CVgenes@target comprises resources that allow progress beyond current standards
Abundant preliminary data are available to each of our three programmes such that we can initiate 6 scientific work packages (WPs) in parallel. Each programme will push the field well beyond the current state-of-the-art.
In Programme 1 (WP1; WP2) we will use world-leading -OMICs platforms as well as in silico approaches to prioritize novel candidate genes for CAD and stroke.
In Programme 2 (WP3; WP4) we will establish in vitro and in vivo models for investigation of pre-selected and novel causal genes and affected pathways and their impact on the development of CAD and stroke.
Programme 3 (WP5; WP6) aims to develop novel assays to monitor the activity of selected pathways and screen libraries to identify small molecules or antibodies that modulate the activity of respective pathways and undertake proof-of-concept studies to test their efficacy against the disease process using both in vitro and in vivo models.
Work-Package 7 deals with all management aspects of the proposal as well as dissemination and Intellectual Property issues.
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The main goal of WP1 is to prioritize genetic risk loci for potential therapeutic intervention and then to assess druggability of protein products of genes. To reach this goal we will utilize the unique resources available in the consortium, apply and optimize the latest computational tools for causal loci and drug target discovery. Specifically, we will reach this goal by identifying and characterizing common and rare variants for CAD and ischemic stroke by 1000 Genomes (1000G) imputation of existing GWAS data sets, as well as on-going analysis of exome chip arrays, and whole-exome sequencing. Furthermore, we will assess, optimize and utilize two- and multi-locus association tests to identify interacting causal loci contributing to both diseases. Moreover, we aim to identify specific tissues and cell- types involved in CAD and ischemic stroke by investigating epigenetic effects of risk alleles.
In this WP we will identify TF (transcription-factor) regulatory gene networks of genome-wide significant CAD and stroke risk loci using co-expression and Bayesian inference algorithms. Identified networks will be further evaluated to determine their enrichments with inherited risk and regulatory elements using the GWA and ENCODE datasets. Next, available datasets (e.g., protein-protein, ChIP-on-chip and DNA methylation datasets) will be integrated to improve network resolution and disease prediction and to assess network activity in atherosclerotic lesions and during atherogenesis. Within the most prioritized networks, novel and established pathways and within these, the most promising druggable targets will be identified.
In this work package, we will use complementary cell model systems and high-throughput analysis to experimentally identify and characterize the molecular and cellular mechanisms through which CAD/stroke associated genetic variants and genes increase cardiovascular risk. We will use SILAC technology to perform quantitative interaction proteomics and identify proteins with allele- specific binding in order to identify functional CAD/stroke variants. Recombinant adeno-associated virus (rAAV) mediated genome editing will be used to precisely knock-in risk variants into cell lines of interest allowing the functional analysis to be performed in a controlled isogenic background and functional consequences of specific variants to be unequivocally linked to gene function and cellular phenotype. High-throughput, systematic RNAi knock-down in appropriate cell types will be used to identify novel genes within CAD-associated loci not previously known to be involved in CAD but which have large cellular effects of known relevance to atherosclerosis when knocked-down. Altogether the outputs of WP3 will increase knowledge of the molecular and cellular mechanisms by which CAD and stroke associated loci mediate their risk as a key step in defining drug targets.
The primary objective of this work package is to demonstrate that the chosen candidate genes affect atherogenesis and -progression in relevant in vivo and in vitro models and that lead compounds developed in WP6 interfere with plaque development in these models. For this work we already have four models in an advanced stage of development (Hdac9-/-, Adamts7-/-, Phactr1- /-, and KIAA1462-/- mice) and we will develop four additional models based on work in WP 1 to 3. Criteria for prioritization will include biological plausibility, prospects for druggability, and fit with the overall strategy of the consortium. These data will be integrated with studies on atherosclerotic phenotypes in humans. By April 2013 GWAS (N=1.700) and methylomics (N=600) data will be available on patients who underwent carotid endarterectomy (AtheroExpress study). We will study allele specific and epigenetic associations with atherosclerotic plaque phenotypes as well as gene, protein and metabolite expression in this and other human material. The outputs of WP4 will provide definitive evidence of the disease relevance of our chosen targets and provide a key step towards the development of novel therapeutic agents.
In this WP we will develop appropriate biological reagents (e.g. cell lines, target and accessory proteins, detection reagents) for HDAC9 and ADAMTS-7 (the 2 precedented targets) and the 2 novel targets. Subsequent to the generation of the biological reagents, we will develop and validate high throughput screening (HTS) compatible assays (typically primary, secondary and counter screens) for all targets and these will be utilized in WP6. In addition, we will develop in silico screening protocols for all targets where appropriate structural information is available. Although P3 and P1 will be the principal initial academic Participants in this WP, other academic Participants will join as appropriate once the two additional targets have been defined.
The main goal of WP6 is to identify lead molecules interfering with pathways leading to CAD and stroke. For this, two approaches will be followed: i) small molecule discovery and lead
optimization program will be initiated and ii) monoclonal antibodies will be generated for the specific targets.
Concerning small molecules, first hit identification and validation will be performed on HDAC 9 and ADAMTS-7, two targets already identified by members of the consortium. Results of precedent work packages will lead to the selection of two further targets, for which also a hit identification and validation campaign will be performed. The campaigns will lead to subsequent lead generation programs which should yield at least 2 compound classes which can be tested in proof-of-concept animal models.
With regard to monoclonal antibodies, mouse monoclonal antibodies against 3 target proteins identified by the consortium will be generated. Batches of monoclonal antibodies will produced and tested for biological activity in assays developed in WP4b. A candidate monoclonal antibody selected for biological and therapeutic activity will be humanized and CHO cell lines producing high levels of the monoclonal antibody will be generated.
WP7 deals with all management aspects of the proposal as well as dissemination and Intellectual Property issues.