Induced Pluripotent Stem Cells for Inherited Optic Neuropathies-Disease Modeling and Therapeutic Development

State-of-the-Art Review Section Editors: Fiona Costello, MD, FRCP(C) Sashank Prasad, MD Induced Pluripotent Stem Cells for Inherited Optic Neuropathies—Disease Modeling and Therapeutic Development Joshua Paul Harvey, MA, BM BCh, Pg Cert, FRCOphth, Paul Edward Sladen, BSc, MRes, PhD, Patrick Yu-Wai-Man, BMedSci, MBBS, PhD, FRCPath, FRCOphth, Michael E. Cheetham, BSc, PhD Background: Inherited optic neuropathies (IONs) cause progressive irreversible visual loss in children and young adults. There are limited disease-modifying treatments, and most patients progress to become severely visually impaired, fulfilling the legal criteria for blind registration. The seminal discovery of the technique for reprogramming somatic nondividing cells into induced pluripotent stem cells (iPSCs) has opened several exciting opportunities in the field of ION research and treatment. Evidence Acquisition: A systematic review of the literature was conducted with PubMed using the following search terms: autosomal dominant optic atrophy, ADOA, dominant optic atrophy, DOA, Leber hereditary optic neuropathy, LHON, optic atrophy, induced pluripotent stem cell, iPSC, iPSC derived, iPS, stem cell, retinal ganglion cell, and RGC. Clinical trials were identified on the ClinicalTrials.gov website. Results: This review article is focused on disease modeling and the therapeutic strategies being explored with iPSC technologies for the 2 most common IONs, namely, dominant optic atrophy and Leber hereditary optic neuropathy. The rationale and translational advances for cellbased and gene-based therapies are explored, as well as opportunities for neuroprotection and drug screening. Conclusions: iPSCs offer an elegant, patient-focused solution to the investigation of the genetic defects and disease mechanisms underpinning IONs. Furthermore, this group of disorders is uniquely amenable to both the disease modeling capability and the therapeutic potential that iPSCs offer. This fast-moving area will remain at the forefront of UCL Institute of Ophthalmology (JPH, PES, PY-W-M, MC), London, United Kingdom; Moorfields Eye Hospital NHS Foundation Trust (JPH, PY-W-M), London, United Kingdom; Department of Clinical Neurosciences (PY-W-M), Cambridge Centre for Brain Repair, University of Cambridge, Cambridge, United Kingdom; and Department of Clinical Neurosciences (PY-W-M), John van Geest Centre for Brain Repair and MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, United Kingdom. The authors report no conflicts of interest. J. P. Harvey and P.E. Sladen co-first author. Address correspondence to Joshua Paul Harvey, MA, BM BCh, Pg Cert, FRCOphth, UCL Institute of Ophthalmology, 11-43 Bath Street, London, United Kingdom EC1V 9EL; E-mail: [e-mail]. Harvey et al: J Neuro-Ophthalmol 2022; 42: 35-44 both basic and translational ION research in the coming years, with the potential to accelerate the development of effective therapies for patients affected with these blinding diseases. Journal of Neuro-Ophthalmology 2022;42:35–44 doi: 10.1097/WNO.0000000000001375 © 2021 by North American Neuro-Ophthalmology Society T he inherited optic neuropathies (IONs) cause severe visual impairment with an estimated prevalence of 1:10,000 (1). They encompass a range of genetically diverse disorders characterized by the preferential loss of retinal ganglion cells (RGCs) leading to optic nerve degeneration and irreversible visual loss. Autosomal dominant optic atrophy (DOA) and Leber hereditary optic neuropathy (LHON) are the 2 most common IONs, sharing overlapping clinical and pathological characteristics despite being genetically distinct conditions (1). About 70% of patients with DOA carry variants in the nuclear-encoded OPA1 gene (3q29; OMIM 605290), which encodes a profusion inner mitochondrial membrane protein. LHON is a primary mitochondrial DNA (mtDNA) disorder, and about 90% of patients harbor point variants in MTND1 (m.3460 G.A; OMIM 516000), MTND4 (m.11778 G.A; OMIM 516003), and MTND6 (m.14484T.C; OMIM 516006), all of which encodes key subunits of the mitochondrial respiratory chain complex I (2–4). Despite the rapidly expanding list of genes that have been identified causing IONs, the pathological hallmark is remarkably similar with early and severe loss of RGCs within the papillomacular bundle, resulting in a dense central or cecocentral scotoma that accounts for the disabling nature of the visual loss experienced by affected patients (Fig. 1) (3,5). The precise pathways linking genetic variants affecting ubiquitously expressed proteins with preferential RGC loss remains unclear, and this lack of mechanistic insight partly 35 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. State-of-the-Art Review accounts for the currently limited treatment options. A key challenge in ION research is the difficulty in obtaining human retinal and optic nerve tissue samples to effectively study the disease. Our understanding of ION disease mechanisms has been derived primarily from nonphysiological models, including patient-derived fibroblasts. Although in vivo models of DOA and LHON have provided key insights, there are limitations because the animals do not fully manifest the human phenotype for its disease progression (6,7). In the face of these challenges, the advent of induced pluripotent stem cells (iPSCs) is an exciting development for disease modeling, possessing great translational potential. This review will explore the current advances and future opportunities that iPSCs offer in the field of ION research. INDUCED PLURIPOTENT STEM CELL DEVELOPMENT Takahashi and Yamanaka (8) revolutionized the disease modeling field through the advent of mouse iPSCs, converting terminally differentiated somatic cells into naïve embryonic stem cell (ESC)-like cells through forced expression of 4 genes, including Oct4, Sox2, Klf4, and c-Myc. Numerous studies have now demonstrated the feasibility of generating human iPSCs (9–12), providing a platform to investigate human disorders that have proven difficult to investigate because of limited access to diseased human tissues, or the unavailability of reliable animal models. Since their discovery, iPSC research has improved exponentially, enabling the efficient reprogramming of somatic cells using a variety of techniques, including episomal vectors and mRNA constructs, which preserve cellular genomic integrity and increase reprogramming efficiency (13,14). In addition, iPSCs can now be generated using cells acquired from noninvasive methods, such as renal epithelial cells from a urine sample, that are particularly useful for children and in circumstances where it is impractical to acquire a biopsy (15,16). In addition, the generation of iPSCs from adult somatic cells removes many of the religious and ethical concerns associated with ESCs. Importantly, iPSCs, like ESCs, provide a near unlimited source of patient-derived material because of their inherent ability to self-renew (9), while also maintaining the pluripotent capacity to generate cells from all 3 developmental germ layers (17). Although the potential of iPSCs has made them the ideal tool for disease modeling, several characteristics currently limit their clinical application. Genomic instability that can occur during reprogramming or subsequent culture needs to be avoided, with evidence for large-scale genomic rearrangements, predominantly in chromosomes 8 and 12 (18,19), copy number variations (20,21), and point variants (22–24) occurring in iPSCs. Studies have also demonstrated that iPSCs can retain gene expression and DNA methylation profiles of their somatic cell of origin, in a state known as partially reprogrammed iPSCs, which reduces their differentiation capacity or limits them to cell fates of the germ line of origin (25–28). As such, careful quality control is required to ensure that the generated iPSCs do not acquire genetic abnormalities before disease modeling or cell replacement therapy. CURRENT PROGRESS IN INHERITED OPTIC NEUROPATHY MODELING iPSC technology provides a gateway to developing improved, physiologically relevant disease models of IONs (Fig. 2). The modeling of inherited retinal diseases (IRDs) has led the way over the past decade with the derivation of 3-dimensional (3D) retinal organoids that recapitulate retinogenesis in a spatiotemporal pattern (29–31). Although retinal organoids are at the forefront of IRD research, their use for IONs is limited because they are a heterogeneous retinal cell culture, containing many cells that are unaffected in ION disease progression and only a limited number of RGCs, which are the target cells of interest. Although studies have begun to investigate the effects of disease-causing variants on RGC biology using 3D retinal organoids (32), there are consistent reports indicating the loss of RGCs FIG. 1. Pattern of retinal degeneration in inherited optic neuropathies. A. Axons of RGCs constitute the retinal nerve fiber layer (RNFL) and they converge, exiting the eye as the optic nerve. B. In patients with inherited optic neuropathies (IONs), preferential degeneration of RGCs occurs resulting in the thinning of the RNFL, the development of optic atrophy, and progressive irreversible visual decline (B). RGC, retinal ganglion cell. 36 Harvey et al: J Neuro-Ophthalmol 2022; 42: 35-44 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. State-of-the-Art Review FIG. 2. Derivation of RGCs from patient-derived somatic cells. Schematic overview of iPSC and RGC generation from somatic cells, such as skin cells, collected from patients with visual loss from an ION. The somatic cells are reprogrammed toward iPSCs using nonintegrative reprogramming and gene editing technologies. The iPSCs are characterized and expanded before being subjected to in vitro differentiation of RGCs through either 3D retina organoids or direct RGC generation through 2D protocols. In vitro RGCs provide a key resource for investigating genetic variant–associated disease mechanisms, therapeutic screening, and potential cell replacement therapies. ION, inherited optic neuropathy; iPSC, induced pluripotent stem cell; RGC, retinal ganglion cell. within the inner layers during retinal organoid maturation, likely because of the lack of a terminal synaptic connections or nutrient deprivation. Although this replicates RGC embryonic development (33), it might limit the use of mature retinal organoids as a model of RGC disease (29,31). To counter these problems, several studies have established 2-dimensional (2D) protocols to generate RGCs (34–39), increasing the specificity and applicability of RGC models and providing an opportunity to carefully dissect the disease mechanisms driving RGC loss in IONs. Induced Pluripotent Stem Cell Modeling of Leber Hereditary Optic Neuropathy The generation of patient-derived iPSCs, in conjunction with 2D RGC models, has proven useful in studying the pathophysiology of LHON-associated RGC loss. 2D differentiation of iPSCs carrying a homoplasmic double mtDNA variant in MTND1 (m.4160T.C) and MTND6 (m.14484T.C) demonstrated that RGCs harboring these mtDNA variants have significantly increased levels of apoptosis when compared with control and isogenic cybrid RGCs (40). RGCs generated from iPSCs derived from an affected patient with LHON harboring the MTND4 m.11778 G.A variant, alongside an unaffected Harvey et al: J Neuro-Ophthalmol 2022; 42: 35-44 carrier with the same MTND4 variant, demonstrated reduced basal respiration and spare respiratory capacity when compared with wild-type (WT) control RGCs. Interestingly, the RGCs established from the affected patient with LHON exhibited enhanced mitochondrial biogenesis, suggesting a potential compensatory mechanism to palliate for the reduced bioenergetic output (41). In addition, RGCs derived from iPSCs carrying the m.11778 G.A mtDNA variant demonstrated a range of cellular defects, including increased apoptosis, increased retrograde mitochondrial transport, decreased levels of stationary mitochondria, and reduced expression of KIF5A, a kinesin required for intracellular organelle transport (42). These studies have brought into focus some of the possible disease mechanisms driven by pathogenic LHON mtDNA variants that eventually lead to RGC death, thus identifying potential therapeutic targets for translational research. Induced Pluripotent Stem Cell Modeling of Dominant Optic Atrophy OPA1 is the major causative gene in DOA. There have been numerous studies that have explored the consequences of OPA1 variants in immortalized cell lines or more accessible human cells, such as fibroblasts and myoblasts (43). To date, 37 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. State-of-the-Art Review a single study has established the effect of an OPA1 variant in 2D RGC cultures. Patient-derived iPSCs were established harboring an OPA1 splice-site variant (c.2496+1 G.T) that is predicted to cause mis-splicing of OPA1 transcripts (44). The OPA1 mutant iPSCs demonstrated increased apoptosis and reduced differentiation competence when compared with WT iPSCs, with an inability to form neural progenitor cells (NPCs) that was rescued with noggin supplementation (44). Two studies have reported the generation of mutant iPSCs, carrying the c.1861C.T (p.Q621*) or c.1635C.A (p.S545R) OPA1 variants, which were derived from patients with a DOA “plus” (DOA+) phenotype (45,46). Although no characterization of OPA1-related function was conducted, both studies confirmed the differentiation potential of the mutant iPSCs through trilineage differentiation assays, providing a platform for further exploring disease mechanisms in DOA. A subgroup of patients carrying OPA1 variants will develop DOA+ with more severe neurological features, including cerebellar ataxia, peripheral neuropathy, and myopathy, in addition to optic atrophy (47). We are gaining a greater understanding of the impact of OPA1 variants on the central nervous system with the differentiation of mutant iPSCs into specific neuronal populations (48). One study generated iPSCs from 2 patients with Parkinson’s disease carrying a 9 base-pair insertion in OPA1 exon 2, which were subsequently differentiated into dopaminergic neurons (49). These neurons showed accelerated cell death with reduced mitochondrial oxidative phosphorylation (OXPHOS) and increased mitochondrial fragmentation when compared with WT cells. NPCs and dopaminergic neurons carrying c.1462G.A (p.G488R) or c.1484C.T (p.A495V) OPA1 variants showed significant reductions in mitochondrial OXPHOS and ATP output and reduced numbers of mitochondria within axonal projections, which also exhibited reduced motility (50,51). Caglayan et al (52) generated an OPA1 heterozygous knockout ESC line through CRISPR/Cas9 gene editing of WT human ESCs (hESCs). In that particular model, OPA1 haploinsufficiency did not result in significant mitochondrial deficits but it did inhibit NPC neuronal specification by altering DNA methylation patterns. Further study of OPA1 variants in iPSC-derived neuronal cell types, other than RGCs, is needed to clarify the mechanisms that contribute to the development of the extraocular features seen in patients with DOA+ phenotypes. Induced Pluripotent Stem Cell Modeling of Syndromic Inherited Optic Neuropathies iPSCs offer an elegant method to create in vitro models of the diverse cell types affected in other inherited diseases where optic neuropathy is a prominent feature, such as Wolfram syndrome (53), Charcot–Marie–Tooth disease (54), and Friedreich ataxia (55–57). iPSCs have been generated carrying variants in WFS1 that account for most cases of Wolfram syndrome. Although there have been no reports of RGCs generated from WFS1 mutant iPSCs, Shang et al (58) 38 have generated iPSC-derived b-islet cells, which accurately model the pancreatic failure seen in Wolfram syndrome. INDUCED PLURIPOTENT STEM CELL THERAPEUTIC APPROACHES Induced Pluripotent Stem Cell Therapy Cell-based regenerative medicine aims to use iPSCs as an autologous or cell banked source to produce specific cell types, which can subsequently be transplanted to replace damaged tissues (59,60). Such an approach is advantageous because it avoids the technical challenges of harvesting neural stem cells (61) and the ethical concerns of ESCs (62). Furthermore, the generation of cells to be transplanted from autologous or closely MHC-matched iPSCs offers the theoretical advantage of minimizing the risk of immune rejection and inflammation. The generation of RGCs from iPSCs is now a reality, but the application of this technology to optic neuropathies poses a number of technical challenges that are unique to the anatomical organization of RGCs and the precise retinotopic connections that need to be preserved from the optic nerve to the lateral geniculate nucleus (63). The integration of RGCs within the inner retina will need to be optimized, and the signaling cues required to guide axonal migration and form the appropriate connections will need to be refined before such an approach can be applied in a clinical setting (64). So far, there have been no animal trials involving the transplantation of RGCs derived from iPSCs. In a proof-ofconcept study, hESC-derived RGCs were injected into the vitreous cavity and analyzed 1 week later, demonstrating the integration of ESC-RGCs into the ganglion cell layer (65). One of the challenges of using iPSC-derived RGCs for optic neuropathies is improving the efficiency of RGC generation, while excluding non-RGC differentiation. Furthermore, it is unknown whether the integration of non-RGC cell types will have a detrimental effect on the functional and clinical outcomes of transplantation. However, animal models have demonstrated that nonhomogenous neural cell populations can integrate into the recipient retina and demonstrate electrophysiological activity (66,67). Promoting the correct retinotopic connections of iPSC-derived RGCs after implantation is the ideal scenario (68); importantly, there is evidence of plasticity within the retinal neural network that could facilitate the integration of iPSC-derived cells into the host retina (69). Nevertheless, the re-establishment of the complex circuitry needed for the proper integration of signals from various pathways to achieve a reasonable degree of visual perception remains an important barrier that will need to be overcome (70). Despite the challenges associated with the transplantation of stem cell–derived cells, there have been several clinical trials for outer retinal diseases. Phase 1 and 2 trials involving the transplantation of hESC-derived retinal pigment epithelium (RPE) cells in patients with Stargardt macular dystrophy and Harvey et al: J Neuro-Ophthalmol 2022; 42: 35-44 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. State-of-the-Art Review age-related macular degeneration (AMD) demonstrated significant visual acuity improvement in the treated eyes (71,72). Mandai et al (73) used an iPSC-derived RPE sheet graft in one a patient with advanced AMD and demonstrated stable vision after transplantation. However, there was evidence of chronic cystoid macula edema 1 year after the initial surgery. A second patient was due to be included in this trial, but the transplantation did not proceed because of the identification of genomic aberrations within the patient-derived iPSC, highlighting the importance of stringent quality control for iPSC populations. A number of safety concerns must be considered in any attempts at clinical transplantation of differentiated cells derived from iPSCs, in particular, the risks of teratogenicity, immunogenicity, and genomic instability (74–76). Reassuringly, no long-term safety concerns were raised in the largest stem cell trial that has reported to date and involved 226 patients with spinal cord injuries (77). Induced Pluripotent Stem Cell Optimization Using Gene Editing The use of gene editing technology, such as CRISPR/Cas9, to correct the causative genetic variants in iPSC-derived cells is particularly appealing for monogenic diseases (5). The correction of variants in nuclear genes causing optic atrophy (Fig. 3), such as OPA1, offers distinct advantages because the replacement cells will be derived from autologous iPSCs, reducing the chance of immunogenicity and graft rejection. Recent studies have demonstrated the feasibility of using CRISPR/Cas9 gene editing to correct variants associated with retinitis pigmentosa (78,79) and Usher syndrome (80). Similarly, CRISPR/Cas9 gene editing has been explored as a potential approach to restore photoreceptor function in Leber congenital amaurosis (LCA), by excising a deep intronic variant in the CEP290 gene that causes aberrant gene splicing (81). The safety and efficacy of this strategy is being evaluated as part of an ongoing clinical trial (ClinicalTrials.gov NCT03872479). Although the CRISPR/Cas9 system has revolutionized gene editing, its applicability to editing mtDNA variants, like those associated with LHON, is more challenging (82). Wong et al (40) replaced defective mitochondria from LHON patient–derived cells carrying homoplasmic double mtDNA variants (m.4160T.C and m.14484T.C) with mitochondria from a WT cell line, and this resulted in a decreased susceptibility to apoptosis (Fig. 3). More recently, CRISPR-free mtDNA editing has been established using a bacterial cytidine deaminase toxin for targeted manipulation and correction of mtDNA variants (Fig. 3) (83). Induced Pluripotent Stem Cell Neuroprotective Strategies Until the integration of iPSC-derived RGCs becomes a realistic possibility, stem cell treatment could still offer advantageous therapeutic benefits by promoting neuroHarvey et al: J Neuro-Ophthalmol 2022; 42: 35-44 protection with the secretion of trophic factors, such as brain-derived growth factor or platelet-derived growth factor (84). Mesenchymal stem cells, which cannot develop into neural tissues, have demonstrated neuroprotective properties in mouse models of optic nerve disease such as glaucoma and traumatic optic neuropathy (85– 87). NPCs derived from iPSCs have also been found to increase RGC survival when transplanted into rats after an optic nerve crush injury (67). This field of research is still in its early stages, and we need a much better greater understanding of the neuroprotective potential of stem cells and how this approach could be optimized for patients with visual loss from IONs. Drug Screening Successful drug development programs often require highthroughput screening of thousands of potential therapeutic agents, and this strategy is being applied to neurodegenerative diseases, including Alzheimer disease and amyotrophic lateral sclerosis (88). In vitro disease models are advantageous because they can rapidly produce large quantities of target cells, whereas high-throughput readouts, such as 96 or 384 well-based assays, increase screening efficiency. For example, one 6-well plate of iPSC-derived RGCs yields the same number of cells (approximately 10 million cells) as the retinal dissection of more than 80 mice (35). This will likely improve with the further optimization and automation of RGC differentiation protocols (89). Differentiated cells from iPSCs have been used in therapeutic screens for ophthalmic diseases, such as nicotinamide in an iPSC model of AMD (54) and antisense oligonucleotide modulation of RNA splicing in LCA (90,91). Although high-throughput screening has not been applied extensively for ION drug development, b estrogen was found to decrease apoptosis in iPSCs harboring OPA1 variants, demonstrating the applicability of such an approach if the proper readouts are used (44). The trilineage differentiation potential of iPSCs is also a major asset for drug screening because they are able to produce a wide range of cell types, including motor neurons and myocytes, which can be affected in patients with more severe syndromic IONs (92–95). There is no doubt that 2D RGC models are efficient tools to study both the therapeutic and toxic effects of drug molecules (96,97); however, they lack the complex cell–cell interactions seen in the native retina and optic nerve. Ideally, this initial stage of drug screening could then be further substantiated within 3D retinal organoids, which more closely match the physiological retinal niche, providing further in vitro evidence before transitioning into more costly in vivo animal model studies (98). Importantly, the use of both 2D and 3D iPSC ION models can facilitate the rapid development of therapeutic strategies, providing a gateway to move into early phase clinical trials. 39 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. State-of-the-Art Review FIG. 3. Schematic overview of gene editing strategies to correct the underlying genetic variants in patient-derived iPSC populations. CRISPR/Cas9 gene editing can be used to edit nuclear encoded variants (purple bars), combined with homology-directed repair (HDR) that uses a repair template encoding the desired base changes (orange bars) to correct the disease-causing variants. Mitochondrial replacement—the mutant mitochondrial DNA (mtDNA) (red mitochondria) in LHON patient-derived iPSCs are depleted and then replaced with mitochondria carrying wild-type mtDNA (green mitochondria) to restore mitochondrial genome integrity and function. mtDNA gene correction—defective mitochondria (yellow mitochondria) harboring mtDNA variants (yellow mitochondria) can be corrected using bacterial-derived cytidine deaminase toxins resulting in respiratory competent mitochondria with wild-type mtDNA (blue mitochondria). iPSC, induced pluripotent stem cell; LHON, Leber hereditary optic neuropathy. CONCLUSION IONs cause severe permanent visual loss, and they represent a major societal burden given their onset in childhood and young adulthood. Although still in its early phase, the application of iPSCs to the field of ION research has tremendous potential both for disease modeling and as a powerful tool for therapeutic drug development and genomic medicine. In the short term, iPSCs provide efficient models that can be applied to study different causative genes and variants, which is a major advantage, given that IONs are genetically heterogeneous. Furthermore, CRISPR/Cas9 correction of disease-causing variants in iPSCs offers the ideal controls to better distinguish genotype–phenotype relations in vitro. The optimization of RGC differentiation protocols will increase the scalability of iPSC-derived RGCs for drug screening not only to assess efficacy but also to exclude possible toxic effects. In the medium term, iPSCs offer the ability to introduce autologous cells within the retina, which may convey neuroprotective effects, while reducing potential adverse immune responses. In the long term, iPSCs combined with gene editing technology could be used for RGC replacement to rescue vision in patients with more advanced optic nerve degeneration. IONs are ideal targets for iPSC-based thera40 peutics, and the future looks bright for much long-awaited breakthroughs for these blinding diseases. STATEMENT OF AUTHORSHIP Category 1: Conception and design: J. P. Harvey, P. E. Sladen, P. YuWai-Man, and M. Cheetham; Acquisition of data: J. P. Harvey, P. E. Sladen, P. Yu-Wai-Man, and M. Cheetham; Analysis and interpretation of data: J. P. Harvey, P. E. Sladen, P. Yu-Wai-Man, and M. Cheetham. Category 2: Drafting the manuscript: J. P. Harvey, P. E. Sladen, P. Yu-Wai-Man, and M. Cheetham; Revising it for intellectual content: J. P. Harvey, P. E. Sladen, P. Yu-Wai-Man, and M. Cheetham. Category 3: Final approval of the completed manuscript: J. P. Harvey, P. E. Sladen, P. Yu-Wai-Man, and M. Cheetham. ACKNOWLEDGMENTS JPH is supported by a Moorfields Eye Charity Research Training Fellowship. MEC is supported by the Wellcome Trust, Foundation Fighting Blindness (United States), Moorfields Eye Charity, and Fight for Sight. PYWM is supported by a Clinician Scientist Fellowship Award (G1002570) from the Medical Research Council (United Kingdom) and also receives funding from Fight for Sight (United Kingdom), the Isaac Newton Trust (United Kingdom), Moorfields Eye Harvey et al: J Neuro-Ophthalmol 2022; 42: 35-44 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. State-of-the-Art Review Charity, the Addenbrooke’s Charitable Trust, the National Eye Research Centre (United Kingdom), the International Foundation for Optic Nerve Disease (IFOND), the UK National Institute of Health Research (NIHR) as part of the Rare Diseases Translational Research Collaboration, the NIHR Cambridge Biomedical Research Centre (BRC-121520,014), and the NIHR Biomedical Research Centre based at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology. 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