, reducing its usefulness for therapies targeted at other sites. Because vascular endothelial cells (ECs) are in contact with the bloodstream and are heterogeneous between organs, they represent an ideal target for site-specific delivery of biological agents. Methods and Results—We isolated human venous EC-targeting peptides by phage display and genetically incorporated them into AAV capsids after amino acid 587. Peptide-modified AAVs transduced venous (but not arterial) ECs in vitro, whereas hepatocyte transduction was significantly lower than with native AAV. Intravenous infusion of engineered AAVs into mice produced reduced vector accumulation in liver measured 1 hour and 28 days after injection and delayed blood clearance rates compared with native AAV. Peptide-modified AAVs produced enhanced uptake of virions in the vena cava with selective transgene expression. Retargeting was dose dependent, and coinfusion of either heparin or free competing peptides indicated that uptake was principally independent of native AAV tropism and mediated via the peptide. Conclusions—AAV tropism can be genetically engineered by use of phage display–derived peptides to generate vectors that are selective for the vasculature.

Identification of coagulation factor (F)X binding sites on the adenovirus serotype 5 hexon: effect of mutagenesis on FX interactions and gene transfer

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.

Analysis of Cell-Specific Promoters for Viral Gene Therapy Targeted at the Vascular Endothelium

Abstract—The use of viral vectors for vascular gene therapy targeted at the endothelium is limited by the promiscuous tropism of vectors and nonspecificity of viral promoters, resulting in high-level transgene expression in multiple tissues. To evaluate suitable endothelial cell (EC)–specific promoters for vascular gene therapy, we directly compared the ability of the fms-like tyrosine kinase-1 (FLT-1), intercellular adhesion molecule-2 (ICAM-2), and von Willebrand factor (vWF) promoters to drive EC-restricted transcription after cloning into adenoviral vectors upstream of lac Z. Vastly different expression profiles were observed. Whereas both FLT-1 and ICAM-2 promoters generated transgene expression levels similar to cytomegalovirus in ECs in vitro, vWF expression levels were extremely low. Analysis of non-EC types revealed that ICAM-2 but not FLT-1 evoked leaky transgene expression, thus identifying FLT-1 as the most selective promoter. With an ex vivo human gene therapy model, the FLT-1 promoter demonstrated EC-specific transgene expression in intact human vein but no detectable expression from infected exposed smooth muscle cells in EC-denuded vein. Furthermore, when adenoviruses were systemically administered to mice, the FLT-1 promoter demonstrated extremely low-level gene expression in the liver, the major target organ for adenoviral transduction in vivo. This study highlights the potential of using the FLT-1 promoter for local and systemic human gene therapy in hypertension and its complications.

Tropism-Modification Strategies for Targeted Gene Delivery Using Adenoviral Vectors

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.

Biodistribution and retargeting of FX-binding ablated adenovirus serotype 5 vectors

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 macrophage-depleted 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 FX binding-ablated Ad5 vectors in vitro and in vivo.

Targeting of Adenovirus Serotype 5 (Ad5) and 5/47 Pseudotyped Vectors In Vivo: Fundamental Involvement of Coagulation Factors and Redundancy of CAR Binding by Ad5

ABSTRACT Vitamin K-dependent coagulation factors can promote adenoviral cell transduction in vitro. In vivo, warfarin pretreatment ablates liver targeting of an adenovirus serotype 5 (Ad5) vector deleted of CAR binding capability. Here, we assess in vivo transduction and biodistribution of Ad5 vectors with nonmodified fibers (Ad5) and a serotype 47 fiber-pseudotyped Ad5 (Ad5/47; subgroup D) virus following intravascular injection. Warfarin reduced liver transduction by both viruses. However, no impact on early liver virus accumulation was observed, suggesting no effect on Kupffer cell interactions. Hence, coagulation factors play a pivotal role in selectively mediating liver hepatocyte transduction of Ad5 and Ad5/47 vectors.

Third-generation lentivirus vectors efficiently transduce and phenotypically modify vascular cells: implications for gene therapy

Grafting of saphenous vein (SV) conduits into the arterial circulation triggers a number of adaptive pathological changes characterized by progressive medial thickening, neointima formation and accelerated atheroma. Previous studies have shown that modification of vein graft biology is possible by adenovirus (Ad)-mediated gene transfer, although gene expression is transient. Advancement of vascular gene therapy to the clinic is compromised by the lack of safe and efficient vector systems that provide sustained therapeutic gene delivery to the vasculature. Due to inadequacies of both Ad and adeno-associated virus (AAV) serotype-2 (AAV-2) systems, we have evaluated gene delivery to endothelial cells (ECs) and smooth muscle cells (SMCs) using alternate AAV serotypes and a third-generation vesicular stomatis virus glycoprotein-pseudotyped lentiviral system. Transduction of both primary human SV EC and SMC was lower using all alternate AAV serotypes compared to AAV-2. However, transduction of both cell types by lentivirus was efficient even at clinically relevant exposure times (15 min), was without toxicity and was promoter sensitive. Transduction levels at lower doses were further enhanced with the addition of the surfactant Poloxamer-407 (P-407). Direct comparison with Ad and AAV-2 confirmed the unique potential for this system. Moreover, we constructed and overexpressed the therapeutic gene tissue inhibitor of metalloproteinase-3 (TIMP-3) using lentivirus and demonstrated transgene production comparable to Ad with concomitant blockade of SMC migration and induction of cell death. We have demonstrated for the first time the potential for third-generation lentiviral vectors, but not alternate AAV serotypes, as efficient vascular gene delivery vectors.