Investigation and manipulation of adenovirus interactions with host proteins

Adenoviruses are the most commonly used vectors for clinical gene therapy applications, accounting for 24% of all clinical trials to date, the majority of which are based on Ad serotype 5 (Ad5). However, the high prevalence of neutralising antibodies and a range of “off target” interactions result in liver sequestration, hepatic transduction and decreased circulation times. Such interactions include Kupffer cell uptake and binding to blood components such as erythrocytes, platelets, complement and coagulation factors. Recent studies have shown that hepatocyte transduction by Ad5 is mediated by a high-affinity interaction between coagulation factor X (FX) and the Ad5 major capsid protein hexon, with FX bridging the virus to heparan sulphate proteoglycans (HSPGs) on the cell surface. This thesis has focused on gaining a greater understanding of the Ad5:FX pathway and potential strategies for its manipulation. FX, a key component of the blood coagulation system, is a zymogen of a vitamin K-dependent serine protease that is primarily synthesised in the liver and circulates in the bloodstream at 8-10 μg/ml. It is composed of a light chain consisting of a domain rich in γ-carboxylated glutamic acid (Gla) residues, two epidermal growth factor-like domains and a serine protease (SP) heavy chain. The Gla domain of FX binds to the virion by docking in the cup formed by each hexon trimer, whilst the SP domain tethers the Ad5:FX complex to the hepatocyte surface through binding HSPGs. Previously, it was demonstrated that pharmacological blockade of the heparin-binding proexosite (HBPE) in the SP domain prevents FX-mediated cell binding. Here, the specific residues of FX which mediate Ad5 attachment to HSPGs were identified. Employing mutagenesis techniques each of the seven basic residues R93, K96, R125, R165, K169, K236 and R240 that were previously shown to bind heparin, were converted to alanine. This mutated FX was termed “SP mutant”. Stable cell lines were generated to constitutively produce the wild-type and SP mutant rFX protein in the presence of vitamin K. The conditioned media was affinity purified using a FX specific mouse monoclonal antibody 4G3 coupled to sepharose. The rFX proteins were quantified by ELISA, had the predicted molecular weight of 59 kDa and were biologically active, as shown by conversion to FXa in the presence of tissue factor and FVIIa. Surface plasmon resonance (SPR) analysis demonstrated the SP mutations had no effect on FX-specific binding to the Ad5 hexon. However the proexosite mutations ablated FX-mediated Ad5 cell surface binding, internalisation, cytosolic transport and gene transfer as shown by confocal microscopy, qPCR and quantification of transgene expression. Assessing the involvement of rFX with single (R125A) and double (R93A_K96A, R165A_K169A and K236A_R240A) point mutations in the SP domain, indicated the residues exhibit different levels of contribution to Ad5:FX complex binding to HSPGs. The seven SP mutations also inhibited FX-mediated Ad5 binding to mouse liver sections ex vivo. Taken together, this study uncovered that basic residues within the HBPE of FX have a fundamental role in Ad5:FX complex engagement with HSPGs at the surface of target cells. This study contributes to the existing knowledge of the FX-mediated Ad5 transduction pathway. Whilst the classical in vitro CAR-mediated Ad5 infection mechanism has been extensively studied, the post-binding events governing FX-mediated Ad5 intracellular transport and gene expression have not been fully characterised. This study employed a panel of small molecule inhibitors of cellular kinases in vitro to investigate cellular and signalling events occurring during FX-mediated Ad5 infection. Blockade of protein kinase A, p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase significantly hindered efficient Ad5 intracellular trafficking and colocalisation with the microtubule organising centres (MTOC), as shown by confocal microscopy, indicating their fundamental involvement in the pathway. Screening a library of 80 diverse kinase inhibitors for effects on FX-mediated gene transfer, highlighted the compound ER-27319 had the ability to prevent Ad5 transduction in vitro. Previous work reported that ER-27319 acts by binding to the immunoreceptor tyrosine based activation motif (ITAM) of the FcεRI receptor gamma subunit in mast cells to prevent spleen tyrosine kinase (Syk) activation. Here, this compound had no effect on FX-mediated cell binding but substantially disrupted intracellular transport at 3 h in the absence of toxicity. It was postulated that this effect may be due to ER-27319 binding to a viral or cellular ITAM-containing protein involved in viral trafficking. Sequence analysis of the Ad capsid proteome for ITAM-like motifs ((D/E)-x-x-Y-x-x-(L/I)-(xn=6-8)-Y-x-x-(L/I)) identified two motifs on the hexon. However neither followed that reported for the FcεRI gamma subunit, instead of the conventional 6-8 amino acid residues between the two Y-x-x-I/L, the hexon ITAM-like sequences expressed 17 or 22 amino acids. Alternatively the ITAM-containing cellular proteins, ezrin, radixin and moesin (ERM) were investigated. The ERM family are key regulators of the cell cortex, capable of interacting with both the plasma membrane and filamentous actin. However, in the time frame imposed by this study this hypothesis could not be studied in depth, but warrants further research to investigate whether ERM proteins have a novel role in FX-mediated Ad5 intracellular trafficking. A wide range of approaches have been investigated to detarget Ad5 from the liver. In this thesis, a pharmacological strategy to preclude FX-mediated liver gene transfer was implemented. A high throughput screening platform was developed to identify a novel small molecule(s) to manipulate the Ad5:FX infection pathway. In addition to the value of such an agent in the gene therapy setting, it may also have potential to treat life-threatening disseminated Ad infections in immunocompromised individuals. Using a fluorescence and cell-based in vitro high throughput assay 10,240 small molecules were screened using the Pharmacological Diversity Drug-like Set library. Initial screening identified 288 compounds that reduced FX-mediated Ad5 gene transfer by > 75% without causing toxicity. Upon further analysis, three compounds, T5424837, T5550585 and T5660138 were identified as consistently ablating Ad5 transduction both in the absence and presence of FX and all had IC50 values < 5.5 μM. These compounds did not directly interfere with Ad5 binding to FX, instead they primarily caused a post-binding stage block of the Ad infection pathway and all affected optimal virus trafficking to the MTOC, as demonstrated by SPR, flow cytometry and confocal microscopy. The candidate molecules have common structural features and fall into the “one pharmacophore” model. Focused mini-libraries were generated relating to these molecules and structure-activity relationship analysis was performed. In vitro screening of the analogues revealed novel hits with similar or improved activity, thereby further validating the initial hits and pharmacophore model. Six compounds, T5550585, its analogue T5572402, T5660138, its analogue T5660136, T5424837 and its analogue T5677956 were tested in vivo. 10 μM T5660138 substantially reduced Ad5 liver accumulation 48 h post-injection and, in addition to its closely related analogue T5660136, significantly reduced transgene expression at 48 h post-intravenous administration of a high viral dose (1 x 1011 vp/mouse). Therefore, this study identifies novel small molecule inhibitors of circulating Ad5 infection. Through investigation and manipulation of Ad5 interactions with host proteins the work presented here, increases the understanding of the key in vivo Ad5:FX tropism determining pathway. In summary, in this thesis the mechanism of FX-mediated Ad5 complex binding to hepatocytes was dissected and potent inhibitors of this important Ad5 infectivity pathway both in vitro and in vivo were identified. This data may contribute to the optimisation of Ad vectors for gene therapy applications and potentially the advancement of anti-adenoviral drug development

Identification of novel small molecule inhibitors of adenovirus gene transfer using a high throughput screening approach

Due to many favourable attributes adenoviruses (Ads) are the most extensively used vectors for clinical gene therapy applications. However, following intravascular administration, the safety and efficacy of Ad vectors are hampered by the strong hepatic tropism and induction of a potent immune response. Such effects are determined by a range of complex interactions including those with neutralising antibodies, blood cells and factors, as well as binding to native cellular receptors (coxsackie adenovirus receptor (CAR), integrins). Once in the bloodstream, coagulation factor X (FX) has a pivotal role in determining Ad liver transduction and viral immune recognition. Due to difficulties in generating a vector devoid of multiple receptor binding motifs, we hypothesised that a small molecule inhibitor would be of value. Here, a pharmacological approach was implemented to block adenovirus transduction pathways. We developed a high throughput screening (HTS) platform to identify the small molecule inhibitors of FX-mediated Ad5 gene transfer. Using an in vitro fluorescence and cell-based HTS, we evaluated 10,240 small molecules. Following sequential rounds of screening, three compounds, T5424837, T5550585 and T5660138 were identified that ablated FX-mediated Ad5 transduction with low micromolar potency. The candidate molecules possessed common structural features and formed part of the one pharmacophore model. Focused, mini-libraries were generated with structurally related molecules and in vitro screening revealed novel hits with similar or improved efficacy. The compounds did not interfere with Ad5:FX engagement but acted at a subsequent step by blocking efficient intracellular transport of the virus. In vivo, T5660138 and its closely related analogue T5660136 significantly reduced Ad5 liver transgene expression at 48 h post-intravenous administration of a high viral dose (1 × 10&lt;sup&gt;11&lt;/sup&gt; vp/mouse). Therefore, this study identifies novel and potent small molecule inhibitors of the Ad5 transduction which may have applications in the Ad gene therapy setting

Retargeting FX binding-ablated HAdV-5 to vascular cells by inclusion of the RGD-4C peptide in hexon hypervariable region 7 and the HI loop

Recent studies have generated interest in the function of human adenovirus serotype 5 (HAdV-5) hexon:  factor X (FX) binding and subsequent hepatocyte transduction and interaction with the immune system. Here, we retargeted adenovirus serotype 5 vectors, ablated for FX interaction, by replacing amino acids in hexon HVR7 with RGD-4C or inserting the peptide into the fibre HI loop. These genetic modifications in the capsid were compatible with virus assembly, and could efficiently retarget transduction of the vector via the αvβ3/5 integrin-mediated pathway, but did not alter immune recognition by pre-existing human neutralizing anti-HAdV-5 antibodies or by natural antibodies in mouse serum. Thus, FX-binding-ablated HAdV-5 can be retargeted but remain sensitive to immune-mediated attack. These findings further refine HAdV-5-based vectors for human gene therapy and inform future vector development

The androgen receptor CAG repeat polymorphism and modification of breast cancer risk in BRCA1 and BRCA2 mutation carriers.

INTRODUCTION: The androgen receptor (AR) gene exon 1 CAG repeat polymorphism encodes a string of 9-32 glutamines. Women with germline BRCA1 mutations who carry at least one AR allele with 28 or more repeats have been reported to have an earlier age at onset of breast cancer. METHODS: A total of 604 living female Australian and British BRCA1 and/or BRCA2 mutation carriers from 376 families were genotyped for the AR CAG repeat polymorphism. The association between AR genotype and disease risk was assessed using Cox regression. AR genotype was analyzed as a dichotomous covariate using cut-points previously reported to be associated with increased risk among BRCA1 mutation carriers, and as a continuous variable considering smaller allele, larger allele and average allele size. RESULTS: There was no evidence that the AR CAG repeat polymorphism modified disease risk in the 376 BRCA1 or 219 BRCA2 mutation carriers screened successfully. The rate ratio associated with possession of at least one allele with 28 or more CAG repeats was 0.74 (95% confidence interval 0.42-1.29; P = 0.3) for BRCA1 carriers, and 1.12 (95% confidence interval 0.55-2.25; P = 0.8) for BRCA2 carriers. CONCLUSION: The AR exon 1 CAG repeat polymorphism does not appear to have an effect on breast cancer risk in BRCA1 or BRCA2 mutation carriers

Over-expression of Eph and ephrin genes in advanced ovarian cancer: ephrin gene expression correlates with shortened survival

BACKGROUND: Increased expression of Eph receptor tyrosine kinases and their ephrin ligands has been implicated in tumor progression in a number of malignancies. This report describes aberrant expression of these genes in ovarian cancer, the commonest cause of death amongst gynaecological malignancies. METHODS: Eph and ephrin expression was determined using quantitative real time RT-PCR. Correlation of gene expression was measured using Spearman's rho statistic. Survival was analysed using log-rank analysis and (was visualised by) Kaplan-Meier survival curves. RESULTS: Greater than 10 fold over-expression of EphA1 and a more modest over-expression of EphA2 were observed in partially overlapping subsets of tumors. Over-expression of EphA1 strongly correlated (r = 0.801; p < 0.01) with the high affinity ligand ephrin A1. A similar trend was observed between EphA2 and ephrin A1 (r = 0.387; p = 0.06). A striking correlation of both ephrin A1 and ephrin A5 expression with poor survival (r = -0.470; p = 0.02 and r = -0.562; p < 0.01) was observed. Intriguingly, there was no correlation between survival and other clinical parameters or Eph expression. CONCLUSION: These data imply that increased levels of ephrins A1 and A5 in the presence of high expression of Ephs A1 and A2 lead to a more aggressive tumor phenotype. The known functions of Eph/ephrin signalling in cell de-adhesion and movement may explain the observed correlation of ephrin expression with poor prognosis

Genomic, Pathway Network, and Immunologic Features Distinguishing Squamous Carcinomas

This integrated, multiplatform PanCancer Atlas study co-mapped and identified distinguishing molecular features of squamous cell carcinomas (SCCs) from five sites associated with smokin

Pan-Cancer Analysis of lncRNA Regulation Supports Their Targeting of Cancer Genes in Each Tumor Context

Long noncoding RNAs (lncRNAs) are commonly dys-regulated in tumors, but only a handful are known toplay pathophysiological roles in cancer. We inferredlncRNAs that dysregulate cancer pathways, onco-genes, and tumor suppressors (cancer genes) bymodeling their effects on the activity of transcriptionfactors, RNA-binding proteins, and microRNAs in5,185 TCGA tumors and 1,019 ENCODE assays.Our predictions included hundreds of candidateonco- and tumor-suppressor lncRNAs (cancerlncRNAs) whose somatic alterations account for thedysregulation of dozens of cancer genes and path-ways in each of 14 tumor contexts. To demonstrateproof of concept, we showed that perturbations tar-geting OIP5-AS1 (an inferred tumor suppressor) andTUG1 and WT1-AS (inferred onco-lncRNAs) dysre-gulated cancer genes and altered proliferation ofbreast and gynecologic cancer cells. Our analysis in-dicates that, although most lncRNAs are dysregu-lated in a tumor-specific manner, some, includingOIP5-AS1, TUG1, NEAT1, MEG3, and TSIX, synergis-tically dysregulate cancer pathways in multiple tumorcontexts

Spatial Organization and Molecular Correlation of Tumor-Infiltrating Lymphocytes Using Deep Learning on Pathology Images

Beyond sample curation and basic pathologic characterization, the digitized H&E-stained images of TCGA samples remain underutilized. To highlight this resource, we present mappings of tumorinfiltrating lymphocytes (TILs) based on H&E images from 13 TCGA tumor types. These TIL maps are derived through computational staining using a convolutional neural network trained to classify patches of images. Affinity propagation revealed local spatial structure in TIL patterns and correlation with overall survival. TIL map structural patterns were grouped using standard histopathological parameters. These patterns are enriched in particular T cell subpopulations derived from molecular measures. TIL densities and spatial structure were differentially enriched among tumor types, immune subtypes, and tumor molecular subtypes, implying that spatial infiltrate state could reflect particular tumor cell aberration states. Obtaining spatial lymphocytic patterns linked to the rich genomic characterization of TCGA samples demonstrates one use for the TCGA image archives with insights into the tumor-immune microenvironment