NIHRDataInsights/HRCSData huggingface dataset:
Title: Deciphering the role of p63 in secondary palate development using systems biology
Funder: Medical Research Council

Abstract: While the descriptive embryology of secondary palate development is well-established, the underlying molecular mechanisms are poorly characterised. Although the transcription factor p63 plays a central role in driving palate development, the gene regulatory networks in which p63 functions remain unknown. The behaviour of these complex networks cannot be predicted by simple intuitive approaches but requires sophisticated inference methods applied at a systems-level to informative datasets. This project will be completed under three objectives. In Objective 1, we will infer the direct, secondary, and downstream targets of p63 activation genome-wide by applying a model-based inference approach to ChIP-seq and RNA-seq time-course datasets. The resulting in silico network predictions will generate experimentally testable hypotheses that will be dissected using a combination of ChIP-qPCR, Nanostring technology, RT-qPCR, in situ hybridisation, and luciferase reporter assays. We will use network predictions to prioritise these experiments with the goal of validating the network links and accelerating the generation of experimental evidence for new genes and pathways involved in palatal development. In an iterative process, these experimental data will feed back into the models to refine the network predictions further. In Objective 2, we will determine the role of p63 during palatal fusion. We will analyse an existing transgenic mouse model which exhibits cleft palate as a result of over-expression of p63 in the palatal epithelia using a combination of RNA-seq, Nanostring technology, and in situ hybridisation. These experiments will be informed by the networks defined in Objective 1. In Objective 3, we will ensure that the data are fully reproducible, improve collaborative working, and make our new methodology widely available, by publishing the full data analysis pipeline as a Galaxy workflow, from raw data processing through to network inference and visualisation.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;2.5 RESEARCH DESIGN AND METHODOLOGIES (AETIOLOGY)
Health categories: ORAL AND GASTROINTESTINAL
Title: Modelling therapies for surgical adhesions
Funder: Medical Research Council

Abstract: Surgical adhesions can cause severe and chronic abdominal pain and life-threatening intestinal obstruction. Although they constitute an enormous socioeconomic burden, there are no effective preventative strategies. Reduced lysis of a post-surgical fibrin-rich clot is an important trigger in the initiation of adhesion development. Critically, a lack of understanding of the aberrant molecular and cellular mechanisms driving mature adhesion formation beyond this first step precludes the rational design of novel biological therapies. Therefore, we will address the following related hypotheses. 1. Aberrant biological signalling pathways mediate key steps in adhesion formation. 2. Peritoneal mesothelial cells contribute to adhesions via epithelial-mesenchymal transition and 3. Inhibiting these key aberrant biological events will prevent adhesion formation. We will combine surgical and transgenic strategies in mice with human culture models to successfully address these questions. First, we will replicate the human clinical scenario in a robust mouse model of surgical adhesion formation. Using Wt1CreERT2;Rosa26mTmG mice will allow us, for the first time, to lineage trace mesothelial cells during adhesion formation. We will define aberrant signalling pathways using biochemical techniques, and localise them to cell populations within the forming scar by immunohistology. Consequently, we will use specific reagents to block adhesion formation in the same in vivo model. In parallel, we will establish monolayer and 3D human cell culture models in which mesothelial cells are triggered to form an adhesion by adding fibrin clot. With these systems, we will establish whether the same biological therapies tested in mice can block human adhesion formation ex vivo. Our experimental strategy will not only illuminate the pathobiology of adhesion formation, but also pave the way for Phase 1 trials of novel biological therapies to prevent adhesions forming in humans.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;5.2 CELLULAR AND GENE THERAPIES
Health categories: ORAL AND GASTROINTESTINAL
Title: Title: Understanding the molecular mechanisms of hyperinsulinaemic hypoglycaemia and developing novel therapies
Funder: Medical Research Council

Abstract: Hyperinsulinaemic hypoglycaemia (HH) is a major cause of recurrent and persistent hypoglycaemia in the neonatal, infancy and childhood periods. So far mutations in ten different genes have been described which lead to HH. The commonest cause is recessive inactivating mutations in ABCC8 and KCNJ11 (which encode the two subunits of the ATP-sensitive potassium (KATP) channels) in beta-cells. In children with medically unresponsive congenital HH mutations in the known genes are found in about 90% of patients. In contrast in children who respond to therapy (diazoxide), mutations in all these genes are only found in about 20% of patients. Thus the aetiology in the vast majority of patients with medically responsive congenital HH is not yet known. We have recruited 1801 patients with HH from 67 countries. 1050 have congenital HH and 1413 were diagnosed in the first 6 months of life. A genetic cause of HH has been found in 43% of patients. We have also described a novel treatment for patients with diffuse HH. The objectives of this study are to use the state of the art techniques in molecular biology (exome and whole genome sequencing) to try and unravel the molecular basis of congenital HH in those patients where no genetic aetiology has been found and to develop novel medical therapies for children with diffuse and focal HH. We have generated a significant amount of preliminary data on some of these rare HH with complex phenotypes and potentially identified novel causes of HH. We will aim to continue the recruitment and phenotyping of patients with HH. All patients will have genetic testing for known causes of HH (SureSelect capture design now includes all known HH genes (ABCC8, KCNJ11, GCK, HNF4A, HNF1A GLUD1, HADH, SLC16A1, INSR, UCP2, HK1 and CACNA1D)) and if no cause if found then exome/genome sequencing will be undertaken to identify novel causes of HH.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: ORAL AND GASTROINTESTINAL
Title: How Paneth cells become a site for intestinal inflammation
Funder: Medical Research Council

Abstract: There is an emerging body of literature highlighting the role of Paneth cells in the pathogenesis of Crohn's Disease (CD). Paneth cells are specialised intestinal epithelial cells (IEC) that are situated at the interface between the intestinal microbiota and our immune system. Recent work has revealed a compensatory role for autophagy in restraining endosplasmic reticulum (ER) stress in IEC. Hypomorphic autophagy in the setting of unabated ER stress within Paneth cells results in spontaneous ileitis that phenocopies human ileal CD. This mouse model may provide the first plausible mechanism of how genetic autophagy defects may lead to ileal CD, affording us an excellent opportunity to investigate the biological mechanisms that link a major genetic risk factor of CD with a specific disease phenotype. This research proposal seeks to address three key questions: 1. What are the inflammatory mechanisms that genetically autophagy-deficient, ER-stressed Paneth cells engage? 2. What is the activation state of bona fide mucosal immune cells in this model of ileal Crohn's disease? 3. Can Crohn's disease patients be stratified based on this disease mechanism? This research proposes flow assisted cytometric sorting of Paneth cells with subsequent transcriptomic and proteomic analysis allowing delineation of the inflammatory mechanisms engaged. Using multi-colour flow cytometric analysis we aim to characterise intraepithelial and lamina propria immune cells and determine their activation state. Using the data deduced from the murine model system, biopsies from patients with Crohn's disease with the same genetic risk alleles will be investigated. The aim is to recapitulate these observations in human samples with the view to demonstrating that patients with ileal CD can be stratified based on genotype, Paneth cell phenotype and markers of activation of mucosal immune cells.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: ORAL AND GASTROINTESTINAL
Title: Stat2 is a therapeutic target in liver inflammation
Funder: Medical Research Council

Abstract: The number of patients dying from liver disease is rising faster than any other cause of illness or death in the UK, with mortality rates increasing 400% since 1970. Inflammation is a key early event and is common to the broad range of causes of liver disease. However, there are currently no drugs licensed for use in liver disease that target inflammation. The PI's recent work demonstrates a novel and unexpected role for Stat2 in governing outcome from inflammation caused by activation of pattern recognition receptors. Preliminary, unpublished data strongly indicate a role for Stat2 in liver inflammation. STAT2 is upregulated in human liver disease and Stat2 loss protects from acute and chronic inflammation caused by carbon tetrachloride in mouse models. The overall aim of this project is to devise therapeutic strategies based on the mechanisms of Stat2-mediated liver injury, using histology, biochemistry and cytokine/chemokine production as endpoints. Our objectives are 1.To determine the tissue compartment and cell type in which Stat2 acts as an inflammatory mediator. The effects of Stat2 deficiency in circulating versus stromal compartments will be assessed in bone-marrow chimera. The main cell types in which Stat2 is expressed will be identified using double-labelled immunohistochemistry and targeted in conditional (floxed) Stat2 knockouts. The effect of Stat2 deficiency on the composition of the host microbiome and its impact on liver injury will be assessed. 2.To determine the downstream mediators and signaling mechanisms for Stat2-mediated liver injury. Based on preliminary data, we will test 3 hypotheses: (a) Stat2 acts as a type I interferon signaling molecule, (b) Stat2 modulates NF-kB-mediated cytokine production and (c) Stat2 modulates cellular migration into injured liver tissue. 3.To devise therapeutic strategies to treat hepatic inflammation based on silencing Stat2 or its the downstream targets using nanoparticle-based siRNA technology.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: ORAL AND GASTROINTESTINAL
Title: MICA: Targeted Regulatory T Cell Therapy for Inflammatory Bowel Disease
Funder: Medical Research Council

Abstract: Crohn's disease (CD) is a chronic, immune-mediated inflammatory bowel disease (IBD) with no known cure, resulting in significant morbidity. Goals of therapy include resolution of symptoms and mucosal healing. Many patients have sub-optimal responses to currently available therapies. This represents a significant unmet medical need. "Thymus-derived" regulatory T cells (tTreg) are effective in modulating immune responses in many pre-clinical models of IBD. Our recent data shows that the starting population for tTreg expansion from the peripheral blood (PB) of CD patients has a critical effect on the phenotype of the expanded cell population. In CD patients, tTregs expanded from a highly pure FACS-sorted "naïve" CD4+CD25hiCD127loCD45RA+ tTreg precursor population have substantial advantages over tTregs expanded from FACS-sorted CD4+CD25hiCD127loCD45RA-, or MACS-enriched CD8-CD25+ precursor populations. These include: 1. Median 98.2% (IQR 97.1-98.4%, n=12) CD4+CD25hiCD127lo purity after 24d expansion, allowing precise definition of each clinical preparation, and increased in vitro suppressive ability in comparison to the other cell preparations described above. 2. 100% FOXP3 TSDR demethylation and reduced Th17 plasticity in vitro, in comparison to other cell preparations, reducing the risk of in vivo conversion to an effector phenotype. 3. A demonstrated ability to traffic to human intestine in vivo, in a humanised mouse model. 4. A demonstrated ability to suppress activation of mucosal T cells in vitro, obtained from inflamed CD intestinal resections. This proposal addresses this unmet need by translating our pre-clinical and patient studies into a clinical-grade GMP flow cytometric sorting solution for the enrichment of highly pure CD45RA+ tTregs from CD patients with GMP cell expansion. This builds on our track record in GMP-Treg manufacture for transplantation and will enable the pivotal Phase Ib/IIa trial of autologous tTregs (TR004) for treatment of CD.


Research activity codes: 6.2 CELLULAR AND GENE THERAPIES
Health categories: ORAL AND GASTROINTESTINAL
Title: The Role of Tissue-resident Hepatic Macrophages in the Resolution of Chronic Liver Injury
Funder: Medical Research Council

Abstract: Hepatic macrophages (MO) play a key role in liver fibrosis resolution. In order to target this therapeutically, a fuller understanding of MO heterogeneity and function is required. The role of embryologically-derived resident hepatic macrophages (RMO; Kupffer cells) in fibrosis resolution has not been defined. Preliminary data demonstrate that during resolution from murine chronic liver injury, hepatic RMO show changes in phenotype, topography and ontogeny to a mixed population of embryologically- (ED) and monocyte-derived (MD) RMO. Aims: 1. To determine the ontogeny of RMO following chronic liver injury 2. To determine the function of RMO during liver fibrosis resolution 3. To perform a comparative systems biology analysis of murine and human hepatic macrophages 4. To analyse the hepatic RMO topographical niche during hepatic fibrosis resolution Methodology: Novel Kupffer cell-specific cre transgenic mice will be used to fate map or selectively deplete hepatic EDRMO during liver fibrosis resolution. A haematopoietic reconstitution mouse model will be used to fate map or deplete hepatic MDMO during liver fibrosis resolution. Sequential intravital microscopy via an abdominal imaging window and laser capture microdissection will be used to characterise RMO motility, fate and topography. Murine and human hepatic RMO subpopulations will be isolated by FACS, with the transcriptome assessed and subjected to systems-biology based analysis. Scientific and Medical Opportunities: By completing this research I will define the role of hepatic RMO in the resolution of chronic liver injury for the first time. Furthermore, by linking functional murine data to the phenotype of human hepatic macrophages using an unbiased systems-biology based approach, I hope to identify conserved pathways which can be targeted therapeutically to modulate macrophage function in vivo, promote liver fibrosis resolution and improve clinical outcomes for patients with cirrhosis.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: ORAL AND GASTROINTESTINAL
Title: The role of mitochondrial Ca2+ uniporter in initiation and development of acute pancreatitis
Funder: Medical Research Council

Abstract: Acute pancreatitis is a frequent, debilitating and sometimes fatal disease. The incidence of acute pancreatitis is rising sharply but there is no specific treatment against this condition. During the last few years a number of research groups (including our own) have identified mitochondrial damage as an important contributing factor to the initiation of acute pancreatitis. We hypothesise that the mechanism of the mitochondrial damage in conditions of acute pancreatitis involves Ca2+ overload of mitochondria in pancreatic acinar cells, which develops due to Ca2+ entry via the mitochondrial Ca2+ uniporter (MCU). In this study we will aim to identify the role of MCU in acute pancreatitis and characterise specific cellular mechanisms that determine this role. In particular, using MCU knockout animals and animal models of acute pancreatitis we will determine if MCU is important for the development of this disease. Standard procedures of histological and biochemical characterisation of acute pancreatitis will be used in these experiments. To determine the putative cellular mechanism of MCU contribution to acute pancreatitis we will characterise its role in mitochondrial Ca2+ entry, bioenergetics, vacuolisation, trypsinogen activation, apoptosis and necrosis of pancreatic acinar cells. We will combine advanced video imaging, electron microscopy and genetics approaches with molecular biology techniques to attain these objectives. We will also test if MCU inhibitors could protect pancreatic acinar cells against damage inflicted by the inducers of acute pancreatitis. The study will involve close collaboration with NIHR Liverpool Pancreas Biomedical Research Unit (NIHR PBRU). In the framework of this collaboration the outcome of our experiments will be shared with clinicians, drug safety researchers and medicinal chemists associated with NIHR PBRU. We anticipate that the results of our investigation will contribute to the translational efforts of this unit.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: ORAL AND GASTROINTESTINAL
Title: Exploring host-gut microbial genetic and immune interactions using twins
Funder: Medical Research Council

Abstract: Human microbiome research is one of the most exciting areas of modern medicine given our ability to manipulate microbes. While studies have found microbial abnormalities in many traits and chronic diseases, these are often inconsistent in the type of microbes implicated. Limitations and biases of current approaches include the fact that case-control studies fail to account for genetic differences in microbiome populations which we have shown are important. Twins are an ideal way to account for genetics. In addition, virtually all studies rely on detecting microbes from the stool sample rather from the gut itself with major assumptions about the faecal sample matching the contents of the lower ileum and colon. Few studies have directly compared faecal and gut wall microbes in healthy humans. Understanding what influences the gut microbes at their site of interaction with the host immune system in normal people is crucial information that is currently lacking. We propose a unique twin study to quantify these changes and provide a resource of the gene-environment-immune relationships at 3 gut sites. 100 extensively genotyped and phenotyped postmenopausal MZ twin pairs aged 50+ will undergo a week's food diary, food frequency questionnaire, followed by stool sampling, bowel preparation and colonoscopy to the terminal ileum. brushings and faecal water will be sampled for 16SrRNA analysis at three sites in the distal gut (terminal ileum, ascending and descending colon). 18 colonic pinch biopsy samples divided equally between 3 sites will be split for RNA sequencing. In a subset of 10 twin pairs Treg cells will be separated and cell specific RNA sequenced to allow comparison and validation of intestinal and cell specific signals. Fresh blood samples will be separated into PBMC for immunophenotyping. We will also create a functional database of gene expression and microbial networks and finally perform a pilot study of causality of key immune microbes in germ free mice.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;1.5 RESOURCES AND INFRASTRUCTURE (UNDERPINNING)
Health categories: ORAL AND GASTROINTESTINAL
Title: Development of rAAV-mediated gene therapy for a severe paediatric metabolic liver disease: Ornithine Transcarbamylase deficiency.
Funder: Medical Research Council

Abstract: The urea cycle is an essential hepatic pathway involved in detoxification of ammonia. Urea Cycle Defects (UCDs) share high rates of mortality and neurodisability. Ornithine Transcarbamylase Deficiency (OTCD) is the commonest UCD (60% of all UCDs). As X-linked inherited disorders, males are severely affected with presentation in the newborn period resulting in coma and death if untreated. Milder cases can present with acute hyperammonaemia at any age particularly in females. Despite best-accepted treatment with ammonia scavengers and protein-restricted diet, acute hyperammonaemic decompensations can occur. Liver transplantation cures UCDs with a risk of mortality and morbidity, including lifelong immunosuppression. The first successful clinical trial of liver-directed GT was led by UCL for Haemophilia B, using AAV2/8. Prof Alexander has shown correction of OTC-deficient mice with an AAV-based GT approach and collaborated in the development of a novel capsid, AAV-LK03, which targets human hepatocytes 12-fold better than AAV2/8. We propose to set up the first paediatric liver-directed GT clinical trial using AAV-LK03 for OTCD. An NHMRC grant (2015-2016) has been awarded to Professor's Alexander & Gissen to optimise vector configuration, and determine efficacy, minimal effective dosing and investigate insertional mutagenesis in a xenograft mouse model engrafted with OTC-deficient human hepatocytes. In this application, we request funding for preclinical studies to determine AAV-LK03 vector pre-existing immunity, biodistribution and formal toxicology. The data will allow preparation of an Investigational Medicinal Product Dossier to support Clinical Trial Authorisation from the Medicines and Healthcare Products Regulatory Agency. This data, along with the proof-of-concept studies by Prof Alexander, will allow an Orphan Designation application and apply for a Protocol Assistance to ensure a complete package for Marketing Authorisation to the European Medicine Agency.


Research activity codes: 5.2 CELLULAR AND GENE THERAPIES
Health categories: ORAL AND GASTROINTESTINAL