Ophthalmology abstracts

The aim of this node is to bring together all the stakeholders in Nucleic Acid Therapeutics (NAT) preclinical and clinical development, engage academic, clinical and industry research and patients, charities and regulatory to establish a national network on NAT and accelerate its preclinical development and clinical translation. Of the estimated ~7,000 rare diseases, 75% affect children, and 80% have a genetic cause. Given the rapid advances in sequencing technologies, coupled with reducing sequencing cost and the implementation of the NHS Genomic Medicine Service, it is becoming increasingly common for rare disease (RD) patients, and their respective physicians, to know the genetic causes of their condition. However, disease modifying therapies are yet to be developed for the vast majority of RDs, resulting in significant morbidity, mortality, and socioeconomic burden. Development and delivery of innovative treatments with new strategies to tackle the genetic causes will overcome the paucity of effective therapies for rare childhood diseases.

People with AMD slowly lose their ability to read, recognise faces and see things in fine detail. There are 600 000 people in the UK with the late stage of the disease, but many more with early disease have poor quality vision and no hope of preventing future vision loss. Superficially we understand what happens in AMD. Providing critical metabolic support for the retina is a single layer of cells, called retinal pigment epithelium (RPE). In AMD the RPE cells die in localised patches, known as Geographic Atrophy or late-stage dry AMD, accompanied by loss of photoreceptors (the retinal cells that receive the light and turn it into the electrical signal the brin uses for vision). The process may also trigger abnormal blood vessel growth, which is called wet AMD. Wet AMD can be partially suppressed with drugs. However, despite decades of research, the cause of the RPE cell loss (dry AMD) is still an enigma and there is no cure.

CLate-Onset Retinal Degeneration (L-ORD) is a rare, autosomal dominant disease that causes blindness due to mutations on C1QTNF5. Most patients carry a single founder mutation (S163R), traced back to Scotland. The mutation causes aggregation and deposition of C1QTNF5 in the Retinal Pigmented Epithelium (RPE), leading to retinal degeneration and vision loss (Hayward et al. 2003). Currently, there are 15 L-ORD patients registered in Moorfields Eye Hospital (MEH), and over 140 published cases (Borooah et al. 2022), whose descendants have a 50% chance to develop the disease due to its dominant trait. While there are no available treatments specifically targeting LORD, the late onset of disease at 40-50 years of age provides a broad window to use preventive therapies. We have developed a CRISPR/Cas9 gene editing approach using patient derived induced pluripotent stem cell (IPSC) RPE. Here we aim to recover C1QTNF5 function, by specifically removing the mutant gene using a dual cutting approach targeting unique single nucleotide polymorphisms (SNPs) on the dominant allele. As a founder mutation, we may be able to develop a single cost-effective therapy suitable for the majority of patients. To achieve this, we aim to identify shared targetable SNPs using Next Generation Sequencing (NGS) in our L-ORD patient cohort. If successful, dual CRISPR SNP targeting could be applicable for all autosomal dominant
diseases, exponentially increasing the global market size.

Leakage and angiogenesis of the microvasculature accompany various retinal diseases such as wet age-related macular degeneration (AMD), diabetic retinopathy (DR), macular oedema and retinal vein occlusion. Anti-VEGF drugs are now a first line treatment for these conditions. Although they have delivered real visual improvements for many patients, they do not work for all patients, or do not always maintain treatment efficacy. Therefore, the need remains to identify targets other than VEGF to effectively treat neovascular retinal disease. This project will determine whether pathological changes in protein glycosylation allow Gal-1 to contribute to VEGF-independent microvascular dysfunction.

Fuchs endothelial corneal dystrophy (FECD) is a common, heritable, and age-related eye disease that can lead to bilateral visual impairment. It is characterised by progressive loss of corneal endothelial cells (CECs), eventually causing corneal decompensation, pain and impaired vision. Up to 80% FECD patients harbour at least one expanded allele (≥50 copies) of a CTG repeat element (termed CTG18.1) within a non-coding intronic region of TCF4. FECD is the most common short tandem repeat (STR)-mediated disease characterised in humans. In more comprehensively characterised STR-mediated diseases e.g. Huntington Disease and Myotonic Dystrophy 1, repeat elements are found to increase and contract in length within somatic tissue in both a tissue-specific and age-dependent manner, and this somatic instability influence disease onset and progression. However, somatic instability in FECD has not been thoroughly interrogated and currently we cannot make accurate prediction on risk for developing this common age-related condition and disease severity based on CTG18.1 genotyping.

Currently, the only definitive treatment for FECD is a corneal transplantation. Multiple innovative therapies are being developed to treat FECD pre-emptively, but the long-term success of these approaches will rely on the ability to accurately identify individuals at risk of developing disease. Thus, there is an urgent need of identifying the most accurate genetic biomarkers predictive of disease onset, progression and severity of FECD to allow for personalised medicine.

The retina is the thin layer of neurons and glia at the back of the eye that functions to detect, process, and relay a stimulus of light. However, as one ages, the physiology and structure of the retina begins to degrade, increasing the susceptibility to retinal diseases including glaucoma and macular degeneration. Despite a significant appreciation for the pathopysiological consequences of ageing on retinal health, we do not understand the aetiology of molecular dysfunctions with advancing age. Animals are widely used in many ageing studies, however ethical considerations, long lifespans (> two years) and associated costs limit their feasibility.

To overcome this limitation we propose to establish the African turquoise killifish (Nothobranchius furzeri), as a novel model for retinal ageing. The Killifish is a freshwater teleost with conserved retinal structure and comparatively short lifespan (4-6 months). The killifish also has a sequenced genome, is genetically tractable, and presents retinal degenerations akin to the aged human retina. In this project we will provide a high-resolution cellular and molecular map of the retina throughout the life span, use established transcriptomic datasets to identify conserved dysregulated genetic pathways and manipulate those pathways in a cell specific manner to determine their role in neuroprotections or neurodegeneration in the ageing retina.

Aging is the most important risk factor for AMD and glaucoma yet relatively little is known about the fundamentals of aging in the retina. This lack of understanding contributes to the remaining major unmet clinical need. Killifish provide the unique novel opportunity understand ageing mechanisms in a rapidly ageing vertebrate retina, identify molecular dysregulation in precise cell types and pair genetic mutations, known to cause retinal degenerations, with ageing to determine their additive effects. This will significantly contribute to our understanding of precise mechanisms as to why age-related retinal degenerations occurs. This project will place us as the leaders in the field using this model and allow us to leverage this position to attract further grant funding and interest from industrial partners. Ultimately, understanding disease mechanism is essential for designing targeted therapies to prevent neurodegeneration, and therefore combat vision decline.

The overarching aim of the project is to establish a means of measuring and manipulating ER: melanosome MCS in RPE cells as a basis for more detailed studies on their molecular architecture and function. Specific objectives are:

1. Quantify the extent of ER: melanosome MCS in cellular models ± POS by EM and relate to equivalent data from mouse eye.

2. Develop/validate light microscopy methods for ER: melanosome MCS analysis in human iPS RPE/ARPE19 cells
- Effects of POS treatments on melanosome positioning and association with ER-targeted fluorescent dyes will be examined by confocal microscopy and compared with data from aim 1.
- Effects of POS treatment on MCS extent by proximity ligation assay (PLA)

3. Determine the role of candidate regulators of ER: melanosome MCS. MCS will be measured following siRNA-mediated depletion or inhibition of eight key candidate MCS regulators.

Dbl3 is essential for RPE function and retinal structure. Dbl3 regulates cytoskeletal remodelling to stimulate epithelial polarisation and RPE phagocytosis. Its overexpression in animal models of retinal degeneration attenuates disease progression by mechanisms that are only partially understood.

Our aims here are (1) to extend the knowledge of Dbl3 functions in the RPE to support the further therapeutic development of Dbl3-based gene therapy, and (2) to establish how Dbl3 signalling regulates mechanical properties of the RPE and responds to pathological stimuli.

We hypothesize that Dbl3 signalling controls RPE maintenance and function by regulating cytoskeletal organisation, cell surface polarity and protein secretion.

Our objectives are to employ gain and loss of function approaches
1) to determine the role of Dbl3 pathway components in RPE polarisation and monolayer structure using differentiated and deficient RPE cell models
2) to establish the role of Dbl3 signalling in polarised secretion and epithelial barrier function
3) to investigate interplay between apical and basal actomyosin activation and the importance of an apical/basal actomyosin gradient for RPE polarity
4) to identify how Dbl3 signalling regulates monolayer tension and responds to mechanical and inflammatory stress
5) to establish the relevance of Dbl3 signalling for RPE polarity in vivo.

We aim to determine how lysosomes of the retinal pigment epithelium (RPE) respond to the remarkable daily influx of photoreceptor outer segment (POS)-containing phagosomes, and to the accumulation of autofluorescent POS-derived material that occurs in ageing and diseased retinae. Our ultimate goal is to identify targets for therapies aimed at limiting the accumulation of POS-derived debris in the retina in age-related retinal degenerative diseases such as AMD. We will:
i) Characterise interactions between lysosomes and phagosomes in healthy RPE in vitro and in vivo and between lysosomes and autofluorescent POS-derived granules in in vitro models.
ii) Determine whether lysosome biogenesis is upregulated in response to phagocytosed POS or accumulation of autofluorescent POS-derived material in cultured RPE.
iii) Determine whether lysosome biogenesis is upregulated in vivo in response to circadian POS phagocytosis.
iv) Determine whether manipulating lysosome biogenesis modulates POS degradation, accumulation of autofluorescent POS-derived granules or the resolution of preformed autofluorescent granules.

These studies will increase our understanding of phagosome: lysosome interactions in health and when phagosome degradation is compromised. They will establish the importance of transcriptional regulation of lysosome biogenesis in phagosome processing and whether this may be exploited to limit accumulation of toxic POS-derived material in the retina.

Aims
1. To identify and quantify common mental health difficulties in a large cohort of teenagers (age 13-19) and young adults (20-30 years) with vision impairment.
2. To explore predictors of mental health outcomes (risk and resilience factors) including characteristics of vision loss, socioeconomic and demographic variables.
3. To develop a service user-designed package of supportive interventions for people with vision impairment, structured around the needs of people with lived experience.

Objectives
a. To use standardised instruments to assess depression, anxiety, post-traumatic stress disorder and day-to-day functioning in 200 young people (100 teenagers, 100 young adults), recruited from Moorfields’ low vision clinic.
b. To explore associations between mental health symptoms / caseness and key predictors: (i) level of vision impairment (visual acuity, visual field); (ii) time of onset of vision loss; (iii) the impact of visual impairment on visual task ability (using standardised instruments); (iv) socio-economic background; (v) age, (vi) gender; (vii) ethnicity; and (vii) previous access to peer support or mental health services.
c. To identify the optimal support desired by people with vision impairment and depression, anxiety or PTSD, using techniques in which people with vision impairment will be ‘experts by experience’.

We will apply cutting-edge imaging technologies to study retinal immune responses to AAV using the mouse. Through a highly multiplexed immunohistochemistry technique called IBEX, we will define cellular alterations in retina and draining lymph nodes at a deeper level than ever before. It will be complemented by high resolution imaging of the dynamic interactions between immune cells and AAV-infected retinal cells in living mice. Using a genetically engineered ‘JEDI’ mouse strain, we can trigger and differentiate responses to AAV capsid versus transgene, to model what is seen in the retina of patients. We will examine the effects of dose or route of administration and test different modifying treatments including steroids and novel depleting antibody injections. Through this project , several widely held assumptions across the field of gene therapy can be tested and the information would be of direct translational relevance to ongoing clinical trials. The richer data obtained using a spatial biology approach will also advance our fundamental knowledge of immune responses in the eye and identify potential avenues for controlling them. 

To improve diagnostics for IRD genetic testing by:
1. Identifying and characterising VUS including non-coding and mutations in patients unsolved by WGS clinical testing. Variants will be selected for blood RNA derived transcript analysis. The functional data will provide the evidence needed to upgrade classification and provide a molecular diagnosis for >20 patients. This will inform current and future genomic studies and clinical diagnostic services.
2. Establishing ONT-SMS for genomic regions not covered by WGS and to enable haplotyping and variant phasing with long-reads. The OPN1LW/OPN1MW gene array and ABCA4 gene will be targeted for Cas9 Targeting of Chromosomal Segments (CATCH) ONT-SMS. We will establish the methods and pilot the assays for a cohort of patients.
3. Investigate the use of adaptive ONT-SMS for retinal gene panel sequencing. Contrary to Illumina based short-read sequencing and gene panel analysis, this will enable phasing of distant variants, characterisation of structural rearrangements and capture of variants in low complexity regions (eg: RPGR). The aim is to pilot this method for future use in clinical diagnostics.

In this collaborative project, Prime Medicine (PM) will generate novel compounds, including tool Prime Editors, to be evaluated, by both teh Davidson lab at UCL and PM, in assays utilizing FECD patient-cell and explant cultures. In turn, UCL will provide patient-derived cells, assay protocols and know-how to enable Prime Medicine to establish FECD patient-derived cellular assays for use in screening novel TCF4 gene correcting prime editors. These collaborative studies will generate data that can be used to determine the therapeutic potential for Prime Medicine’s TCF4-correcting prime editors.

Macular degeneration remains the leading cause of blindness in the developed world, and there are to date no treatments to reverse its effects on vision. In its end-stage, it develops with roughly equal probability into either a ‘wet’ exudative condition or a ‘dry’ geographic atrophy. The wet form can be somewhat modelled in mice by inducing choroidal neovascularization, for example by laser lesions. Instead, we lack an animal model of dry AMD. This limits the understanding of the pathology, as well as the testing of therapeutic candidates. We propose to develop a mouse model of geographic atrophy, by depleting RPE cells in a patch of the mouse retina. We will achieve this by expressing the molecule DTA in an AAV vector, which will be delivered subretinally in the mouse eye. The focal depletion of RPE cells, which is known to occur in dry AMD, will allow us to study the survival of different photoreceptor cells with this region. We will compare these results with available data from human geographic atrophy in order to evaluate the quality of our model. In the future, this approach could be extended to testing molecules and gene therapy vectors for the treatment of dry AMD.

We propose to replace and combine our current Phoenix Micron III and Bioptigen SD-OCT system, with the latest Micron IV system. The fully operational system will provide a single unified mouse and rat imaging platform. It will be able to perform colour imaging of both the retina and cornea, fluorescein angiography, OCT, laser CNV and electroretinography. The additional speed, reliability and ease of use will accelerate the progress of many research programs studying eye diseases at Moorfields.

Diabetes and its complications cause a significant public health burden in Sri Lanka. Blindness and visual impairment due to diabetic retinopathy is one of the key complications that needs further attention as it is an avoidable condition with adequate screening and timely treatment. Out of the 15.4 million adults in Sri Lanka, it is estimated that 2.3 million have diabetes and there is no way of stratifying the people most at risk of blindness to offer immediate treatment. Therefore, in this study we aim to develop and validate a diagnostic model which can then be used as a screening tool in mass screening of diabetes patients to identify, educate and/or triage those who are at risk of diabetic retinopathy and thereby delay/prevent visual impairment.

We propose to apply newly developed approaches and tools to test a new model of how spatial cytoskeletal remodelling orchestrated by apical Cdc42 signalling drives epithelial morphogenesis and regulates gene expression and epithelial homeostasis. The expected results will enhance our understanding of the principles and molecular mechanisms involved in epithelial tissue morphogenesis and homeostasis, and impact on future work on a wide range of proliferative, degenerative and age-related diseases affecting the retina and other tissues and organs.

Support will allow us to construct this platform by funding the required THUNDER Imager.

This project will reveal the collective function of one million neurons in the mouse cerebral cortex by pairing a transformational new imaging method to powerful assays of behavior and neural function that we have perfected. Brain function is organized by the collective firing of a myriad neurons working in concert, but understanding this organization has been hampered by our inability to measure the concurrent activity of a sufficient number of neurons. This limitation can now be overcome thanks to a transformational new method of 2-photon imaging developed in the USA, called light beads microscopy, which can image the activity of one million neurons simultaneously in the brain. First, we will implement this method and image the activity of a million neurons in the mouse visual cortex during visual behavior, providing an unprecedented understanding of their collective function. Second, we will use our techniques of rabies tracing to relate the activity of a single neuron with that of thousands of presynaptic and neurons across all areas of the visual cortex. Third, we will go beyond visual cortex and image the activity of all presynaptic boutons, coming from the whole brain, impinging of the dendrites of a single cortical neuron, and make causal manipulations to understand the laws of integration of synaptic inputs. The results will provide an unprecedented view onto the collective function of a myriad neurons in the brain, providing an experimental test of fundamental theories of brain function.

Aims of the PhD: This project will develop new MRI and functional testing methodologies to improve our understanding of the neurofunctional profile of inherited retinal disorders (IRD), leading to better prognosis, application of therapeutics and more reliable outcome metrics for clinical trials. Four aims of the PhD will be:
(1) establishing a set of age-appropriate tests suitable for clinical trial endpoints.
(2) use these tests to characterise the early signs of disease
(3) determine disease progression in the paediatric cohort providing insight into prognosis
(4) assess for novel genotype-phenotype correlations

Age-related macular degeneration (AMD) is a leading cause of vision loss in the ageing population. AMD occurs due to progressive degeneration of the light-sensitive cone photoreceptor cells within the central retinal region known as the macula. Cone loss causes irreparable vision impairment, including visual acuity decline, colour vision disturbances, and central blind spots. Currently, there are no effective treatments for preventing AMD progression or resulting vision loss. Development of effective treatments relies on a thorough understanding of underlying causes of cell dysfunction and degeneration. Animal models are required to gain this insight, however many available models do not accurately recapitulate AMD. African turquoise killifish are a model of accelerated ageing that naturally develop AMD-like retinal degeneration. This project aims to utilise the killifish model to decipher the mechanisms underlying cone death in the ageing visual system. Many fishes related to killifish have a macula-like region with increased cone density compared to peripheral retina; killifish will be investigated for a similar high-acuity area. Killifish retinas will be examined throughout their short 6-month lifespan to determine when ageing degeneration occurs and assess AMD-like manifestations, including subretinal lipid deposits and inflammation. TIMP3 is an AMD-associated gene of unclear function during AMD progression. TIMP3 levels will be increased in killifish retinas to exacerbate disease and evaluate its role in the AMD retina. Finally, the neuroprotective gene BDNF will be overexpressed in the killifish retina to determine whether increased BDNF can alleviate age-related retinal disease and act as a potential AMD therapeutic. This work provides the unique opportunity to develop killifish as a retina ageing model, determine the cellular mechanisms that lead to AMD, and assess how healthy retinal ageing can be promoted in a model of age-related retinal degeneration.

We will evaluate best practices in curating datasets for training efficient and ethical clinical AI models, and explore the factors influencing performance in retinal image classification by clinicians with AI-based decision support.

Plate readers use different modes to automatically detect, measure and quantify light emitted by biological samples. Plate reader modalities include the measurement of absorbance, fluorescence and luminescence. Typical applications include routine functions like protein and nucleic acid quantification, microbial growth assays, cell viability assays, reporter assays and enzyme activity assays to more complex functions (e.g. fluorescence and luminescence assays to measure molecular interactions on a microscopic scale). Plate readers are therefore a core instrument in a laboratory setting that are routinely used in experimental assays to underpin fundamental research from probing basic molecular or cellular functions to clinically or therapeutically relevant screening approaches. Current plate readers at the Institute include a Berthold luminometer (luminescence), a Tecan Safire (absorbance and fluorescence) and a BMG Labtech FLUOstar Optima (absorbance, fluorescence and luminescence). However, these machines are reaching the end of their lifespan (17-20 years), and should they break down, are irreparable. This would have an immediate and significant impact on the ability of researchers at the Institute to conduct their experiments. It is thus critical that we replace our current plate readers with a new, modern multimodal plate reader.

A new multimodal plate reader will facilitate experiments that lead to new scientific discoveries, therefore impacting a wide range of research relating to the different cells and tissues of the eye (e.g. lens, cornea, retina, choroid, retinal pigment epithelium) and eye diseases (e.g. inherited retinal degenerations, corneal dystrophies, age-related macular degeneration, diabetic retinopathy and uveitis). To name but two of many examples, advanced multimodal plate reader functions will be used for the development of new therapies to measure interactions of novel drugs with their cellular target protein and for quantitative assessment of the effect of non-coding genetic variants on gene transcription.

Our long-term objective is to use information about gene loci identified genetic studies (GWAS) to be associated with increased intraocular pressure (IOP) and the development of glaucoma to develop algorithms to predict genetic tendency to raised IOP via trabecular meshwork pathology or/and SC/collector pathology in order to sub-classify patients into groups that will respond differentially to certain treatments and to identify new molecular mechanisms underlying IOP increases as a basis for new therapies.

This application focuses on the first steps of this research programme: the development of the necessary experimental approaches and workflows to study the biological functions of identified genes and to determine how they cooperate in pathophysiologically important processes. Our aims are

1) to use existing GWAS, expression, and biological function data to predict hypothetical functional networks of genes that may cooperate in specific cell types to regulate IOP, generating a framework into which future data can be integrated;
2) to establish in vitro systems modelling trabecular meshwork and Schlemm’s canal cells (collaboration Derryl Overby);
3) to analyse the function of two Rho GTPase activators associated with glaucoma to establish workflows required for gene analysis and as a first step towards identification of new disease-relevant molecular mechanisms.

Our cells contain structures (organelles) that perform essential functions e.g., energy production. Transport is vital for organelle function and defects cause diseases e.g., neurodegeneration and cancer. Transport is driven by the cytoskeleton, a system of proteins that act as motors and tracks. Two types of tracks; microtubules and actin filaments, are thought to co-operate to regulate fast long-distance and slow local transport, respectively. However, we recently discovered a suite of proteins, conserved among animals, that empower short actin filaments to deliver fast long-distance transport. This project will determine how this occurs and thus will address a fundamental biological question.

The aim of this program is tailored towards understanding mechanism of disease and development of a therapeutic intervention, building on our recent discovery that elucidated the genomic cause of the previously unexplained locus for dominant retinitis pigmentosa (adRP) type 17. In this program, we propose three interconnected modules to classify SVs as disease-causing or benign, reveal the mechanism of altered gene regulation in patient-derived photoreceptor cells, create in vivo models of RP17 and assess GDPD1 toxicity, and to combine this knowledge to test different therapeutic approaches to intervene in disease for RP17-affected individuals.

The retinal pigment epithelium (RPE) is a single-layered epithelium that forms the outer blood/retinal barrier is essential for survival and function of the neural retina. RPE degeneration leads to major disease that cause reduced or loss of vision, such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy. The healthy RPE is non-proliferative and requires effective stress-response mechanisms to remain functional. This project focuses on such a stress-response mechanism that we have originally identified to link signalling mechanisms connected to cell-cell junctions with tissue integrity. Recent transcriptomics data suggest that the central component of this mechanism, Apg-2, is downregulated in the RPE of patients suffering from early AMD. Pilot studies indicate that downregulation of Apg-2 leads to activation of inflammatory signalling mechanisms and induction of cellular senescence, a signalling mechanism that includes the transcription factor ZONAB and that leads to a similar senescence phenotype in endothelial cells. We propose to determine the function, regulation and disease-induced deregulation of Apg-2 and ZONAB in the RPE and endothelial cells.

Our understanding of brain function is fundamentally limited by our inability to read out the collective firing of vast populations of neurons. This collective firing constitutes a code that we have been able to read out only in tiny fragments. Fortunately, a transformative new technology has now appeared that greatly increases our ability to read the neural code: the Light Beads Microscope (LBM), which allows recordings from ~1 million neurons in the cerebral cortex (Demas et al, Nature Methods, 2021). This feat is unprecedented: the previous record, obtained with standard two-photon microscopy, was 50,000 neurons. The LBM is a modified two-photon microscope that images an entire volume in the time it takes to scan a plane. It was developed in the laboratory of our collaborator Alipasha Vaziri at Rockefeller University, and will next be deployed in key laboratories across the United States. Thanks to our close collaboration with the Vaziri laboratory, we have the opportunity to deploy the first such device outside the USA. We will install it in our laboratory at UCL, where we will provide ample access to UK colleagues to perform, pilot, or observe transformational experiments that would not have been possible even a year ago. An LBM is a combination of three components: (1) a specialized source of brief, frequent, and strong light pulses; (2) a multiplexing module that sculpts these pulses into a line of beads; (3) a two-photon mesoscope that projects this line vertically and scans a horizontal plane, thus imaging a volume. We have already secured the second and third items thanks to separate funding, and are here requesting funds to purchase the first item, the light source: a powerful laser feeding into a high-repetition-rate femtosecond Optical Parametric Amplifier (OPCPA) system. With this light source, we will obtain a transformative new microscope that will allow unprecedented volumetric measurements of the activity of ~1 million neurons in the living brain. At higher magnification, such volumetric measurements will also provide unprecedented measurements at subcellular scale, for instance to reveal the activity of the entire dendritic tree of a single neuron.

The retinal pigment epithelium (RPE) is a single-layered epithelium that forms the outer blood/retinal barrier is essential for survival and function of the neural retina. RPE degeneration leads to major disease that cause reduced or loss of vision, such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy. The healthy RPE is non-proliferative and requires effective stress-response mechanisms to remain functional. This project focuses on such a stress-response mechanism that we have originally identified to link signalling mechanisms connected to cell-cell junctions with tissue integrity. Recent transcriptomics data suggest that the central component of this mechanism, Apg-2, is downregulated in the RPE of patients suffering from early AMD. Pilot studies indicate that downregulation of Apg-2 leads to activation of inflammatory signalling mechanisms and induction of cellular senescence, a signalling mechanism that includes the transcription factor ZONAB and that leads to a similar senescence phenotype in endothelial cells. We propose to determine the function, regulation and disease-induced deregulation of Apg-2 and ZONAB in the RPE and endothelial cells.

The aims of this project are to:

1. To quantify the impact of inherited macular disease (IMD) on vision-related quality of life, mental health, and wellbeing
2. In collaboration with people with inherited macular disease, to develop a user-led integrated support programme for people with IMD,
3. To evaluate the impact of this integrated support programme for the first year after diagnosis of inherited macular disease.

The overarching aim of this work is to develop thin electronic SmartPatches embedded into current treatment patches, which will provide feedback to parents/carers and children about the daily patching dose they have achieved. This may improve adherence, shorten overall treatment duration, and improve visual outcomes. 

Over the last years I identified a pathway through which focal loss of photoreceptors affects remaining vision. This pathway involves laterally projecting inhibitory cells, particularly H1 horizontal cells, and is particularly relevant for loss of central vision, such as following age-related macular degeneration. Building on this finding, I developed a gene therapy approach to restore this lateral input and improve vision. During the period funded by the Moorfields Eye Charity Springboard award, I want to investigate whether this therapeutic approach improves image forming vision. I aim to combine behavioural and functional techniques in mice to measure the improvement in complex visual processing following restoration of H1 lateral input

The primary aim of our research is to further our understanding of the molecular mechanisms that permeability factors utilise to induce leakage in the retina. To identify which calcium signals initiate permeability will achieve two goals: 1) it will uncover new therapeutic targets and possibly find a common leackage meadiator to all permeability factors, allowing to treat a wider percentage of patients; 2) It will Improve specificity of the anti-VEGF theapies that will target permeability specifically leaving other physiologically essential roles of VEGF-A intact

With Biomed Engineering at UCL, we have developed the technology to view mitochndrial respiration and oxygenated and deoxygenated blood flow in the choroid of the retina. This uses real time, non-contact cutting edge infra-red spectroscopy in the analysis of optical signals hidden in reflected light from the back of the eye. In mouse models these signals clearly differentiate retinal age and also reveal metabolic retinal decline long before pathology is present. This project will translate this technology to the human situation providing a direct metric of critical use in the clinic. This will be the first time choridal flow amd mitochndrial respiration have been viewed directly in the human eye. As both are key agents in disease, early detection of changes in them is of paramount importance.

Dominant optic atrophy (DOA) is the most common inherited optic neuropathy and is caused by mutations within
the OPA1 gene. This studentship is focused on investigating the potential of CRISPR activation (CRISPRa) to
enhance levels of OPA1 and restore mitochondrial homeostasis. This will be achieved through the following
goals:
1. Optimisation of CRISPRa mediated upregulation of OPA1 in cell culture models
2. Investigation of OPA1 upregulation in DOA iPSC-derived retinal ganglion cells (iPSC-RGC)
3. Testing if OPA1 upregulation can rescue mitochondrial dysfunction
These goals will highlight the potential of CRISPRa to treat DOA, mitochondrial dysfunction and
haploinsufficiency genetic disease.

Photoreceptors are sensory devices of the eye that help convert light to vision. Photoreceptors become damaged due to light exposure. The damaged outer segment portions are shed when we open our eyes every morning and 'eaten' by retinal pigment epithelial (RPE) cells facing them by a process called phagocytosis, which allows photoreceptors to renew themselves. The damaged material is then passed to a destruction machinery called lysosomes that clear the material from the RPE to keep the RPE healthy. In age-related diseases of the eye, the material taken in by the RPE is not cleared efficiently. Since the regulatory mechanism that ensures efficient clearance of internalizing material is unknown, we cannot design effective therapeutic tools to repair this process. The programme of study aims to identify the precise components that ensure engulfed material passes onto the clearance machinery efficiently and to gain insight into how they work together to form a mechanism that can be therapeutically targeted in patients with retinal disease. Primary RPE cells will be used to identify components of the clearance mechanism by functionally supressing each candidate protein, using a gene knock down library screen approach. A novel 3D in vitro model system will be set up in this programme, comprising RPE cells attached to an engineered 3D gel encapsulated vascular layer representing the RPE-Choriocapillaris (CC) complex of the eye. This model mimics key features of normal and AMD patient eyes, that will enable characterization of how the identified regulatory mechanism is dysregulated in disease and testing novel therapeutic targeting approaches.

Defective lung vascular development causes congenital lung disease with significant perinatal morbidity and mortality but also predisposes to adult lung disease. Therefore, elucidating the molecular and cellular mechanisms that underlie lung vascular development should increase our understanding of the aetiology of such conditions and help identify molecular targets for diagnosis and treatments. However, specific knowledge of lung vascularm development and its integration with airway architecture and epithelial branching patterns remains scant. Further, for many molecular pathways identified in model organisms, the relevance to human lung development and therefore disease remains poorly established. Here, we propose to analyse lungs from genetically engineered model organisms to systematically define roles of SEMA3 signalling in lung vascularisation, epithelial branching and epithelial endothelial cross talk. Further, we will apply this knowledge to examples of human congenital lung disease. Thus, we will increase our understanding of the mechanisms that govern lung development and identify candidate molecular pathways that may serve as diagnostic or therapeutic targets.

Our growing understanding continues to accentuate the central role of the immune system in almost every aspect of health and disease. Beyond autoimmunity, allergy and infection, dysregulated immunity is seen universally from dementia to atherosclerosis to cancer. New immunotherapies such as checkpoint inhibitors are delivering stunning outcomes for cancer that illustrate the potency of the immune system. The COVID-19 crisis has also acutely demonstrated our incomplete knowledge of immune system responses, to either emerging viruses or the vaccines to counter them.”

Genetically unexplained disease, also known as missing heritability, in human diseases represents a major challenge. More than 25 distinct genetic causes of inherited corneal disease (ICD) have been characterized to date; however, a considerable number of ICD patients remain genetically unsolved. This studentship aims to elucidate the genetic causes of ICD in a genetically pre-screened subset of patients who lack a molecular diagnosis. The student will utilise recently generated corneal-specific omics datasets derived from healthy and patient tissues, to design bespoke bioinformatic pipelines to interrogate genomic sequencing data to enhance mutation detection rates and identify novel genetic causes of ICD."

Defective lung vascular development causes congenital lung disease with significant perinatal morbidity and mortality but also predisposes to adult lung disease. Therefore, elucidating the molecular and cellular mechanisms that underlie lung vascular development should increase our understanding of the aetiology of such conditions and help identify molecular targets for diagnosis and treatments. However, specific knowledge of lung vascularm development and its integration with airway architecture and epithelial branching patterns remains scant. Further, for many molecular pathways identified in model organisms, the relevance to human lung development and therefore disease remains poorly established. Here, we propose to analyse lungs from genetically engineered model organisms to systematically define roles of SEMA3 signalling in lung vascularisation, epithelial branching and epithelial endothelial cross talk. Further, we will apply this knowledge to examples of human congenital lung disease. Thus, we will increase our understanding of the mechanisms that govern lung development and identify candidate molecular pathways that may serve as diagnostic or therapeutic targets.

Animals constantly make decisions, such as how to respond to a threat or where to look for food. Critically, the same external environment can drive different decisions on different occasions, even in the same animal: thus, the animal’s internal state interacts powerfully with external inputs to determine decisions. Our goals are to understand: how internal states influence decision-making behavior; what are the neural bases of these effects; and how these mechanisms change during learning.

The project focuses on investigating the missing heritability of inherited eye disease. We are performing studies to identify novel genes, investigate candidate non-coding variants, develop tools to aid interrogation of NGS intractable genes and functionally test cryptic splice variants.This will provide a molecular diagnosis where otherwise not possible and we aim to develop this for use in the clinical diagnostics laboratory in collaboration with the North Thames Genomic Laboratory Hub and Great Ormond Street BRC.

Nystagmus is a condition involving abnormal, rhythmic movements of the eye away from and back to the visual target. The lack of image stability leads to a worsening of the quality of vision, comparable, or even worse than that imposed by macular degeneration. Nystagmus is associated with several retinal dystrophies, including, in its congenital form, mutations in a large number of genes. Therefore, treating nystagmus by resolving each disorder is impractical and finding a universal therapy that could treat several of these conditions would instead be ideal. Recent work has suggested that nystagmus is caused by over-activity in a specific class of retinal ganglion cells. This class of motion-sensitive ganglion cells is specifically labelled in the Hoxd10-GFP transgenic mouse. We propose to target AAV-mediated expression of optogenetic molecules to this class of ganglion cells, in order to reduce their aberrant activity and reduce or resolve the pathological nystagmus. To achieve this, we will develop a promoter of a size suitable to AAV vectors and specific for Hoxd10-GFP+ ganglion cells. The specificity of this vector, together with recent positive results in clinical trials targeting optogenetic molecule stimulation to ganglion cells, would give our strategy a strong potential to be translated to the clinic.

We introduce the Efficient Medical record Manager & Administration tool “EMMA”. EMMA will be a web-based software only accessible to NHS staff that will scan patients’ electronic health care records and clinical notes to autofill preclinical forms used in elective, non-urgent, procedures. EMMA is an innovative tool that will reduce administration time associated with clinical appointments and improve healthcare capacity in the NHS.

This project aims to understand the events leading to retinal disease caused by mutations in the IFT140 gene. Since some IFT140 mutations can cause an isolated retinal disease, whereas others can cause syndromic disease affecting several tissues, the project will have broader implications for different systemic ciliopathies that affect the retina and other ciliated organs. The project will contribute to the latest research in the field of retinal development and degeneration using in vitro retinal models and in vivo mouse models. We will also test different therapeutic approaches to help alleviate the cellular defects that later on may be translated to practical therapies.

1. Collect clinical information on 111 patients with confirmed CRB1 mutations and examine the time course of the disease and establish any connections between the exact genetic change and the clinical features
2. Invite 30 patients, divided into equal groups of 10 with LCA, RP and cone-rod dystrophy and investigating them closely with state-of-the-art imaging tests over a 2 year period to determine vision-related measures that can be used in future clinical trials.
3. Currently, we tend to use adult tests to measure a child’s vision but this is often inaccurate due to their level of attention, working memory and comprehension. Hence we will compare a series of child-friendly vision function tests with standard investigations to see if there are more accurate, reliable and repeatable alternatives that can be used effectively in the future.
4. Investigate whether mutations in the CRB1 gene have an impact on brain structure and function in RP and LCA groups of patients

The retina is one of the most energy demanding tissues in the body. Glial cells are the critical support cells for neurons and require large amounts of energy (adenosine-5′-triphosphate) to carry out these roles. Disruptions in retinal glia can cause neuronal dysfunction and degeneration resulting in severe visual impairment and even blindness. Mitochondria are the power plants of the cell and are key for a wide variety of crucial cell functions, including respiration and energy production. Mitochondria can move around inside a cell in response to stimulation. The dynamic behaviour of these organelles is important for mitochondria distribution and potentially glial function. This project grant will support me to build a platform to study these dynamic events in the living zebrafish retina using advanced microscopy and computer programmes. I aim to watch these mitochondria move around in glial cells in the retina of the zebrafish. We will also manipulate neuron function (stimulation) to see if this affects the mitochondrial position within glial cells. By doing so we can describe their dynamics and how they support neurons in the eye, which will be critical for understanding their importance for a healthy retina and eventually what goes wrong in disease.

Defective pre-mRNA splicing is one of the most common causes of dominant inherited retinal dystrophy (IRD) and variant induced altered splicing (exon skipping or pseudo-exon inclusion)causes many IRDs. Transcriptomic data has highlighted a retina specific splicing programme that leads to “retinal-specific exons” that are excluded in non-retinal cell-types. However, we are only just beginning to understand how this splicing programme is regulated. This studentship aims to investigate the RNA processing machinery in the human retina through the use of retinal organoids and gene editing. The student will address the following specific hypothesis driven objectives:
a) Investigate the role of specific RNA-binding proteins in retinal differentiation and splicing.
b) Delineate the retinal targets of the spliceosome and test if haploinsufficiency affects
spliceosome retinal function.
c) Determine if cilia associated proteins regulate splicing.
d) Investigate if the cilium functions as a sensor to regulate alternative splicing in the retina.
These independent but complementary aims will test a series of hypotheses to build a comprehensive study of human retinal splicing and its regulation. The generated data will enhance our understanding of this fundamental and disease relevant process.

We propose the first clinical trial to answer the question “Does NAM treatment protect against glaucoma worsening?” in people newly diagnosed with glaucoma. The trial will be a placebo-controlled randomised trial. ‘Placebo-controlled’ means that half the participants will get a pill with NAM and half will get a ‘sugar pill’. ‘Randomised’ means the type of tablet each participant gets happens by chance alone. We aim to establish how NAM works, answering the questions “Can we predict, from mitochondrial function measurements, which people with glaucoma will worsen more quickly?”, “Does NAM result in better mitochondrial function in people with glaucoma?” and “Do people with glaucoma and poor mitochondrial health benefit most from NAM?”

There is an urgent need to rapidly expand clinical capacity in response to the huge backlog in outpatient activity for Moorfields patients caused by the restrictions in clinical activity during the Covid pandemic, while safeguarding staff and patient safety through minimisation of infection risk throughout future waves of COVID-19, influenza and other airborne and surface transmissible pathogens.

The retina is the light-sensitive tissue at the back of our eyes which transforms light into electrical signals to your brain and is responsible for vision. Unfortunately, over 1 in 3000 people in the UK, will have an inherited retinal disease (IRD), a genetic condition which causes a dysfunction of the retina, and the most common cause of blindness in young people in the UK. Accordingly, IRDs account for a leading cause of severe visual impairment in the working age population. Depending on the genetic condition, some may be blind from birth, while others may have their peripheral or central vision progressively deteriorate over several decades. Treatments are emerging for some IRDs but these require identification of the precise causative genetic mutation.Mutations in over 300 genes are associated with IRDs and identifying the affected gene in a patient is the first step towards diagnosis, prognosis and treatment. Currently, IRDs are detected first by community opticians and referred to ophthalmology for retinal imaging and diagnosis with a subsequent referral to specialist eye hospitals, such as Moorfields Eye Hospital, for further imaging and a genetic test.However, because of limitations in the availability of IRD expertise, detection and diagnostic rates remain poor, with most individuals having to wait more than 5 years for a diagnosis. This delays possible treatment pathways and assistance with sightloss. Our plan is to prepare images of IRD patient retinal scans (datasets) from four leading eye hospitals, Moorfields Eye Hospital, Oxford, Liverpool and Tokyo (Japan). We will use these retinal scan datasets to benchmark, further train and then test Eye2Gene, a deep-learning algorithm which is able to detect and diagnose IRDs from a patient’s retinal scan.If Eye2Gene is successful, we aim to provide detection and diagnosis of IRDs through non-specialist hospitals within months instead of years, thus democratising expert IRD knowledge widely across the NHS for patient benefit, by enabling expertise to be delivered locally

Age-related macular degeneration (AMD) is a complex disease and the leading cause of blindness in the western world. Over time, changes in the retinal pigment epithelium (RPE) leads to the damage of light-sensing cells in the retina and central vision loss. There is strong evidence that the immune system plays an important role in the development of AMD. Ageing and stress cause RPE cell damage and the build-up of debris in and around the RPE, resulting in chronic inflammation and an immune response in the macular region. Investigating the events that lead to local tissue inflammation and an immune response in AMD will provide us with a greater understanding of early AMD pathology and could allow us to develop new therapies to treat or prevent vision loss.

Retinoic Acid (RA) is a morphogen involved in macular development1 and has recently been shown to be dysregulated during retinal degeneration, which may significantly contribute to dysfunction and vision loss in many eye diseases2. In the healthy retina, RA is locally downregulated in the macula by the expression of Cyp26a1 in Muller glia. This project will identify the consequences of RA up-regulation on macular function and investigate the role of Cyp26a1 in preventing macular damage. Thus, this project will increase our understanding of how eye diseases start and explore the use of RA inhibition as treatment for many degenerative diseases

Objectives:
1. Differentiate wildtype and choroideremia (CHM) patient derived retinal pigment epithelium (RPE) from existing induced pluripotent stem cell (iPSC) lines (0-6 months)
2. Transfect the pS/MARt-CHM-GFP vectors to into CHM patient derived iPSC RPE and assess whether there is functional rescue through western blot for REP1, rescue of prenylation function, and improved phagocytosis and compare this with viral vectors (3-12 months)
3. Use various nanoparticles to try to compare transfection rates of non-viral vector on iPSC derived RPE

Glaucoma and trachoma are leading causes of blindness worldwide, with over 80M people affected and close to 10M at immediate risk of permanent sight loss. For both diseases, surgical treatment success is directly dependent on the avoidance of postoperative scarring. However, there is no treatment to prevent scarring in trachoma and the current drugs used to prevent scarring following filtration surgery for glaucoma can have serious blinding side effects. We have designed an innovative medicinal product consisting of biodegradable microparticles loaded with doxycycline, a common drug, for local delivery at the time of surgery to achieve safe, targeted and sustained anti-scarring action. These have shown remarkable anti-scarring efficacy in laboratory tests, and we now propose to evaluate their effectiveness in an animal model of post-surgical ocular scarring. The successful completion of this project will allow us to develop this treatment towards clinical trials, with a potential benefit to millions worldwide.

An artificial eye (AE) is worn after enucleation or evisceration of the eye for a range of diagnoses such as tumour, severe trauma, infection or painful blind eye or for congenital deformity of the eye. The global need is for 7.6 million individuals wearing an AE and the current time-consuming, artisan process, of handmade “analogue” AEs by an ocularist has not changed in more than 100 years.

We have developed a digital end-to-end solution for the production of 3D printed AEs. To replace invasive impression moulding of the socket, we have developed non-invasive anterior segment optical coherence tomography (OCT) to fulfil this role and to measure the anatomy and colour gamut of the fellow eye to serve as a template for the AE. This information is converted by computer assisted design into a 3D printable file, so that the AE is 3D printed in less than 2 hours to be fitted into the patient. We have manufactured our first AE prototypes using this technology.

In this application for a Springboard Award, we will be generating preliminary data on the use of prime editing to rewrite the two most common mutations in the USH2A gene, c.2277G>T and c.2299delG. These mutations are a common cause of Usher syndrome and autosomal recessive non-syndromic retinitis pigmentosa. We will be using CRISPR/Cas9-mediated homology directed repair to introduce the two mutations into human cell lines. Thereafter, prime editing guide RNA (pegRNA) for each mutation will be optimised for use with the prime editor (PE2 system) or in conjunction with a strategy favouring incorporation of the edit during DNA repair (PE3 and PE3b system). The efficacy, precision and accurace of prime editing of these mutations will be assessed by highthroughput sequencing (HTS) on a MiSeq (Illumina) platform. Thereafter, delivery routes for the optimal editors will be explored, including an AAV-mediated delivery of a split-intein prime editing system or lipid-based delivery of mRNA-encoded prime editor together with chemically modified synthetic pegRNA. This preliminary data will quide the strategy for prime editing of the USH2A mutations in human photoreceptor cells. Finally, a new disease model of USH2A associated disease will be developed. Patient-derived induced pluripotent stem cells (iPSC) will be differentiated to 3D retinal organoids and the disease phonotype fully characterised as a readout for rescue by prime editing.

Work package 1. Comprehensive systematic review of adverse events (AE) of minimally invasive glaucoma surgeries.
Work package 2. Longitudinal study using linked population data from UK to assess resource utilisation and 5-year risk of revision surgery or secondary glaucoma procedure and serious adverse events following conventional glaucoma surgery and MIGS to inform health economic evaluations. Work package 3. Health economic evulation of minimally invasive glaucoma devices

Glaucoma is a common eye condition that can lead to loss of vision if not identified and treated early. One factor which can increase the chance of developing glaucoma is high eye pressure (ocular hypertension), which is usually detected during an eye health test at the optometrist. Eye pressure is considered to be high if it is above 21 mmHg.

This project will reveal how neural activity is structured across the brain to support diverse behaviors, using powerful techniques that we developed to probe mouse behavior and to record and interpret the activity of large neuronal populations. Behaviors emerge from the coordinated activity of vast populations of neurons distributed both within and across brain regions in ways that have been hitherto impossible to measure and understand.

Aims:
1. To generate patient-derived induced pluripotent stem cell (iPSC) RDH12 (recessive and dominant) and WT retinal organoids
2. Undertake global transcriptomic profiling using RNA-Seq on RDH12 (recessive and dominant) and WT retinal organoids to further understand molecular disease mechanisms
3. Test 5 compounds for efficacy using the stable wildtype and mutant RDH12 HEK293 cell lines

In this pilot project we will determine the acceptability of using a digital phenotyping smartphone app (called mindLAMP) among patients with vision impairment. We will recruit 20 participants with retinitis pigmentosa, the leading cause of severe sight impairment in working age adults, for a 3-month pilot study using mindLAMP.

Human ESCs represent a promising source for cellular replacement therapies owing to their availability, pluripotency, and unlimited self-renewal capacity. However, they also carry risks of neoplastic change, uncontrolled proliferation, and differentiation to inappropriate cell types1,2. The eye is advantageous in investigating hESC-based cell therapy as it is accessible and confined, and the transplanted cells can be monitored directly in vivo, with the possibility of being removed or destroyed if there is evidence of neoplastic change3,4. Furthermore, long-term immunosuppression can be delivered locally.
Late AMD is characterized by irreversible cell loss, initially of RPE cells and subsequently of neuroretinal and choroidal cells5, and thus may be amenable to hESC-based cell therapy4. The disease process includes damage to the RPE’s specialized basement membrane, Bruch’s membrane5. Currently, treatments exist only for the exudative or ‘wet’ form of AMD. These treatments rely on angiogenesis inhibitors6 or indirect transplantation of an autologous RPE–Bruch’s complex (retinal translocation surgery)7. However, the former treatment only suppresses the disease, requiring long-term repeat delivery, and the latter, although restoring the macular anatomy, does not prevent disease recurrence.
There is no treatment for atrophic ‘dry’ AMD, which is characterized by RPE loss and progressive neuroretinal cellular dysfunction. Suspensions of hESC-derived RPE (hESC-RPE) cells have been transplanted in human subjects with dry AMD and Stargardt’s disease, but the extent of cell survival and restoration of vision remains ambiguous8. A recent, single-patient report described transplantation of an autologous induced pluripotent stem cell (iPSC)–derived RPE patch on its own secreted basement membrane9. The iPSC-RPE survived with maintenance, but no improvement, of visual acuity at 12 months.
We have developed a technique to create sheets of hESC-RPE10 and propose that it may be possible to alter the disease process in severe wet AMD and dry AMD with transplantation of a hESC-RPE cell patch under the macular. In order to test this hypothesis and demonstrate safety and potential efficacy of the treatment we carried out a series of preclinical animal studies.

Continuation of funding for 10-10-10 challenge.

The Cells for Sight Advanced Therapies manufacturing facility has been and continues to be central to the delivery of the joint UCL-Moorfields strategy for the development and manufacture of novel cell-based therapies for blinding eye diseases.

Strategic Initiative Grant

Currently, there are no treatments for L-ORD and patients are faced with losing central vision in both eyes if they inherit a faulty copy of the gene. In this project we will test whether CRISPR genome editing can be used to switch off the faulty copy of the gene. The onset of L-ORD occurs from 40 years of age, this provides us with a large window of opportunity to assess whether a patient has inherited the disease-causing mutation, and use CRISPR editing as a preventative measure for vision loss. As L-ORD is most commonly caused by a founder mutation the therapy could be used to treat the majority of patients. This study will provide proof of principle for genome editing as a potential treatment for L-ORD. This approach could also be used for other dominant diseases affecting, not only the eye but any tissue in the body. 

The eye has particularly stenuous metabolic demands and metabolism is distrubed in a wide variety of important, blinding, retinal diseases. Direct assessment of metabolism within the eyes of patients is not possible but with advances in 'omics of healthy and diseased ocular tissues along with novel approaches to model metabolism computationally it is now in principle possible to predict cellular metabolism within specific groups of cells in considerable detail. We have generated a series of retinal metabolism models but these require further work and validation in order to provide a convincing basis for large-scale funding (which we have attempted to secure). The main goal of this project is to consolidate work to date and create a computational toolbox that will make it possible to gain funding addressing disease prediction and intervention in age-related macular degeneration, diabetic retinopathy and inherited retinal degenerations. The tools will also have relevance to our understandings of optic nerve metabolism in glaucoma.

We now understand the genetic cause of disease in that majority of FECD patients. Remarkably, up to 80% of white European patient populations share the same genetic cause of disease; a genetic spelling mistake termed a ‘repeat expansion mutation’ in a gene called TCF4. Similar types of ‘repeat expansion mutations’ located elsewhere in the human genome are also known to be responsible for more than 40 human diseases including Huntington’s disease, Myotonic dystrophy and amyotrophic lateral sclerosis (ALS). In this research proposal we aim to provide in-depth molecular characterisation of toxic cellular events that specifically drive FECD pathology in the corneal endothelium to facilitate and enhance the design and development of gene-directed FECD therapies.

The growth of the human eye is carefully controlled to ensure the correct length of the eyeball. Severe problems with vision can occur when the eye is too short (e.g. microphthalmia) or too long (e.g. myopia or ‘short-sightedness’). Microphthalmia has been found in up to 11% of blind children. Short-sightedness is a very common problem throughout the world affecting almost 50% of all UK adults, and as high as 90% in children from East Asia; it can lead to other sight threatening complications. This project aims to better understand what genetic factors may determine the length of our eyes, and manipulating them to induce or reduce growth accordingly, to normalise the size of the eyeball.

Dominant optic atrophy (DOA) is the most commonly diagnosed inherited optic neuropathy (ION) in the United Kingdom and there are currently no approved treatments. The progressive visual loss in DOA is caused by the loss of function, and ultimately the death of, a population of specialised nerve cells, called retinal ganglion cells (RGCs). We have developed a way to study DOA in the laboratory dish using cells from patients to make RGCs; however, we have not yet been able to study their function to test if it is compromised, or if these cells die. This proposal aims to develop functional tests of RGC health, complementing our previous studies, and produce a complete model of DOA in a dish. 

The growth of the human eye is carefully controlled to ensure the correct length of the eyeball. Severe problems with vision can occur when the eye is too short (e.g. microphthalmia) or too long (e.g. myopia or ‘short-sightedness’). Microphthalmia has been found in up to 11% of blind children. Short-sightedness is a very common problem throughout the world affecting almost 50% of all UK adults, and as high as 90% in children from East Asia; it can lead to other sight-threatening complications. This project aims to better understand what genetic factors may determine the length of our eyes, and manipulating them to induce or reduce growth accordingly, to normalise the size of the eyeball.

This study will mine the available data on patients with ABCA4 retinopathy to determine the detailed haplotypes in those with retinopathy as well as population data from the following sources: Moorfields clinical cohort of patients, Genomics England (cases and controls), NIHR Bioresource )(cases and controls), UK Biobank (controls). Existing clinical data will be analysed also. This will allow the full understanding of genetic variation in the gene that contributes to disease and identify those that may be appropriate for clinical trials.

Call for clinical cohort samples for ‘omics analysis

Macular diseases are a leading cause of vision loss in the UK, and affect a layer of cells at the back of the eye called retinal pigment epithelium (RPE). Degeneration of these cells results in the death of the light-sensitive retina, causing central vision loss. Dr Carr and her colleagues are interested in looking at early disease mechanisms in RPE cells to find out how macular disease develops and to identify pathways that could be targeted therapeutically to prevent sight loss in patients.

The London Project has created an induced pluripotent stem cell bank at the UCL Institute of Ophthalmology, containing cells donated from patients with age-related macular degeneration (AMD) and inherited macular diseases. These stem cells contain the same genetic background as the patient and can be used to make diseased RPE cells in a dish, to study macular degeneration. Genes play an important role in the development of macular disease. If we know the full genetic background of patient cells, we can work out how genes impact on the development of macular disease in our patients’ RPE cells, and use this knowledge to potentially develop personalised medicines.

This project aims to
1) Identify common genetic variants that increase the risk of AMD in our patient cells, and use this information to assess the effects of genetics on early AMD.
2) Provide genetic information that will be used to develop new genome editing therapies for patients with inherited macular disease.

To help achieve this, the team require state-of-the-art Nanopore sequencing technology that would enable Dr Carr and her team to sequence 12 inherited macular disease patient and AMD patient cell lines..

Gene therapy is an emerging field that has shown early success in several branches of medicine, including ophthalmology. Most existing gene therapy approaches have focused on replacing expression of identified genes. Early data from clinical trials has validated this strategy to rescue genetic conditions. A major challenge is now to pursue gene therapy with the aim of treating a wider range o retinal disorders including age-related conditions. Replacement of a gene will not be sufficient to tackle these conditions. Instead, the development of novel gene therapy approaches will
be required.

This project Mutations in the long-wavelength (LW) and middle-wavelength (MW) cone opsin gene array are associated with a wide range of visual defects including red-green colour vision deficiency, blue cone monochromacy and cone dystrophy. Advances in our understanding of the genetic basis of disease has revealed different genetic mechanisms, and differential effects on cone photoreceptor viability and function. Interchange haplotypes are the most common cause of disease, resulting in aberrant splicing and exon 3 skipping in vitroin minigene assays. Here, we will create models with LW and MW interchange haplotypes in genomic and cellular context by differentiating patient iPSC derived 3D retinal organoids. With these models, we plan to investigate the effect of interchange haplotypes on the extent of aberrant splicing in the human retina, and to assess if this correlates
with cone photoreceptor differentiation. In addition, we will determine the relative expression of genes in multigene arrays, that may influence cone photoreceptor mosaic patterning. Proof of concept in minigene assays has shown the potential of antisense oligonucleotides (AONs) to suppress exon 3 skipping, and these AONs will be tested in retinal organoids as a therapeutic approach, in addition to an alternative CRISPR-based RNA processing modification approach. We will compare these data with patient phenotypes, to assess if cone photoreceptor survival, or the rate of photoreceptor loss and functional deterioration, correlates with cone opsin genotype. This knowledge is important for selecting the target cone opsins most likely to respond to a therapeutic intervention for potential restoration of visual function in patients. 

This project will generate new procedures for cross-site MRI research, that will facilitate acquisition of the large, statistically comparable imaging datasets needed for understanding mechanisms of rare eye disease. Generate the first test of visual cortex function in LHON, a method demonstrated to have high sensitivity to novel treatment outcomes in ocular gene therapy, thus providing valuable information for treatment development. Develop the largest MRI dataset of LHON to date, allowing us to start addressing outstanding questions in the literature about mechanisms of LHON, including (1) which neural changes result directly from the acute disease stage and which changes reflect later-stage knock-on effects from altered neural input, (2) which of various candidate mechanisms (i.e., myelination or axonal death) cause these changes, (3) how do these changes in post-retinal brain measures relate to visual function, and (4) which genotype/phenotype relationships predict disease onset and recovery. Provide proof-of-concept and benchmark data for future research on treatment outcome.

The blood-brain barrier (BBB) prevents most drugs from accumulating at therapeutically required levels in the brain. We have identified a Methamphetamine (METH)-inducible transportation pathway, which strongly enhances transit of chemicals and proteins from the blood to the rodent brain. Here, we propose to investigate this pathway for therapeutically significant delivery of oligonucle tides or viral vectors, namely adeno-associated virus 9 (AAV9). Our findings will facilitate pre-clinical evaluation of novel CNS treatment modalities and potentially the clinical treatment of a wide variety of neurological diseases.

Inherited retinal dystrophies (IRDs) are an opportune target for novel gene therapies, but information on their natural history is limited. In this study we plan to retrospectively analyse the natural history and pathogenic variants of CNGB1, CYP4V2 and RP2 retinopathies and a fourth indication which is still to be confirmed.

Further funding for the CFS facility. 

This project aims to be the first to investigate islet cell transplants into the anterior chamber of the eye as a treatment for diabetic retinopathy (DR). We aim to use state-of-the-art retinal biomarkers and a novel imaging technique to detect real-time apoptosis of retinal cells to examine early neurodegenerative changes and treatment effect in a well established animal model of diabetic retinopathy. This study will investigate whether this novel therapy has the potential to treat both diabetic retinopathy and cure systemic disease simultaneously and whether there is any additional local neuroprotection from insulin in transplanted eyes.

The project proposes to use hiPSCs-RPE cells derived from CHM patients to examine the molecular processes underlying the cell death mechanisms involved in CHM. hiPSCs-RPE cells have been shown to exhibit key physiological functions and it is one of only a few cell types derived from hiPSCs that have met standards for use in human clinical trials. We will use hiPSC-RPE derived from two CHM patients and a male commercial hiPSC line (RBi001-A) that will be genetically engineered by CRISPR/Cas9 tool. We plan to use these models to examine the distinct pathways of cell death in RPE, including apoptosis and non-apoptotic forms such as necroptosis. Cell death pathways will be evaluated using established molecular and morphological markers.

Ophthalmology has the highest outpatient activity of any NHS speciality (7.9 million episodes; 2018-19). The COVID-19 pandemic has unmasked structural weaknesses in UK hospital eye care. We will soon enter a “recovery” phase with a surge in need for outpatient capacity. Without novel and efficient care pathways incorporating social distancing, the current service model will rapidly be overwhelmed. This represents a high-risk scenario for patients, with the potential for thousands of people to become blind from avoidable causes. The mental health consequences of COVID19 and subsequent lockdown for both NHS staff and patients are unknown but probably significant. The impact on human suffering, individuals’ livelihoods and quality of life, healthcare resources and healthcare spending and the wider economy are considerable.

We will deliver a blueprint for socially-distanced outpatient care employing proven multiply-parallel linear flow examination systems. This exemplar will provide a toolkit for reactive capacity expansion across the NHS during recovery from COVID19 peak 1. We will rapidly train a healthcare naïve workforce to carryout sophisticated medical examinations. Mental health assessments will identify anxiety or depressive symptoms in staff and patients, with support and interventions provided if required. UCL design and modelling expertise will optimise both safety and efficiency of movement of patients and staff. Commercial digital providers will develop open-source enhancements for enabling technology. We will assess safety and efficiency of our new model of care. Ultimately, our goal is to develop a cost-effective and sustainable model for healthcare globally, overcoming existing deficits in NHS care.

Prospective discovery and validation study of diagnostic biomarkers.

This partnering award is to generate an international network of research groups with laboratories based in the USA. We will combine the expertise of three research laboratories to create a synergistic output: MacDonald lab (BBSRC funded; UCL) - transcriptomics in the ageing zebrafish retina with the Clark laboratory (Washington University; USA) - sequencing techniques and mammalian retinal transcriptomics and the Ruzycki laboratory (Washington University; USA) - single-cell sequencing techniques and comprehensive comparative analysis of retinal transcriptomic datasets. The output of this work will be closing the existing gap between transcriptomic datasets and comparative genomics in the fields of retinal development and ageing. This work will also increase the skillset of early career researchers involved in this collaboration, and importantly enhance the UK bioeconomy.

The primary aim of this study is to assess whether NAD+/NADH ratio measured in peripheral blood can be used as a clinically useful biomarker for glaucoma susceptibility/progression. The secondary aim is to improve mitochondrial function and ultimately halt glaucoma progression in NTG patients, by increasing the NAD+/NADH ratio with Vitamin B3 supplementation. This study will therefore consist of two parts. Part 1. Measuring NAD+/NADH ratio in 30 NTG patients with various levels of glaucoma severity/progression and 30 controls, and correlate this to mitochondrial function and glaucoma progression. Part 2. Establish fibroblast lines form 6 NTG patients with the worst mitochondrial function and 6 controls. Following this, fibroblast lines will be supplemented with Nicotinamide to improve NAD+/NADH ratio and mitochondrial function. This study will form pilot data which will help towards setting up a larger study looking into vitamin B3 improving mitochondrial function and more importantly halting glaucoma progression.

Glaucoma comprises a heterogeneous group of diseases, characterised by progressive optic neuropathy and visual field loss, and is the most common cause of irreversible blindness worldwide. Primary open-angle glaucoma (POAG), accounts for 70% of all cases. The development of glaucoma is strongly associated with raised intra-ocular pressure (IOP) but precise pathophysiological mechanisms remain unclear. Lifestyle factors have been implicated in the pathogenesis of a number of chronic diseases and represent potential therapeutic targets. The lifestyle determinants of IOP and POAG are less clear. Aims are to describe and compare patterns of IOP and incident POAG in a cohort of UK adults; investigate the associations between various lifestyle factors, including alcohol, cigarette smoking, adiposity, exercise and dietary factors, with IOP and incident POAG; analyse the links between lifestyle factors, IOP and incident POAG to determine whether any potential associations with POAG risk are mediated through IOP or other mechanisms.

A key line of research at UCL Institute of Ophthalmology (IoO) is to understand the function of visual circuits and how diseases affects it. In order to achieve this, it is necessary to observe and study neurons embedded in their intact networks. Two-photon microscopy is a specialized technique that makes use of long wavelength, near infra-red, excitation light. This minimizes light scattering and allows deep imaging inside neuronal tissue, that is not possible to achieve using other equipment.  The Institute aims to set up a flexible, multi-user facility that will provide two-photon microscopy suitable for the diverse interests of several research groups. The equipment will enable unparalleled functional imaging and targeted electrical stimulations and recordings for a diverse range of models, including dissected retinas, ‘retinas in a dish’ derived from human stem cells, and transgenic zebrafish and mouse visual cortex and retina

Membrane contact sites, where the membranes of neighbouring organelles are tightly apposed, provide an important interface for non-vesicular transport and inter-organellar communication. The endocytic pathway introduces huge amounts of lipids and proteins into cells for sorting and degradation in the lysosome. By providing platforms for lipid exchange, calcium signaling and protein interactions, membrane contact sites between lysosomes and the ER control the fate of large pools of intracellular lipids.

We have recently identified a role for the late endosomal sterol binding protein Niemann Pick type-C protein-1 (NPC1), in tethering lysosome-ER contact sites through interaction with an ER-localised lipid transfer protein, Gramd1b. NPC1 is required for egress of dietary cholesterol from lysosomes for transport to the ER; NPC patient cells deficient in functional NPC1 protein accumulate cholesterol in the lysosome. We found that artificial expansion of the lysosome:ER interface in NPC1-inhibited cells restored cholesterol transport to the ER and corrected the cholesterol accumulation phenotype.

Here we aim to define the molecular architecture of NPC1-regulated contact sites, including how patient mutations in the sterol-sensing domain might affect Gramd1b interaction. Cells lacking functional NPC1 accumulate sphingolipids as well as cholesterol and our preliminary data suggests that sphingolipid egress from lysosomes may be mediated by NPC1-dependent contact sites. We will determine the role of lysosome:ER contacts in the transport of cholesterol and other lipids including sphingolipid. Finally, we will develop ways to expand the lysosome: ER interface and, in zebrafish models of NPC, assess the impact of contact site expansion/mobilizing lipids on other NPC phenotypes including ataxia.

After early development, the mature brain gradually stabilizes and retains only certain level of plasticity, the ability to exhibit long-term modification of its neural response to adapt to the external environment. This limited degree of plasticity in the adult brain is responsible for reduced learning ability and incomplete recovery from brain injury, diseases, and ageing, so there is critical need to identify ways to enhance adult plasticity and elucidate its underlying neural mechanism.

A series of recent works uncovered that running enhances visual responses and promotes plasticity in the adult brain, suggesting a potential of recruiting neuromodulatory pathways to regulate cortical plasticity in the mature circuit. In this project, we propose to apply in vivo systems approaches to pinpoint the local and dispersed brain circuits that enable this interactive and tightly regulated process: we will identify the long-range neuromodulatory pathways that convey behavioural state information to visual cortex and drive neuroplasticity and investigate how these projections influence local neurons in the primary visual cortex to accomplish the circuit remodeling. In the process, we hope to uncover the principle governing neuroplasticity in the adult brain and establish a mechanistic framework for potential therapeutic interventions to promote rehabilitation and perceptual learning.

The purchase of a new confocal microscope will protect and extend the research that the Institute and MEH can perform on a wide range of eye disease. 

Major current hurdles for wide clinical use of AAV vectors are attributable primarily to: (i) host elimination by both immune and non-immune sequestering mechanisms – such neutralization by host antibody responses critically limits the possibility of repeated AAV delivery; (ii) AAVs are prevalent in the environment and hence a large proportion of the population carry AAV antibodies (up to 80%)– this pre-existing immunity renders AAV unable to infect target cells forcing substantial patient cohorts to be excluded from clinical trials. The current proposal is founded on compelling track record in the field and brings together a ‘best-with-best’ multidisciplinary team of international leading academic and EFPIA partners with complimentary expertise in gene therapy, immunology, chemistry, engineering, biotechnology, drug safety, viral vector production, regulatory and clinical trials. The overall goal is to analyse the currently available clinical data and then design preclinical and clinical studies to fill the knowledge gaps in advanced therapies development. Our main aims are to: 1) Develop improved model systems for predicting product immunogenicity in humans. This will be achieved by generating human and NHP 3D hepatic models; 2) Enhance our understanding of gene/cell therapy drug metabolism inside a host of cell types. The plan is to define metabolism of the therapeutic vector genome in different cell types to understand whether rates of degradation, episomal maintenance, or integration, and metabolic stress induced by AAV vector transgene expression vary from cell to cell. We will then adopt strategies to mitigate the loss of vector genomes and improve persistence; 3) Use diverse clinical expertise to establish the clinical factors around pre-existing immunity limiting patient access to advanced therapies therapy; 4) Engage regulators to ensure that the concepts and the data generated through this IMI programme will fill the gaps and support future trials.. 

Glaucoma is the commonest cause of incurable blindness globally. Population screening tests are inadequate, resulting in frequent late detection. Even with current therapy, many patients continue to lose vision. Compounded by the exponentially increasing glaucoma prevalence, we urgently need to innovate our management strategy.
My research will: 1) elucidate the genetic architecture and biological pathways underlying glaucoma; 2) discover novel environmental and blood biomarker associations, determining if they modify genetic risk; and 3) develop prediction models to enable efficient population screening and personalised glaucoma treatment. Building on my recent study examining intraocular pressure, genetic work will focus on factors underlying retinal ganglion cell susceptibility to pressure. This understudied area is critical to determining individual risk. Single cell sequencing studies will elucidate the functional role of discoveries. Analyses identifying non-genetic predictors of glaucoma will use data from 18 population studies. Causality will be examined using Mendelian randomisation and gene-environment interactions will be tested.
I will then develop and validate prediction models to: 1) identify people at highest risk of glaucoma in the population, enabling targeted screening; 2) identify glaucoma clinic patients at highest risk of blindness, enabling stratified care; and 3) predict response to first-line treatments, improving efficacy. To achieve this, I will develop a world-leading resource, combining longitudinal clinical data with genotypes. Ultimately, the models will be developed into clinical tools and examined in prospective cluster-randomised trials.
This research has the potential to transform glaucoma care, reducing blindness while providing an innovative strategy to address the rapidly increasing burden. Furthermore, the fundamental discovery will facilitate multiple streams of glaucoma-related research, including novel drug development.

Neuroprotective agents can promote neuronal survival with the potential to protect against vision loss and retinal cell death. They have been shown to hold promise for slowing sight loss in patients with different forms of inherited retinal disease. Choroideremia (CHM) is an X-linked recessive chorioretinal dystrophy caused by mutations in the CHM gene, which encodes rab escort protein 1 (REP1, a protein involved in lipid prenylation and intracellular trafficking), affecting males with a prevalence of 1 in 50-100,000. Gene therapy has made significant in-roads, with phase 3 clinical trials for CHM underway. But alternative therapies must be developed so patients have a choice of treatment, a less invasive approach, where gene therapy may not be possible, or it fails. In this proposal, a well-characterised zebrafish model of CHM will be used to test the following selected neuroprotectants: Curcumin, levodopa (L-DOPA), N-acetylcysteine amide (NACA) and tauroursodeoxycholic acid (TUDCA) for safety and therapeutic outcomes. These neuroprotectants have been tested in patients with other ocular or systemic disease, so their safety profile is established. If we show proof-of-principle they are successful at slowing the retinal degeneration in choroideremia, we will be able to move to a clinical trial for patients rapidly. Successful neuroprotective strategies could provide months or years of useful vision that would allow an individual to continue to be gainfully employed and maintain their independence and quality of life.

There have been significant advances in gene therapy for Choroideremia (CHM), however, at the 2019 International Choroideremia Symposium in Philadelphia, Pennsylvania, hosted by the Choroideremia Research Foundation, with over 20 international experts, it was strongly suggested that alternative therapeutic approaches should be investigated. Neuroprotective agents were considered to hold promise for slowing sight loss in patients. Using existing disease models of choroideremia (zebrafish, mouse and patient cells with the genetic defect), this proposal will test the following selected neuroprotectants: Curcumin, levodopa (L-DOPA), N-acetylcysteine amide (NACA) and tauroursodeoxycholic acid (TUDCA) for safety and treatment outcomes. These neuroprotectants have been tested in other disease models and patients such as retinitis pigmentosa, so their safety profile is established. If we show they are successful at slowing the retinal degeneration in choroideremia, we will be able to move to a clinical trial for patients quickly.

We have developed a non-viral gene therapy for the USH2A gene, which causes type 2 Usher syndrome and isolated retinitis pigmentosa. USH2A is at least 3 times bigger than the capacity of viral gene therapy delivery systems (vectors). We have proof-of-principle for the successful application of USH2A non-viral vectors in patient skin cells and zebrafish models, but because the vectors lack the viral component, they cannot infect cells easily. Hence, to improve delivery to the light-sensing retinal cells (photoreceptors) we will use nanotechnology (microscopic needles to deliver an electric pulse) to improve vector uptake and investigate the photoreceptor's immune response towards this gene therapy for improved outcomes. Primary research questions: 1. Can we improve uptake of the USH2A non-viral gene therapy vectors into mouse photoreceptors using a modified electric pulse to the retina? 2. Are there any immune factors that can be manipulated to encourage more USH2A to be produced in the photoreceptors?

Continuation of project aimed to derive GMP grade cells for a glaucoma therapy.

Continuation of project aimed to derive GMP grade cells for a glaucoma therapy.

Addressing the function of the genes that control organ development is a fundamental goal in biology. However, because of genetic robustness and compensatory mechanisms, mutations in many genes show no phenotype, limiting our ability to understand their function. To circumvent this problem, I developed a pioneering genetic modifier screen approach in zebrafish that enables finding eye development genes that would otherwise remain unnoticed (Young et al., 2019). The genes we find will be screened for mutations in patients with eye defects to find new genes of diagnostic value associated with these pathologies. By combining the genetic analysis of patients with developmental eye globe defects with research in zebrafish , this pilot research programme will identify and validate new genes involved in eye formation and growth compensation.

Purpose: Assess colour vision and cone structure in patients with diabetic retinopathy (DR) and retinal diabetic neuropathy (RDN). Methods: 50 patients with DR/RDN and 10 controls will undergo comprehensive colour vision assessment using the Advanced Vision and Optometric Test (AVOT) system and high-resolution retinal imaging using adaptive optics scanning light ophthalmoscopy (AOSLO). Hypothesis: An inverse relationship between severity of disease and both colour discrimination and cone density. Implications: Establish the relationship between cone loss and colour vision in progressive retinal disease.

Inherited optic neuropathies are an important cause of blindness in children and young adults. This group of disorders can be caused by spelling mistakes (mutations) in a number of genes that lead to the irreversible loss of specialised retinal ganglion cells that constitute the optic nerve. The optic nerve transmits images from the eye to the brain and these genetic changes damage the optic nerve causing visual loss, especially in the central part of the field of vision. A number of genes have been identified that cause inherited optic neuropathies and rather remarkably, all of them affected mitochondria, which are the tiny batteries within cells that produce the energy needed for them to survive. Despite these advances, the mutations causing disease have yet to been identified in a significant proportion of individuals who have been given a diagnosis of inherited optic neuropathy. In these cases, genetic counselling for affected individuals and their families remains challenging as it is not possible to accurately predict the risk of passing the condition to the next generation. It is also not possible to contemplate treatment approaches, such as gene therapy, if we do not know the gene that is at fault and how it causes damage to the optic nerve. The central aim of this project is to identify the genes that are responsible for disease in individuals with visual loss with a diagnosis of inherited optic neuropathy in whom known genes have already been excluded. Where applicable, we will also use well established laboratory techniques to confirm how these genetic defects affect mitochondrial function.

Myopia prevalence has increased dramatically in recent years. About 30% of adults are affected worldwide, and in South-East Asia up to 95% young adults have myopia, making it the leading cause of irreversible blindness. In the UK, almost one in five teenagers are now myopic. Up to 20% of the myopes develop high/progressive myopia, with irreversible axial elongation of the eye and an increased risk of sight-threatening conditions such as retinal detachment, choroidal neovascularization and glaucoma. The efficacy of currently available interventions is limited, as the mechanisms driving myopia progression are still unknown. Myopia onset and progression occur exclusively during childhood and adolescence. Light of high intensity, i.e. sunlight, is thought to regulate eye growth by inhibiting scleral remodelling and axial elongation via dopaminergic retino-scleral signalling pathways. These signals may be altered in myopia, leading to defects in scleral biomechanics (altered thickness, elasticity and extracellular matrix composition). The light/dopamine pathway is thought to involve the release of growth factors by the dopamine-stimulated retinal pigment epithelium (RPE), which diffuse through Bruch’s membrane and choroid to act on the sclera. The choroid also undergoes stretching and thinning during myopia progression and produces growth factors and matrix metalloproteinases, which have been linked to scleral remodeling and/or eye growth regulation. Studies in the marmoset myopia model have shown that these biomechanical changes are accompanied by gene expression changes in the choroid/RPE, including genes linked to tissue mechanics (TGF; FGF-2). This suggests that mechanical changes to the choroid are translated into biochemical signals, with a modified secretome that could alter scleral cell behaviour. We hypothesize that signals from the choroid are crucial to the regulation of scleral biomechanics and contribute to myopia development and progression. This project proposes to test this hypothesis by investigating scleral fibroblast - choroid cell interactions using cells isolated from human pediatric donor tissue (we have already successfully established primary cultures of choroid and scleral fibroblasts). We will construct a biomechanically relevant 3D sclera biomimetic and will test the effect of choroidal cells’ secreted factors on the properties of scleral fibroblasts, and how this is affected by mechanical stress or biochemical stimulation of the choroidal cells. We will use cytokine arrays and/or proteomics to identify the secreted factors involved. This work will elucidate the choroid role in ocular growth regulation and characterize choroidal signalling molecules that participate in scleral remodelling, potentially identifying new drugable candidates for progressive myopia management

1. To discover novel genetic variants underlying glaucoma and RGC susceptibility.
2. To determine the functional consequence of the identified genetic associations with glaucoma and to identify the key biological pathways that may serve as novel treatment targets.
3. To develop genetic prediction models which identify patients at highest risk of blindness from glaucoma and predict individual response to treatment, enabling personalized management.

There have been significant advances in gene therapy for Choroideremia (CHM), however, at the 2019 International Choroideremia Symposium in Philadelphia, Pennsylvania, hosted by the Choroideremia Research Foundation, with over 20 international experts, it was strongly suggested that alternative therapeutic approaches should be investigated. Neuroprotective agents were considered to hold promise for slowing sight loss in patients. Using existing disease models of choroideremia (zebrafish, mouse and patient cells with the genetic defect), this proposal will test the following selected neuroprotectants: Curcumin, levodopa (L-DOPA), N-acetylcysteine amide (NACA) and tauroursodeoxycholic acid (TUDCA) for safety and treatment outcomes. These neuroprotectants have been tested in other disease models and patients such as retinitis pigmentosa, so their safety profile is established. If we show they are successful at slowing the retinal degeneration in choroideremia, we will be able to move to a clinical trial for patients quickly.

The intrinsically photosensitive retinal ganglion cells (ipRGCs) survive preferentially compared to other retinal ganglion cells (RGCs) in Leber's Hereditary Optic Neuropathy (LHON) and Dominant Optic Atrophy (DOA). Therefore, the objective of this study is to determine what is different about the transcriptomic profile of these cells that allows them to survive compared with other RGC subtypes in inherited optic neuropathies? Our a priori hypothesis is that differentially expressed genes in these ipRGCs contribute to their selective neuroprotection against mitochondrial dysfunction.

Current therapies for retinal degenerative eye diseases aim to slow progression but do not restore vision or repair damage. There is therefore a need for novel therapies to repair cellular loss and restore vision. Müller cells found in the retina, have the unique ability to regenerate all cell types in the zebrafish retina after injury. Although the human retina harbours a population of Müller glia with stem cell properties, there is currently no evidence for regeneration, partly due to the difficulty of using human tissue for these types of investigations. The aim of this study is to investigate the regenerative potential of human Müller glia native to retinal organoids grown from human pluripotent stem cells. Using human stem cell derived retinal organoids, damage to these retinae will be induced in vitro. Various growth and differentiation factors will then be added to the damaged retina to promote ‘endogenous regeneration’ as reflected in the ability of Müller cells to proliferate and become neurons within the organoid. This will be investigated by protein and gene expression studies. It is expected that the results will increase our knowledge of mechanisms involved in the ability of human retinal Müller cells to regenerate the retina. This will be of benefit to the research community and therefore lead to further investigations relating to the promotion of endogenous regeneration in the human retina which would be of benefit to many people.

We will examine the ultrasound characteristics of 4 major intraocular tumour types: choroidal naevus, melanoma, haemangioma and metastasis. We will study the dimensions of each type to correlate them and we will study the internal blood flow characteristics using colour flow mapping and Dopller ultrasound techniques. Unique features of this study are the numbers of patients at Moorfields, the technical ability and equipment of the ultrasound department, and evaluating all four tumour types in one study, side by side. In the sort term we will validate our clinical impressions from managing these patients in the clinic. In the medium term we will publish the results to disseminate the findings to the ophthalmic community. In the long term we will use the findings to help diagnose the 1400 referrals that are seen in the oncology service annually at Moorfields.

The project will trial two home-monitor measures in children (6—12 years) with chronic visual impairments (a key clinical need). Specifically:
1. A home monitoring device for amblyopia, using a tablet-computer measure of contrast sensitivity
2. A home monitoring device for paediatric glaucoma, using a tablet-computer measure of visual field loss
The work is designed to evaluate the feasibility of paediatric home-monitoring in general, to provide pilot data for two novel tests, and to foster closer collaborations across UCL and MEH.

The project - Some inherited retinal diseases (IRD), including eye diseases, are the result of altered genes. It is now possible, in some cases, to improve vision for patients with these diseases by injecting drugs that target the faulty genes. Once such drug, Luxturna, is already in use and others are being trialled. We need to understand exactly how these diseases alter the eye so we can measure exactly what effect the new drugs are having. This project will analyse images from one of the world’s largest collections of retinal images of rare eye diseases from Moorfields Eye Hospital and other leading European Centres, and use artificial intelligence to predict how the structure of the retina may change over time.

The project - The retina is the light-sensitive tissue lining the back of our eye. To maintain the stable  environment needed for normal visual function, retinal blood vessels have evolved to form a highly specialised cellular barrier that regulates the passage of many substances. This vascular barrier is formed principally by the endothelial cells that line the vessel wall but its function is under the influence of other associated cells, including pericytes, neurons, and glia, which together form what is called the neurovascular unit (NVU). In the eye, the NVU regulates the retinal vessels that constitute the inner blood-retinal-barrier (iBRB). Currently it is unclear how and when NVU components are integrated during development and how this relates to iBRB function. We will address this lack of knowledge by quantifing the temporospatial organisation of NVU components and iBRB function using biomedical image analysis. We will then extend the study to investigate the impact of diabetes on NVU formation and function in the contecxt of diabetic retinopathy. We will use zebrafish as a preclinical model as zebrafish share 70% genomic similarity with human, establish a basic body plan within 24 hours, are embryonically transparent, and tissues/cells of interestcan be visualized using transgenic lines. Thus, we will be able to study specific NVU components non-invasively over time to unravel iBRB function.

The project - Glaucoma is the commonest cause of incurable blindness globally, affecting over 80 million people. Current population screening tests are ineffective and many patients have irreversible vision loss at diagnosis. Even after diagnosis, many patients continue to lose vision despite current therapy. Compounding this, the number of people affected by glaucoma will increase by 50% in the next 20 years. Already stretched glaucoma services are therefore compelled to innovate to minimise blindness. This project aims to develop easily adoptable tools, based on genetic and artificial intelligence (AI) algorithms, that will enable earlier detection and more effective glaucoma care.

The project - Leber hereditary optic neuropathy (LHON) and autosomal dominant optic atrophy (DOA) are two diseases where vision is lost due to damage to the optic nerve at the back of the eye. The optic nerve connects our retina (the film at the back of the eye, responsible for ‘developing’ pictures) to the brain, similar to the cable linking your camera to your laptop.     The optic nerve is made of specialised nerve cells and in both LHON and DOA, these nerve cells die due to problems with their mitochondria. Mitochondria are the cell’s ‘power stations’ converting food (such as sugar) into energy that can be used by the cell. As the process of transmitting light signals to the brain demands a lot of energy, these cells are particularly sensitive to any problems with their mitochondria.
A particular type of nerve cell within the optic nerve (the ‘ipRGC’) is much more robust to poor mitochondrial function compared with other nerve cells. These cells survive late in the course of both LHON and DOA, when most other nerve cells have been lost. We predict that there are a number of genes, active in ipRGCs, which protect them from the damage caused by poorly functioning mitochondria.

The project - Inherited optic neuropathies (ION) are an important cause of blindness in children and young adults. Spelling mistakes (mutations) in many different genes have been found to cause this group of disorders, which is characterised by the loss of specialised cells within the retina known as retinal ganglion cells (RGC). When damaged, this leads to severe irreversible vision loss.  Finding the mutation that cause disease is essential for patients and families to be able to access the best clinical care including family testing and counselling, prognosis and importantly, access to possible treatments. Current genetic testing strategies for ION in the UK are targeted and do not find the mutation in a significant proportion cases.

Diabetic macular oedema (DMO) is a major severe complication associated with the metabolic disorder of diabetes and the foremost cause of central vision loss and blindness. Around 30 million people world-wide are suffering from DMO and not all of them benefit sufficiently by the current lines of treatment. Recent proteomic data suggest that plasma and vitreous concentration of the glycoprotein LRG1 increases with disease progression and our data indicate that LRG1 is a trigger of microvascular dysfunction. Based on this evidence, we are requesting fundings to test whether LRG1 primes the retinal microvasculature to hyper-permeability characteristic of DMO and, consequently, whether LRG1-blockade could prevent the onset of DMO. 

The mortality rate for pancreatic cancer remains high. We have observed that in the KPC model of pancreatic ductal adenocarcinoma (PDAC) knockout of the gene encoding the secreted glycoprotein LRG1 enhanced survival. PDAC is characterised by a dense stroma and hypovascularisation, which limits the immune response and delivery of drugs. We aim to test the hypothesis that LRG1 is involved in PDAC-associated desmoplasia through investigating its effects on pancreatic stellate cells and establishing the therapeutic potential of a function-blocking antibody. If successful, this study could define a novel strategy to target fibrosis in PDAC and ameliorate outcomes of current therapies. 

Juvenile macular dystrophies are a group of diseases affecting children and young adults that lead to the loss of central vision as a result of damage to the macula, a special region of the light-sensing retina that enables us to see fine details clearly. A number of juvenile macular dystrophies, including Doyne honeycomb dystrophy, Best vitelliform macular dystrophy, Sorsby fundus dystrophy and Pattern dystrophy are inherited in an autosomal dominant fashion, meaning that the affected individual need only inherit one faulty copy of the gene for the disease to manifest. Unfortunately, there is currently no cure or treatment for these juvenile macular dystrophies. One approach involves the use of antisense oligonucleotides (ASO) to specifically target the faulty copy of the gene leaving the normal copy intact. In this study, we wish to investigate the utility of ASO therapy for the treatment of juvenile macular dystrophies exemplified by Doyne honeycomb macular dystrophy. Doyne honeycomb macular dystrophy is caused by an autosomal dominant mutation, c.1469C>T, p.(Arg345Trp), in the EGF containing fibulin extracellular matrix protein 1 (EFEMP1) gene. The mutation leads to misfolding of the resultant protein (fibulin 3) and consequently its retention in the retinal pigment epithelium (RPE) from which it is normally secreted. The accumulation of the faulty fibulin 3 protein in the RPE leads to the formation of sub-RPE lipoproteinaceous deposits called drusen near the macula, which impedes central vision. Recently, it was shown that skins cells (fibroblasts) from patients harbouring the EFEMP1 mutation can be reprogrammed to induced pluripotent stem cells (iPSC) that in turn can be differentiated or made into RPE.1 This iPSC-RPE model recapitulates important features of the disease, including dysfunction of the RPE cells that in turn alters the extracellular matrix (ECM) and initiates the formation and deposition of sub-RPE drusen.1 This human model of EFEMP1-associated maculopathy is therefore a powerful tool to investigate novel therapeutic approaches. In this project, we will use the iPSC-RPE model to investigate the therapeutic potential of ASO therapy to specifically target the mutant allele and thereby rescue the disease phenotype.

The intrinsically photosensitive retinal ganglion cells (ipRGCs) survive preferentially, compared to other RGCs in Leber’s Hereditary optic neuropathy (LHON). Therefore, the aim of this study is to determine What is different about these cells in terms of their transcriptomic profile that allows them to survive compared with other RGC subtypes in the inherited optic neuropathies? Our a priori hypothesis is that differentially expressed genes in these ipRGCs contribute to their selective neuroprotection against mitochondrial dysfunction. The overarching objective of this study is therefore to identify such “neuroprotective” candidate genes so that they can guide the development of novel mutation independent therapeutic approaches, applicable not only to inherited optic neuropathies, but more generally to both common optic neuropathies and degenerative neurological disease in general. RGCs will be isolated by thy1 immunopanning of digested retinas obtained directly after death from LHON model and wildtype control mice. These isolates will then be used to obtain single cell transcriptomic profiles (scRNAseq) using the Chromium 10X platform. ipRGC profiles will be isolated bioinformatically from the general RGC population by defining them as those cells expressing the melanopsin (OPN4) gene. Gene expression in these cells can then be compared to that of RGCs more widely, in both LHON and wild type mice. This approach overcomes both the limitations of immunopanning for Opn4 and provides data on all subtypes of RGCs (not only ipRGCS) in the context of LHON, which will allow further future research questions to be answered in silico.

High myopia significantly increases the risk of blinding complications such as glaucoma or retinal detachment. However, only little is known about the underlying molecular mechanisms. Patients, mice and zebra fish lacking the endocytic receptor megalin display a very severe congenital myopic phenotype suggesting that megalin serves a key role in normal ocular development. Investigations of megalin-deficient mice show that the endocytic apparatus is severely affected in both retinal pigment epithelium and proximal tubular epithelium cells. Exosomes are small secreted extracellular vesicles originating from fusion of multivesicular bodies/late endosomes with the cell membrane. Exosomes have been proposed to harbour important roles in communication between the retina pigment epithelium (RPE) and the neuro retina, an essential part of ocular development. Based on the overall well-established importance of megalin for the endocytic system, We hypothesize, that megalin dysfunction leads to high myopia through aberrant formation of the multivesicular bodies and consequently exosome release and RPE-retinal signalling. The overall goal of this study is to improve our understanding of the molecular mechanisms underlying congenital severe myopia. We will do this through detailed ultrastructural investigations of the endocytic system of normal and megalin-deficient primary porcine RPE cells. This study will provide important information on the molecular machinery involved in exosome formation in the RPE and determine if megalin holds a key role in this and may be a potential future therapeutic target.

This project will establish the brain pathways that combine information from different sensory modalities in order to make decisions. The neuronal populations supporting this combination and determining the subsequent choice and actions can be distributed widely and sparsely across the brain. Understanding their activity requires recording large neural populations throughout the brain while animals respond to multisensory stimuli. With our newly developed audiovisual task for mice and the recent advances in large-scale electrophysiology recordings, these experiments are now possible. First, we will record from early auditory and visual cortices to establish whether these are unisensory or, as some recent studies suggest, contain behaviourally relevant multisensory signals (Objective 1). We will then perform paired recordings from sensory cortices and frontal regions known to be involved in decision-making (Objective 2). Building on these results, we will establish a brainwide map of activity, comprising ~100,000 neurons, to map the evolution of audiovisual integration throughout the brain (Objective 3). Taken together, these experiments will provide the first brainwide map of a multisensory network at the neuronal level and answer long-standing questions about audiovisual processing

Glaucoma, the leading cause of irreversible blindness worldwide affects almost 65 million people worldwide aged 40-80 years. Though IOP is the main risk factor, in Normal tension glaucoma (NTG) the characteristic neuropathy occurs with IOP measurements within the normal healthy population range. While lowering IOP can be beneficial to slow progression, patients may still deteriorate, suggesting other factors confer susceptibility. Mitochondrial dysfunction is believed to contribute to an increasing number of ageing-associated neurodegenerative diseases and growing evidence suggests that it contributes to glaucoma development and progression. The high energy demand of the unmyelinated retinal ganglion cells (RGCs)— demonstrated by the presence of abundant mitochondria in their axons, makes the optic nerve head (ONH) susceptible to mitochondrial dysfunction. Animal studies have found that localised loss of metabolic support from astrocytes on the ONH results in damage to the RGCs. These astrocytes also have a high energy demand, provided by their giant mitochondria. Initial findings from our team in peripheral blood lymphocytes of patients with non-progressive Ocular Hypertension (OHT) have indicated that increased mitochondrial function confers resistance to glaucoma. Furthermore, we conducted a pilot study on 10 High Tension Glaucoma (HTG) and 10 Normal Tension Glaucoma (NTH) age matched Caucasian patients, with a range of glaucoma severity. Data so far show that NTG patients and those with the most severe glaucoma have basal and maximal mitochondrial respiration rate in peripheral lymphocytes almost 60% lower compared to HTG and mild/moderate patients respectively.
Aim: to define a subset of glaucoma patients with mitochondrial dysfunction who will be suitable for personalised (stratified) therapeutic intervention in the future. Hypothesises: mitochondrial dysfunction will be more frequent amongst patients with NTG and advanced/more rapidly deteriorating HTG.

Cardiovascular leakage underlies and accompanies diseases ranging from cancer to stroke. Leakage occurs through openings in the endothelium via loosened paracellular junctions. However, increasing evidence, in particular during a pathogenic stimulation of the vasculature lining neural tissues, also points to leakage occurring transcellularly, via caveolae-mediated transcytosis, either simultaneously to paracellular leakage or in sequence. Our lab showed that each leakage pathway can be studied in isolation following vascular stimulation with lysophosphatidic acid (LPA), which exclusively mediated paracellular leakage, or methamphetamine (METH), which exclusively triggered caveolar transcytosis. Here, we propose to use LPA and METH as paradigms in cultured brain endothelial cells and perfused ex vivo brains to characterise mechanisms of para- and transcellular leakage. First, underlying signal transduction networks will be determined by comparative phosphoproteome analysis. Second, we will determine molecular determinants and differences of each pathway and develop strategies to inhibit its key steps. Lastly, we will determine the relative contribution of para- and transcellular leakage in response to typical pathological leakage factors such as VEGF, bradykinin and thrombin.
This research will have particular impact on our understanding of stroke, where vascular leakage appears to be transcellular in the initial and paracellular in the more chronic phase of reperfusion.

We aim to correct inherited retinal dystrophy (IRD)-associated mutations in situ and rescue retinal degeneration in human cell-based models. We will combine advances in base editing technology, stem cell biology and gene therapy delivery systems to provide proof-of-principle evidence that the base editing machinery can i) be delivered to human photoreceptor cells in retinal organoids modelling IRD using a dual adeno associated virus (AAV) system encompassing a split CRISPR base editing platform, ii) efficiently repair genomic mutations causing retinal degeneration, and iii) rescue the pathogenic retinal phenotype. This will advance genome editing technologies towards their therapeutic application in the clinic.

This project will use use an assortment of retinal imaging techniques to measure the density of the blood vessels, local retinal thickness, and make measurements about how the blood moves through the vessels. This project will focus on watching how blood flows down the vascular tree from the large vessels as they enter the eye, to the smallest capillaries. We will look at how the vessels respond to the pulse rate, making measurements of how they expand and contract. The project will measure how fast the blood moved through the vessel in relation to cardiac cycle. These meausrements will be made while the eye is ‘at rest’ and during stimulated conditions.

Diabetic retinopathy (DR) is a complication of long-term diabetes, which can lead to blindness. Despite many years of research there are no good treatments available. One of the reasons for this lack of therapeutic options is our limited understanding of the pathobiology of DR. Although it is well known that blood vessels become abnormal, it is less well understood what happens to the rest of the retina. Furthermore, much of our current knowledge about potential disease processes is based on rodent models. However, to fully understand what happens in the human disease, it is essential to study human patients.

This project will use an assortment of retinal imaging techniques to measure the density of the blood vessels, local retinal thickness, and make measurements about how the blood moves through the vessels. This project will focus on watching how blood flows down the vascular tree from the large vessels as they enter the eye, to the smallest capillaries. We will look at how the vessels respond to the pulse rate, making measurements of how they expand and contract. The project will measure how fast the blood moved through the vessel in relation to cardiac cycle. These meausrements will be made while the eye is ‘at rest’ and during stimulated conditions.

The primary objective of this project is to further our understanding of the pathophysiology of dominant optic atrophy (DOA), the most common inherited optic neuropathy in the population and a cause of progressive blindness in children and young adults. The hallmark of this disorder is the irreversible loss of nerve cells, specifically retinal ganglion cells (RGCs), and optic nerve degeneration. The majority of patients with (DOA) harbour pathogenic mutations in the OPA1 gene (3q29), which encodes for a mitochondrial inner membrane protein. In this project, I will investigate how OPA1 mutations contribute to nerve cell loss and human disease.

The aim of this project is to test the hypothesis that the secreted glycoprotein leucine-rich alpha-2-glycoprotein-1 (LRG1) plays a role in vascular dysfunction in retinal ischaemic disease. We will:
1. Investigate how the Lrg1 gene is induced and regulated in an ischaemic setting
2. Investigate the temporo-spatial induction of Lrg1 and the associated vascular pathology in animal models of ischaemic retinal disease
3. Determine the potential beneficial impact of inhibiting LRG1, or its regulators, on vascular function in vivo.
These studies will pave the way for the potential application of new tharapies to block the vascular disruptive properties of LRG1 in retinal ischaemic disease. 

The aim of this PhD project is to generate a zebrafish model of age-related macular degeneration (AMD) by precisely knocking in a homologous disease associated mutations in the zebrafish genome. Pairing the novel genetic model with the many advantages of the zebrafish retina we will uncover cellular and molecular events leading to degeneration and the pathogenesis of AMD.

Keratoconus (KC) is a leading cause of blindness in young adults globally.
However, suspected KC is difficult to diagnose in the early stages of disease.
Integration of imaging and genetics has the potential to revolutionise early detection and prevention of disease and inform development of personalised treatments.
This research will generate and evaluate a new algorithm for early detection of KC from longitudinal Pentacam data that we anticipate will;
(i) reveal new insights on specific features of KC that will enable earlier diagnosis
(ii) inform recommendations for corneal collagen cross linking to preserve vision at an early stage of disease
(iii) enable evaluation of whether a patient is eligible for laser refractive surgery or should be referred to a KC clinic for continued assessment
Through the integration of the outcomes above, with our new data for genomic risk we plan to further develop and refine diagnosis and prognosis of KC leading to better outcomes for patients.

Addressing the function of the genes that control organ development is a fundamental goal in biology. However, because of genetic robustness and compensatory mechanisms, mutations in many genes show no phenotype, limiting our ability to understand their function. To circumvent this problem, I developed a pioneering genetic modifier screen approach in zebrafish that enables finding eye development genes that would otherwise remain unnoticed (Young et al., 2019). The genes we find will be screened for mutations in patients with eye defects to find new genes of diagnostic value associated with these pathologies. Overall this research programme will elucidate the gene regulatory network and cell signalling events underlying eye formation and growth compensation by combining research in zebrafish with the genetic analysis of patients with developmental eye globe defects.

Regulation of communication between the ER with endosomes and lysosomes at membrane contact sites (MCSs) is a recent concept that has major implications for our understanding of the dynamic state of cellular compartments and signaling. We have shown that these MCSs are sensitive to endocytic organelle calcium stores and mediate both downregulation of EGF receptor signaling and bi-directional cholesterol transport.

Epithelia and endothelia form cellular barriers that line organs and blood vessels to separate different body comportments. The retina is separated by two such barriers from the blood: an epithelium at the back of the eye and the endothelium that lines the retinal blood vessels. Barrier formation requires cells to interact with each other via adhesion complexes. Such adhesion complexes do not just function as a glue that sticks cells together but also control cell behaviour, function and survival. In chronic and inflammatory diseases, disruption of adhesion complexes can lead to barrier failure, and reduced or loss of vision. Such conditions are associated with diabetes, chronic inflammation, inherited disorders and age-related loss of vision. Here, we ask how mechanisms-associated with such adhesion complexes regulate the cellular response to stress and barrier integrity, and, thereby, help to maintain a functional retina.

The retinal pigment epithelium is a layer of cells at the back of the eye, the health of which is essential for normal vision. Many blinding eye diseases are due to defects in the normal functioning of the retinal pigment epithelium. This includes inherited diseases that lead to loss of vision often at a young age but also diseases associated with aging that cause reduced or loss of vision. In the UK alone, more than 600,000 people suffer from age-related macular degeneration, a disease associated with malfunctioning of the retinal pigment epithelium and for which now cure exists. The importance of the retinal pigment epithelium for vision is because of its support functions for photoreceptors, the cells that harvest light and stimulate the transmission of signals to the brain. Photoreceptors develop a specialized domain that senses light. To remain functional, photoreceptors constantly renew this domain, which involves the shedding of old material. This old material is internalized by retinal pigment epithelial cells in a process called phagocytosis. If the retinal pigment epithelium is defective, phagocytosis does not occur, the shed photoreceptor material accumulates, and the retina degenerates. Malfunction of the phagocytic machinery leads to retinal degeneration and blindness, with retinitis pigmentosa associated with the Mer tyrosine kinase (TK) being the best-known case in this disease category. We have identified the mechanism that drives the first steps of phagocytosis of shed photoreceptor outer segments (POS). This enables the exploration of therapeutic approaches to rescue phagocytosis in deficient retinal pigment epithelial cells. In vitro studies with patient-derived RPE cells deficient in phagocytosis led to the identification of viral vectors expressing the key component of that new signalling mechanism which can rescue phagocytosis efficiently. The current project is a proof of concept study to test such vectors in vivo to determine potential toxicity and therapeutic effectiveness.

Although over 250 genes are implicated in inherited retinal dystrophies (IRDs), approximately 40% of individuals with IRD lack a molecular diagnosis. Molecular diagnoses provide patients and families with accurate prognoses, recurrence risk and improved access to clinical trials. Genetic findings also inform our understanding of disease mechanisms, leading to new treatments. However, new genes/variants are likely to only account for a few rare cases, and are often hard to detect or unequivocally prove. This necessitates a consortium approach, pooling data and preliminary findings to expedite discovery and validation. The UKIRDC shares data and expertise, and is in a unique position to address these difficulties. We have an unprecedented opportunity to discover the genetic cause of disease in families that lack a molecular diagnosis by interrogating and interpreting the genome and exome data we have already generated. Through data sharing of large cohorts of unsolved patients (446 recruited to the UKIRDC), and the ability to cross-interrogate other large UK IRD genome datasets (NIHR-Bioresource and GEL >2,200 affected individuals), we will identify previously intractable disease mechanisms; for example, structural variants in non-coding regions (disrupting gene expression or DNA architecture), intronic and putative regulatory region variants (disruption of splicing or regulatory elements), and novel rare disease genes with variants present in only a few families. Once identified, variants will be further characterised using the collective functional expertise of the consortium, detailed in this proposal, for example generating cellular models by applying stem cell technologies and genome editing. By addressing IRDs as a consortium, we aim to ensure that all UK patients have the best possible diagnostic/prognostic information and access to clinical trials.
28 January 2020

I will investigate how glial cells find their correct position and shape in the retina. I aim to characterise the specific glial-neuronal cell interactions and determine intrinsic molecular pathways regulating the distinct glial morphological responses. Combining confocal time-lapse imaging, cell specific transgenes and genetic ablation of specific neuron types I will investigate the nature and necessity of glial-neuronal interactions in real time in vivo. I will also use cell-labelling techniques to validate the glial specific expression of candidate genes identified in our previous transcriptomic dataset. The top three candidates will be selected for CRISPR-Cas9 mutagenesis.
12 December 2019

Mutations in the AIPL1 gene cause Leber's congenital amaurosis (LCA), the most severe and rapid inherited retinal degeneration resulting in the loss of vision within the first few years of life. Currently, there is no cure or treatment for LCA caused by the loss of AIPL1, which thus has a devastating impact on the quality of life. A promising therapy for the treatment of LCA is gene therapy. Landmark clinical trials to treat LCA caused by mutations in the RPE65 gene provided proof-of-principle evidence of the safety and potential of gene therapy, and led to the approval of the first gene therapy (voretigene neparvovec-rzyl (Luxturna)) to treat LCA. The potential of gene therapy to treat LCA caused by AIPL1 mutations is supported by encouraging results of visual functional rescue in transgenic models of AIPL1 deficiency. However, the window of opportunity for therapeutic intervention in AIPL1 LCA patients is within 5 years after birth, during which time residual morphological and functional preservation of retinal photoreceptors is retained. This presents a significant challenge for AIPL1 gene replacement therapy, as longitudinal studies in RPE65 LCA patients revealed that efficient rescue requires sufficient transgene expression in the specific cell type affected prior to the onset of retinal degeneration. Therefore, in this study, we will test the efficiency and specificity of the transduction of AIPL1 adeno-associated viral (AAV) vectors in a human photoreceptor model. We have isolated renal epithelial cells from the urine of LCA patients harbouring molecular confirmed AIPL1 mutations, reprogrammed these cells to induced pluripotent stem cells (iPSC), and differentiated these iPSC to three-dimensional retinal organoids. Moreover, we have used CRISPR/Cas gene edition technology to 'knockout' the AIPL1 gene in a control iPSC, thus generating isogenic AIPL1 WT and AIPL1 KO iPCS from which retinal organoids have been differentiated. In this study, we will treat the patient derived retinal organoids and the AIPL1 KO organoids with AIPL1-AAV that have been prepared for a first-in-man gene therapy trial. We will assess the efficacy and specificity with which the AIPL1 transgene is delivered to and expressed in the photoreceptor cells. We will moreover determine the efficiency with which the AIPL1-AAV can rescue the disease phenotype of the organoids. This will be the first study to investigate the delivery and effect of AIPL1-AAV in human photoreceptor cells, providing critical information for the first-in-man gene therapy trial.
11 September 2019

My research program will 1) identify novel genetic mechanisms and genes associated with CEDs, 2) probe the pathophysiology and 3) facilitate the development of new therapies and diagnostic methods. Investigating the molecular mechanisms underlying TCF4 triplet repeat-mediated FECD, the most common genetic cause of CEDs and the most prevalent triplet repeat-mediated disease in humans, will be a major focus, with wide reaching implications. This will be accomplished by utilising patient-derived corneal endothelial cells to study the pathological consequences of the repeat expansion within its genomic and cellular context, in combination with a broad range of functional genomic approaches. Furthermore, a gene-targeted therapy for this condition will continue to be developed in partnership with clinical and Biotech collaborators. The resources and expertise I develop will also complement investigations of other genetically distinct forms of CEDs, such as PPCD, that we have recently attributed to non-coding mutations in epithelial-tomesenchymal (EMT) regulator encoding genes.
28 August 2019

"Eupatilin (brand name ‘Stillen’) has been widely used in the clinic as a drug for gastritis and peptic ulcer and has relatively few and mild reported adverse effects. Therefore, eupatilin could be used as a mutation independent approach for treating ciliopathy patients carrying CEP290 mutations, either in combination with sepofarsen or gene editing (most individuals only have one allele of c.2991+1665A>G which could respond to these approaches), or as an option for individuals with different CEP290 mutations, for who there is currently no treatment. It is the aim of this research to independently validate the published findings on the effects of eupatilin and extend the research into a human 3D retinal organoid model to show the potential to treat inherited retinal dystrophies associated with loss of CEP290 function."
21 August 2019

The two most common causes of death in England are dementia and cardiovascular disease (CVD). Dementia affects more than 800,000 people in the UK while every three minutes, someone has a heart attack. Given that we are living longer, more people are likely to be affected by these diseases over the next decade and it is therefore important that we develop effective strategies to identify those who are at risk. For CVD, current screening tests involve blood tests, measurement of blood pressure and asking questions about factors such as smoking and family history. In dementia, the diagnosis is challenging and relies on filling in a cognitive questionnaire. It has been estimated that more than 50% of cases in the developed world of the most common type of dementia, Alzheimer's disease (AD), are not picked up. The eye is the only part of the body where we can directly see blood vessels and the nerves. The eye develops from the same tissue as the brain and therefore changes in the nerves of the brain are often reflected in the eye. It has been shown that thinning of the nerves at the wallpaper lining the back of the eye, the retina, happens in people whose cognitive function is worsening. Similarly, changes in the blood vessels of the body, such as those that come with high blood pressure or diabetes, are also often visible in the eye. Researchers have found that specific features on retinal photographs can identify who might develop a stroke or heart attack within 5 years. In the last decade, a particular type of retinal scan, called optical coherence tomography (OCT), has revolutionised the study of eyes. OCT takes seconds to capture, is risk-free to patients, and has a resolution less than one hundredth of a millimetre. Over 30,000 OCTs are done each month at Moorfields Eye Hospital alone and many community eye services, such as high street opticians, now offer OCT scanning on their own devices.
23 July 2019

In industrialized countries, age-related macular degeneration (AMD) is now the leading cause of untreatable blindness. In addition to an age-related disease etiology, there are also inherited forms of macular degeneration, such as juvenile-onset Stargardt disease. These conditions, for which there are currently a lack of effective treatments, involve the loss of photoreceptor cells in the central retina, where a high cone photoreceptor density is responsible for effecting high resolution vision. We recently discovered that cone photoreceptors can modulate the sensitivity of the surrounding rod photoreceptors to allow them to function more effectively in daylight conditions. In retinal disorders involving degeneration of the macular cones, this lateral interaction is impaired, leading to saturation of the rod photoreceptors dynamic range and impaired vision in daylight. We have also recently discovered that direct modulation the neurons underlying this lateral interaction, the horizontal cells, improves quality of vision in mice lacking functional cones. Together, our results identify a specific circuitry underlying rod-mediated vision as a potential therapeutic target following macular degeneration. In this project, we aim to exploit these new findings to re-establish the rods’ diminished ability to function in daylight using two distinct approaches. Firstly, we will use direct modification of the rods to permanently shift and extend their light sensitivity into the daylight range. A small area of modified rods that are effective in daylight, likely with a higher temporal resolution, would improve extra-foveal fixation and vision. Secondly, we intend to establish a system that confers light sensitivity onto the horizontal cells, to replace the normal light-mediated input from the cones. We will thus restore the natural horizontal cell-derived modulation of light sensitivity to the rod photoreceptors, allowing them to function in daylight. Thus by utilizing our knowledge of specific aspects of retinal circuitry, we aim to develop effective novel therapies for improving vision in patients with advanced macular degeneration.
22 July 2019

Of particular significance, I am using the Cre-­Lox recombination system to provide lineage tracing and definitive proof of the fate acquired by any newly-­generated Müller cell-­derived neurons, a strategy that is sorely missing from the field at present and has raised questions about the veracity of some previous reports (see Pearson and Ali, Neuron, 2018). However, progress on this element of the project has been hampered by frustrations in the murine breeding programs, since I require double homozygote mice, bearing both the retinal degeneration gene in question (I am using two models) and the floxed reporter gene. I have now generated these lines and they are working well for acute injury. However, since my research question specifically focusses on progressive degeneration, I must wait a number of months for these animals to be at the correct stage for my investigations. I therefore seek further support for an additional 6 months to allow me both to complete my Fellowship investigations and leave sufficient time to complete a project grant application round, with submission planned for Autumn 2019, decision due Spring 2020.
4 July 2019

Progressive inherited retinal degenerative disorders (PIRDDs), such as age-related macular degeneration, are the leading cause of untreatable blindness in the Western world. PIRDDs are caused by gradual irreversible apoptosis of retinal neural cells resulting in dysfunction and vision loss. In the UK these diseases currently affect approximately 600,000 people at an estimated cost to the NHS of £1.6 billion per year. The incidence of PIRDDs increases exponentially with age. As the population is ageing worldwide, and 5% of people over 75 will be affected, the identification, and potential treatment, of the underlying cellular and molecular dysfunctions will be critical for global health going forward.
4 July 2019

AMD is the most common cause of vision loss in the elderly, yet there is no treatment for “dry” AMD (over 80% of cases). In dry AMD, progressive degeneration of RPE is associated with an accumulation of intracellular lipid-rich material. Every day the RPE phagocytoses the distal 10% of photoreceptor outersegments (POS), shed daily for essential renewal3. POS are densely packed with lipid-rich membranous disks that the RPE must digest and dispose of. Failure of the RPE to efficiently clear this material is associated with retinal toxicity4. Importantly, reducing cholesterol by the addition of cyclodextrins prevented RPE cell damage in a model of lysosome dysfunction5, strongly implicating cholesterol accumulation in the RPE with cell damage and loss of vision.
4 July 2019

This UTF Licensing Project investment is aiming to use syngeneic and humanised mouse models to evaluate the effect of anti-LRG-1 mediated vascular normalisation on the efficacy of adoptive T cell therapies and anti-PD1 immunotherapy. In addition to using tumour growth inhibition and overall survival plots to investigate the impact of anti-LRG-1 on the efficacy of these therapies, flow cytometry and immunohistochemical analysis on tumour samples will enable the evaluation of the effect of vascular normalisation on anticancer tumour-infiltrating lymphocytes.
2 July 2019

Genetic diseases of the retina cause severe impairment of sight. The aim of our research is to develop new treatments for the benefit of people affected. In the proposed project we will investigate selected blinding diseases by generating model retinas in the laboratory from stem cells. Using these models we will find out how the genetic defect can cause harm to the retina. The information acquired will enable us to design targeted treatments to correct the gene defect and thereby protect the retina against harm. By testing the impact of new treatments in the new laboratory model we will be able to prioritise candidates for further development and testing in human clinical trials for the benefit of people affected.
26 June 2019

"Artificial Intelligence (AI) has sparked significant interest in recent years for its potential impact in multiple aspects of life. AI is particularly suited for automatically recognising patterns in complicated scenarios and drawing conclusions based on these patterns. Deep Learning (DL), a branch of AI, has shown promise in many aspects of Ophthalmology, not only for its diagnostic potential but also its prognostic and screening capabilities. Reliable screening allows pathologies to be diagnosed at an early stage, when treatment is more likely to be successful. Inherited retinal diseases are a common cause of blindness; a tool that allows early diagnosis would be of great benefit, not only for prognostic and counselling purposes, but also in the context of novel therapeutic strategies like gene therapy. We aim to apply DL to electroretinography, a test which measures the electrical activity of the eye and is used to evaluate many causes of inherited retinal diseases."
25 June 2019

Age-related Macular Degeneration (AMD) is the most common cause of vision loss in the elderly. Though widespread, there is no treatment for “dry” AMD, which accounts for over 80% of cases. Dry AMD is characterised by the accumulation of lipid-rich material, largely derived from photoreceptor outer segments phagocytosed by the retinal pigment epithelium (RPE). Phagocytosis of photoreceptor outer-segments introduces huge amounts of lipids and proteins into the RPE for sorting and degradation in the lysosome. In AMD, some of this material escapes complete degradation in the lysosome, resulting in the accumulation of lipid-protein aggregates, termed lipofuscin, in the RPE. Lipofuscin is associated with lysosomal cholesterol accumulation and promoting cholesterol efflux has been proposed as a therapeutic approach for early AMD. It is becoming increasingly clear that many organelles, including the lysosome directly contact the endoplasmic reticulum (ER) at regions known as membrane contact sites. These sites are not only essential for the transfer of lipids, ions and metabolic intermediates but also the regulation of organelle function. We have uncovered a role for lysosome-ER contact sites, in the egress of endo/phagocytosed cholesterol out of lysosomes. Remarkably, using the lysosomal storage disease Niemann Pick type-C (NPC) as a model of lysosomal cholesterol accumulation, we found that expansion of ER-lysosome contact sites restored cholesterol transport in NPC. Building on this exciting and unexpected finding, I now propose to screen for FDA-approved small molecules that expand ER-lysosome contacts to mobilise lipids in the RPE. Preventing the accumulation of lipid-rich material in the RPE by contact site expansion could provide an exciting novel therapeutic strategy for AMD and other retinopathies.
20 June 2019

Blood vessels distribute oxygen and nutrients to all cells in the body. Accordingly, their abnormal development or function causes congenital and acquired cardiovascular disease. Blood vessels are lined by vascular endothelial cells (ECs), which contain the blood and serve as hubs to integrate systemic and organ-derived signals. The EC transmembrane protein NRP1 promotes vascular development and pathology by conveying signals from its ligands VEGF and SEMA3. Nevertheless, NRP1’s importance for blood vessel growth is not fully explained by it binding these known ligands. In non- ECs, NRP1 has been also implicated as a regulator of cell-cell adhesion. However, NRP1’s adhesion ligands have remained elusive and the importance of NRP1-mediated adhesion for vascular growth has not yet been investigated. We found that disrupting NRP1’s cell adhesion function impairs tissue vascularisation without compromising VEGF and SEMA3 binding. We therefore propose to (a) define the precise vascular phenotypes caused by loss of NRP1-mediated adhesion, (b) elucidate the underlying cellular mechanisms and (c) identify NRP1’s elusive adhesion ligand(s). Defining the role of NRP1-mediated adhesion in normal and abnormal blood vessel growth will increase our understanding of normal development and, in the long run, help improve therapeutic strategies that promote tissue vascularisation in ischemic diseases.
10 June 2019

Intraocularly administered antibody-based medicines have revolutionised the treatment of chronic blinding conditions. Current treatments require intravitreal injections approximately every other month. A key driver for developing new intravitreal antibody-based medicines is they must have a longer duration of action to reduce the number of intravitreal injections over time, which will be preferable for patients, caregivers and healthcare providers. We have developed stable IgG antibody mimetics called FpFs for intraocular use and a 2-compartment aqueous outflow model of the eye (PK-Eye) which accurately estimates the human clearance times of therapeutic proteins which we use to evaluate new molecules and formulations. An intractable problem for developing longer acting protein therapeutics for ocular use is that animal models develop antibodies against the candidate drug, so the use of the PK-Eye is critical to accelerating preclinical development of this class of medicines. The PhD project is focused on further developing antibody mimetics that will display prolonged duration of action in the vitreous. FpFs which are prepared by the chemical conjugation of different antibody-based fragments will be evaluated using the PK-Eye to determine structure-function correlations needed to optimise properties required to extend the duration of action in the vitreous cavity. Interested students should have knowledge of ocular pharmacokinetics and the pharmaceutical sciences, in particular (i) the design and use of in vitro models, (ii) protein modification and (iii) basic drug delivery principles, in particular affinity drug delivery.
26 April 2019

"Age related macular degeneration (AMD) is the major cause of blindness in those >60 in Western societies and a growing problem as populations age. In both mouse models (CFH-/-) and patient retinal tissues, mitochondrial decline is a key feature (Calaza et al 2015; Terluk et al 2015). As complement polymorphisms are present in 50% of patients, immune vulnerability is also characteristic. There are multiple points of interaction between innate immunity and mitochondria that could drive disease. The importance of immune vulnerability is highlighted by the failure of pathological development in retinae of CFH-/- mice kept in clean environments where weakened immunity is unchallenged. We will explore the relationship between immune vulnerability, mitochondria, stress and inflammation using CFH-/- and CFH-/+ mice defining the roles and interactions of each in disease develop as a platform for targeted therapy. CFH-/+ more closely mirror the human condition, while CFH-/- provide stronger phenotypic expression. We will run multiple commercial mouse protein arrays to measure retinal stress, inflammation and mitochondrial integrity as a function of age and environment. These will be used to address the following: Is there inherent tissue stress and mitochondrial weakness in clean ageing CFH-/- and CFH-/+mice retinae imposed by their genotypes that can be exploited by open environments? If so, what are these vulnerabilities? We will measure stress and mitochondrial function in ageing clean and dirty genotypes and wild type controls, profiling aged changes and their temporal sequence. For mitochondria we will also measure ATP and complex activity. We will then move clean animals at progressive ages into open environments known to drive inflammation. Re-measuring the above metrics will show what changes occur in stress markers and mitochondrial function, prior to and during the establishment of inflammation. These data provide a road map for cellular changes that are a platform for disease in this model. We will use cytokine arrays as markers of inflammation as these have been successfully employed by us. These will also be used for bloods to provide a comprehensive picture of systemic changes in mice as they are moved between environment identifying key agents that respond to environmental changes. We have paid little attention to the role of environmental pathogens as potential drivers of AMD. In part this is because disease development is slow and human environmental exposure so varied. This highlights the importance of this proposal using mice."
25 April 2019

Retinal dystrophies are a major cause of untreatable sight-loss. Transplantation of healthy photoreceptors to replace lost cells in end-stage disease is showing significant promise. At present, however, production of stem cell-derived retinal cells in vitro is costly and time-consuming, and there may be immune rejection. An attractive, but unproven, alternative is to try to unlock the potential for endogenous regeneration in patients earlier in the disease process, by recruiting cells within the retina with stem-like properties. Müller glia (MG) can reacquire a stem-like state and regenerate new neurons following retinal injury. MG -dependent spontaneous regeneration is remarkable in lower vertebrates but is very limited in mammals. Key to the lower vertebrate response is the activation of Lin28 and suppression of Let-7. Here, we seek to determine the regenerative capacity of mammalian MG during progressive retinal degeneration and the potential for exploiting this for retinal repair by targeting the Lin28/Let7 pathway.
24 April 2019

"To validate and apply an artificial intelligence (AI)-derived, optical coherence tomography (OCT) segmentation algorithm to the “Moorfields AMD Database”, the largest single centre database of eyes with neovascular (“wet”) age-related macular degeneration (AMD) in the world. This segmentation algorithm will provide objective, quantitative, and novel measures for a range of retinal morphologic parameters in an automated manner, and will provide new insights into: 1) differential responses to anti-vascular endothelial growth factor (VEGF) therapy in “wet” AMD, 2) the natural history of “dry” AMD, including risk of conversion to “wet” AMD, and 3) novel prognostic indicators. Perhaps most importantly, we will aim to facilitate worldwide clinical research in AMD by making the anonymised raw data from our study openly available to the academic community via an open source digital repository."
10 April 2019

In this article we describe the application of a new and innovative method to sequence the disease-associated TCF4 repeat expansion. The method termed ‘No Amp Targeted Sequencing’ provides a robust and accurate method for genotyping clinically relevant samples and overcomes the limitations of alternative approaches available. Furthermore, our study has revealed that the TCF4 mutation behaves in a dynamic way, provide novel insights into the cellular mechanism responsible for the disease.
4 April 2019

In this article we describe the application of a new and innovative method to sequence the disease-associated TCF4 repeat expansion. The method termed ‘No Amp Targeted Sequencing’ provides a robust and accurate method for genotyping clinically relevant samples and overcomes the limitations of alternative approaches available. Furthermore, our study has revealed that the TCF4 mutation behaves in a dynamic way, providing novel insights into the cellular mechanism responsible for the disease.
22 March 2019

Conditions such as advanced retinitis pigmentosa, age-related macular degeneration and diabetic retinopathy are the predominant causes of registered blindness and are characterized by photoreceptor loss. Photoreceptors are terminally-differentiated neurons, and once lost, they are not replaced. Cell replacement therapy offers the potential to reverse the associated sight loss by replacing the dying cells with healthy ones. A fundamental requirement for the development of effective photoreceptor transplantation is the establishment of new synapses between transplanted donor photoreceptors and their targets in the host retina, the bipolar cells (BCs). Recent reports indicate the potential for rescuing visual function in advanced disease and we now have exciting new data to strongly indicate that photoreceptors transplanted into models of end-stage disease can elicit reproducible improvements in retinal function. While very promising, the degree of improvement in visually-evoked activity requires improvement and defining ways to promote donor/host connectivity will be key to achieving robust restoration of visual function. Axonal pathfinding and synaptogenesis are mediated by a variety of diffusible and contact-mediated signalling pathways. Recent reports indicate that, in normal development, Wnt5a/5b is produced by rod BCs and acts to promote rod-BC synapse formation. In this project, we seek to apply these findings to the transplantation paradigm in order to enhance photoreceptor connectivity. Specifically, we will examine the effects of Wnts on rod neurite outgrowth and synaptic contact with BCs in vitro. Factors shown to promote photoreceptor axonal pathfinding in vitro will be expressed in models of end-stage degeneration using AAV vectors and combined with photoreceptor transplantation to determine whether they promote donor/host synaptic connectivity. Synaptic connectivity will be assessed using established trans-synaptic labelling techniques while visual function will be assessed using multielectrode array recordings, electroretinogram recordings and behavioural tests, where appropriate.
22 March 2019

Molecules that are upregulated in disease are potentially therapeutic targets as they can be inactivated with low risk of affecting normal healthy tissue. GEF-H1 is a protein upregulated in several diseases such fibrosis, chronic inflammation and infection leading to tissues dysfunction. We have developed and validated in cell culture and in vivo ocular disease models the first generation of permeable peptides to inhibit GEF-H1. Most pharmaceuticals are small molecules, although there are some exception, small molecules are more likely to be absorbed, locally and/or after oral administration if given as prodrugs. One advantage small molecule drugs have over peptides is that many small molecules can be taken orally whereas peptides generally require injection or another type of parental administration. Thus, based on the knowledge obtained from the develop and validation of permeable peptides to inhibit GEF-H1, we propose here the development of small molecules to inhibit GEF-H1.To ensure a robust set of inhibitors is discovered with potential for future drug development, we will develop at least 3 series small molecules. During the first year, the PhD student will design small molecular inhibitors based on modelling and/or crystal structures and will test them in bioactivity assays using surface plasmon resonance in comparison to the peptide inhibitors. During the second year, at least 10-20 compounds per series of the key small molecules will be synthesized and tested in bioactivity assays using surface plasmon resonance in comparison to the peptide inhibitors. During the third year the lead small molecules will be tested in cell culture models of ocular fibrosis, inflammation and infection. This research will lead to new treatment for the most common ocular diseases.
22 March 2019

The aim of this application is to request funds that will support clinical and fundamental research on visual development and plasticity in healthy vision and eye disease at the IO / Moorfields. My group develops novel tests that elucidate how vision develops across the lifespan in children and adults with healthy vision and eye disease, and the neural mechanisms that support this development. To address this important question, we use a combination of pioneering behavioural and neuroimaging-based tests. These tests incorporate the latest advances in test optimisation technology to keep tasks short and patient-friendly, eye-tracking to improve ease of use for those who have difficulty keeping the eyes fixed in one place or with providing button-press responses (babies, young children, patients with low vision), and virtual reality to test effects of visual impairment on everyday visual functioning. We use state-of-the-art neuroimaging methods that use machine learning and other computational innovations, to understand visual impairment at the level of its underlying processes in the brain, and to predict the scope for visual recovery after treatment. This multidisciplinary approach makes us world-leaders in transforming the latest computational and technological advances in visual neuroscience into new child- and patient-friendly vision tests that are more sensitive and informative than the current gold-standards used in clinics. Our tests address an urgent need in clinical studies that test new innovative treatments of eye disease with challenging populations such as children, elderly, or rare patients at Moorfields. The MEC Career Development grant is crucial to the continuation of this important work, as it will provide me with the opportunity to build on my group’s successful scientific track-record (made possible for a large part by MEC) and raise further funds that will consolidate us for the long term. It will also provide me with the research funds needed to collect crucial proof-of-concept data that will form the basis for a grant application that seeks to improve vision tests for children and low-vision patients using powerful new neuroscience tools.
22 March 2019

The retina is the layer of nerve cells at the back of the eye that senses light and converts light into electrical signals that are sent to the brain allowing us to see. Diseases of the retina are one of the biggest causes of blindness in the UK and globally. We are now able to generate images of the cells of the retina with extremely high resolution, which gives us the ability to see the structure of the retina and which cells have died away in various diseases. However, these imaging techniques do not always tell us if the cells that have not yet died away are working properly. We can measure the electrical signals generated by the cells of the retina in response to light flashes in a non-invasive way, by placing electrodes around the eye (a bit like an electrocardiogram (ECG) recording from the heart). This tells us about the function of cells that have not died away, which is important as many treatments will only work if problems are detected before the cells have died, i.e. before changes are seen on imaging. Whilst these electrical signals have been recorded for decades, more recent advances in our understanding of how the retina works, and also advances in mathematical modelling techniques allow us to develop newer techniques to better and more precisely evaluate aspects of retinal function. It is anticipated that these new techniques will help us better understand inherited retinal disease (the largest cause of blindness in the working age in England and Wales) and age-related macular degeneration (the largest cause of blindness in the elderly). Also, they will allow us to evaluate whether newer treatments are working or not, and also to choose the right patients for the right treatment, so that futile or harmful treatments can be avoided. Finally, it might be possible to incorporate some of the techniques into portable devices which can be used in clinic or remotely, so that patients do not need an extra hospital visit.
22 March 2019

Early onset inherited retinal diseases (IRD), such as Stargardt disease and retinitis pigmentosa (RP), can lead to permanent vision loss over a period of 10 to 30 years and are cumulatively the leading cause of blindness in the working-age population in the UK.

Moorfields Eye Hospital (MEH), Europe’s largest eye hospital (2M patients), has the largest and best characterised, genetically and phenotypically, cohort of IRD patients in the world (>9000), which includes 800 Stargardt and 600 RP patients. Discovering the causal genetic mutations in IRDs is a prerequisite to determining prognosis and inclusion in any gene-directed clinical trials, such as gene therapy.

IRDs often have characteristic patterns of progression due to gene expression timing and distribution in the different types of retinal cells. Experienced clinicians learn to diagnose these, using various imaging modalities, longitudinal information on the patient’s symptoms, and genetic screening. However, the process is time-consuming and expensive, as it requires access to specialist centres, expensive clinical and genetic tests, and specialist training in electrophysiology, image interpretation and bioinformatics. Moreover, the spectrum of disease-causing mutations, which may be non-coding, and the genetic heterogeneity of similar clinical phenotypes is still poorly understood. Consequently, 40% of IRD patients do not have a diagnosis because of lack of data or insight.

My solution is to develop a Machine Learning (ML) system, trained on the wealth of high-dimensional patient data available at MEH, capable of (i) predicting genetic diagnosis in IRD (ii) predicting patient outcome (iii) recommending treatments and assessing their efficacy.

In order to achieve this, I will create a longitudinal dataset from the available data at MEH, incorporating, imaging, electroretinograms, quantitative and qualitative phenotypes extracted from free-text clinical records, genetic data, along with self-reported patient data. Human Phenotype Ontology (HPO) terms will be used to annotate patient records in a standardised manner. Much of the groundwork for this has already been completed, firstly, through my development of the Phenopolis platform (www.phenopolis.org) for analysing genetic and HPO data, and secondly my contribution to an image processing pipeline for MEH.

I will use this dataset to train Convolutional Neural Networks, a ML approach that successfully solves complex classification problems on high-dimensional and imaging data, to predict causative genetic mutations. I first propose to evaluate my methods on three categories of IRD patients with known causative genes: i) ABCA4 for Stargardt, ii) USH2A for RP, and iii) other 10 genes in similar IRDs. The networks will be tested first on i) vs ii) and later on the harder problem of i) vs ii) vs iii). I will assess the predictions to discover what is the pertinent clinical information used by the network and whether this sheds new insights into the phenotypic and genetic heterogeneity of these diseases, in particular with regards to incomplete or age-related penetrance.
22 March 2019

Aniridia is a genetic eye disease caused by mutations in PAX6. It is characterised by variable iris and foveal hypoplasia, nystagmus, cataract, glaucoma and limbal stem cell deficiency leading to corneal keratopathy. Over 40% of mutations are nonsense and therefore amendable to nonsense suppression therapy. We have identified a generic drug called amlexanox, previously used for treating mouth ulcers and asthma, that can be repurposed for dual inhibition of nonsense-mediated decay and nonsense suppression. This drug has been shown to be successful at halting the retinal degeneration and blindness in choroideremia (caused by mutations in CHM) and improving protein prenylation function by 40%. We have peer-reviewed sight-loss charity funding to test the efficacy of amlexanox in aniridia using PAX6-patient derived induced pluripotent stem cell optic cups. But as this condition has a variable course of disease, with some patients more severely affected than others, we do not know the most suitable outcome measures for a clinical trial.
14 February 2019

The lymphatic system is a network of vessels that carries excess fluid from tissues into the bloodstream. In people with lymphatic disease, the lymphatic system cannot drain fluid properly, causing uncomfortable tissue swelling that is difficult to treat. Moreover, the skin overlying the swollen tissue can become infected, and the transport of immune cells to lymph nodes can be impaired. These conditions can be inherited, for example in a condition known as lymphedema, or can be acquired, for example due to lymph node resection after cancer surgery. Here, we will investigate a novel mechanism by which lymphatic vessels form through the recruitment of specialised cells in the blood. Understanding more about the process by which lymphatic vessels normally form may uncover processes that can be stimulated to grow new lymphatic vessels in patients with lymphatic disease.
12 February 2019

Rare diseases affect approximately 7% of the population ( https://www.raredisease.org.uk/what-is-a-rare-disease/ ). For these, it is harder to pool data for research purposes, as, unlike other common disorders, the pertinent data is highly specialised, embedded and inaccessible within hospital networks (images, radiographs, electrophysiology, molecular diagnosis). How then do we collate person-specific clinical information from multiple locations and across time for the purposes of clinical care and research? A standard strategy might be to link data within the NHS Data Spine, and then access the data en masse for research. This is technically challenging and ethically difficult without explicit patient consent.  

MyEyeSite explores a different tack – give the job to the patient! Our unique insight is to start with highly motivated patients and their medical community, within a specific disease group, and support them with new, accessible technology. 
23 January 2019

Over the past 40 years, there has been a dramatic increase in our understanding of the molecular process involved in vesicular transport, which has lead to a ‘vesicle centric’ model of protein transport where organelles are thought to function in isolation. However, it is becoming increasingly clear than many organelles directly contact each other and that these sites are not only essential for the transfer of lipids, ions and metabolic intermediates but also the regulation on organelle function. We have uncovered an exciting and unexpected role for the R-SNARE VAMP4 in endosome-ER contact site formation and have evidence to suggest that these contact sites play an important role in cholesterol transport in vitro and its homeostasis in vivo. We propose that by studying the function of VAMP4 we will gain important insight into the molecular processes underpinning membrane contact site formation and the mechanisms regulating cholesterol physiology.
15 January 2019

Lens opacity is the most common cause of blindness worldwide. The world health organization (WHO) estimates 18 million people are bilaterally blind from cataract, representing almost half of all causes of blindness globally. Congenital cataracts are seen in 1-6/10,000 births in the UK and 5-15/10,000 births in developing countries and are a significant cause of visual impairment in infants and children. In addition to surgical challenges, there are multiple short-term and long-term post-operative issues including; secondary glaucoma, retinal detachment, complex optical rehabilitation and prevention / management of amblyopia. Hence, frequent post-operative assessments are needed throughout childhood and life-long follow-up to check for glaucoma. 

To better understand the underlying molecular mechanisms of lens opacification, we need to improve our knowledge of genes expressed in the lens, including identifying new genes for diagnosis purposes, elucidating their function, and thereby also identify potential pathways for nonsurgical treatment for cataract. Our laboratory has made substantial contributions to the molecular genetics of inherited cataract by identifying six important genes causing a wide range of isolated congenital cataract subtypes.

• gap-junctional proteins (connexin 50 and connexin 46), 

• water channel protein (AQP0), 

• heat shock protein (Alpha-B crystallin), 

• developmental gene (PITX3) 

• wolfram syndrome gene (WFS1) 

• and recently many novel and recurrent mutations in known genes causing congenital cataract.

These genes are already being used for diagnosing congenital cataracts.

Our main aim to identify genes in three large families with autosomal dominant and recessive congenital cataract through an advanced technology called next generation sequencing (NGS) and to study functional consequences of the first mutation found in WFS1, causing an isolated congenital cataracts.

However, gaining an understanding of the molecular and biochemical/functional mechanisms underlying different genetic forms of cataract may give us clues for the development of potentially non-surgical treatments and prevention of cataract surgery in children, so alleviating an enormous public health problem.
14 January 2019

The hallmarks of neovascular age-related macular degeneration (nAMD) are pathological neovascularization of the outer retina and subretinal fibrosis as a result of a wound healing response that follows choroidal neovascularization. Anti-VEGF therapy has become a standard treatment that improves visual acuity in many nAMD patients; however, unsuccessful treatment outcomes have often been attributed to the progression of subretinal fibrosis. Our previous unpublished work demonstrates that GEF-H1 inhibitors rescue fetures of fibrosis and inflammation in in vitro models of eye disease as well as in models of allergic and autoimmune disease, i.e.,. ocular surface conjunctivitis and experimental uveoretinitis. Thus, our aims are to test whether GEF-H1 plays a role in pathological fibrosis due to neovascularization of the outer retina in in vitro and in vivo models of nAMD. Our objectives are to test the effect of GEF-H1 inhibitors in laser induced choroideal neovascularization (CNV) and in retinal in neovascularisation in the oxygen induced retinopathy (OIR) model . Our results will demonstrate whether GEF-H1 inhibitors can reduced choroidal and retinal NV, as well as fibrosis in animals and will allow to assess their therapeutic potential in neovascular diseases in the eye.
27 December 2018

Cardiovascular disease with compromised blood supply to crucial organs is a major cause of morbidity and mortality in the Western world. Stimulating new blood vessel growth by therapeutic administration of angiogenic factors or cells holds much promise to treat tissue ischemia in these conditions. Accordingly, research investigating the molecular and cellular mechanisms of blood vessel growth is of great clinical importance. New blood vessels are normally generated when endothelial cells (ECs) in pre-existing blood vessels proliferate and migrate into hypoxic tissues that release angiogenic factors. Additionally, prior studies have proposed that circulating blood cells can contribute ECs to further enhance blood vessel growth and may be administered to help compensate for dysfunctional pre-existing endothelium in patients with cardiovascular disease. However, the identity and existence of circulating endothelial precursors in the adult has remained controversial. We have recently shown that early embryonic hematopoietic cells termed erythromyeloid progenitors circulate in the embryo and can differentiate into ECs to promote blood vessel growth. Our new pilot data suggest that similar precursors exist after birth and can give rise to ECs that incorporate into growing vasculature during normal development and in ischemic disease. We propose to define the prevalence and their contribution of these precursors to vessel growth in development and disease.
20 December 2018

Patient information website/app on genetic eye disease for public engagement
19 December 2018

Over the past 40 years, there has been a dramatic increase in our understanding of the molecular process involved in vesicular transport, which has led to a ‘vesicle centric’ model of protein transport where organelles are thought to function in isolation. However, it is becoming increasingly clear than many organelles directly contact each other and that these sites are not only essential for the transfer of lipids, ions and metabolic intermediates but also the regulation on organelle function. We have uncovered an exciting and unexpected role for the R-SNARE VAMP4 in endosome-ER contact site formation and have evidence to suggest that these contact sites play an important role in cholesterol transport in vitro and its homeostasis in vivo. We propose that by studying the function of VAMP4 we will gain important insight into the molecular processes underpinning membrane contact site formation and the mechanisms regulating cholesterol physiology.
18 December 2018

Background: Fuchs endothelial corneal dystrophy (FECD) is a common, age-related, sight threatening condition that primarily affects the corneal endothelium. Surgical interventions are currently the only treatment option available to patients with advanced disease to restore vision. Corneal transplantations are invasive procedures that rely upon specialist skills and facilities. They can be associated with both intra and post-operative complications and are dependent on the availability of healthy donor material, of which there is currently a global shortage. These issues, coupled with the global aging population, highlight the need for alternative and effective treatment strategies to be developed for FECD.

A triplet repeat expansion (CTG18.1), situated within a non-coding, intronic, region of TCF4, has been associated with FECD. We have now demonstrated that CTG18.1 confers significant (p=5.69 x10-71) risk for FECD (>76-fold) in our large (n > 450) British patient cohort. 
Targeted treatment strategies for FECD are now a realistic goal given our emerging understanding of the underlying genetic mechanism of disease. 

Project Aim: To utilise an ex vivo patient-derived cell model to investigate FECD disease mechanisms and test the utility of targeted therapeutic approaches. This project represents a continuation of the testing of the ability of therapeutic oligonucleotides directed at the TCF4 transcript with >50 repeats to reduce the presence of RNA foci and downstream toxic effects in cultured corneal endothelium derived from Fuch’s patients.
1 December 2018

To explore, develop, and apply the latest developments in automated machine learning (“AI that can build AI”) to medical image classification.
23 November 2018

Docosahexaenoic acid is a dietary acquired fatty acid essential for healthy vision that is taken up and utilised by cells in the retina. It is particularly important as reduced levels have been associated with common eye diseases including age related macular degeneration and retinitis pigmentosa, but little is known about how it is handled by retinal cells. Development of a method known as click-chemistry, has the potential to provide an accurate way to examine fatty acid uptake and movement within cells. This method requires two components that work together like two pieces of a jigsaw, with the fatty acid having a small modification allowing another component, a label, to be ‘clicked’ on. The label allows the position of the fatty acid to be detected using a specialised light or electron microscope. The methods optimised in this study will be invaluable for future studies to examine the role of the fatty acid in eye disease.
22 November 2018

Aniridia is an orphan heritable disease (1:100,000 incidence) in which PAX6 gene mutations cause severe developmental eye problems. Aniridia-related keratopathy (ARK) manifests as persistent, chronically painful defects of the outer epithelial layer of the cornea, vascularisation and scarring. This correlates with dysfunction in the ability of limbal epithelial stem cells (LESC), stromal cells and matrix to maintain normal corneal epithelium. ARK- induced light sensitivity can lead to social exclusion. Current treatments include whole tissue transplantation and cultured LESC therapy which have both shown poor long-term outcomes. This may in part be due to 1) antigen presenting cell presence in whole tissue transplantation and 2) stromal cell absence in cultured LESC therapy. An optimal ARK treatment would include transplantation of both healthy LESC and stromal cells. Our proposed solution utilises our patented technology known as RAFT (Real Architecture for 3D Tissues) in which LESC and stromal cells are co-cultured in a transplantable type I collagen-based tissue equivalent. Good manufacturing practise (GMP) protocols have been established in our pre-GMP laboratories and RAFT has undergone pre-clinical safety studies in a rabbit model. Having sought regulatory advice (MHRA) we now need to undertake 1) GMP protocol validation in our Cells for Sight MHRA/HTA licensed manufacturing facility, and then 2) submit a Investigational Medicinal Product Dossier to proceed to first in human RAFT transplantation studies in patients with ARK. In this phase I / II clinical trial we will perform RAFT transplantation in one eye of 21 systemically immunosuppressed ARK patients to assess RAFT safety, tolerability and preliminary efficacy. The outcome measures will include corneal surface normalisation and stability (lack of conjunctivalisation, vascularisation and epithelial defects, and improvement in corneal clarity).
5 November 2018

Research Question: Is prophylactic laser iridotomy in PACS cost effective compared to no treatment?
Aim: To perform a cost utility analysis to determine the cost effectiveness of prophylactic LPI in the UK.
Background: Primary angle-closure glaucoma (PACG) affects 20 million people worldwide. Primary angle closure suspects (PACS) have a higher but poorly quantified risk of developing glaucoma. Laser peripheral iridotomy (LPI) is widely practiced as prophylaxis against PACG but its efficacy is unproven. (1)
2 October 2018

In AMD, as with many degenerative diseases, cells are lost leading to a critical loss of function. In AMD the main cell that is initially affected is the retinal pigment epithelium cells (RPE) the support cells for the light sensitive cells of the eye. The stem cell approach aims to replace cells in the eye that are either damaged or missing. In the first of the trials developed by the London Project (a clinical/science collaboration aiming to deliver a stem cell based cure for AMD), we are using human embryonic stem cells (hES) that have been transformed into RPE cells. These RPE cells will then be transplanted as a sheet on an artificial membrane under the patient's retina. In order to manage this, we have designed a specially engineered ‘patch’ consisting of the artificial membrane coated by a carpet of RPE cells within a specially designed surgical delivery tool. The trial has received regulatory approval and funding to commence in 2015.

During the preparatory studies for this trial, it was noted that over time the eye reacted to the presence of the artificial membrane with fibrosis. This did not affect the beneficial outcome of replacing the cells, but is likely to limit optimal function and physical orientation. As it is critical to deliver the cells as a single continuous sheet, the use of a membrane is fundamental for the survival of the cells and ultimately to the success of this treatment strategy. As such, we are exploring ways to inhibit the fibrosis that is encountered. It is hoped to combine the knowledge and understanding of reducing or eliminating fibrosis gained from Prof Khaw’s work in enhancing stem cell treatments for AMD.
27 September 2018

The replacement of tissues lost due to disease is a major avenue of investigation for the treatment of diseases such as age-related macular degeneration (AMD). Currently, the production of replacement retinal pigment epithelial (RPE) cells from human embryonic stem cells (HESC) holds great promise for the treatment of those affected by this degenerative disease. However, HESC-derived cells are not patient specific and there is a risk of rejection of these non-related donor tissues.
Recent advances in stem cell technology have led to the development of a new type of stem cell, known as induced pluripotent stem cells (iPSC). These cells, which can be created from the skin or blood cells of patients, have the ability to turn into any cell type of the body, and therefore offer new hope for the production of patient specific tissues for transplantation.
We have previously shown that iPSCs can be used to derive RPE cells that are functional and rescue failing vision in rats suffering from retinal degeneration.
27 September 2018

1) To develop a photoreceptor/bipolar cell co-culture system to assess the effect of candidate molecules on photoreceptor axonal pathfinding
2) Determine whether or not Wnt signalling promotes rod photoreceptor neurite outgrowth and rod-bipolar cell synaptogenesis in vitro and in transplantation
3) Apply the co-culture system to screen potential candidates for cone synaptogenesis

Currently, best-corrected visual acuity (BCVA) is the only validated end-point that is accepted as a visual function clinical end-point in clinical trials in retinal diseases. However, changes in BCVA do not parallel disease progression from intermediate to late AMD or the progression of non-central geographic atrophy (GA) to fovea-involving GA as BCVA is only affected when the disease involves the fovea. Therefore, there is an unmet need to capture early changes in visual function that are experienced by subjects that strongly correlate to the anatomical changes. Developing and validating these visual function end-points will enable the evaluation of novel therapeutic agents to prevent or delay the progression of AMD, a disease of paramount public health importance with significant societal burden. Furthermore, studies evaluating visual function changes systematically in ageing and eyes that are genotypically or phenotypically at risk of development or progression of advanced AMD or with a family history of AMD are lacking. With the recent evidence that photoreceptors may be affected very early in these eyes, it is important to find out the most relevant visual function tests that can detect these early changes accurately.
12 September 2018

Lysosome dysfunction is implicated in lysosomal storage diseases, atherosclerosis and multiple age-related neurodegenerative diseases, including Parkinson’s, Alzheimer’s and age-related macular disease. Lysosomes are not simply terminal degradative vacuoles but are signalling organelles central to maintaining cellular homeostasis. Nutrient sensing on the lysosome can lead to nuclear translocation of the transcription factor, TFEB, and transcription of lysosomal genes. However, it remains unclear how newly synthesised lysosomal proteins are packaged into new lysosomes and how old/damaged lysosomes are cleared. We recently established models of lysosomal aging/dysfunction in retinal pigment epithelial cells, taking advantage of the huge degradative burden of these post mitotic cells, and identified conditions that upregulate lysosome biogenesis and activity. We propose to identify the trafficking steps and molecular machinery underlying lysosome biogenesis and test the relative contributions of lysosome exocytosis and lysophagy (autophagy of lysosomes) to cellular clearance of aged/damaged lysosomes. Our ultimate goal is to exploit these mechanisms to promote damaged lysosome clearance or rejuvenation and/or promote lysosome biogenesis for the treatment of diseases where lysosome activity is compromised. The efficacy of potential treatments in upregulating activity, whilst readily measurable in culture, is difficult to assess in vivo. We will establish a molecular signature of aged/damaged lysosomes in cultured RPE cells and determine whether that signature can be used to identify damaged lysosomes in aged/diseased retinae and other disease settings where lysosome dysfunction is implicated.
30 August 2018

This award recognises the UCL Institute of Ophthalmology and its National Health Service partner Moorfields Eye Hospital as the United Kingdom's national centre of excellence for eye research. The focus of this Centre grant is human translational studies (pre-clinical and early-phase in man) and together, UCL and Moorfields are the world's most productive hospital/university partnership in ophthalmology (Boston Consulting 2013; Elsevier 2015). Our foremost aims are to deliver innovative research that rapidly translates into improvements in visual health, quality of life and prosperity. Our strategy is delivered through cross cutting research themes that are underpinned by excellent science and interdisciplinary approaches; committed to driving innovation and targeting unmet clinical need particularly in common diseases; providing distinct but inter-dependent therapeutic modalities, technologies and investigative approaches with highly specialist infrastructure that, in many cases, is unique in Europe.
24 August 2018

RB1 mutation is very frequently associated with retinoblastoma initiation/progression. Although importance of mutation of other genes have been suggested for malignant progression and for phenotypic differences among retinoblastoma patients. The difference include sensitivity to chemotherapy and malignant status. This is associated with lack of genomic information of retinoblastoma. Although mutation analysis of RB1 gene have been expensively performed, the exome sequences of Retinoblastoma is limited. Especially there is no report of whole genome sequence analysis. Furthermore, no paper has not been published about classification of retinoblastoma based on mRNA expression profiling and whole genome sequencing.

We have started the whole genome sequencing analysis of human retinoblastoma samples, which include fresh samples and historic ones in collaboration with Ashwin Reddy (MEH, Barts), Serena Nik-Zainal (Sanger Institute, University of Cambridge) and Zerrin Onadim (Barts) under ethical approval. Also, in fresh retinoblastoma samples, we also determine mRNA expression profiling, aiming to correlate genotype, cellular properties and clinical phenotypes. We have completed 17 whole genome sequences and 7 expression profiling.
23 July 2018

Background 
The cornea is the transparent tissue situated at the front of the eye. It protects the eye from the external environment and focuses light onto the retina. The innermost part of this tissue is comprised of a specialised layer of corneal endothelial cells. These cells perform a pump-like mechanism removing water from the outer layers of the cornea, which, if left to accumulate, causes corneal swelling and clouding leading to loss of vision and/or blindness. 

Fuchs endothelial corneal dystrophy (FECD), characterised by corneal endothelial cell death, is a common, age-related, disease estimated to affect more than 4% of individuals over 40 years of age. A genetic mistake, termed ‘mutation’, in a gene called TCF4 is the most common cause of FECD. We have recently discovered that approximately 75% of FECD patients in the UK have a mutation in the TCF4 gene. 

Need for research 
Invasive corneal transplantation surgery is currently the only treatment option available to restore vision and prevent blindness for FECD patients. This treatment relies upon specialist facilities and is dependent on the availability of healthy donor material, of which there is currently a global shortage. Graft rejection and the need for systemic immunosuppression in some individuals, coupled with the global aging population, highlight the need for alternative and effective treatment strategies to be developed for FECD. 

Aim of the study
1.    To further understand the relationship between mutations in the TCF4 gene and FECD. 
2.    To investigate the biological reasons for the disease, using a model system that we have developed using donated corneal endothelial cells removed from FECD patients as part of their planned surgery. 
3.    To use the cell model to test new therapies for FECD designed to target the common TCF4 mutation that causes disease. 

Expected outcomes 
Cutting-edge technology will be used to develop a genetic test for FECD that will have the potential to accurately identify pre-symptomatic individuals so that we have a window of opportunity to prevent and treat the condition before sight loss. The cell model of FECD will be used to enhance our understanding of the biological reasons for disease, and enable us to test potential therapies that could be rapidly translated into clinical trials for this sight threatening condition.
20 July 2018

Measuring photoreceptor activity in vivo through electroretinogram (ERG) is an essential method to understand the pathogenesis of retinal dystrophy and test potential treatments. Recent technical advances in rodent ERG machines offer improved performance. In particular, the Diagnosys Celeris offers more reproducible and faster data acquisition. This equipment is approximately £49,000 and would ensure rapid and robust characterization of rodent retinal dystrophy models and the validation of potential therapies.
11 July 2018

The front surface of the eye is covered by two types of cells: corneal epithelial cells form a sheet centrally surrounded by a ring of conjunctival epithelial cells. There is a clear separation of these two cell types at a region known as the limbus. This clear separation between these two cell types breaks down in the disease of limbal stem cell deficiency. The corneal epithelium becomes invaded by conjunctival epithelium and blindness results. Dr Tiago Ramos, the post-doctoral research associate named in this proposal, as part of his recent PhD studies has been studying the reasons for this clear separation between the two cell types. He has identified small capsules (called exosomes) that are released by conjunctival epithelial cells containing the molecule microRNA-9 (miR-9). The aim of this proposal is to study the role played by miR-9 in maintaining the two cell types on the surface of the eye.
9 July 2018

We will identify the cellular and geographic sequence of cell death in the retina in patients with intermediate age- related macular degeneration (AMD). This will pinpoint what makes AMD progress towards irreversible visual loss. Our goal will be achieved via machine learning, genotyping and high resolution phenotyping of intermediate AMD patients using both retrospective and prospective data. Patients will undergo state-of-the-art imaging of the major tissues; neurosensory retina, retinal pigment epithelium (RPE) and choriocapillaris (CC), to identify fast progressors. We will control for genetic risk by genotyping all individuals to infer ancestry and to identify individuals with extreme polygenic risk scores. This will account for confounder effects of cryptic genetic diversity. Machine learning will be utilized to identify novel structural biomarkers on a population basis. Genetic and structural biomarkers will be validated in already collected genetic biobanks and extensive imaging databases of aging patients. These validated biomarkers will be key to:
1) detect conversion to late AMD earlier;
2) discriminate slow and fast progressors
3) identify novel therapeutic targets. This data may improve clinical trial design by better characterizing the study population and result in novel therapies by addressing the underlying mechanisms of one of the largest unmet medical needs.
2 July 2018

The focus of Dr Arno’s research programmes is to analyse existing whole genome sequencing (WGS) data and emerging data from the 100,000 genomes project to: (1) Unpick the rare non-coding disease mutations from the background of benign variation in known IRD genes and determine their effect on the protein using various laboratory techniques. (2) Identify new IRD genes and investigate their function in the retina using molecular biology techniques. (3) Understand why some gene mutations can cause different or unexpected clinical outcomes. (4) Use new technology to investigate certain un-readable regions of the genome that are known to harbour IRD mutations in a subgroup of patients. This is an ambitious programme of research questions but with exciting potential to advance our use, analysis and interpretation of this data towards the advancement of diagnosis, understanding and treatments.
1 July 2018

Dr Nikolas Pontikos is a post-doctoral fellow in computational biology and data science. His project will utilise the latest technologies and his skills in computer science to make sense of data using Artificial Intelligence. Work by Dr Pontikos will aim to apply machine learning so that retinal images and genetic data can help clinicians and scientists understand and potentially apply a diagnosis with the help of Artificial Intelligence. He will collect significant amounts of data from the project which will be analysed and shared with collaborators at the UCL Institute of Ophthalmology.
1 July 2018

We plan to dose our cellular disease models with ataluren (control) and amlexanox, then measure levels of PAX6 (both through immunostaining and western blot) and characterise the cellular phenotype. The human transcription factor VSX2 (previously known as CHX10) is a downstream target of PAX6 that is expressed in retinal progenitor cells and aids differentiation of bipolar cells. PAX6 knockout human retinal models are expected to show a reduced expression pattern of VSX2 and aberration of retina-specific cell differentiation and consequent laminated structure.
6 June 2018

Coversin, derived from a protein in the saliva of the Ornithodoros moubata tick, represents a second-generation complement inhibitor, which acts on complement component-C5, preventing release of C5a and formation of C5b–9 (also known as the membrane attack complex or MAC). Under complement dysregulation, the C5 protein is cleaved by C5 convertases into proinflammatory signalling molecules C5a and C5b, which are thought to contribute to conditions such as uveitis, an inflammatory condition of the eye. Coversin has been proven efficacious in several animal models of complement-mediated diseases and clinical trials. However, it’s application to the eye including PK/PD has not yet been fully investigated. This study seeks to assess the ocular penetration of Coversin and assess the tolerability and pharmacokinetics of this drug when administered via topical and intravitreal administration to rabbit eyes.
30 May 2018

Content The StarT Innovative Training Network (ITN) aims to train a new generation of vision researchers specializing in inherited retinal diseases (IRD). It combines the expertise of eight academic and six private sector partners. The prime deliverable will be 14 highly trained early starting researchers (ESRs) with unique skills for academic or private sector careers within the European job market.

Eye diseases are among the most common inherited human disorders. Around one third of the known genetic defects or syndromes involve the eye. Eye research has often blazed a trail for many disciplines to follow, giving a lead in genomics, transcriptomics, functional genomics, genome editing, stem cell biology, animal models of disease, and the development of novel therapeutic approaches such as gene therapy. In recent years, geneticists (some involved in this ITN) have identified a large proportion of the genes underlying IRD. However, the mutations identified do not account for all IRD cases, exemplified by Stargardt disease (STGD1), by far the most common IRD affecting ~1/8.000 persons (925.000 affected individuals worldwide; more than one-third of all IRDs). New classes of mutations in the disease gene ABCA4 which are either largely undetected by conventional genetic strategies or the significance of which is unclear have been discovered. The objectives of StarT are twofold: 1) to address these knowledge gaps through cutting edge research using innovative approaches related to genomics, transcriptomics, functional genomics, genome editing, the development of animal and cell-based disease models, stem cell biology, and the development of different therapeutic strategies; and 2) to give the next generation of European ophthalmic researchers unparalleled opportunities to be trained by a consortium of the best IRD research labs in Europe.
30 May 2018

1. What is the early ocular aniridia phenotype that arises from a human PAX6-deficient optic cup?
2. Which molecular mechanisms are disrupted in the early differentiating retina?
3. Will the aniridia-iPSC optic cup provide insights into clinical phenotypes such as retinal lamination defects that have previously not been described?
4. Can we restore functional PAX6 protein following administration of amlexanox- a dual nonsense-mediated decay inhibitor and translational readthrough drug?
Aims and Objectives:
1. Model a human 3D optic cup up to 17 weeks from a patient with nonsense-mediated PAX6-aniridia and a normal unaffected (wildtype) control using induced pluripotent stem cell (iPSC) technology
2. Fully characterise the human PAX6-deficient optic cup using histology, cell death/proliferation assays, immunostaining and RNASeq
3. Test the efficacy of amlexanox treatment for aniridia by assessing structural and functional PAX6 restoration
24 May 2018

The aim of this project is to provide proof-of-concept for the use of CRISPR/Cas9 gene editing as a therapeutic tool for autosomal dominant bestrophinopathies. The main focus of this project is to identify the molecular conditions where we can specifically edit the region around the BEST1 gene so that the dominant mutant allele is disrupted whilst leaving the healthy allele intact. We will utilise CRISPR/Cas9 technology as a molecular scissor to selectively “cut out” the dominant negative BEST1 allele and test the physiological implications of gene editing in induced pluripotent stem cell (iPSC)-derived retinal pigment epithelial (RPE) cells created from patients.
11 May 2018

CLN5 Batten disease is a lethal paediatric inherited neurodegenerative lysosomal storage disorder caused by mutations in the CLN5 gene. Children with CLN5 Batten disease suffer progressive and devastating dementia, motor decline, epilepsy, visual failure and ultimately premature death. There is no treatment for CLN5 disease and there is a desperate need for a novel effective therapy. We propose a preclinical assessment of AAV-mediated gene therapy to restore expression of CLN5 in both the brain and eye to prevent lethal neurodegeneration and maintain quality of life through prevention of blindness, respectively. This will be assessed using a CLN5 deficient mouse model that presents both the brain and eye symptoms observed in patients. We will also examine safety associated to over-expression of the CLN5 gene and biodistribution. Two vector iterations will be tested side-by-side administered either via intravenous or intracerebroventricular injection to verify which has the greatest therapeutic efficacy in the brain and by subretinal administration to the eye in pre-symptomatic neonatal mice as proof-of-concept. When optimisation of the individual treatments has progressed, combination therapy of both brain and eye will be performed in older early symptomatic mice to mimic the clinical scenario of treatment following diagnosis. A number of key factors support this proposal: (A) CLN5 protein is a soluble enzyme that can be secreted from a cell and taken up by another, amplifying the therapeutic effects of gene delivery, (B) The applicants and collaborators have significant experience in rescuing mouse models of neurodegenerative diseases using gene therapy, and (C) encouragingly, there is evidence supporting the therapeutic efficacy of AAV9-mediated gene therapy in the brain of a sheep model of CLN5 disease. This project is an essential first step towards treatment for CLN5 disease.
8 May 2018

Thanks to a series of projects previously funded by the BBSRC, we have gained important new insights into the workings of the human visual system. First, we have developed a new method of dissecting the visual pathways at the stage at which the signals are separated into incremental (ON) and decremental (OFF) pathways; the crucial methodological advance is that by manipulating the harmonics of a flickering light we can produce radically different signals in the ON and OFF pathways, so allowing us to probe the pathway at several stages quasi-independently. Second, we have developed a simple new model in Laplacian space with which we can predict visual performance over six decades of intensity. Third, we have developed a new model of lateral interactions in the early retina. The purpose of this proposal is to further develop and test these models in a novel series of experiments, and also to integrate them to produce a coherent, unified model of human vision.
27 April 2018

Inherited retinal diseases are now the most common cause of blindness in working age adults in England and Wales, and the second commonest in childhood. Currently, there is no cure or specific treatment available to patients. There are currently ~250 known disease causing genes, so developing a gene therapy for each would be extremely challenging, time consuming and costly. The degeneration of rods and cones is central to visual impairment arising from many inherited and acquired retinal degenerations. Recent advances in the understanding of the mechanisms of photoreceptor cell death have emphasized the role of failure of bioenergetics, and in particular, a failure of glucose uptake into photoreceptors associated with impaired aerobic glycolysis.
In this proposal, we seek to test the hypothesis that enhancing photoreceptor aerobic glycolysis can rescue photoreceptors from accelerated cell death, and therefore, lead to the development of a universal treatment for both rod and cone dystrophies regardless of their genetic basis. We will use two zebrafish models rpgrb-/- and pde6c-/- (human equivalent PDE6C and RPGR) manifesting a rapid retinal degeneration before 5 days post fertilisation, and will enhance glucose uptake and utilization by over-expressing key glucose transporters (RDCVF and GLUT1) and metabolic enzymes (LDH, G6PD and PFK) using microinjection of capped RNA. To assess glucose uptake we will dose the mutant zebrafish embryos with 2- NBDG (2-(N -(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose), a fluorescent glucose analog that can be used to monitor glucose uptake into live cells, and provide a quantitative measure following overexpression of key transport genes.

The impact of these interventions on photoreceptor metabolism will be assessed using established metabolic assays (ATP levels and oxygen consumption using the Seahorse XF Analyzer) and gene expression analysis. We will map metabolic networks using flux balance analysis (FBA) to provide insights into how metabolism in cells of a particular tissue shifts from one state to another in a more comprehensive way than is classically possible. Photoreceptor survival will be assessed using a combination of histology, cell death assays and functional vision optokinetic reflex testing. If successful, further funding or partnership with industry will be sort to either develop a gene therapy using the most efficacious candidate or a pharmaceutical agent to enhance glucose uptake or metabolism in higher order animals, such as the RPGR canine model. This project has the potential to prevent, halt or slow photoreceptor cell death by altering retinal metabolism in a vast number of patients with incurable blinding disease.
25 April 2018

Fuchs endothelial corneal dystrophy (FECD) is a common, age-related, condition estimated to affect more than 4% of individuals over 40 years of age in the United States. For patients with advanced FECD, corneal transplantation is currently the only treatment option available to restore bilateral vision and prevent blindness. Despite FECD being considered to be a complex genetic disorder, huge strives have recently been made in understanding the genetics of this disease. In 2010 a genome wide association study provided the first hint that FECD may not be as genetically complex as originally anticipated. ). A subsequent study identified a CTG repeat expansion, situated on within a non-coding region of TCF4 (termed CTG18.1) to be disease-associated, remarkably linking the initial GWAS hit with a putative functional variant. There is an absence of FECD animal models to facilitate translational research studies and we therefore aim to demonstrate that patient-derived corneal endothelial cell (CEC) cultures offer an ideal model for probing disease mechanism and testing therapeutic approaches for CTG18.1-related pathology, crucially within its native cellular and genomic context.
25 April 2018

Regulation of communication between the ER with endosomes and lysosomes at membrane contact sites (MCSs) is a recent concept that has major implications for our understanding of the dynamic state of cellular compartments and signalling. We have shown that these MCSs are sensitive to endocytic organelle calcium stores and mediate both downregulation of EGF receptor signalling and bi-directional cholesterol transport. 

Sphingolipids are one of the major lipid components of cell membranes and are increasingly appreciated as key modulators of diverse physiologic and pathophysiologic processes. Sphingosine kinase (SphK) phosphorylates sphingosine to form sphingosine-1-phosphate (S1P) a key bioactive signalling sphingolipid implicated in tumourigenesis. Intracellular S1P is downregulated by dephosphorylation or degradation by ER-localised enzymes. Our preliminary data implicates SphK in MCS regulation, either directly, in a tethering complex, or by downstream effects on acidic organelle calcium stores. Here we investigate the role of SphK in MCS formation and our predicted novel function for MCSs in S1P transport from endosomes to the ER for dephosphorylation/degradation.
24 April 2018

This study has three aims:

• To determine if a C3b deposition threshold needs to be reached to trigger phagocytosis

• To assess the impact of energy deficiency on C3b deposition at the cell surface 

• To demonstrate that APL-2 could prevent C3b-mediated phagocytosis in energy deficiency condition

This Hub builds on the significant successes of the UKRMP1 Acellular Approaches for Therapeutic Delivery Hub that provides a platform for a new transformative and fresh translational focus forward towards the clinic. The overarching goal in Acellular UKRMP2 is to develop state of the art new smart material technologies and take them through a robust translational gated pipeline to pre-clinical testing in three important clinical areas, namely the eye, the liver and musculoskeletal applications. The proposed Hub has outstanding cross-disciplinary scientific and clinical investigators, links to industry/manufacturing, and a strategic gate structure that provides a coherent structure with clear leadership. The pre-clinical WPs will provide clinical user-pull to inform the materials technologies and accelerate the impact of the Hub.
12 April 2018

Glaucoma is a complex disease and the mechanisms of its initiation and progression are still largely undetermined although several potential mechanisms have been proposed. Glaucoma is thought to be initiated from a discrete sub-population of cells in the eye, if this sub-population is very small compared with surrounding of normal healthy cells, the large number of normal cells may mask the detection of the underlying pathological mechanisms. Also, in a clinical context, the discrete sub-population of glaucoma initiating cells might be the ideal targets of any potential treatments. However, very little is known about the context of this heterogeneity, and how this drives the progression of glaucoma. An ideal way to investigate the potential pathological heterogeneity within a cell population is by employing single cell based analysis technologies. We have a Fluidigm C1 single cell analysis platform, which has the potential to simultaneously analyse 800 single cells for DNA sequencing or RNA expression. Using this approach, we will determine the cellular heterogeneity in glaucoma at a single cell level, and aim to identify novel glaucoma initiation and progression cell types, glaucoma associated biomarkers, and protein targets for its treatment.
5 April 2018

The aim of this PhD project will be to 
(1) prepare and characterise novel protein-fluorophore conjugates in vitro, 
(2) investigate the use of these tools to monitor the rate of apoptosis progression using well-established in vivo models of glaucoma and Alzheimer’s Disease and
(3) Building on recent work by the lab develop novel formulations of protein-fluorophore conjugates to facilitate the topical delivery of these contrast agents.
(4)During this project, you will have the opportunity to gain experience in the following techniques
5 April 2018

In this PhD studentship, we will develop a non-viral plasmid vector system with unlimited cloning capacity (it has already successfully accommodated the LDLR gene consisting of 135kb) that incorporates a human DNA motif called the scaffold/matrix attachment region (S/MAR), which confers (i) episomal maintenance, thus eliminating the risk of insertional mutagenesis, (ii) mitotic stability, and (iii) protection against epigenetic silencing permitting long-term expression. We will deliver the USH2A-S/MAR vector to USH2A-/- patient fibroblasts and the ush2a-/- transgenic zebrafish (generated through CRISPR/Cas9 gene editing by our group previously) to assess safety and therapeutic efficacy; ultrastructural analysis of photoreceptors and stereociliary bundle in neuromasts, cell death assays, immunostaining of Usherin, GPR98 and Whirlin to assess localisation to the connecting cilium, optokinetic reflex testing in zebrafish, and long-term gene expression with germline transmission capacity.
5 April 2018

Manufacture of the devices for the study including ensuring stability of the devices throughout the study.
Psychological tests for the patients including calibration of the psychophysics device.
1 April 2018

Primary cilia are hair-like protrusions on most cells, which function as environmental sensors that are built and maintained by intraflagellar transport (IFT), but the precise function of IFT proteins is not well understood. The photoreceptor outer segment is a highly specialised light sensing primary cilium. Disruption of IFT as a result of IFT gene mutations cause a range of disease manifestations from isolated retinal degeneration to pleiotropic, systemic ciliopathies. Importantly, different mutations in the same IFT gene can cause different ciliopathy phenotypes. For example, mutations in IFT140 cause Mainzer-Saldino syndrome (MSS) and Jeune Asphyxiating Thoracic Dystrophy (JATD), but also isolated non-syndromic retinal degeneration. The underlying mechanism for this phenotypic variability is unknown and it is unclear why retinal degeneration is such a highly penetrant phenotype of ciliopathies. The major aims of this research are to understand the disease mechanism(s) and retinal susceptibility to different IFT140 mutations. We will test potential therapeutic approaches for different mutations, to rescue the IFT mutant protein and alleviate the cellular phenotypes. We will address these questions through heterologous expression of IFT140 variants in stable cells and by modelling disease in ciliated retinal pigment epithelium cells and three- dimensional retinal organoids by differentiating induced pluripotent stem cells from patient tissue and investigating isogenic IFT140 knock-out lines. This study will greatly enhance our understanding of IFT140-mediated retinal disease and disease mechanisms in ciliopathies, and will form the basis to translate promising drugs and compounds that alleviate the cellular defects into practical therapies.
28 March 2018

Choroideremia (CHM) is a genetic condition that causes progressive vision loss mostly in men and is due to degeneration of the retina and choroid. Over time, the visual field narrows and progresses to tunnel vision and blindness commonly occurs in late adulthood. It is an X-linked recessive disease caused by functional defects in CHM/REP1, a chaperone protein for Rab GTPases, which are critical modulators of multiple steps in membrane traffic pathways. Thus, retinal gene therapy with an adeno-associated viral vector encoding REP1 in patients with CHM is a very promising therapeutic approach. However, alternative approaches towards a cure should be also explored. Here, we propose to study the mechanisms of cell death in CHM, focusing on the retinal pigment epithelium (RPE). We aim to dissect the molecular and cellular pathways leading to RPE cell death using human stem cell-derived RPE that will be genetically modified to carry the CHM mutation and/or from induced pluripotent stem cell RPE derived from a CHM patient. We foresee that unravelling these mechanisms will open new avenues for the development of novel therapeutic strategies for CHM.
28 March 2018

Recent years have seen a dramatic increase in the prevalence of myopia worldwide, particularly in East Asian populations. Myopia now affects 30-50% of adults in Europe, America, Australia and Asia1,2. Epidemiological studies have confirmed its onset and progression during childhood and adolescence, with 3 to 7.3% of teenagers now requiring glasses for myopia1. Progression to high myopia and its associated axial elongation of the eye with the risk of sight-threatening conditions such as retinal detachment and choroidal neovascular membrane formation has led to myopia being considered a public health problem2. Research has moved from concentrating on the role of near work and accommodation associated with increasing educational demands in childhood and adolescence to the role of outdoors light exposure (limited by increasing educational demands and indoor activities) and retinal release of dopamine in response to light, inhibiting scleral remodelling and axial elongation3,4. Clinical trials are exploring pharmacological treatments such as the use of diluted topical muscarinergic receptor inhibitor atropine5. The efficacy of currently available interventions is limited.

All known pathways triggering axial elongation converge on one common final tissue, the sclera. Understanding of the biological processes governing sclera biomechanics is limited, although a number of studies points at defects in secretion and remodelling of extracellular matrix (ECM) in myopia6-9. Our previous work in tissue biomechanics in another ocular disease with similar mechanical features (Floppy Eyelid Syndrome) suggests that the mechanical properties of scleral fibroblasts could be key to the maintenance of the tissue integrity and stiffness10, 11. We propose to develop a human sclera biomimetic to explore fibroblast scleral fibroblasts-matrix interaction in a pseudo-physiological 3D model, using scleral fibroblasts from human donor eyes (Moorfields Eye Bank), including child donor eyes. We will use this model to a) study sclera biomechanics, with the specific aim of developing new treatment targets and b) in combination with our miniaturized soft-tissue mechanical force analyzer12 to screen novel and existing molecules (e.g. Rho kinase inhibitors, commercially available as topical eye medication) to prevent and/or correct scleral remodelling.
22 March 2018

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