Community Research and Development Information Service - CORDIS

Periodic Report Summary 4 - SYBIL (Systems biology for the functional validation of genetic determinants of skeletal diseases)

Project Context and Objectives:
Skeletal diseases range in prevalence from rare monogenic phenotypes (<1/2000 – <1/100,000) to highly prevalent but genetically complex conditions such as osteoarthritis (OA) and osteoporosis (OP). Moreover, many patients with rare skeletal diseases develop OA or OP early in their life when compared to the normal population. In addition, disease-causing determinants in a subset of patients at the severe end of the ‘common disease spectrum’ are likely to be representative of mono- or oligo- genetic conditions and resulting from one or more disease-causing genetic variant with high pathological relevance and known as the ‘rare-among-the-common’ concept.

Despite decades of disease gene identification and characterization there are still very few therapeutic interventions to prevent, halt or modify skeletal disease progression and therefore the identification and application of new and effective treatments requires novel and innovative ‘large-scale research’ that can identify tractable therapeutic targets and biomarkers of genetic skeletal diseases.

In this context, the overarching aim of SYBIL is to perform in-depth functional validation of the genetic determinants of both rare and common skeletal diseases and the epigenetic and age related factors contributing to the morbidity of this diverse group of extremely painful and highly debilitating conditions.

Through an integrated and multidisciplinary approach the main objectives of SYBIL are to develop a comprehensive range of cell and animal models that are representative of the diverse range of gene products and genetic pathways implicated in both rare (RSDs) and common skeletal diseases (CSDs). Validation of these model systems through deep-phenotyping approaches will allow their in-depth characterization at the genetic, molecular and metabolic level by cutting-edge -omics-based technologies. Moreover, analysis of these data through powerful and all encompassing ‘Systems Biology’ and ‘in silico modeling’ approaches will provide valuable and exciting new knowledge of skeletal pathobiology and by the simultaneous modeling of fundamental and complex physiological pathways will identify potential therapeutic targets for further validation. Finally, this large-scale approach will identify disease mechanisms and processes common to different disease subsets, and will provide a unique opportunity to exploit the distinct characteristics of monogenic skeletal diseases by using these genetic disease models to shed light on the more complex and widespread polygenic human skeletal diseases.
Project Results:
Underpinning the entire SYBIL work programme is the identification and prioritization of genetic variants of skeletal diseases (WP1), which will be modelled and studied in-depth throughout the duration of the project. The prioritization of genetic variants is an evolving process as new genes and mutations are identified by SYBIL partners. These numerous genetic variants are representative of the diverse range of gene products and genetic pathways that are fundamental for skeletal development and relevant pathologies.

A carefully selected cohort of variants was defined during the first years of the project (M1-M36) that has now been supplemented by newly identified disease-causing variants of scientific importance. During the 1st reporting period 40 different genes and associated variants were identified as SYBIL priority targets and this has been enhanced by an additional 15 variants during the 2nd reporting period. During the 3rd reporting period a further set of variants was added to the database, some of which are novel variants recently identified (e.g. TCIRG1 and NRP3). The database currently contains 100 unique genetic variants in > 50 different genes.

More importantly, several of these new variants (IFITM5, miR-31, FBN2, SLC10A7 POP1) were immediately selected for mouse model generation in WP3 due to their novelty and scientific importance. This capacity to rapidly develop gold-standard mouse models highlights the advantage of longer-term and large-scale EU funding for delivering new knowledge in a competitive and timely manner.

In the 1st reporting period 60 new cell models were developed by SYBIL; ranging in complexity from relatively simple cell models to more complex patient derived cell lines. We now generated an additional cell bank of more than 40 cell lines during the 3rd and 4th reporting periods. However, notably, SYBIL partners have started to derive the first iPS cells from murine and human peripheral tissues and human peripheral blood. This important technical development will ultimately bring human relevance to cell culture models and allow direct comparisons with mouse models. The 3rd reporting period has seen the successful generation and validation of human iPS cell lines from patients with various GSDs (ODCD, MED, SEMD-JL). These iPS cells have been derived from urine, blood and ligament/fibroblasts and SYBIL partners have adopted a standard protocol. Moreover iPS cells have been differentiated along chondrocyte and osteoblast lineages. In parallel, we have also established cell lines from wild-type zebrafish and Chihuahua model of OI for further characterisation. These novel cell lines have been studied in detail during the 4th reporting period and are shedding new knowledge on the disease mehcanisms of skeletal condtions. Moreover, protocols for the differentiation of human and murine iPSC towards osteoclasts, mesenchymal cell lines including chondrocytes now have been established and validated during the 4th period. Overall, this broad coalition of different cell models for genetic skeletal diseases will allow disease mechanisms to be defined in detail in relevant cell types and also to act as important pre-clinical models for later work packages.

To complement the cell model approach an extensive (and responsive) program of novel animal model generation and validation (WP3) has provided the gold standard in disease modelling for deep-phenotyping and generating definitive -omics-based data for Systems Biology. In the 1st reporting period SYBIL partners validated 4 existing mice models of genetic skeletal diseases and embarked on the generation of 8 novel mouse models, which were prioritized at the start of the project. During the 2nd reporting period SYBIL partners have started to validate and deep-phenotype these 8 novel mouse models (WP4), whilst an additional 6 important mouse models are currently being generated for validation and phenotyping in the 3rd reporting period. To broaden the portfolio of animal models CRISPR/Cas9 mediated genetic modification has been established and will now be applied to the generation of novel zebrafish models. The 3rd reporting period has also seen the application of CRISPR/Cas9 technology for the generation of knockout zebra fish and knock-in mouse models. At the end of the 4th reporting period SYBIL has now generated over 25 mouse models of skeletal disease. Morever, the application of CRISPR/Cas9 technology for the generation of mutant zebrafish and mouse lines resulted in the generation of 12 knockout/mutant zebrafish and mouse lines by several partners in this 4th period..

Deep-phenotyping (WP4) of cell and animal models of skeletal diseases is a major objective of SYBIL and in the 1st reporting period we proceeded with the initial characterization of both existing models and new models, including establishing standard protocols to ensure a common description. These activities have continued in the 2nd reporting period as more cell and animal models enter the SYBIL pipeline including the novel mouse models develop in the 1st reporting period as part of WP3. The 3rd reporting period has seen the in-depth phenotyping of a broad group of 15 mouse models of GSDs that will enter the –Omics part of the SYBIL pipeline of functional validation during the 4th reporting period. In addition there was continued phenotyping of cellular models of GSDs in the 3rd reporting period, which included newly established iPS cell lines that had been differentiated into bone and cartilage cells. During the 4th reporting period, WP4 performed the deep phenotyping of 5 new animal models and of fibroblasts from three new groups of OI patients. Furthermore, the deep-phenotyping of existing animal and cellular models has continued successfully. Results have been published, or submitted for publication and are expected to be published in P5.

The -Omics Knowledge Factory (WP5) generated one proteomic and four transcriptomic profiles in the 1st reporting period. In the 2nd reporting period transcriptomics and proteomics profiles have been generated, or are currently being processed, for several of zebrafish and mouse models developed in the 1st reporting period. Significantly, during the 2nd reporting period a proof-of-concept study has been performed showing that metabolomics allows for reproducible detection of characteristic profiles upon induction of ER-stress. The generation and validation of an extensive portfolio of novel cell and animal models during the 2nd reporting period will now allow extensive –omics analysis to be performed in the 3rd reporting period that will lead to the characterisation of disease mechanisms and identification of new therapeutic targets for further validation. In the 3rd reporting period an increasing number of transcriptomic, proteomic and metabolomic profiles have been derived from both cell and animal models that had been generated and phenotyped in the first two reporting periods. During the 4th reporting period extensive transcriptomic profiling of several mouse models revealed important pathways for the skeletal disorders under investigation as exemplified by the Notch2 model for Hajdu-Cheney syndrome, which has just been published. To better understand how these transcriptional changes are brought about a genome-wide mapping of enhancers relevant for skeletal development was performed. By comparison with profiles for DNA-binding proteins two modes of promoter-enhancer interaction were discovered. This funding period has also seen a large boost in metabolomics analyses of cell culture models and of body fluids of mouse models. A potential metabolic signature for ER-stress has emerged and metabolomics changes in differentiated iPS cells have been detected. Furthermore, in mouse mutants from a model for osteogenesis imperfecta a reproducible metabolite pattern was detected in urine, which may serve as a biomarker.

A systematic analysis of the Omics data has been made possible by the integration of the Galaxy computing platform. Moreover, the SYBIL central data repository has been extended to cover the entire breadth of data, documentation using the SYBIL standard procedures and ontologies. The capturing of this new knowledge will enhance the activities of down stream work packages (WP6-8).

The use of Systems Biology (WP6) to integrate phenotypic, molecular and -omics-derived data is a major strength of SYBIL and initial work in the 1st reporting period focused on developing Systems and in silico models of the major cell types and protein networks involved in skeletal development: osteoblasts, osteocytes and chondrocytes. During the 2nd reporting period transcriptomics data from a mucolipidosis type II mouse model were integrated with the network in order to detect modules of interacting proteins that are differentially expressed in the disease. During the 3rd reporting period new predictions of disease-related proteins and disease modules were generated by UNIMAN using systems biology analysis of omics data for mucolipidosis type II, osteogenesis imperfecta, Desbuquois dysplasia and Xbp1 knock-out, and results were released on the SYBIL knowledgebase. During the 4th reporting period SYBIL partners have enhanced their systems biology analysis pipeline to rapidly and consistently analyse large amounts of transcriptomics data generated through SYBIL, and to allow subsequent integration into network models. The analysis pipeline was also applied to publicly available data making it possible to gain new insights into common mechanisms occurring in different skeletal diseases. A web application was developed to allow interactive searching of the expression responses and altered pathways in these datasets. Reconstruction, testing and refinement have further improved the ModCellTM system. New molecular targeted drugs were added to the ALACRIS’ drug database, which now contains 539 drugs, each described by their targets and affinity metrics, of which 60 drugs may be relevant for predictive modelling of therapeutic effects for skeletal diseases, and which includes a wide range of drugs for repositioning.

Biomarker development (WP7) in the 1st reporting period aimed at identifying potential biomarkers for skeletal diseases using existing model systems. Five candidate proteins underwent preliminary analysis and peptide antibodies were generated for validation in the 2nd reporting period. In the 3rd reporting period significant progress has been made in establishing reliable technologies for biomarker analysis (e.g. GAG sulphation, MRM-MS/MS) and a broad group of new molecules are being investigated and validated (e.g. secreted proteins, vesicles and circulating miRNAs) including GAG composition. The most important aim for the ongoing period is to utilize the data obtained in WP1 to WP6 in order to establish a large number of additional biomarker candidates and to analyze their diagnostic, prognostic and therapeutic value in CSDs and/or RSDs.

Identification of therapeutic targets (WP8) commenced in the 1st reporting period with the validation of previously characterized small molecules that target specific cell functions and pathways involved in skeletal diseases. Both cellular and mouse models were tested with a range of compounds and the deep-phenotyping of these models has been undertaken in the 2nd reporting period. The studies in the 2nd reporting period were performed in animal models of skeletal disorders available in SYBIL or in primary cells transfected with mutant proteins. The effects of small molecules were evaluated in the 2nd reporting period by histology, X-rays, micro-CT, skeletal staining, biochemical studies, expression at the mRNA and protein levels and bone resorption assays. During the 2nd reporting period over 20 different therapeutic approaches have been investigated in a broad range of genetic skeletal diseases. The 3rd reporting period has seen the testing of a broad range of therapeutic approaches, whilst commercialisation of 2nd reporting period deliverables is underway and both patents and orphan drug designation has been granted. Period 4 has seen the repurposing of carbamazepine for MCDS and the granting of orphan drug designation. This will allow a phase 1/II clinical trial starting in early 2018.

In the 1st reporting period SYBIL undertook a diverse range of dissemination activities (WP9) which include providing a regularly updated website and quarterly newsletters. Moreover, partners regularly presented SYBIL-related posters and platform presentations at National and International meetings including a special SYBIL satellite meetings (e.g. MBE 2014). Several SYBIL research studies were also been published in relevant academic journals. During the 2nd reporting period the open access project website was expanded to include educational material and webinars with a detailed description of the SYBIL project. In addition the password protected SYBIL portal was improved and subsequently updated to enable better communication between SYBIL members. A detailed program of training visits and workshops has been developed and implemented throughout the 2nd and 3rd reporting periods. SYBIL members organised and/or participated in events designed specifically for public and patient groups, at which SYBIL activities were presented and discussed, thus promoting cartilage and bone research to the wider communities and stake holders. During the 2nd and 3rd reporting periods SYBIL research was presented at national and international (European, Asian and American) research meetings and numerous articles published in high impact factor journals. The WP9 work is progressing well in the 4th period with all partners actively engaged in dissemination of SYBIL results and studies to the wider audiences, both scientific and lay. The staff and student exchanges have been successful and resulted in significant knowledge transfer leading to high impact publications expected soon. The training programme of SYBIL staff is also successful and will be continued via collaborative meetings and online webinars. Moreover, SYBIL activities resulted in an approved clinical trial and a potential clinical application.

A large integrating project such as SYBIL requires a robust management platform (WP10) and this was in place during the 1st reporting period. Procedures for the efficient coordination and administration of SYBIL, such as the operational committees, are now established and functioning efficiently.
Potential Impact:
During the course of this project, SYBIL will create a comprehensive portfolio of cellular and animal models of skeletal diseases that will be extensively characterised and validated by a broad range of state-of-the-art techniques. These ‘gold standard’ pre-clinical models will be complemented by an exemplary set of new biomarkers of skeletal diseases. This unique and very valuable resource and associated knowledge will have an immediate and major impact on developing and delivering new therapies for patients across the full spectrum of skeletal diseases. Indeed, new knowledge generated by SYBIL will invigorate current research programmes and rapidly open up new research avenues to stimulate the discovery of novel drug candidates and the implementation of new therapies. These SYBIL activities will help reap the benefits of –omics technologies for advances in healthcare; indeed, the ‘rare disease market’ represents an area of enormous unmet medical need, both in the EU and globally.

It is well known that the socio-economic costs of genetic skeletal diseases (like many different rare disease groupings) is a significant part of the ageing-related problems in the European Community and worldwide. Unfortunately, there are currently no therapeutic interventions to prevent, halt or modify disease progression and therefore the generation of new and effective treatments requires novel and innovative research that can identify tractable therapeutic targets and biomarkers of the disease. SYBIL will provide the molecular information that can be synthesised to provide the knowledge base upon which more clinically relevant and cost effective management practices can be developed.
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United Kingdom


Life Sciences
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