NIHRDataInsights/HRCSData huggingface dataset:
Title: Transfusion and Treatment of severe Anaemia in African Children: a randomised controlled trial (TRACT)
Funder: Medical Research Council

Abstract: TRACT is a multicentre randomised controlled trial to identify optimal transfusion strategies and supportive treatment for 3700 African children, aged 2 months to 12 years, presenting to hospital with severe anaemia (defined as haemoglobin (Hb) <6 g/dl). Children will be enrolled at admission to hospital over 2 years in Malawi and Uganda and followed for 6 months. The trial has been designed to address the poor outcome of this condition including high rates of in-hospital mortality, 6-month case fatality and chronic morbidity. Factors associated with poor outcome include potentially treatable co-morbidities such as recurrent infection and multiple vitamin deficiencies, which are not addressed in current guidelines. TRACT is designed to probe each of these multiple contributing factors directly. TRACT will simultaneously evaluate three ways to reduce short and longer-term mortality (primary endpoint) and morbidity following severe anaemia in Sub-Saharan Africa using a factorial design: 1/ Liberal transfusion (30ml/kg) versus conservative transfusion (20ml/kg) vs no transfusion (last strategy for children with Hb>4 g/dl and uncomplicated severe anaemia only) 2/ Multi-vitamin multi-mineral (MVMM) supplementation (including folate/iron) for 3 months versus routine care (folate/iron alone) 3/ cotrimoxazole prophylaxis for 3 months versus no antibiotic prophylaxis. The primary outcome is cumulative mortality to 4 weeks (transfusion comparisons) and to 6 months for the nutritional support/antibiotic prophylaxis comparison. Secondary outcomes include mortality at 48 hours, 4 weeks, 3 months and 6 months (cumulative) (where not the primary outcome); development of new profound anaemia (Hb<4g/dl) during acute admission or development of severe anaemia (Hb<6g/dl) post discharge; readmission to hospital; proportion achieving correction of anaemia (defined by WHO as Hb>9g/dl); adverse events relating to transfusion.


Research activity codes: 6.4 SURGERY
Health categories: BLOOD
Title: Engineering a new generation of blood substitutes
Funder: Medical Research Council

Abstract: There is a clinical need, and a financial market, for a long-lasting sterile red blood cell substitute to be used in environments or patient groups where homologous blood is not available or appropriate. Synthetic hemoglobin (Hb) is the best candidate for the oxygen-carrying component of a blood replacement. However, cell-free Hb displays an inherent toxicity relating to its capacity to induce oxidative reactions, causing damage to DNA and cell membranes. We have shown that tyrosine residues can enhance the safe removal of these oxidative intermediates through the formation of safe electron transfer pathways from the external environment of the protein to the heme iron. We have patents pending on novel bioengineered Hb mutants with additional electron pathways resulting in rapid and safe removal of toxic intermediates. Furthermore, we recently discovered an intrinsically less reactive form of Hb that is a better starting molecule to engineer a safe product. The first aim is to generate new tyrosine mutants of this novel Hb species, optimising the ability of plasma reductants such as vitamin C to decrease oxidative toxicity as measured by cell free liposome studies and damage to primary aortic endothelial cells. (Milestone 1). Additional mutations, known to decrease NO scavenging at the heme oxygen binding site, will then be added. A lead candidate will emerge from this process and will then be converted into a candidate blood substitute via globin PEGylation to increase circulation time. We will upgrade protein production (Milestone 2) to a scale for testing in models of trauma and transfusion. Real time monitoring and ex vivo biochemistry will be undertaken to obtain the maximal amount of information from each study. Succesful tests in the model systems (Milestone 3) will greatly enhance our goal of securing further funding to further optimise our product for clinical trials.


Research activity codes: 5.1 PHARMACEUTICALS;5.2 CELLULAR AND GENE THERAPIES
Health categories: BLOOD
Title: MICA: An open label dose-escalation study of a novel adeno-associated viral vector for gene transfer in subjects with haemophilia A
Funder: Medical Research Council

Abstract: The goal of this proposal is to conduct a Phase I/II gene therapy trial for haemophilia A (HA), the most common inherited bleeding disorder, which results from mutations in the human factor VIII (hFVIII) gene. The same serotype 8 pseudotyped adeno-associated virus vector (AAV8) that mediated stable (>3 years) therapeutic expression of transgenic protein without long-lasting toxicity in subjects with haemophilia B (HB), a related condition, will be used to evaluate a novel FVIII expression cassette: HLP-codop-hFVIII-V3. This expression cassette (5.2kb) has been engineered so that it is efficiently packaged into AAV8 virions, despite being larger than the normal packaging capacity of this vector (4.6kb). Extensive testing in animals showed that in the context of AAV8 this oversized hFVIII cassette was at least 20X more potent than vectors encoding wild-type FVIII. Importantly, AAV8 HLP-codop-hFVIII-V3 had an excellent safety profile even at doses that were 10X higher than planned for use in clinical studies. A first in man, Phase I/II dose escalation study of the oversized hFVIII expression cassette is now proposed in adults with severe HA. The primary objective is to establish the safety and efficacy of systemic administration of AAV8-HLP-codop-hFVIII-V3 vector over three different dosage levels: 2e11, 6e11 and 2e12 vector genomes/kg. This is the same dose range tested in the HB clinical trial. We therefore predict that systemic administration of the oversized hFVIII vector will be safe, with absence of persistent Grade III or greater dose-limiting toxicity (according to modified CTCAE v.4). The key secondary objective is to define the dose of vector that mediates expression of hFVIII at > 5% of normal in peripheral blood; a level that can significantly ameliorate the severe bleeding phenotype. Successful HA gene therapy will transform the treatment paradigm for this disease and impact on a variety of other disorders affecting the liver.


Research activity codes: 6.2 CELLULAR AND GENE THERAPIES
Health categories: BLOOD
Title: Recombinant proteins for GMP-compatible niche creation to optimize in vitro platelet production for human transfusion
Funder: Medical Research Council

Abstract: WP1: Identification of candidate membrane-expressed proteins (MExPs) The secreted and membrane-tethered proteins for cell lines that support proplatelet (ProPt) formation by MKs in cocultures that promote have been identified from expression arrays and proteomics analysis. The ectodomain of 400 candidate proteins will be cloned into expression vectors by a gene synthesis company. WP2 Synthesis of recombinant (rec)MExPs. The recMExPs will be expressed in a mammalian system using the existing infrastructure of Dr Wright's laboratory (Sanger Institute). The recMExPs will be produced in two forms: a soluble pentamer for the initial screening (WP3) and a tagged monomer for immobilization onto a 2D/3D substrates (WP4). WP3: High throughput screening of candidates recMExPs. Phase I: ProPlt assay with single recMEXPs: Cultured MKs will be harvested and plated into fibrinogen-coated 96-well plates with pentameric recMExPs. ProPlt formation will be analyzed 48 hours later by means of immunohistochemistry and automated image analysis. Phase II: ProPlt assay using a combinatorial algorithm: Preliminary data have shown that concurrent recMExPs have a synergistic effect on ProPlt formation. A combinatorial screen of all positive recMExPs identified in Phase I will be carried out using an "overlapping blocks" mathematical model to identify the best combinations of 3 recMExPs. WP4 Immobilisation of candidate recMExPs onto substrates for niche creation. WP4 will validate the results obtained in WP3 with non-soluble immobilized forms of the same recMExPs. Monomeric recMExPs with a biotin tag will be immobilized on a plastic surface coated with fibrinogen and streptavidin in equimolar ratios. The ProPlt assay will be carried out on these functionalized plates as described above. We will also determine the range of optimum surface density for each recMExP by ELISA. Finally we will generate appropriately-tagged recMExPs for functionalisation of collagen-based 3D scaffolds.


Research activity codes: 5.2 CELLULAR AND GENE THERAPIES
Health categories: BLOOD
Title: ADAMTS13 structure and the molecular basis of VWF recognition and cleavage
Funder: Medical Research Council

Abstract: Von Willebrand factor (VWF) is a large multimeric plasma glycoprotein that is critical for platelet recruitment to sites of blood vessel damage. VWF platelet-tethering function is highly dependent upon its multimeric size, with larger VWF multimers being more haemostatically competent. VWF plasma multimeric size, and consequently its platelet-tethering function, is regulated in the blood by proteolytic processing by the plasma metalloprotease, ADAMTS13. Deficiency in VWF causes the most common inherited bleeding disorder, whereas people with ADAMTS13 deficiency suffer from life-threatening thrombotic thrombocytopenic purpura. Increasing VWF and decreasing ADAMTS13 levels in plasma are risk factors for both myocardial infarction and stroke. ADAMTS13 is very highly specific as it cleaves only one known substrate protein, VWF, and does so at just a single site - between Tyr1605 and Met1606 in the VWF A2 domain. Physiologically, proteolysis can only occur when VWF is unravelled by shear as the scissile bond and cryptic exosites in VWF are only revealed when VWF unravels. The holy grail of research into ADAMTS13 and VWF is to understand the complex mechanism of VWF force-induced VWF unfolding and highly single substrate specific cleavage by ADAMTS13. To address this, we will express, purify and characterise a panel of ADAMTS13 domain fragments to understand their interaction with and proteolysis of VWF and the importance of Ca2+ binding in this process. We will also determine the crystal structures of these fragments, in isolation, in complex with anti-ADAMTS13 Fab fragments and in complex with VWF. The results from these studies will not only provide detailed information upon ADAMTS13 function, it will provide insight in to thrombotic disease, provide insight into the structure and function of other ADAMTS family members, and perhaps more importantly provide the opportunity to rationally modify/improve ADAMTS13 for use as a therapeutic agent.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: BLOOD
Title: The role of von Willebrand Factor in endothelial platelet capture
Funder: Medical Research Council

Abstract: This study will use an in vitro system to study the role of VWF in platelet capture by endothelial cells(EC) and thus its role in atherogenesis. Epidemiological studies of patients with CVD or genetic defects of VWF addressing this have given conflicting results. Animal studies have been suggestive but again incomplete. A novel aspect of this proposal is the use of primary (progenitor) endothelial cells cultured from peripheral blood and mutant VWF. We previously showed that these cells synthesise, store and release VWF in a physiological manner and that they provide a unique demonstration of how defects in VWF synthesis affect the function and behavior of endothelial cells. Moreover, using state of the art perfusion systems and bespoke perfusion chambers with differing geometries we are able to reproduce the patterns of flow encountered in vivo and which appear to be a major determinant of plaque formation. Indeed a key theme throughout this study is the importance of shear in VWF biology: both for synthesis and for function. Variable distribution of VWF within the vascular tree in part reflects these differences in flow and is reflected in their different susceptibility to atheroma. This study will provide precise detail on this relationship and map VWF expression through narrowed and branching vessels where turbulent flow can produce large changes in shear over small distances. The critical measure in all cases will be platelet capture on the surface of the EC and a secondary measure will be evidence of resulting EC activation. In both cases the key question will be whether this is mediated by circulating VWF or by VWF emerging from the EC. Our experimental approach is designed specifically to give a precise answer to this question in a physiologically representative system, but also determine mechanisms and consequences for EC.The answers will be potentially crucial factors in planning to exploit the central position of VWF for therapeutic effect.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: BLOOD
Title: Experimental manipulation of indices of lesion severity in multi-vessel coronary artery disease: Quantitative exploration of collateral influences.
Funder: Medical Research Council

Abstract: Aims: To test the hypothesis that collateral mediated changes in coronary flow pattern can alter the physiological severity of stenoses in multi-vessel coronary artery disease. Objectives: -To understand how 'two-for-one' stenting occurs (how the severity of a stenosis can be improved without a need for further stenting or bypass surgery). -To quantify the role of the collateral circulation in this clinically important process. -To incorporate an assessment of collateral function into the physiological diagnostic approach to patients with multi-vessel disease. Methodology: -40 patients with multi-vessel CAD (30-40% of all CAD patients) will be recruited over 2.5 years. All angiography will be clinically indicated. -Patients will undergo baseline intracoronary pressure and velocity measurements using a 0.014" pressure and flow sensor tipped wire. Assessments will be made to all moderate-severe stenoses. -One wire will be placed in one vessel (A) and a second wire in another vessel (B). -An intracoronary balloon will be positioned over the wire in a proximal, non-diseased segment in (A). It will be inflated to low-pressure (to avoid endothelial damage) for 60-90 seconds to transiently occlude flow. Pressure and flow will be measured distally in (A). -Pressure and flow will be measured simultaneously in (B) to provide data on collateral flow modulation between (A) and (B). -Measurements during balloon occlusion will be compared to baseline to quantify flow redistribution in response to an acute change in antegrade flow in (A). -The process will be repeated with hyperaemia to determine FFR and changes from baseline. -Wave intensity analysis will be applied to determine the effect of hyperaemia on collaterals. Scientific and medical opportunities: Findings from this study will impact across the entire spectrum of the disease, with mechanistic insights that can be readily translated into improved clinical practice.


Research activity codes: 4.2 EVALUATION OF MARKERS AND TECHNOLOGIES
Health categories: BLOOD
Title: Mechanisms and functions of CRACR2A-L Rab GTPase in vascular endothelial cells
Funder: Medical Research Council

Abstract: Objective 1: To determine the contribution of CRACR2A-L to Weibel Palade body trafficking. The general strategy for this project will be to use high resolution imaging to quantify and measure the distribution of CRACR2A-L and vWF in endothelial cells. Contribution of CRACR2A-L to Weibel Palade body trafficking in response to thrombotic stimuli will be assessed by overexpressing wtCRACR2A-L or GTP/Ca2+-binding mutants or alternatively, cellular levels of protein will be reduced by short interfering RNA. Secretion of vWF as a measure of Weibel Palade body exocytosis will be assayed using ELISA. The effect of CRACR2A-L on other endothelial functions such as apoptosis and proliferation will also be assessed. Objective 2: To discover the fundamental mechanisms by which CRACR2A-L regulates Weibel Palade bodies. Affinity chromatography followed by LC-MS/MS will be used to identify downstream effector proteins. Potential effector proteins will be identified and ranked according to peptide abundance and pathway analysis. Confirmed effectors will be verified by co-immunoprecipitation and rescue of function experiments using high resolution imaging. To be able to understand the structural mechanisms of CRACR2A-L action, structural models will be created by X-ray crystallography and homology modelling. Objective 3: To verify the in vivo role of CRACR2A-L using an endothelial specific knockout mouse. First, production of a CRACR2A global 'knockout first' (with conditional potential) mouse will be outsourced. Subsequent breeding with FLP deleters and endothelial specific Cre recombinase mice will generate an endothelial specific CRACR2A-L knockout mouse. The in vivo contribution of global and endothelial CRACR2A-L to Weibel-Palade body exocytosis will be analysed by measuring clotting in response to tail injury. In addition, real time intravital imaging of clot formation will be performed in the femoral artery and in the mouse ear. Angiogenesis will also be assessed.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: BLOOD
Title: Hypoxic pre-conditioning of seeded tissue engineered scaffold to improve in vivo neovascularisation
Funder: Medical Research Council

Abstract: Aim: To determine if hypoxic pre-conditioning of seeded decellularised trachea scaffold will promote the release of pro-angiogenic cytokines and accelerate angiogenesis. Objectives: 1. Optimising hypoxic pre-conditions - Determine if hypoxic preconditioning of seeded cells promotes pro-angiogenic factors which accelerates neoangiogenesis 2. Testing hypoxic pre-conditioning response in in vivo study - Using heterotopic transplant of seeded scaffolds on mice model to demonstrate accelerated functional angiogenesis in vivo Methodology: 1. Determine if hypoxic preconditioning enhances angiogenesis of seeded scaffolds by using CAM assay to delineate the best conditions 2. Perform cytokine analysis of conditioned medium to interrogate for pro-angiogenic factors 3. Ascertain functional characteristics of angiogenesis by using in vivo murine model for heterotopic transplant of scaffold accompanied by histological and SEM analysis of explanted scaffolds 4. Establish if a novel photoacoustic imaging technique can provide non-invasive analysis of microvasculature Scientific and medical opportunities: 1. Improve the understanding of angiogenesis process in implanted trachea scaffolds in vivo 2. Improve angiogenesis in seeded scaffolds by determining the right hypoxic environment for MSC production of pro-angiogenic factors 3. Potential amalgamation of this research with other on-going parallel research on optimisation of tissue engineered trachea in vivo which will facilitate the development of GMP grade scaffold for clinical use 4. Validation of the use of photoacoustic images to monitor angiogenesis in vivo will allow for reduction in the use of more invasive methods and possible pre-clinical applications


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: BLOOD
Title: The genetic basis and biology of dyskeratosis congenita and bone marrow failure
Funder: Medical Research Council

Abstract: Dyskeratosis congenita and bone marrow failure are a heterogeneous group of life threatening disorders affecting children and adults. They are unified by the inability of the bone marrow to make an adequate number of mature blood cells and patients die prematurely from infection or bleeding. We hold a large unique international registry of bone marrow failure patients, with samples stored from 643 patients with dyskeratosis congenita and 766 patients with bone marrow failure. In approximately 30% of these dyskeratosis congenita patients the genetic basis is unknown and in bone marrow failure patients the primary pathology remains unknown in over 80% of cases. The main aim of the research is to determine the genetic basis and primary pathophysiology of dyskeratosis congenita and bone marrow failure. To facilitate this goal we will undertake genetic analysis (principally based on next generation sequencing) in families we have collected with two or more affected individuals with bone marrow failure, but which do not meet the criteria of known bone marrow failure syndromes. Characterization of the familial forms of bone marrow failure will lead to the identification of new genes and biological pathways that are likely to be important in the idiopathic forms of these disorders. Elucidation of the genetic basis will provide new diagnostic and therapeutic strategies that are highly desirable since treatment remains unsatisfactory for many patients with bone marrow failure.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: BLOOD
Title: Development of a novel therapy for Beta-Thalassaemia using CRISPR/Cas9 to edit the major alpha-globin enhancer
Funder: Medical Research Council

Abstract: Beta-thalassaemia is an inherited anaemia caused by reduced or absent production of the haemoglobin component beta-globin. Despite being one of the first molecular diseases to have its pathophysiology characterised, the management of beta-thalassaemia is far from optimal, limited to supportive therapy for the majority. We know that the unbalanced production of globin chains and subsequent excess of alpha-globin chains are the central causative factors in the pathophysiology of this condition. Currently, most attempts to ameliorate beta-thalassaemia aim to resolve globin imbalance by increasing gamma-globin production. In this project we aim to take a complementary approach; the reduction of excess alpha-globin chains, as natural reductions of alpha-globin output, in the form of co-inherited alpha-thalassaemia, ameliorate the disease phenotype in beta-thalassaemia patients. To achieve this reduction, the major regulatory element controlling alpha-globin expression will be deleted using the CRISPR/Cas9 system. Proof-of-principle experiments have demonstrated the efficacy of this approach. We hope to develop the efficient editing of long term repopulating haematopoietic stem cells (LT-HSCs) and have two complimentary aims: 1) To optimise gene-editing of the MCS-R2 enhancer by identifying the key sequences that need to be disrupted to achieve the ideal level of alpha-globin knock-down. This will be done through generating a guide-RNA library to edit various transcription factor binding sites, using a newly developed Cas9 derivative that allows for flexibility in the PAM specificities required for CRISPR/Cas9 editing. 2) To identify which sub-population of stem cells have long-term repopulating ability. Evidence suggests that the CD49f+ fraction of CD34+ cells are enriched in LT-HSCs. We will use a xenograft mouse model to evaluate whether improved editing efficiency and long-term repopulation can be achieved by editing this purified CD49f+ sub-population alone.


Research activity codes: 5.2 CELLULAR AND GENE THERAPIES
Health categories: BLOOD