Chronic hepatitis B (CHB), caused by hepatitis B virus (HBV), is a leading cause of liver cirrhosis, liver failure and hepatocellular carcinoma (HCC). Of the 350 million chronic HBV carriers worldwide ~20% will die from terminal liver disease. 14 million Europeans live with CHB; the associated mortality rate is ~36,000 deaths/year. Current treatments offer at best control of infection but no cure. Chronicity relies on a unique intracellular viral replication intermediate termed covalently closed circular (ccc) DNA which ensures progeny virus production; its eradication is the prerequisite for a cure. However, how cccDNA is generated from the relaxed circular (RC) DNA genome present in infectious virions is not known, except that the multiple conversion steps required must largely depend on host factors. To identify such host factors as novel antiviral targets, this proposal combines the complementary competences of four HBV-experienced partner labs (Baumert & Brino, France; Bielawski, Poland; Nassal, Germany) using state-of-the-art technologies and most recent virological advances/findings. These include RNAi-based screening and preclinical validation of targets by advanced cell-culture systems, including HBV-infectable cell lines and novel HBV reporter vectors. HBV-relevant host factors will be explored as antiviral targets. For most rapid translation into the clinic, a lab renowned for development of antivirals (Neyts & Dallmeier, Leuven, Belgium) will join as non-funded associate partner to initiate HTS screening for small-compound host factor inhibitors. Target discovery will be complemented by identification of pertinent viral and host factor polymorphisms in clinical HBV patient sample repositories (Baumert & Bielewski), using advanced mass spectrometry (MS). Patient-specific polymorphisms plus clinical data will be merged into a correlative virus/host factor/disease data base to reveal novel biomarkers for progression of liver disease and personalized treatment approaches. To help exploit project-specific R&D results, the partner SME VIRONEXX (Strasbourg, F) will professionally assess market potential via established contacts with major pharma; this feedback will help early-on to focus the program on targets with the highest foreseeable impact for biomarker and drug development. We expect this project to substantially advance basic knowledge of a key issue in HBV pathogenicity and allow identification of new targets that are crucial for the eventual cure of chronic hepatitis B. Moreover, the project contributes to developing and jointly using EU-wide data bases and technology platforms.

Despite the fact they constitute two thirds of the human genome, repetitive sequences are largely ignored. FSHD is an autosomal dominant disorder with a strong epigenetic component. Unlike the majority of genetic diseases, FSHD is not caused by mutation in a protein-coding gene. Instead, the disease is associated with a reduced copy number of the D4Z4 macrosatellite repeat mapping to 4q35. Despite years of intensive research, the molecular pathogenesis of FSHD remains largely unknown. We recently identified DBE-T, a chromatin-associated lncRNA produced preferentially in FSHD patients. DBE-T mediates a Polycomb to Trithorax epigenetic switch at the FSHD locus, driving chromatin remodeling and de-repression of 4q35 protein-coding genes in FSHD patients. In FSHD, up-regulation of multiple 4q35 candidate genes has been reported. Based on this, it has been suggested that FSHD could be considered a continuous gene disease in which the epigenetic alteration of multiple genes contributes to the final outcome. Since DBE-T behaves as a master regulator of the FSHD locus being required to activate all FSHD candidate genes, it is a very intriguing candidate to develop therapeutic approaches aimed at normalizing 4q35 gene expression in FSHD patients. Nevertheless, DBE-T mechanism of action is poorly understood. Here we propose to tackle these issues by addressing the following questions: - Is DBE-T responsible for the enhanced disease penetrance of FSHD in muscle? - How is DBE-T tethered to chromatin? - How does DBE-T activate FSHD candidate genes?

The Fragile X gene (FMR1) is polymorphic for the number of CGG trinucleotide repeats in its 5’-untranslated region. Repeat sizes in the general population range between 5-55 CGG repeats. In Fragile X syndrome repeat expansions exceed 200, silencing expression of FMR1, resulting in intellectual disability. Carriers of the Fragile X premutation have between 55-200 repeats in the FMR1 gene and are at risk for developing Fragile X-associated tremor/ataxia syndrome (FXTAS). FXTAS is a late onset neurodegenerative disorder causing tremor, ataxia, brain pathology, cognitive loss, dementia and early death in some individuals. The proposed pathological mechanism is a toxic RNA gain-of-function model in which mRNAs containing expanded CGG repeats accumulate in neuronal nuclear aggregates. These RNA aggregates sequester specific RNA-binding proteins, thus impairing their normal cellular functions and ultimately resulting in neuronal death. Currently, no treatment exists for FXTAS. As the potential molecular target is well defined (i.e. the mutant FMR1 mRNA), FXTAS is highly amenable to the development of gene targeting therapy. Therefore, the primary objective of this proposal is (1) to establish critical developmental periods when disease might be halted or reversed and (2) to identify pharmacological and molecular compounds to alleviate expanded CGG-induced toxicity. Collectively, the partners of this multidisciplinary consortium have excellent in vivo and in vitro models of FXTAS, valuable resources and state-of-the-art and emerging technologies available.