NIHRDataInsights/HRCSData huggingface dataset:
Title: Ultra-high resolution optical coherence tomography (UHR-SD OCT) in infants and children: characterisation of normal and abnormal foveal development
Funder: Medical Research Council

Abstract: Recently, optical coherence tomography (OCT) has significantly improved diagnosis and therapy of retinal diseases in adults. However, OCT is not yet available clinically for infants and young children. Histological studies suggest that the fovea develops until at least 45 months of age but this has never been visualised in vivo. We aim to use a recently developed handheld ultra-high resolution spectral domain OCT (UHR-SD OCT) designed for children to (i) characterise normal and abnormal development of the fovea, (ii) improve early diagnosis of infants and children with foveal hypoplasia, (iii) better understand mechanisms of nystagmus and (iv) estimate or predict later visual acuity by also characterising eye movements in nystagmus. Approximately 320 OCT images of control subjects and patients with idiopathic infantile nystagmus (IIN), albinism, PAX6 mutations and achromatopsia will be obtained using cross-sectional and longitudinal sampling (from birth to 6 years of age, in 10-12 year old children, and in young adults). OCTs will be obtained using a spectral domain handheld OCT (3.2micro meter resolution, 1000 A-scans x 100 B-scans, 10x10mm, Bioptigen Inc, NC) Analysis will include measurements of the thickness of the nerve fiber layer, all processing and photoreceptor layers and retinal pigment epithelium. Foveal grading will be based on total retinal thickness, foveal pit depth, and widening of the outer nuclear and outer segment layers at the fovea. Eye movements of children with nystagmus will be recorded with head free high resolution infrared oculography (EyeLink 1000) suitable for infants and children. Eye movement characteristics such as amplitude, frequency, intensity, foveation characteristics and waveform will be analysed using Spike 2 Software. We anticipate that this study will help establish OCT as a key investigational and diagnostic tool for children.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;4.2 EVALUATION OF MARKERS AND TECHNOLOGIES
Health categories: EYE
Title: Proof of Concept for Real Architecture for 3D Tissues in the Cornea
Funder: Medical Research Council

Abstract: 10 million people worldwide are blinded by scarring of the cornea. The ability to engineer biomimetic corneal components could improve quality of life. As a first step, we are working to treat limbal epithelial stem cell (LESC) deficiency of the cornea, which causes painful, blinding surface failure. We have transplanted cultured LESCs to improve vision in patients with chemical burns. However, many patients have challenging diseases requiring a substrate to aid cell survival, in our case human amnion. This approach can improve vision, however, we have found the long-term therapeutic benefit to be variable. In part, we suspect, because cultured LESC sheets do not restore the normal LESC 'niche' destroyed by disease. We have made simple constructs of the human corneal surface. Novel technology (Real Architecture for 3D Tissues - RAFT) is used to make a fibroblast-seeded collagen I construct with surface topology that mimics the in vivo stem cell niche, with an epithelium on the surface. Our data are very promising, the epithelial cells grow successfully and produce an epithelium. RAFT constructs are reproducible and have significant advantages over human amnion for LESC culture. Amnion is biologially variable and often (40% of cases) does not support the growth of healthy LESCs. Here we will quantify RAFT mechanical strength, using a culture force monitor, as this must be sufficient to withstand surgery. Spectral transmission (transparency) will be measured as RAFT should ideally be at least as transparent amnion. We will test if RAFT is safe to use and if it can improve the surface of a cornea with LESC deficiency with our Japanese collaborators using an established rabbit model of LESC deficiency. If successful this data will inform GMP compliant standard operating procedures for therapeutic RAFT production. IoO have the experience and capacity to manufacture GMP compliant RAFT constructs in the 'Cells for Sight Cell Therapy Research Unit'.


Research activity codes: 5.2 CELLULAR AND GENE THERAPIES
Health categories: EYE
Title: Deciphering molecular pathways and cellular events involved in uveal coloboma
Funder: Medical Research Council

Abstract: In this research proposal we plan to perform an expression analysis using RNA sequencing to identify genes that are differentially expressed in the anterior optic fissure (OF) in wild-type and Foxg1-/- null embryos and to investigate the role of Wnt8b in OF closure. For our expression analysis experiment, we will dissect tissue from wild-type and Foxg1-/- null mutant mice using laser capture microdissection (available at Roslin Institute) at two different developmental time points (before and just when the two tips of the OF meet). The sequencing and statistical analysis of results will take place at GenePool (University of Edinburgh). Crucial to our selection of coloboma-candidate genes will be our collaboration with Prof FitzPatrick, who will provide us with data that will arise from a human exome analysis screen of patients with coloboma. Expression of genes shared by the two different studies will be examined in wild-types and Foxg1-/- nulls. Using ex-vivo electroporation we will alter expression of these genes in wild-type optic cups and the phenotype will be evaluated by qualitative and/or quantitative techniques. Our strongest candidate genes will be sequenced to screen for mutations in samples of coloboma patients in the MRC-HGU. In parallel and based on our pilot data, we will study the functional relationship between Foxg1 and Wnt8b in the developing optic stalk and OF. To establish this, we will study: -When Wnt8b expression becomes defective in the developing eye of Foxg1-/- nulls. -How expression of downstream targets of the Wnt/beta-catenin and planar-cell-polarity pathway changes in our Foxg1-/- and Foxg1-/-;Wnt8b-/- mutants. -What the OF phenotype is at later stages of development in our double mutants. We will finally generate a double mutant strain for the Wnt8b and Pax2 genes and study the eye phenotype in these embryos. Once we publish our results, our RNA sequencing and optic fissure gene expression data will become publicly available.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: EYE
Title: Hypoxia-inducible factors in age-related macular degeneration
Funder: Medical Research Council

Abstract: In this fellowship I will investigate the hypothesis that hypoxia-inducible factor (HIF) activation in myeloid cells is responsible for neovascular age-related macular degeneration. The aim is to define the molecular and cellular processes responsible for choroidal neovascularisation (CNV) by investigating the role of HIFs in myeloid cells during experimental CNV. I will investigate the specific roles of HIF1a and HIF2a in myeloid cells in normal retina and during experimental CNV by manipulating HIF pathways using highly targeted transgenic approaches. I will use transgenic mice that express cre-recombinase under the control of the myeloid specific promoter lysM. This mouse line facilitates the efficient cre-mediated deletion of HIF-related target genes in myeloid cells and is therefore a suitable tool to investigate the role of circulation-derived and resident myeloid cells in the development of CNV using immunohistochemistry, flow cytometry and in vivo angiography. The objectives are: 1) To determine the distribution of myeloid cells in the normal retina and in experimental CNV 2) To characterize these cells by immunophenotyping in vitro and in vivo 3) To determine the impact of HIF activation on the extent of CNV 4) To determine the role of HIF activation in recruitment of myeloid cell populations to CNV By defining the role of HIF in myeloid cells in the development of experimental CNV I aim to identify new opportunities to improve the efficacy of therapeutic intervention.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: EYE
Title: Optimising RPGR gene replacement as a potential treatment for X-linked retinitis pigmentosa
Funder: Medical Research Council

Abstract: Retinitis pigmentosa (RP) represents a heterogeneous group of inherited disorders with great genetic and phenotypic variability. Indeed, many different genes have been identified to cause RP, however, approximately 10-20% of all cases feature mutations in the coding sequence for Retinitis Pigmentosa GTPase regulator (RPGR). The overall goal of the proposed project is to lay the ground for a phase I / II clinical trial on the safety and efficacy of gene replacement therapy in patients with mutations in RPGR. As expression levels need to be high enough to convey a therapeutic effect, this project will emphasize the optimisation of transfection and expression efficiency. To achieve this, I will optimise the expression cassette of human cDNA sequence to increase expression efficiency within the transfected cells by codon optimisation of the therapeutic gene sequence. I will analyse performance of resulting sequences through quantitative expression essays in cell culture, human tissue samples and in vivo experiments. Secondly, I will test transfection efficiency of modified vectors with targeted alterations of vision capsids, which are reported to escape proteosomal degradation in the target cell to further optimise transfection and expression efficiency in vitro and in vivo. Once the AAV vector is optimised in terms of expression efficiency, I will use clinical grade viral vectors for in vivo experiments in the existing genetically engineered knockout mouse model. In vivo analysis will use the same state of the art diagnostic equipment and protocols as commonly implemented in clinical trials, such as non-invasive retinal imaging (SLO) and functional testing (ERG). Ex vivo methods will include analysis of transfection and expression efficiency by means of western blot and realtime-qPCR. Rescue of the mouse model phenotype (opsin mislocalization) will be analyzed by immuno-histochemistry.


Research activity codes: 5.2 CELLULAR AND GENE THERAPIES
Health categories: EYE
Title: Investigating age-dependent regulation of complement in human eye tissues: implications for Age-related Macular Degeneration
Funder: Medical Research Council

Abstract: Dysregulation of the complement system is thought to underlie Age-related Macular Degeneration (AMD), where the Y402H polymorphism in the complement factor H (CFH) gene is a major risk factor for this common form of blindness. Our previous studies have shown that this polymorphism adversely affects the interaction of the disease-associated 402H variant with sulphated glycosaminoglycans (e.g. heparan sulphate (HS) in the human Bruch's membrane), which might result in local chronic inflammation promoting the initiation and progression of tissue damage within the macula. More recently we have obtained preliminary evidence that age-related changes in Bruch's membrane (e.g. in HS composition) may specifically influence the binding of CFH 402H. On this basis, we have hypothesised that normal changes in the macula occurring during ageing contribute to the development of AMD, in particular in individuals carrying the CFH 402H allele. Therefore, the aim of this research is to test this new hypothesis by analysing human eye tissues from donors of different ages (i.e. 30-80+ years); we will characterise how the Bruch's membrane changes with age and how this influences the functions of CFH (in a 402H/402Y allotype-specific manner). This multidisciplinary project will involve immunofluorescent microscopy, HS compositional analysis, mass spectrometry, complement assays, affinity chromatography, microtitre plate-based binding assays and biophysical techniques; here we will utilize the human eye samples we have collected (over the last 3 years), with additional samples to be provided by the Manchester Eye Bank. The project is focused at understanding early changes in the human eye that may be responsible for the initiation/progression of AMD, rather than the later stages, i.e. once tissue damage has occurred. In this regard, our studies have the potential to facilitate the design of improved treatments for AMD, which could potentially be targeted early in the disease process.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: EYE
Title: Developing gene therapy to treat blindness caused by Stargardt Disease
Funder: Medical Research Council

Abstract: ABCA4 is a protein expressed in photoreceptor cells which is vital for normal photoreceptor function. Mutations in ABCA4 are the commonest cause of monogenic inherited retinal degeneration and usually present in childhood. Although gene therapy with AAV vectors has been shown to be safe and effective at photoreceptor gene replacement, the ABCA4 coding sequence at 6.7 KB is known to be too large to be packaged effectively into standard AAV vectors. We have recently developed an AAV transgene sequence which includes the full length ABCA4 and various regulatory elements to enhance gene expression. We have developed a method of packaging this transgene in two parts into AAV5 and AAV8 capsid serotypes. Through a complex method of intracellular DNA recombination, transduction with these vectors leads to effective expression of ABCA4 protein in human cells in vitro. We wish to take these vectors along a developmental pathway that would result in a Phase 1 clinical trial to treat Stargardt disease. We have a considerable amount of experience with the processes required by GTAC and the MHRA in gaining clinical trial approval for AAV gene therapy First in Man studies. The method of delivery of the ABCA4 transgene is technically challenging and several steps are now required to optimise vector efficiency and the targeting of photoreceptors. We propose to test several compounds which may improve the efficiency of recombination within photoreceptors and would therefore reduce the total vector dose administered to each patient. We will also confirm functional activity of the ABCA4 protein in the knockout mouse in our laboratory. Whilst lentiviruses provide an alternative way of delivering large genes, these vectors have additional safety concerns for human use, such as the risk of insertional mutagenesis and also an increased risk of immune reactions. AAV vectors in contrast have been shown to be safe and effective for human retinal gene delivery.


Research activity codes: 5.2 CELLULAR AND GENE THERAPIES
Health categories: EYE
Title: Investigation of factors that prevent endogenous regeneration of the retina by Müller stem cells
Funder: Medical Research Council

Abstract: Degeneration of retinal neurons is the ultimate cause of blindness resulting from prevalent conditions, such as age related macular degeneration (AMD), diabetic retinopathy (DR), end stage glaucoma, retinal detachment and retinitis pigmentosa. Although extensive research is being undertaken to replace damaged retinal neurons by neural stem cell transplantation, stimulation of endogenous proliferation and neural differentiation of retinal stem cells would be a preferred alternative to the transplantation approaches currently being investigated. The research therefore aims to identify factors that may inhibit regeneration of diseased retina, and whose expression may be potentially altered to promote the endogenous proliferation and neural differentiation of Müller stem cells in situ. Using proteomic approaches, we will investigate the total protein and cytokine expression profile of fragments of human gliotic retina, surgically removed as part of the treatment for complicated retinal detachment, and will compare this profile with that of normal cadaveric retina. Factors selectively upregulated or downregulated in gliotic retina will be examined for their ability to inhibit or promote Müller stem cell growth and differentiation in vitro. Candidate factors found to be active on the inhibition or promotion of these cell functions, will be further examined for their ability to block or promote stem cell activities using agonist or antagonist molecules for these factors. The work will be undertaken in collaboration with Prof. Sun Xinghai and Dr Lei Yuan from the Eye Hospital at Fudan University, Shanghai. The proteomic analysis will be performed at the UCL Institute of Ophthalmology and is expected to identify candidate factors which can be examined by both the UK and the Fudan group on Müller stem cell growth and differentiation. The project will form part of a PhD training program for a Chinese student who will visit the UCL Institute of Ophthalmology for a year.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: EYE
Title: Modulation of limbal niche stiffness to regulate stem cell differentiation
Funder: Medical Research Council

Abstract: We seek to prove that it is largely the mechanical properties (stiffness) of the limbal niche which maintains stem cells in an undifferentiated state and that disease/wounding affects the niche's mechanical properties prohibiting stem cell survival and secondly that we can alter the niche's mechanical properties by precise delivery of collagenase, using a medically approved hydrogel, prior to stem cell therapy. This study is important because a) it is the first to investigate the mechanical properties of a stem cell niche, b) it is the first to consider modification of tissue stiffness as an adjunct to stem cell therapy and c) the anticipated treatment is simple, cheap and applicable to a wide range of stem cell therapies. We have previously shown that the mechanical properties of a collagen substrate can be tuned to enable differentiation of limbal stem cells. Similarly we have found that collagenase can reduce the substrates stiffness in a dose dependent manner producing an improved level of undifferentiated limbal stem cells. We propose to gauge the impact of elastic modulus on limbal stem cell niche function and whether the precise application of collagenase as a surgical pre-treatment can confer a significant improvement to the maintenance of undifferentiated limbal stem cells in culture. The elastic-modulus of the cornea will be measured by the application of acoustically induced inelastic light scattering (Brillouin microscopy) that allows non-contact, direct readout of the corneas mechanical properties. The translational outcome of this proposal would be the attainment of critical safety and efficacy data required for a first-in-man trial of collagenase for the restoration of the limbal stem cell niche (collagenase is already used clinically in skin wound management).


Research activity codes: 5.2 CELLULAR AND GENE THERAPIES;5.4 SURGERY
Health categories: EYE
Title: Development of biological and synthetic substrates for the ex-vivo expansion of conjunctival epithelium for ocular surface reconstruction
Funder: Medical Research Council

Abstract: Aim: To develop a novel tissue-derived and synthetic substrate with novel surface chemistry to support the ex-vivo expansion of conjunctival epithelium. Objectives 1. Develop a range of expanded polytetrafluoroethylene (ePTFE) substrates with novel surface chemistry through gas plasma treatment and adsorption of proteins including fibronectin and laminin IV. Determine the surface chemistry through x-ray photoelectron spectroscopy and contact angle analysis. 2. Optimise the decellularisation process for human conjunctiva by determining acellularity using a quantitative DNA assay. 3. Determine the optimal substrates by propagating a human conjunctival cell line and determine adhesion/density (quantitative LDH assay), morphometric analysis, and phenotype of the resulting cell populations. 4. Propagate primary conjunctival cells on the optimised substrates and characterise their phenotype. Perform more detailed analysis including immunohistochemistry, histology and confocal analysis. 5. Determine tensile strength of the synthetic and biological cultured constructs. Cell phenotype will be analysed by immunocytochemistry and flow cytometry to quantify cell populations using the following cell markers: CK4/13/8/19 (conjunctival epithelial surface markers); connexin 43 (gap junctional protein); involucrin (terminally differentiated cells); beta integrins; caspase 3 (apoptotic cells); MUC 5AC/CK7 (goblet cells); Ki67, P63alpha, ABCG2 (progenitor cells). Scientific and medical opportunities This study will lead to biological and synthetic cultivated constructs for use as grafts for ocular surface reconstruction. This could profoundly improve clinical outcomes in patients with blinding ocular surface diseases. These developments will also represent novel developments in the field of regenerative medicine with applicability to many diseases in which cellular replacement therapies may hold the only hope for cure.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: EYE
Title: Cognitive, behavioural, environmental and genetic associations of myopia in the Twins Early Development Study
Funder: Medical Research Council

Abstract: Aim: To examine the environmental and genetic factors for myopia using the Twins Early Development Study (TEDS) Objectives: 1. Obtain a prevalence estimate of school-onset myopia in the UK 2. Examine the relationship between refractive error and educational, behavioural and cognitive traits 3. Determine myopia heritability at age 18 and investigate the relative effect of genes and environment 4. Perform a genome wide association study (GWAS) for myopia at this age 5. Examine the role of common variants identified from adult GWAS in myopia 6. Assess gene-environment interactions in myopia Methodology: Refractive data, obtained from 5,000 of the 18-year-old TEDS cohort using optometry assessments, will enable a current estimate of myopia prevalence in this age group. Measures of cognition, behaviour and education over childhood development, uniquely studied by TEDS researchers, will allow detailed exploration of environmental influences on myopia. Quantitative genetic techniques (maximum likelihood, multivariate structural equation twin models) will be used to investigate heritability and whether genetic influences on myopia and intelligence, and other factors, are shared or separate. GWAS will be used to search for novel common genetic variants for myopia in this age group and the role of variants identified in older populations will be tested. Identified genetic polymorphisms and high quality data on environmental factors during childhood, will enable examination of gene-environment interactions. Scientific and medical opportunities: This project will provide data, currently unavailable, on myopia prevalence in the UK young adult population, and a unique opportunity to explore aetiological factors in this age group. Exploring environmental influences on myopia, together with novel or replicated genetic variants, and their interactions, will have significant implications for future treatment development to slow progression and visual impairment.


Research activity codes: 2.3 PSYCHOLOGICAL, SOCIAL AND ECONOMIC FACTORS;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: EYE