NIHRDataInsights/HRCSData huggingface dataset:
Title: Understanding and Ameliorating Pathogenesis in FSHD
Funder: Medical Research Council

Abstract: Hypothesis: DUX4 leads to down-regulation of PAX7 target genes, resulting in sustained HIF1alpha activity that both facilitates apoptosis and suppresses canonical Wnt/beta-catenin signalling to affect muscle differentiation and function in FSHD. Research Objectives 1: Understand DUX4-Pax7-HIF1alpha axis. Express a panel of DUX4 or PAX7 constructs via viral-mediated delivery (including constitutively-active and dominant-negative versions) in FSHD/control myoblasts to assay effects on HIF1alpha and its downstream target genes (RT-qPCR, Western and immunostaining). 2: Determine if Pax7 interferes with HIF1alpha levels. Investigate whether constitutive Pax7 expression will suppress HIF1alpha activity in FSHD myoblasts. Conversely, inhibit Pax7-target genes using dominant-negative PAX7 or DUX4 constructs to examine effects on HIF1alpha activity. 3: Determine if elevated HIF1alpha contributes to apoptosis in FSHD. Inhibit HIF1alpha with specific inhibitors, and siRNA and/or CRISPR\Cas9 in FSHD myoblasts to determine if cell survival is improved. Examine interactions between HIF1alpha and P53. 4: Inhibit/overexpress/stabilise Hif1alpha in myoblasts and examine if canonical Wnt signalling is altered using TopFLASH/FopFLASH luciferase activity assays. Determine whether increased canonical Wnt signalling/beta-catenin activity affects HIF1alpha and rescues myogenesis/survival under oxidative stress. 6: Determine if antioxidants including vitamin C, vitamin E, CoQ10 reduce HIF1 activity while improving myogenic differentiation in FSHD myoblasts. Is vitamin C as effective when HIF1alpha activity is inhibited/enhanced/stabilised? 7: Perform RNASeq on healthy and FSHD myoblasts exposed to antioxidants to further decipher the signalling pathways that are altered to improve myogenic differentiation/survival under oxidative stress. This research will both further reveal disease mechanisms and also allow better understanding of potential therapies for FSHD.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: MUSCULOSKELETAL
Title: Injectable hydrogel for the regeneration of the intervertebral disc: evaluation using an ex vivo loaded disc culture model.
Funder: Medical Research Council

Abstract: The treatment of degenerative disc disease (DDD) has advanced little in the last 20 years with current treatments addressing symptoms, rather than the underlying pathology. Thus there remains a large unmet clinical need and market opportunity for effective therapies to treat DDD. The NPgel developed by our group displays temperature dependant gelation and is fully compatible with a range of cells. The properties of NPgel give it the potential for launch as a standalone therapy, in the first instance, to provide improved therapeutic options for patients through regulation as a class III device. This can be combined, if necessary, with minimally manipulated BMPCs. We have completed initial in vitro cell behaviour studies demonstrating BMPC differentiation towards NP like cells, where they produce an appropriate matrix. We have also demonstrated in vivo safety of NPgel. However, prior to full efficacy testing within a large animal model it is essential to firstly determine the behaviour of NPgel and incorporated BMPCs under mechanical loads which mimic those seen in the human IVD. The complex loading pattern seen in humans is difficult to represent within animal models. In addition it is unethical to progress to large animal efficacy studies without firstly understanding this key behaviour. Therefore, we replace animal models with post mortem human tissue for this stage of the work, in accordance with the principles of the 3Rs which are strongly supported by the MRC. This project aims to investigate the mechanical properties of the NPgel under loads which mimic those seen in the human IVD. In addition we aim to address the behaviour of minimally manipulated BMPCs incorporated within NPgel and NPgel alone following injection into IVD tissue and cultured under loads which mimic those seen in the human IVD. Understanding this behaviour will then enable progression to pre-clinical efficacy testing in a large animal model, which we anticipate will lead to clinical trials.


Research activity codes: 5.2 CELLULAR AND GENE THERAPIES;6.2 CELLULAR AND GENE THERAPIES
Health categories: MUSCULOSKELETAL
Title: Validation of neutrophil elastase as a target to improve muscle regeneration in Duchenne muscular dystrophy
Funder: Medical Research Council

Abstract: Duchenne muscular dystrophy (DMD) is a genetic disorder caused by loss of function mutations in the gene that encodes the cytoskeletal protein dystrophin. In the absence of dystrophin muscle fibres become more prone to damage, which induces muscle fibre necrosis, and in turn inflammation and fibrosis. Muscle regeneration is impaired in DMD leading to loss of muscle mass and strength. Children diagnosed with DMD have shorter life expectancy and undergo progressive loss of muscle mass and strength that will lead them to be wheelchair bound by the age of 12 and often require external respiratory support by the age of 20. There is no cure for DMD and the treatments currently in place, which are mostly based on corticosteroids, can only mildly delay diseases progression but also produce significant adverse effects. In a mouse model of DMD, the mdx4cv mouse, we have discovered that the chronic presence of neutrophils in the muscle leads to accumulation of neutrophil elastase, which impairs muscle stem cell proliferation and differentiation ex vivo. Thus, neutrophil elastase might play a key role in the impairment of muscle regeneration observed in DMD and therefore stands out as a potential therapeutic target to enhance muscle regeneration and prevent muscle loss in DMD. The goal of this project is to validate neutrophil elastase as a therapeutic target for DMD by assessing muscle regeneration, muscle pathology and muscle function in dystrophic mice treated with a selective neutrophil elastase inhibitor. More specifically, we will first select a lead compound amongst three candidates by using an ex vivo assay that we have developed. For this purpose we will use myoblasts (which are the myogenic progeny of muscle stem cells) derived from patients with DMD. This compound will be then taken further into animals and tested for its efficacy at improving muscle regeneration, histopathology and function in dystrophic mdx4cv mice.


Research activity codes: 5.2 CELLULAR AND GENE THERAPIES;6.2 CELLULAR AND GENE THERAPIES
Health categories: MUSCULOSKELETAL
Title: Investigating the role of the primary cilium in muscle regeneration
Funder: Medical Research Council

Abstract: To investigate effects of primary cilia-loss on SC function, Ta3-floxed mice were crossed with Pax-7CreERT2 mice, leading to tamoxifen-inducible Cre recombinase-mediated deletion of Ta3 in Pax7-expressing postnatal muscle SCs. Pilot data show that Ta3-loss significantly impairs muscle repair after injury with a myotoxin (cardiotoxin). Underlying mechanisms will be investigated using: -IN VITRO assays of primary SCs in culture to examine proliferation/differentiation/ migration/self-renewal and apoptosis (Obj 1a). -EX VIVO analysis of single muscle fibres will use time-lapse microscopy and immunostaining to determine effects on proliferation/differentiation/ migration/self-renewal and apoptosis (Obj 1b). Both assays will also be performed in the presence of a Hedgehog (Hh) pathway agonists (purmorphamine), which may overcome the requirement for a cilium/Ta3 (Obj 3). In addition to pharmacological activation of Hh-signaling, we will use a Ta3 expressing retrovirus to restore function in isolated SCs. -IN VIVO injury by cardiotoxin injection followed by repeat injury after one cycle of regeneration will determine whether Ta3-loss leads to a diminished stem cell pool (Obj 1c). In vivo rescue experiments will examine the effects of systemic delivery of the Hh pathway agonist, purmorphamine, and of microRNA: miR-133 (Obj 3) -The impact of Ta3 loss on the stem cell niche will be investigated with a focus on basement membrane components and receptors (Obj 2). -Global molecular changes resulting from Ta3-loss will be analysed by transciptomics (Obj 4). The project is underpinned by our finding that muscle regeneration is affected in the absence of Ta3 function. It benefits from complementary expertise in myogenic signalling and mouse genetics, from existing mouse lines, established cell and molecular biology approaches and expert collaborators, who support different aspect of the project.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: MUSCULOSKELETAL
Title: Agrin, a unique growth factor for cartilage regeneration in osteoarthritis
Funder: Medical Research Council

Abstract: Osteoarthritis (OA) is a leading cause of disability for which we do not have a cure. We discovered that the proteoglycan Agrin is a chondrogenic factor, unique for its capacity to induce cartilage formation without features of hypertrophy or calcification. Agrin also suppresses the cartilage degrading enzyme MMP-3. The first aim of this application is to test the hypothesis that administration of Agrin reverts cartilage and bone changes and improves symptoms in animal models of osteoarthritis. To this end we will use an inducible, cartilage-specific agrin transgenic model. Such transgenic mice and controls will be subjected to two different established models of instability-induced osteoarthritis. We will also deliver full-length Agrin or stabilized mutants that cannot be cleaved by MMP-3 or deletion mutants by adenoviral vectors intra-articularly in wild type mice to assess whether delivery to all joint tissues (including synovial membrane and bone) ameliorates the symptoms and structural changes in OA. We will investigate the signalling mechanism downstream of Agrin with particular emphasis on our recent discovery that Agrin is a potent inhibitor of the WNT signalling pathway, which, if over-activated, predisposes patients to osteoarthritis. This will be achieved with molecular tools in vitro and, in vivo, testing the capacity of Agrin to antagonize primary axis duplication in xenopus embryos. We will determine at which level (receptor, cytoplasm or nucleus) and through which mechanism Agrin inhibits WNT signalling and we will characterize the composition of the Agrin receptor complex in chondrocytes using immunoprecipitation.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: MUSCULOSKELETAL
Title: The role of syndecan-3 in bone metabolism
Funder: Medical Research Council

Abstract: In ageing the balance between bone formation and resorption is skewed towards bone resorption, resulting in a decrease in bone mass and strength leading to fragility fractures and deformities. With increasing life-expectancy and an ageing population, there is a clear need to understand the mechanisms underlying age-related bone diseases. Syndecan-3 (Sdc3) is a transmembrane heparan sulfate proteoglycan receptor involved in the development of the central nervous and the muscular systems, and previous research suggests that Sdc3 may regulate signalling pathways important in bone homeostasis. In pilot studies we have found that adult mice lacking the Sdc3 gene (Sdc3 KO) are characterised by a 40% decrease in trabecular bone volume. However, we do currently not know whether this is due to a defect of bone metabolism during bone growth, low adult peak bone mass, or premature age-related bone loss. The aim of this proposal is to understand how Sdc3 regulates bone development and homeostasis. We will study bone architecture and metabolism in Sdc3 KO mice from early postnatal, to 12-month old osteopenic mice, by microCT, dynamic bone histomorphometry and measuring bone turnover markers. We will investigate the role of Sdc3 in mechanical loading induced bone formation by performing in vivo loading studies of the tibia. To determine whether the bone defect is due to a direct effect on bone formation we will a Sdc3 KO mouse with rescue of expression in the osteoblast lineage. We will study Sdc3 involvement in osteoblast differentiation and bone matrix deposition and mineralisation, as well as signalling of important osteoblast regulators: HB-GAM, FGF2 and Notch in tissue culture. These studies will increase our understanding of bone metabolism, and may identify Sdc3 mediated pathways as targets for novel bone anabolic strategies.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: MUSCULOSKELETAL
Title: Sarcomere proteostasis in titinopathies
Funder: Medical Research Council

Abstract: This project will use a cross-disciplinary approach using to understand the interplay between the sarcomeric M-band and muscle protein quality control pathways controlling the turnover of myofibrils, and how mutations in the major sarcomeric protein titin affect this interplay of cellular structure, mechanics and signalling. Biochemical studies will measure the impact of missense mutations on titin domains for paediatric and adult myopathies. We will use cell and animal models to inform on the impact of pathogenic titin mutations on the basic biological mechanism of sarcomere proteostasis and to discover cellular pathways that lead to the tissue-specific impact of these mutations. Cardiomyocyte cultures from neonatal and adult rodents, iPSC- derived myocytes and skeletal myocyte cultures will be used as easily manipulated models for studying cellular responses to physiological and pathological effects on proteostasis stimuli. This will be complemented by super- resolution confocal microscopy and advanced image analysis to visualize with resolution approaching the dimension of individual M-band components the spatial relations of these events, and the changes induced by mechanical activity or genetic disruptions in animal models or patient biopsies. An underpinning activity will be the protein biochemistry necessary to generate the enzymes and structural proteins required for functional and kinetic studies. Structural analysis by X-ray crystallography, small-angle X-ray scattering and NMR will be highly dependent on our proven ability to generate highly functional proteins for these types of studies, which will not only show basic molecular principles of M-band protein interactions with components of the protein turnover machinery, but reveal the mechanisms of pathogenic mutations and guide the development of small molecule compounds that could target such interactions interfaces or active sites.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: MUSCULOSKELETAL
Title: The regulation of collagen (I) homotrimer synthesis and its role in musculoskeletal dysfunction
Funder: Medical Research Council

Abstract: Type I collagen is the major structural component of vertebrate tissues. The COL1A1 and COL1A2 genes encode the alpha-1(I) and alpha-2(I) polypeptides which form collagen heterotrimers. Abnormal homotrimeric collagen (I) derived from COL1A1 alone is associated with age-related musculoskeletal diseases and fibrosis which are major contributors to morbidity and pose huge clinical and societal challenges. This project aims to determine how COL1A1 and COL1A2 mRNA levels influence collagen (I) homotrimer synthesis, the direct effect of collagen (I) homotrimer on the structure and biomechanical properties of musculoskeletal tissues and whether synthesis causes detrimental cellular stress. The first objective will test the hypothesis that there is a finite cellular buffering mechanism controlling collagen (I) heterotrimer synthesis and determine whether miR-133 or miR-129 are negative regulators of collagen (I) homotrimer synthesis. Over-expression of COL1A1 and knock-down of COL1A2 will be carried out in osteoblasts and tendon fibroblasts. Collagen (I) mRNA and polypeptide chain ratios will be measured using qRT-PCR and metabolic labelling respectively. Transfection of osteoblasts and tendon fibroblasts with miR mimics and antagomirs will be used to determine whether miR-133/-129 are negative regulators of COL1A1, but not COL1A2 synthesis and may be suitable pharmacological targets. The second objective will examine how the structure and biomechanical properties of bone, joints and soft-tissues are altered by collagen (I) homotrimer. Biomechanical properties of bone and tendon will be assessed by 3-point bending, nanoindentation and tensile testing. Osteoporosis and osteoarthritis will be assessed by micro-computer tomography and histological scoring and correlated to collagen fibril structure and organisation. Cellular stress will be evaluated in the third objective in cell cultures and tissues by Western blotting, imaging and qRT-PCR for stress markers.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: MUSCULOSKELETAL
Title: Damage mechanisms in tendon disease
Funder: Medical Research Council

Abstract: We will firstly explore the role of HMGB1 as a tenocyte subset DAMP effector pathway. Human tendon biopsies will be evaluated for HMGB1/TLR4 mRNA/protein expression followed by in vitro interrogation of the impact of exogenous HMGB1 on normal/diseased tendon via cytokine manipulations (rhHMGB1/anti HMGB1/TLR4 siRNA/HMGB1 chemical inhibitors) with the primary readout by RNAseq. Thereafter we will investigate the functional capacity of HMGB1 defined tenocyte subpopulations (FACS sorted HMGB1+/-) in vitro in the context of their ECM regulation (collagen synthesis/RT-PCR) and inflammatory potential (multiplex cytokine/chemokine assays) in tendon disease. Finally, to understand the net contribution of HMGB1 blockade on tendon injury we will utilise our previously published murine tendon injury model whereby neutralising antibodies to HMGB1/ HMGB1 inhibitors will be injected directly into the tendon immediately post injury in WT BALB/C mice. We will additionally carry out transfer adoptive transfer of HMGB1+/- tenocytes directly into WT injured tendon lesions with readouts as above and biomechanical assessment of tendon ex vivo. Next,we intend to use two complimentary strategies to investigate tenocyte subpopulations obtained after tissue disaggregation.1)fluorescence sorting using a set of candidate protein markers followed by bulk transcriptomics (microarray/low input RNA seq) of gated populations. 2)unbiased single cell transcriptomics without gating-384 cell RNA sequencing to 6 independent tendon biopsy samples providing an in-depth transcriptional profile of tenocyte subpopulations across the spectrum of tendon pathology. Finally, we will investigate the functional capacity of subpopulations in vitro in the context of their ECM regulation/ inflammatory potential. Tenocyte subpopulations will be FAC-sorted by virtue of surface markers derived and undergo mechanical/inflammatory stimuli with primary readouts on measures of tenocyte function as detailed above.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: MUSCULOSKELETAL
Title: Osteoarthritis: Going back to basics
Funder: Medical Research Council

Abstract: Osteoarthritis (OA), characterised by articular cartilage loss and pathological ossification, affects almost 9 million people in the UK and is a major financial, social and healthcare burden. If we improve our understanding of the mechanisms underpinning disease aetiology this will enable us to deliver new paradigms to identify those at risk of OA, and to deliver personalised interventions to treat patients with OA. My previous research has shown that in a murine model of ageing-related OA, the articular cartilage chondrocytes undergo a transition from their inherently stable phenotype to a transient one, characteristic of the chondrocytes in the growth plate. Moreover, I revealed an association between growth abnormalities and OA development. This project will build upon this, to translate these pilot data to the human condition, and to examine the hypothesis that: Retaining a stable articular cartilage phenotype protects against OA RESEARCH PLAN By merging my expertise in endochondral bone growth and OA, this NIRG will deliver data describing whether abnormal growth dynamics can predict OA onset, and whether their stabilisation can protect against disease development. Importantly, the proposed studies will assess translation efficacy in terms of human health at all stages. To deliver this, I will answer: 1) Do altered growth dynamics underpin OA predisposition? 2) Can a switch to a transient chondrocyte phenotype contribute to OA? 3) Does targeted stabilisation of the chondrocyte phenotype protect against OA? Together, this will permit us to identify at risk individuals, and consider therapeutic approaches based on mechanistic understanding. This will likely lead to long-term patient benefit and societal impact, essential to reduce the burden of this globally relevant disease.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: MUSCULOSKELETAL
Title: Understanding skeletal diseases using human induced pluripotent stem cells
Funder: Medical Research Council

Abstract: We have generated iPSCs from patients with two closely related rare skeletal dysplasias, multiple epiphyseal dysplasia and pseudoachondroplasia caused by mutations in matrix proteins, Matrilin3 (MATN3) or COMP. We have differentiated these through a mesenchymal stromal cell route to chondrocytes to produce growth plate-like cartilage pellets. Pellets from MATN3 mutant iPSCs are larger than related healthy pellets, with altered expression of cartilage matrix proteins, transcription factors (also for COMP mutants) and increased sensitivity to BMP2 . Building on this we will generate further MATN3 and COMP mutant iPSC lines, then using CRISPR-Cas9 gene editing correct and create the mutations in mutant and healthy iPSC lines respectively, to eliminate molecular changes due to other genetic factors. RNAseq will reveal aberrant molecular pathways of COMP and MATN3 mutant chondrocytes. Mutant cartilage pellets show regional differences from wt; the nature of which will be evaluated by laser capture-RNAseq, compared with cartilage and validated using qRT-PCR, RNAscope multiplex in situ hybridisation and immunocytochemistry (for protein). We will determine if the phenotype is caused by abnormal protein within the cell or abnormal matrix and signalling outside the cell by 1) BMP2 interaction analyses with mutated and wt MATN3, and COMP if implicated in BMP signalling; 2) determining the effect of mutant protein on wt cells by i) adding purified mutant MATN3 or COMP to wt cells, ii) mixing chondroprogenitors overexpressing mutant protein with wt cells carrying a BMP-Smad1(or other appropriate) reporter; 3) determining structural differences in the extracellular matrix by serial blockface-SEM imaging/ electron tomography. We will integrate all data, validating the role of key aberrant pathways using knock down and small molecule/antibody inhibition. We will determine similarities between MATN3 and COMP mutations and suggest new drug targets for alleviation of patient symptoms


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES
Health categories: MUSCULOSKELETAL