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Periodic Report Summary 2 - SYNSIGNAL (Synthetic Cellular Signaling Circuits)

Project Context and Objectives:
Cellular signaling systems are crucially important for a broad range of critical health and disease areas and high value industrial applications. Signaling systems are the target for more than half of the medicines marketed by the pharmaceutical industry, and form the main R&D area for the nutrition, flavour and fragrance industries. SynSignal is a multidisciplinary high-tech consortium working in synthetic biology's area of greatest untapped potential, delivering a synthetic biology toolbox and finished products custom designed for major present and future industrial applications of cellular signaling.

Synthetic Cellular Signaling Circuits of interest for SynSignal. Natural cellular signaling cascades are comprised of multiple functional components, with each individual component of the system typically being a protein, or a multiprotein complex. Traditional biochemical and molecular biology methods focused on studying the individual component parts of signaling cascades in isolation, while more modern approaches including systems biology, genomics and proteomics have provided insight into the function of signaling cascades as a whole. Synthetic biology provides a powerful and new perspective - bringing additional methodologies and thought processes to bear- particularly classical engineering disciplines. From a synthetic biology perspective, synthetic cellular signaling circuits are perceived as being analogous to electronic circuits. Each component of the circuit, encoded by a DNA sequence of defined structure and function, is physically interchangeable with compatible modular building blocks of similar or dissimilar function, which makes the system on the whole designable and thus accessible for engineering. The cellular context of the signaling circuit, the "chassis", is considered as being similarly modular and designable. This way of thinking is beginning to provide powerful new tools and methods to understand, and more importantly to control and modulate the complex signaling systems of cells.

Industry and Cellular Signaling. Development times for new products across a broad range of industries that focus on cellular signaling are excessively long, with associated high costs. In addition, the lack of effective screening platforms for a number of high value industrial applications involving signaling has a severely adverse affect on innovation and discovery, impeding the delivery of new products. These deficiencies create an economic bottleneck that makes the search for promising technologies to overcome this bottleneck a high priority. Therefore, design of synthetic cellular signaling circuits is one of the key areas of development in synthetic biology, with a number of high value industrial applications, spread across several key European industry sectors. SynSignal will address this urgent and imposing bottleneck, by providing new and sophisticated synthetic biology tools to overcome the challenges facing signaling-based product development in the pharmaceutical, flavour, fragrance and nutritional industries.

Essential Objectives of SynSignal are:

1. Implement a development program consisting of an iterative cycle of i) Design & Engineering, ii) DNA Assembly and Protein Production, and iii) Testing to accumulate a toolbox of synthetic parts (bio-bricks), cell lines (chassis), and complete signaling circuits.

2. To develop tools for our signaling toolbox with broad combinatorial potential applicable for different types of signaling cascades.

3. To develop whole synthetic signaling pathways that are applicable as screening platforms for creating new medicines in key disease areas, particularly Cancer and Diabetes.

4. To develop synthetic signaling cascades which are transferrable into human cells for the treatment of specific diseases.

5. To develop human signaling pathways are applicable as screening platforms for creating new Flavour, Fragrance and Nutritional ingredients.

6. To generate technologies and intellectual property that is widely disseminated to the broader European SME and large industry community.

7. To identify societal perceptions and concerns about synthetic biology in Europe in order to ensure long-term impact and sustainability of research efforts in the field.

Project Results:
Major achievements are grouped according to thematic work packages (WP)

WP2 (Computer Aided Design): (i) Mechanistic models of the GPCR activation cycle, a synthetic yeast pheromone pathway in mammalian cells (WP5) and a synthetic MAPK/MP1 signalling cascade have been developed. Software packages were developed to be used for development and analysis of pathway models. (ii) EPFL developed a method to characterise the mobility of single GPCR in the plasma membranes at different stages of cellular signalling. (iii) GB developed several assays measuring interactions between proteins using BRET.
WP3 (Taste) produced a cell line expressing TRPC1channel and a chimeric Gα subunit coupling to taste GPCRs. The cells were tested for improved Ca2+ signaling with a wide array of TAS2R constructs. Crosstalk between Ca2+ response and cAMP level was studied. Furthermore, new taste-modulating substances were identified and a majority of mouse Tas2rs were deorphaned. Partner GB has established methods to measure taste receptor-mediated signaling in insect cells via BRET. Partner NUID-UCD has developed generic mathematical model of GPCR activation. Partner EPFL develops methods for the production and analysis of micro vesicles expressing taste pathway.
WP4 (Olfaction) (i) A heterologous expression system was established for reproducible production of functional active ORs. (ii) The combined in-silico and cell-based screening project has been extended to olfactory and non-olfactory GPCRs present in pancreatic and muscle cells. (iii) Synthetic olfactory signalling cacscades have been established to measure signalling reactions between GPCRs and various downstream signalling proteins. (iv) Novel miniaturized bioanalytical methods have been established using mass spectrometry, cryo-electron microscopy, nanopore-based electrical recordings of native vesicles and cell-derived plasma membranes on beads. (v) Generally applicable open source software for designing and building synthetic signalling circuits and models of synthetic signalling cascades have been developed.
WP5 (Cancer) engineered a Galpha chimera that functionally connects yeast growth to CXCR4 signaling. A functional gene expression/luciferase circuit based on the Ste12-VP16 fusion protein has been generated in HEK293 mammalian cells. Transcriptional activation can be regulated through upstream proteins (Dig1 and/or Dig2). A strategy has been set up for the baculovirus-mediated transplantation of the complete yeast mating pathways hooked-up to a cancer-related GPCR (MultiPrime/ I. Berger). Together with WP1, two mathematical models of the yeast pheromone synthetic pathway were built and tested.
WP6 (Diabetes) improved the in vivo imaging platform to assess the effects of ligands and of the implementation of synthetic signalling pathways on beta cell function and replication. Candidate receptors for improved beta cell signalling have been selected, based on vasopressin, taste, and olfactory receptors. Novel ligands targeting receptors OR1Q1 and V1b have been tested and induced insulin release from beta cells. Furthermore, a methodology has been developed to replace endogenous pancreatic islets by islets transplanted into the anterior chamber of the eye, allowing to assess the physiological effects of synthetically engineered beta cells in vivo.
WP7 (Functional antibody fragments): 31 receptors and 50 signaling proteins have been defined as targets. Stable cell lines for expression of 30 receptors have been created. 16 receptors and signaling proteins have been expressed and purified. Camel antibodies for V1b and 5HT2c receptors and ApocIII and Mek1 signaling proteins have been produced. Functional characterization of the anti-V1b and anti-ApocIII has been performed. Further, we have developed a generic assay to probe activation of GPCRs in membrane fractions immobilised on magnetic beads using flow cytometry to monitor the separation of trimeric G proteins coupled with fluorescent antibody.
WP8 (Dissemination & Exploitation) analyzed in detail the public discourse on synthetic biology in seven European countries. These country reports were discussed with a stakeholder committee at the Third Project Meeting. Dissemination activities include press releases, a software, presentations of the consortium at different scientific academies and organizations as well as several big pharma companies. Scientific dissemination activities include numerous conference presentations and several scientific publications. Further, several partners have started with the first exploitation of project results. Partner GB has conducted a FTO analysis.

Potential Impact:
A central objective of SynSignal is to radically improve synthetic biology technologies and to create novel tools for a wide range of applications to discover molecules for health and industrial purposes. The SynSignal collaborative teams have implemented this mission and achieved significant results in the first 18 months of the work program. We have created several synthetic multicomponent heterologous signaling cascades, which to our knowledge are the highest complexity synthetic signaling systems ever produced. We have created, tested, and implemented several different readout strategies that provide unique benefits for molecule discovery from different industrial purposes including pharmaceutical drug development, and flavor and fragrance molecule development. SynSignal particularly focuses on i) designing individual synthetic signaling building blocks, ii) combinatorially assembling them, and iii) testing signaling of the cascade in low background cell lines. The aim is to produce disruptive next generation high-throughput compatible synthetic signaling regulatory circuits which provide uniquely powerful signal to noise profile over state of the art systems.
Notably, the work plan of our consortium answers the call to focus on new technologies that will greatly accelerate discovery in the life sciences. Our joint effort will open entirely new avenues for advancing large-scale European and global efforts that aim at catalyzing and exploiting the emerging field of synthetic biology, and industrial biotechnology. SynSignal tackles and will overcome clear and present challenges and bottlenecks, which impede these fields in Europe and worldwide, to the benefit of industrial and academic R&D, and human health. SynSignal stands for innovation, catalyzed by drawing on the best available in European SMEs and the academic sector for its ambitious endeavor. Our work plan fosters a productive mindset, which fully supports fluid exchange between the academic and private life science sectors, and embraces the entrepreneurial spirit.
SynSignal Impact: The work plan In the Europe 2020 strategy for growth, competitiveness in the future requires first and foremost a “strengthening of the sources of growth in Europe's industrial base“ (Barroso, 2009). The European pharmaceutical and biotech industries face increasingly difficult challenges from countries with significantly lower labor costs. To maintain on European soil highly paid, highly skilled jobs in these industries, we must immediately develop competitive advantages that compensate for our higher labor costs. The vision of SynSignal is to provide European pharmaceutical and industrial biotechnology companies with these competitive advantages through development of innovative technology platforms which change the way we discover and produce novel products. SynSignal‘s high throughput, synthetic biology-based technology platforms will boost both the speed and efficiency at which essential signaling pathways that dictate cellular processes can be modified, modulated and interfered with, thereby reducing cost of product development, and speeding the time of delivery of new products to market.
Our technologies will make accessible, for the first time, cellular signaling and metabolic pathways that are currently impossible to address using established technologies, thereby creating opportunities to develop entirely novel classes of potent and efficient therapeutics, and new and better molecules for the fragrance, flavor and nutritional industries, opening multibillion € markets. Strengthening of these markets with innovative technologies such as those SynSignal will provide is imperative to strengthen and maintain Europe’s leading position in this large and growing sector, thus maximizing the impact of research and innovation on European societies and economies. Synthetic Biology is an emerging technology with the potential to be transformational in a large number of key areas of important socioeconomic challenges in healthcare, nutrition, green technology, and manufacturing. The United States has led the way in synthetic biology as judged by publication output and investment, outcompeting Europe in this viral and emerging field. This can be countered only with highest level technology development by the best European research teams and SMEs. We formed the SynSignal team and created the development plan of this proposal exactly with the objective to close this competitive gap.

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