NIHRDataInsights/HRCSData huggingface dataset:
Title: A mechanistic understanding of corneal pathobiology and the development of therapeutic strategies for the treatment of connective tissue disorders
Funder: Medical Research Council

Abstract: As the primary refractive component of the eye, the well-functioning cornea must be precisely curved, strong and transparent. Our previous studies have shown that these intrinsic qualities are governed by the hierarchical ultrastructure and complex microanatomy of its cells and extracellular matrix. Building on our existing knowledge of healthy corneal function, our main objectives are to (1) understand the structural basis of normal corneal function and dysfunction, and elucidate the role of a previously identified but as yet, uncharacterised corneal elastic fibre system that we believe may help to restore shape when the cornea is distorted; (2) increase our understanding of the role of extracellular vesicle signalling in the development, regeneration and repair of the cornea; (3) develop photochemical therapies for corneal ectasia through laboratory studies and the collection of clinical data into our UK cross-linking consortium audit tool; (4) develop synchrotron x-ray scattering and microscopy techniques to carry out time-resolved biomechanics/structure correlations to study in three-dimensions, at high-resolution, and in an orchestrated, hierarchical manner, the response of the normal and pathological corneas to different loads; (5) collaboratively demonstrate the applicability of these new techniques to other areas of connective tissue research. Most of these will be achieved by combining state-of-the-art imaging techniques, including microfocus synchrotron x-ray scattering, x-ray spectroscopy, optical coherence tomography, multiphoton laser microscopy, high-pressure cryopreservation electron microscopy and volume serial block face scanning electron microscopy, immunofluorescence microscopy, with established biomechanical and biochemical testing procedures (extensometry, interferometry, inflation testing and immunohistochemisty) to study karyotypically normal and abnormal human foetal and adult corneas, pathological corneas and corneal tissue alternatives.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: EYE
Title: Improving functional connectivity following transplantation of cone photoreceptors
Funder: Medical Research Council

Abstract: Retinal diseases due to photoreceptor degeneration are the leading cause of untreatable blindness in industrialised countries. We seek to develop a new clinical therapy by transplanting healthy photoreceptors to replace the cells that are lost. Whilst CNS repair is an ambitious goal, photoreceptor transplantation is one of the most feasible types of repair, as transplanted cells only need to make short-distance synaptic connections to host bipolar cells in order to connect to the remaining circuitry and relatively few new functional cones may be required for useful vision. Towards our aim, we have developed robust protocols that generate large numbers of bona fide photoreceptors from mouse and human pluripotent stem cell (PSC) sources and shown that transplantation of healthy photoreceptors into models of partial degeneration can rescue vision. However, an unequivocal demonstration of functional connectivity in models of advanced degeneration has yet to be achieved despite its clinical importance; the first clinical trials of photoreceptor transplantation will be in patients with near-complete photoreceptor loss. We have continued to improve our transplantation approach and now have important data demonstrating rescue of retinal function in a mouse model of advanced retinal disease following the transplantation of human PSC-derived cones. Moreover, the data strongly indicate that this is mediated by synaptic connectivity between donor and host. Here, we will characterise the extent of functional connectivity that can be achieved in advanced disease using current methods. We have developed a number of tools and will use synaptic tracing, electrophysiology and behavioural tests to assess function from cell to behaviour. Next, we seek to develop methods to improve donor/host connectivity by manipulating the host environment and by altering the ability of donor cells to respond to cues (positive & negative) within the host and to assess their ability to improve vision.


Research activity codes: 5.2 CELLULAR AND GENE THERAPIES
Health categories: EYE
Title: Resolving the basis of phenotypically variable hereditary abnormalities of eye formation
Funder: Medical Research Council

Abstract: Although abnormalities of eye formation are a major cause of blindness, the genetic bases of such phenotypes are poorly understood. The list of genes implicated in microphthalmia, anophthalmia and coloboma (MAC) is growing but only accounts for a minority of cases. This proposal will identify new genes involved in eye formation and will elucidate why MAC phenotypes show variable penetrance. Our proposal builds on ongoing MRC Programme Grant research which has enabled us to establish novel zebrafish models in which to explore phenotypically variable MAC phenotypes. We will identify novel genetic interactions contributing to MAC phenotypes. Due to the robustness of eye formation, MAC phenotypes may only be revealed when embryos carry more than one genetic mutation. Consequently, novel geneticially sensitised zebrafish lines showing low penetrance phenotypes will be subject to Crispr/Cas9 gene targetting to assess the consequences of systematically disrupting hundreds of potential genetic interactions upon the penetrance and expressivity of MAC phenotypes. We also aim to understand the molecular and tissue level functions of selected genes that are critical in the expression of MAC phenotypes. We will clarify the conserved roles for Yap and Mab21l1/2 proteins in eye morphogenesis using genome wide screens in worms to complement novel transgenic approaches in zebrafish. Finally, we will elucidate the epistatic interactions which affect severity of MAC phenotypes. We will exploit novel fish models in which we can vary the penetrance of MAC phenotypes to study why genetic lesions can result in different outcomes between individuals and even between left and right eyes. This research will both inform and be informed by large-scale human genomics studies, and will deliver a comprehensive analysis of genetic interactions that build the eyes and will provide a wealth of information to inform diagnosis and understanding of debilitating abnormalities of eye formation.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: EYE
Title: Restoration of vision with new generation Artficial Theranostic Cornea
Funder: Medical Research Council

Abstract: During MRC Career Development Award, my aim is to develop a clinically applicable host acceptable Assisted-Regeneration artificial Corneas (ARCs) focused on requirements of high-risk patient to combat with infection, inflammation, and will test to evaluate and establish this as cell-free pro-regenerative corneal implants for custom-fit personalised medicine. The ARC with its built-in monitoring system to alert these high-risk patients and doctors of potential problems like rejection before irreversible damage or failure occurs represents the future of personalized medicine. Over the 5 years, I aim to complete: 1. To an immune-compatible nano toolbox of materials that can be used to develop theranostic nanoparticles. 2. To integrate the nanoparticles into an implant, i, e this case the ARC. 3. To completely characterise the function of ARCs in vitro, focussing perfecting the diagnostics aspects, and test potential incorporation into a clinically used prosthesis, the Boston KPro. 4. To evaluate clinical feasibility of ARCs by monitoring the safety and efficacy of ARCs with both diagnostics and therapeutic functions with established models within the state-of-the-art artificial cornea facilities of my mentioned collaborators. We will develop our nano toolbox mainly with gold, iron and self-assembled small peptides for nanoparticles that will incorporate drugs and bioactive anti-infective and anti-inflammatory peptides such as KR12. The loaded nanoparticles will then be incorporated into constructs made mainly from collagen and collagen-mimetic peptides. The implants will be tested in vitro for biocompatibility and efficacy. The best composite ARC implants will then be tested with pre-clinical model with our collaborator Prof Grifith and finally evaluate the clinical feasibility with Prof Chodosh. Successful testing of ARC will provide knowledge for development of further theranostic systems that can be extended to use in other target organ systems.


Research activity codes: 5.2 CELLULAR AND GENE THERAPIES
Health categories: EYE
Title: Development of retinal gene therapy to treat dominantly inherited disease using a novel RNA-based silencing system
Funder: Medical Research Council

Abstract: Inherited retinal degenerations such as retinitis pigmentosa (RP) are the most common cause of untreatable blindness in the young. Recently great progress has been made with gene replacement therapy using adeno-associated viral (AAV) vectors following FDA approval of Luxturna to treat Leber congenital amaurosis, but therapies for more common dominantly inherited conditions remain an unmet need. Dominant mutations in the rhodopsin gene are overall the most common cause of RP and although rhodopsin is only expressed in rods, the unique photoreceptor architecture means that once rods are lost, cones also degenerate through secondary mechanisms, leading to complete blindness. Gene silencing approaches using sequence-specific short hairpin (sh)RNA molecules are not new, but a key problem in translation to clinical trial is limiting off-target effects, particularly in cones which are essential for human vision but do not express mutant rhodopsin. The Pol III promoters (e.g. U6 or H1) that drive siRNA expression are not cell-specific. Hence following AAV gene therapy the shRNA would also be expressed in cones. This would increase the risk of off-target effects and activation of the interferon pathway caused by uncontrolled expression of double-stranded RNA. To overcome this problem, we have for the last 4 years worked an interference pathway in which the RNA is released from an intron within the gene being expressed - known as a 'mirtron'. A key advantage is that RNA expression is regulated by the same rod-specific promoter and is not therefore induced in cones. We co-express a codon-optimised rhodopsin transgene that replaces the mutant rhodopsin knocked down by the mirtron. Preliminary work has identified suitable artificial mirtrons that are correctly spliced and which do not affect the codon-optimised gene. Furthermore mirtron-derived pri-miRNA transcripts do not require Drosha/DGCR8 and thus bypass a potential bottleneck in RNA processing.


Research activity codes: 5.2 CELLULAR AND GENE THERAPIES
Health categories: EYE
Title: Preclinical evaluation of a new chemical cross-linker for the treatment of keratoconus
Funder: Medical Research Council

Abstract: Keratoconus is a progressive condition, affecting young and working age people, in which the cornea becomes misshapen significantly disrupting the refraction of light into the eye. It is a lifelong condition and a significant health burden in work-age adults, and is a leading cause of corneal transplantation in the UK. One of the key features of keratoconus is a loss of corneal mechanical stability, with the stiffness of the cornea reducing by up to 40 % compared to a normal cornea. Corneal collagen cross-linking increases the stiffness of the cornea and can reduce the progression of this disease by preserving corneal integrity due to strong bonds formed within the collagen. Over the past decade, collagen cross-linking using ultraviolet A (UVA) radiation combined with the photosensitiser riboflavin has been used but it has several limitations. In particular, the exposure to UVA radiation risks toxicity to cells in the stroma and the corneal endothelium. Also for effective stiffening it is necessary to remove the corneal epithelium to allow penetration of the photosensitiser which is painful for the patient and increases the risk of infection. We have developed a cross-linking solution and demonstrated that it stiffens the cornea in ex vivo porcine and human tissue to a similar extent to the existing treatment. Also we have shown that it penetrates into the cornea without needing to remove the epithelium. Furthermore, in contrast to the UVA treatment, we have demonstrated no toxicity to the cells in the cornea. It is a simple chemical solution that is dissolved at the time of use and delivered to the patient's eye after mixing. This study is designed to develop our novel patented technology along the translational pathway through an in vivo preclinical trial using rat and rabbit models. During this time we will build a partnership with a commercial partner and MHRA approval for a phase 1 safety trial to follow.


Research activity codes: 5.1 PHARMACEUTICALS;5.3 MEDICAL DEVICES
Health categories: EYE
Title: Clinical trial of cone photoreceptor transplantation for the treatment of retinal degeneration
Funder: Medical Research Council

Abstract: Loss of cone photoreceptors is a common feature of a range of inherited retinal dystrophies, including cone and cone-rod dystrophies (CD), where only the photoreceptors are affected, as well as Stargardt disease and age-related macular degeneration (AMD), in which both the photoreceptors and the RPE are compromised. The prevalence of these conditions varies from 1:30,000 for CD to 1:150 for advanced AMD. Endstage disease for all these conditions is characterised by a lack of central vision, due to the absence of surviving cones in the macula. We hypothesise that replacement of the photoreceptors, either in isolation or with RPE, can restore vision in all these conditions, despite the different disease aetiologies. In a previous MRC-funded study (MR/M007871/1) we developed a GMP-compatible protocol that allows the differentiation of human embryonic stem (ES) cells to cone precursors. We have recently shown that transplantation of these cells into mouse models of endstage retinal dystrophy allows them to form functional connections with the underlying retina and restore vision. The objective of this study is to scale-up the developed GMP protocol, produce clinical grade ES-derived cone photoreceptor precursors and conduct a combined phase I/II clinical trial of cell therapy for the treatment of cone dystrophies. The objectives of the clinical trial will be to establish the safety and efficacy of ES-derived cone photoreceptor transplantation in patients with a cone dystrophy in whom there is a central scotoma due to cone photoreceptor loss without overt damage to the underlying retinal pigment epithelium (RPE). Following transplantation, we will characterise retinal structure using retinal imaging and will determine whether there is an improvement in visual function using electrophysiology and psychophysics.This study will pave the way for the development of therapies for Stargardt disease and AMD which require co-transplantation of cone photoreceptors and RPE.


Research activity codes: 6.2 CELLULAR AND GENE THERAPIES
Health categories: EYE
Title: Investigating the role of perivascular mesenchymal stem cells in macular fibrosis secondary to neovascular age-related macular degeneration
Funder: Medical Research Council

Abstract: Intravitreal injection of VEGF inhibitors is the standard of care for nAMD patients. However, ~50% of treated eyes develop macular fibrosis, which causes anti-VEGF resistance and eventually irreversible blindness. Myofibroblasts drive new blood vessels into fibrovascular lesion in nAMD by producing excessive amounts of extracellular matrix proteins but their source is unknown. Recent studies suggest that Gli1+ perivascular mesenchymal stem cells (Gli1+ pMSCs) can differentiate into myofibroblasts and contribute critically to tissue fibrosis in the lung, liver, and heart. We found that the retina and choroid also contain a network of Gli1+ pMSC. In retinal injury and inflammation, they detach from blood vessels, migrate to the site of damage, and participate in retinal scar formation. This proposal aims to understand the mechanisms by which Gli1+ pMSCs differentiation into myofibroblasts in nAMD. We have found that myofibroblasts in macular fibrosis express Gli1. We will first characterise the phenotype of myofibroblasts obtained from a cohort of donated nAMD eyes. We will then induce retinal fibrosis in Gli1-CreERt2:tdTomato mice and isolate Gli1-Tomato cells from the retina and choroid at different stages of the disease for single-cell RNA sequencing. Bioinformatic analysis will be conducted to understand the trajectories of retinal and choroidal Gli1+ pMSCs as they transition into myofibroblasts, and key pathways involved in this process. These pathways will be confirmed in retinal and choroidal pMSC cultures from Gli1-CreERt2:tdTomato mice, and bioinformatic analysis will also uncover high impact hub genes. We will use the acquired knowledge to design strategies to prevent or reverse myofibroblast differentiation. These strategies will be tested in pMSC cultures from mouse retina and choroid, bone marrow-derived human MSCs, and an in vivo model of retinal fibrosis in Gli1-CreERt2:tdTomato and CreERt2:iDTR mice.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: EYE
Title: Bio-synthetic corneal endothelial grafts for transplantation
Funder: Medical Research Council

Abstract: Diseases of the corneal endothelium, such as Fuchs' endothelial corneal dystrophy (FECD) and pseudophakic bullous keratopathy (PBK), result in significant loss of vision and are the commonest reasons for corneal transplantation. Damage to this endothelial layer leads to oedema and thus loss of transparency. Replacement of the endothelial layer with a corneal transplant reduces the oedema and restores transparency. Although corneal transplantation is more than 80% successful at 1 year, five-year graft survival rates are significantly reduced to 70% for FECD and 52% for PBK, meaning that patients often require a second transplant. There is a global shortage of corneas with only one available for every 70 required, therefore, there is an opportunity to combine biomaterials with in vitro expanded corneal endothelial cells (CECs) to produce multiple bioengineered grafts from each donor cornea. These biomaterials not only serve as a carrier for CEC transplantation but may also enhance cell function to increase the long-term success of transplanted grafts. We have developed a poly-epsilon-lysine based hydrogel with excellent optical and mechanical properties that can be controlled by the nature and percentage of the cross-links and the density of the peptide. We have data that show primary CECs adhere and proliferate on the surface of the gel to produce a confluent monolayer and that this graft has the physical integrity to be manipulated for transplantation using a clinical delivery device. Furthermore, the graft functions to restore corneal thickness and transparency in an in vivo rabbit model of endothelial failure. This study will develop our technology along the translational pathway by improving the crosslinking method to reproducibly allow production of thinner hydrogels, optimising the manufacture to allow scale up and culture of human CECs in a GMP aligned protocol and evaluating the safety and efficacy of these optimised grafts in an in vivo rabbit model.


Research activity codes: 5.2 CELLULAR AND GENE THERAPIES
Health categories: EYE
Title: Investigating the pathogenetic mechanisms leading to progressive scarring and glaucoma surgery failure and development of new targeted therapeutics
Funder: Medical Research Council

Abstract: Glaucoma is the leading cause of irreversible blindness worldwide. Glaucoma filtration surgery to reduce the intraocular pressure is the mainstay of surgical treatment in glaucoma, but it fails primarily due to scarring in 50% of patients after 5 years of follow-up. I will use state-of-the-art computational genomics techniques to gain novel mechanistic insights into why a large subgroup of patients develops progressive scarring and irreversible loss of vision after glaucoma surgery. Using this knowledge, I will develop new treatments to prevent this from happening. KEY GOALS: 1) Prospective longitudinal study of 300 patients after their first glaucoma surgery looking for clinical evidence of progressive scarring over an 18-month period. I aim to: (A) unravel the complex pathogenetic mechanisms leading to progressive scarring through detailed longitudinal and cross-sectional transcriptomic analyses and detailed longitudinal phenotyping; and (B) identify biomarkers of progressive scarring for clinical risk stratification. 2) Develop novel targeted therapeutics to silence the principal pathogenetic pathways leading to progressive scarring and to prevent failure of glaucoma surgery. I will deliver siRNAs using different concentrations and targeted nanoparticle formulations, and test their biophysical properties, gene silencing efficiency and cytotoxicity. I will also validate the most efficient siRNA nanoparticle formulations using an established pre-clinical rabbit model of glaucoma filtration surgery. These studies will help to inform future clinical trials of targeted RNA therapeutics in glaucoma. BENEFITS: This project is innovative, interdisciplinary and will directly improve the management of glaucoma patients. A better understanding of the factors leading to progressive scarring will allow for more refined risk stratification and importantly, it will translate into new avenues for targeted anti-fibrotic therapeutics and precision medicine.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES;5.1 PHARMACEUTICALS
Health categories: EYE