KLF1 is a transcription factor (TF) specifically produced in developing red blood cells (RBCs) where it is essential for regulating the expression of many genes, and thus the proteins synthesized in these cells. Indeed, KLF1 is considered a master regulator of red blood cell production and function. It would therefore be anticipated that mutations in KLF1 have adverse outcomes, and this is indeed the case with the number of individuals identified with RBC disorders associated with mutations in KLF1 increasing rapidly over recent years. Of these the heterozygous E325K mutation (substitution of glutamic acid with lysine at amino acid 325) within the DNA binding domain of KLF1, is causally linked to a severe form of RBC disease. However, how this mutation effects the function of KLF1 in vivo to cause the disease phenotype is currently not known. Studying the defects behind many RBC diseases is severely impeded by paucity of suitable, and adequate quantities of material from anaemic patients. Hence suitable model cell systems are required that accurately mimic RBC diseases, but to date have not been available. We have recently developed technology and generated the first human immortalized adult RBC lines. The cells undergo the normal process of RBC synthesis in vitro, and provide a sustainable supply of cells. We have also developed a platform, allowing us to introduce mutations in specific positions in the genome, producing a sub-line and supply of cells with the mutation for study. We thus have the unique opportunity to create model cellular systems of RBC disease. We propose to create a line from a patient with the E325K mutation, and introduce the E325K mutation into one of our existing lines, recreating the disease genotype and phenotype. We will use these systems to (i) obtain a comprehensive map of the complete repertoire of proteins aberrantly expressed in cells with E325K KLF1 using comparative proteomic techniques, to determine the extent of the disordered proteome. These data will also serve to facilitate, and thus improve diagnosis of further patients with the mutation, and may reveal overlap with profiles of RBC disorders of unknown etiology, prompting screening for KLF1 mutations (ii) delineate the molecular mechanisms by which the E325K mutation results in disrupted gene regulation, and thus altered protein expression and the disease phenotype using genome-wide analysis techniques. KLF1 binds to the regulatory regions of the genes it controls to, in most cases, induce their expression. We will therefore determine if E325K KLF1 interferes with the binding of normal KLF1 to such regulatory regions, and conversely if E325K KLF1 binds promiscuously to the regulatory regions of genes not normally expressed in RBCs. However, expression of a gene is often controlled by multiple regulatory regions that may lie at a distance from each other in the DNA, and from the target gene, which must interact via alterations in the 3D chromatin (DNA) structure, facilitated by TF binding, for gene expression. Therefore, to determine how E325K KLF1 may distort the genetic readout of cells we will analyse its effect on such chromatin configuration at the loci of selected KLF1 regulated genes. Aberrant binding of E325K or normal KLF1 to regulatory regions, and impeded or incorrect interaction between such regions would serve to prevent production or reduce the level of proteins required by RBCs, whilst potentially cause proteins not normally present in RBCs to be produced (iii) determine if the mutation perturbs the interaction between KLF1 and co-factors required for its activity, and if so the effect of the loss of such factors on the binding of KLF1 to gene regulatory regions. As well as revealing the molecular mechanisms by which the E325K KLF1 mutation results in the observed disease phenotype, the data will also provide further insight into the regulation of gene expression and thus red blood cell production by KLF1.

Synaptic terminals are the connections that allow information to flow from one nerve cell to the next. Synaptic terminal proteins, including one called SV2a, are key to this information flow & consequently for overall brain function. Loss of synaptic terminal proteins is thought to contribute to a number of illnesses, including schizophrenia. This highlights the importance of understanding potential mechanisms & effects of synaptic terminal protein loss. We focus on schizophrenia, although findings are likely to be relevant to other brain disorders, normal neurodevelopment & ageing as well. Schizophrenia affects 1 in 100 people & is characterized by psychotic & negative symptoms, & cognitive impairments. It is a top ten cause of disability in working-age adults. A leading hypothesis proposes that synaptic terminal protein loss underlies impaired brain function to lead to the cognitive & other symptoms of schizophrenia. Complement proteins are produced by brain cells & regulate levels of synaptic terminals by tagging them to be broken down by immune cells called microglia. Some complement proteins are elevated in schizophrenia, & it is thought this leads to loss of SV2a, & other synaptic terminal proteins, in schizophrenia. However, it remains unknown if the complement & SV2a changes underlie the symptoms & cognitive impairment in schizophrenia, or if further SV2a loss occurs during the course of the disorder. Moreover, it is not known if modulating SV2a impairs brain function in schizophrenia, as predicted. Finally, we lack approaches to measure synaptic terminal proteins at multiple time points, or to measure post-synaptic proteins. We plan to address these critical gaps in understanding in three related work-packages. The first tests whether SV2A levels are altered at illness onset in schizophrenia relative to controls and if they reduce further during the course of schizophrenia using brain scans. It will also test if complement levels at presentation predict increasing cognitive impairment & other symptoms over time & if this is linked to altered SV2a levels. The second tests if reducing SV2A activity impairs brain function & leads to symptoms in schizophrenia. This is important to understand the functional consequences of reductions in SV2A. We will use a drug called levetiracetam to reduce SV2A activity and compare its effects on brain function against a placebo using brain scans. The third involves developing new approaches to image synaptic proteins. The current approach to image SV2A involves a small amount of radiation. This limits the number of scans someone can have, particularly in adolescence. It is necessary to scan adolescents & at multiple time-points to fully understand what happens to synaptic terminals during brain development & many brain disorders, which often begin in adolescence. To overcome this limitation, we aim to develop an ultra-low radiation approach & compare it to the current standard scan approach. This will involve scans in healthy volunteers. In addition to synaptic terminal proteins, post-synaptic proteins are also important to brain function, and affected in schizophrenia & other brain disorders. However, there is currently no way of studying them in live humans, so it is not possible to test if post-synaptic proteins are involved in these disorders. To address this, we aim to develop a new PET tracer for post-synaptic markers. We will evaluate potential ligands & select the most promising ligand to take forward. If this experiment supports progression, we will then conduct a study to determine the reliability of the ligand in humans. These studies have the potential to identify new approaches to treat schizophrenia, & other disorders with similar cognitive impairments & symptoms, including Alzheimer's disease, mood & autistic disorders. They also have the potential to develop new tools to further understanding of brain disorders.

Inflammation is a healthy response to infection or physical damage, which helps to eliminate harmful microbes. However, many of the factors released during an inflammatory response cannot discriminate between self and microbe, and therefore risk causing collateral damage to the inflamed tissue. For this reason, inflammation is usually very tightly regulated. A healthy inflammatory response has a rapid onset and an orderly resolution phase, in which activated immune cells exit the inflamed tissue or return to their resting state. This allows normal function of the affected tissue to be restored with minimal damage. An inflammatory trigger can be thought of as an accelerator pedal, and resolution as the brake: safe driving requires judicious use of both. Inadequately controlled, damaging inflammation is the defining characteristic of chronic diseases like rheumatoid arthritis, chronic obstructive pulmonary disease, inflammatory bowel disease and many others. Uncontrolled inflammation also strongly contributes to cardiovascular disease, neurodegenerative conditions like Alzheimer's disease, and many forms of cancer. For decades the main focus of researchers on these diseases has been to identify triggers of inflammation and try to block their effects. This approach has met with only moderate success, and the overall economic, societal and personal burdens of chronic inflammatory disease continue to grow in the developed world. The focus on triggers of inflammation risks overlooking the equally important process of resolution. Evidence both from genetic studies of human disease and from animal experiments clearly shows that inflammatory disease can be caused or made worse by defects in the "braking" mechanisms that underlie resolution. More and more researchers are now trying to understand the biological processes involved in the resolution of inflammation. It is thought that reinforcement of resolution mechanisms may be an effective way to treat inflammatory diseases. Our research on a protein called tristetraprolin (TTP) develops the concept of reinforcing resolution. Mice that cannot produce TTP develop severe, spontaneous inflammatory disease, therefore we know that TTP is an important brake to inflammation. We have also learned that the function of TTP is controlled by a molecular switch that converts it between active and inactive states. We can detect a lot of TTP protein in chronically inflamed joints of patients with rheumatoid arthritis, but it seems to be in the inactive state. We suspect that the persistent inactivation of TTP prevents resolution of inflammation, much like a faulty brake. We believe it will be possible to reduce inflammation by restoring the function of TTP, effectively repairing the damaged brake. To do this, we plan to use two different drugs that we predict will switch TTP from inactive to active state. One of these drugs is already used to treat multiple sclerosis, whilst the other is being investigated as a potential treatment for cancer. If this work is successful it may lead to new clinical trials, and ultimately to an entirely new type of treatment for inflammatory diseases, one that is based on promoting resolution rather than blocking inflammatory triggers.

Red blood cells (RBCs) are essential for life as they carry oxygen to all tissues of the body and are produced at a rate of over two million per second. RBC deficiency and life-threatening anaemia are caused by genetic disorders, chronic infection, inflammation and exposure to radiation and drugs for cancer treatment. Anaemias are treated by transfusion of RBCs collected from healthy donors but this is only effective in the short term and significant problems arise in patients who require repeated transfusions. The limited number of drugs that are used to treat anaemia, including erythropoietin stimulating agents, act by enhancing RBC production but the majority are not directed to the underlying cause of the disorder. This project aims to gain a better understanding of RBC development and maturation that could lead to improved strategies for producing RBCs in vitro and more targeted drug treatment for congenital anaemia. A significant number of RBC disorders, from relatively benign blood group variants to severe cases of anaemia, have been associated with mutations in the erythroid transcription factor, KLF1. KLF1 regulates the expression of genes associated with the structure and function of RBCs. Recent studies have shown that KLF1 also plays a role in macrophages associated with the erythroid island (EI) niche where RBCs develop and mature. Deep within the bone marrow and spleen, the human EI niche is inaccessible for study so we developed in vitro model of the EI niche using genetically programmed induced pluripotent stem cell-derived macrophages (iPSC-DMs). Activation of KLF1 in iPSC-DMs enhanced their ability to support RBC proliferation and maturation and we showed that the mechanism of action involves both factors involved in cell-cell contact and factors that are secreted. The first aim of this proposal is to assess the effect of candidate EI niche-associated factors on erythroid cell proliferation and maturation. From our existing dataset of KLF1 target genes, we will test the secreted and membrane-associated factors for their ability to enhance the in vitro production and maturation of RBCs using recombinant proteins and synthetic mono-biotinylated peptides. This will lead to improved protocols for the production of RBCs from limitless sources such as iPSCs where current protocols fail to produce fully mature, enucleated cells. As blood transfusion is the first line of treatment for RBC disorders this alternative source will overcome problems associated with donor-derived transfusion such as but limitations in supply and transfusion-transmitted infection. Our second aim is to assess how mutation in KLF1 affects the erythroid island niche and to identify factors that are aberrantly expressed within the genetically defective niche. We will use iPSCs derived from congenital anaemia (CDA) patients carrying the KLF1-E325K mutation and we will generate iPSCs carrying an inducible form of the mutant protein. These iPSCs will be differentiated into EI-like macrophages and we will then test their ability to support the proliferation and maturation of RBCs. We will discover factors that are aberrantly expressed in KLF1-E325K "diseased" iPSC-DMs compared to control iPSC-DMs. Mixed co-cultures will be used to define the intrinsic and extrinsic effects of the E325K mutation and we will identify macrophage-specific targets of KLF1-E325K by RNA sequencing, proteomic analyses and chromatin immunoprecipitation. These studies will identify novel drug targets that would lead to the development of new treatments for congenital anaemia as well as those caused by infection, inflammation and exposure to anti-cancer drugs. The action of novel drugs will be tested using our novel in vitro culture system.

Regenerative medicine aims to develop biomaterial and cell-based therapies that restore function to damaged tissues and organs. It is a cornerstone of contemporary and future medicine that needs a multidisciplinary approach. There is a world-wide shortage in scientists with such skillsets, which was highlighted in 2012 by the Research Councils UK in their 'A Strategy for UK Regenerative Medicine" which promotes 'training programmes to build capacity and provide the skills-base needed for the field to flourish'. The major clinical need for regenerative medicine was highlighted by the Science and Technology Committee (House of Lords; July 2013), who identified that 'The UK has the chance to be a leader in [regenerative medicine] and this opportunity must not be missed', and that 'there is likely to be a £44-54bn NHS funding gap by 2022 and that management of chronic disease accounts for around 75% of all UK health costs'. Vascular diseases are the leading cause of death and disability worldwide, musculoskeletal diseases have a huge burden in pain and disability, diabetes may be the 7th leading cause of death by 2030, and peripheral nerve injuries impair mobility after traumatic injuries. There is a pressing need for commercial input into regenerative medicine. Whilst the next generation of therapies, such as stem cells and biomaterials, will be underpinned by cutting-edge biology and bioengineering, strong industrial-academic partnerships are essential for developing and commercialising these advances for clinical benefit. We have established strong industrial partnerships which will both enhance the CDT training experience and provide major added value to our industrial partners. Regenerative medicine is a top priority for the University of Manchester (UoM) which has excellence in interdisciplinary graduate training and a critical mass of internationally renowned researchers, including newly appointed world-leaders. Our regenerative medicine encompasses physical, chemical, biological and medical sciences; we focus on tissue regeneration and inflammation, engineering and fabrication of biomaterials, and in vivo imaging and clinical translation, all on our integrated biomedical campus. We propose a timely Centre for Doctoral Training in Regenerative Medicine in Manchester that draws on our exceptional multidisciplinary depth and breadth, and directly addresses the skills shortage in non-clinical and clinical RM scientists. Our expertise integrates tissue regeneration & repair, the design & engineering of biomaterials, and the clinical translation of both biological and synthetic constructs. Our centres of excellence and internationally-leading supervisors across this multidisciplinary spectrum (details in Case for Support and UoM Letter of Support) highlight the strength of our scientific training environment. Defining CDT features will be: integrated cohort-based multidisciplinary training; skills training in engineering, biomedical sciences and pre-clinical translation; imaging in national Large Facilities; medical problem-solving nature of clinically co-supervised PhD projects, including in vivo training; comprehensive instruction in transferable skills and commercialisation; outward-facing ethos with placements with UK Regenerative Medicine Platform hub partners (UoM is partner on all three funded hubs), industrial partners, and international exchanges with world-class similarly-orientated doctoral schools; presentations in seminars and conferences. In this way, we will deliver a cadre of multidisciplinary scientists to meet the needs of academia and industry, and ensure the UK's continuing international leadership in RM. Ultimately, through training this cadre of doctoral scientists in regenerative medicine, we will be able to improve wound healing, repair injured nerves, blood vessels, tendon and ligaments, treat joint disease and restore function to organs damaged by disease.