RUNNING TITLE: GLAUCOMA NEUROPROTECTION CLINICAL TRIALS  



19
GLAUCOMA NEUROPROTECTION CLINICAL TRIALS  
TITLE PAGE
Title: Neuroprotection in glaucoma: towards clinical trials and precision medicine

Authors and affiliations:
Tasneem Z Khatib1,2,3 and Keith R Martin1,2,4,5,6,7

1. Centre for Brain Repair, University of Cambridge, UK
2. Eye Department, Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK
3. Medical Sciences Division, University of Oxford, UK
4. Cambridge NIHR Biomedical Research Centre, UK
5. Wellcome Trust – MRC Cambridge Stem Cell Institute, University of Cambridge, UK
6. Department of Ophthalmology, University of Melbourne, Australia
7. Centre for Eye Research Australia, Melbourne, Australia

Corresponding author: Keith Martin, +61 3 9929 8429 (tel), krgm2@cam.ac.uk, Centre for Eye Research Australia, Level 7, Peter Howson Wing, 32 Gisborne St, East Melbourne VIC 3002
Acknowledgements: 
This work was supported by grants from Fight for Sight, Addenbrooke’s Charitable Trust, the HB Allen Charitable Trust, the Cambridge Eye Trust, the Jukes Glaucoma Research Fund and core support grant from the Wellcome Trust and MRC to the Wellcome Trust – Medical Research Council Cambridge Stem Cell Institute.

Conflict of Interest:
KRM is co-founder of the gene therapy company Quethera Ltd and a consultant to Astellas Ltd. 
Running title: glaucoma neuroprotection clinical trials  

Abstract
Purpose: The eye is currently at the forefront of translational medicine and therapeutics. However, despite advances in technology, primary open-angle glaucoma remains the leading cause of irreversible blindness worldwide. Traditional intraocular pressure (IOP)-lowering therapies are often not sufficient to prevent progression to blindness, even in patients with access to high-quality healthcare. Neuroprotection strategies, which aim to boost the ability of target cells to withstand a pathological insult, have shown significant promise in animal models but none have shown clinically relevant efficacy in human clinical trials to date. We sought to evaluate the current status of neuroprotection clinical trials for glaucoma and identify limitations which have prevented translation of new glaucoma therapies to date. 
Methods: Literature searches identified English language references. Sources included MEDLINE, EMBASE, the Cochrane Library and Web of Science databases; reference lists of retrieved studies; and internet pages of relevant organisations, meetings and conference proceedings, and clinical trial registries. 
Results: We discuss six key neuroprotective strategies for glaucoma that have reached the clinical trial stage. Delivery of neurotrophic factors through gene therapy is also progressing towards glaucoma clinical trials. Refinements in trial design and the use of new modalities to define structural and functional endpoints may improve our assessment of disease activity and treatment efficacy. Advances in our understanding of compartmentalised glaucomatous degeneration and continued progress in the molecular profiling of glaucoma patients will enable us to predict individual risk and tailor treatment. 
Conclusion: New approaches to future glaucoma neuroprotection trials could improve the prospects for new glaucoma therapies. Glaucoma treatment tailored according to an individual's unique risk profile may become increasingly common in the future. 

Introduction

Adult-onset primary open angle glaucoma (POAG) is associated with optic nerve head damage, retinal ganglion cell death, axonal degeneration and progressive visual loss. There are multiple underlying genetic and environmental factors that contribute to the development of POAG, but how these interact to determine the disease course in each individual with glaucoma remains incompletely understood. 

The mainstay of current treatment is to lower the intraocular pressure (IOP) which has been established as the strongest modifiable risk factor associated with disease progression1,2. IOP can be lowered medically, using laser therapy or by surgical intervention but remains the only treatment strategy across all POAG populations regardless of the underlying pathophysiology.  

Recent clinical trials3 have shown that IOP lowering with topical anti-hypertensives such as the prostaglandin analogue Latanoprost in the United Kingdom Glaucoma Treatment Study ((UKGTS) trial registration number ISRCTN96423140) can effectively reduce the rate of POAG progression. However, the clinical need to develop additional strategies remains, as a proportion of patients may continue to worsen despite IOP lowering treatment. Even in populations with good access to glaucoma care, an estimated one in eight patients will still eventually become blind in at least one eye due to glaucoma progression over 20 years4. Of 2208 patients under UK hospital glaucoma care and receiving regular follow up and full conventional treatment to lower IOP followed for mean of 6.7 years, 21.2% progressed at greater than 0.5 dB/year, 10.7% progressed at more than 1.0 dB/year and 2.1% progressed at more than 2.0 dB/year in their better eye. If these results were extrapolated to England and Wales, with a glaucoma population of >500,000 diagnosed patients under conventional care, one would predict approximately 10% of treated glaucoma patients (50,000) are progressing at >1dB/year.  Approximately 15%5-46%6 of patients who underwent maximal medical and surgical intervention continued to progress to blindness at up to 20 years follow up despite low IOPs and it has been estimated that 60 million people worldwide will be affected by 20207.

Alternatives to IOP-lowering approaches for POAG have focused on neuroprotection. Neuroprotection strategies typically either exploit signalling pathways to stimulate cell survival or inhibit cell death pathways as a way to boost the cells ability to withstand a pathological insult. A large number of studies have demonstrated effective neuroprotection for glaucoma in animal models8-19. However, demonstrating that agents which have achieved neuroprotection in animal models of glaucoma have translational potential in human POAG clinical trials has presented several major challenges to date. 

Firstly, accurately modelling a variable and complex multifactorial disease process such as POAG, where the underlying pathology is not fully understood, is difficult. Available animal models replicate some aspects of the disease, but all have significant limitations. As an example, many glaucoma models involve induced elevation of IOP; such models may be helpful when assessing the site of injury, mechanisms of damage and gene expression changes,  but do not model the increased susceptibility to glaucomatous damage that may be a contributing factor in many glaucoma patients. Factoring in this increased susceptibility is particularly relevant when distinguishing glaucomatous pathology from that seen in ocular hypertensive patients, where the IOP is elevated but who do not have detectable structural or functional deficit, or normotensive patients who display a characteristic glaucomatous phenotype.

The degree of induced IOP elevation has recently been shown to elicit differential and often opposing changes in the transcriptome profile of retinal ganglion cells, according to whether IOP elevation is mild (elevation between 1-4 mmHg) or moderate (elevation  4mmHg)20. For example, activation of neuronal death pathways was seen following sustained ‘mild’ IOP elevation over a period of two weeks, whereas there was an increased activation of cell survival and neurite outgrowth pathways following ‘moderate’ IOP elevation. The authors postulated that this might indicate a selective impact on retinal ganglion cell somas or axons and dendrites according to the degree of IOP elevation, suggesting an additional level of complexity when selecting models of experimental glaucoma and selecting outcome measures to assess efficacy. 

Chronic models of acquired or spontaneously developing glaucoma, such as the beagle or basset hound canine glaucoma model have a protracted time course, are expensive and at present telemetry is not advanced enough to monitor the level and time course of IOP elevation to quantify the level of exposure to injury. Advanced glaucoma in these models can subsequently develop into a secondary angle closure with lens dislocation which also limits their usefulness in evaluating therapies for POAG. 

Acute IOP elevation models21 are more reliable and reproducible, with the potential for repetitive injury and strain to modulate the degree of induced damage. The increased predictability also confers a reduction in the number of animals required to detect evidence of efficacy but has limitations in the degree of relevant information provided when modelling a chronic, progressive condition such as POAG. Acute studies also fail to take into account the fluctuations in IOP frequency and magnitude that are typical of the glaucomatous disease process.

Differences in outcome measures used when assessing laboratory studies and human patients are another source of variability and neuroprotective agents are often administered either prior to or concurrently with the onset of damage in animal studies which limits their relevance when comparing any effect to therapeutic intervention in patients who already have the disease. 

Below we summarise some of the neuroprotection strategies for POAG that have reached the clinical trial stage to date, potential pitfalls with lessons learned from trials for other diseases and attempts that have been made to refine POAG clinical trial endpoints.  

Clinical trials for glaucoma neuroprotection

Brimonidine
Laboratory studies have demonstrated that systemic administration of brimonidine provides retinal ganglion cell neuroprotection in animal glaucoma models independent of its effect on IOP10,22. This neuroprotective effect has been postulated to be mediated via a variety of mechanisms including brain derived neurotrophic factor (BDNF)23 and basic fibroblast growth factor upregulation, nitric oxide synthase 3 metabolism and retinal vasomodulation24, the activation of cell survival signalling pathways and prevention of apoptosis and 2 modulation of N-methyl-D-aspartate (NMDA) receptor function25,26. Brimonidine administered topically twice a day for 2 weeks prior to elective pars plana vitrectomy achieved 2nM in the vitreous27 which was at a sufficient concentration for neuroprotection in the preclinical studies28. 

Brimonidine monotherapy lowered the incidence of visual field progression compared to timolol treated patients (9% vs 39%) in the Low Pressure Glaucoma Study Group (ClinicalTrials.gov identifier NCT00317577)  over a period of 30 months in patients who were able to tolerate the treatment29. However, in this trial, the progression rate in the timolol cohort was worse than control untreated group of other trials such as the Collaborative Normal Tension Glaucoma Study30. This raises the possibility that the difference reported between the two arms in the Low Pressure Glaucoma Treatment Study was due to timolol enhancing progression rather than brimonidine reducing progression, or a combination of the two. Brimonidine has a greater incidence of topical side effects such as hyperaemia, discomfort and hypersensitivity compared to other topical anti-glaucoma medications and the results reported could have been skewed by a selectively higher dropout rate in the brimonidine arm compared to the timolol arm. 

Cell therapy
Local administration via intravitreal injection to target retinal ganglion cells theoretically minimises unwanted side effects seen with repeated topical application. Single injections into the eye are well tolerated by patients with other chronic eye diseases such as age-related macular degeneration and may have a sustained therapeutic effect. 

Intravitreal administration of mesenchymal stromal cells (MSC) has been shown to be strongly neuroprotective in experimental glaucoma31–33. Advantages of MSCs compared to other types of cells include the fact that they are easy to obtain from a variety of sources including adult bone marrow, avoid ethical concerns and can be used without immune suppression. An analysis of the protective factors produced by MSCs strongly implicated platelet-derived growth factor (PDGF) and subsequent blockage of PDGF signalling prevents MSC-mediated neuroprotection in animal models34. We have also demonstrated a dose dependent protection against apoptosis with PDGF treatment with greater protection from MSCs in human post mortem retinal tissue35. 

However, despite these encouraging results, PDGF and MSCs can induce reactive gliosis in retinal Muller cells and astrocytes with upregulation of intermediate filament proteins and retinal folding36. There have been similar recent reports of proinflammatory vitreous clumping37 of MSCs injected intravitreally as well as thick epiretinal membrane formation following MSC administration in humans38. These adverse effects may also be due in part to the inconsistency in MSC isolation and preparation. An attempt to standardise the identification of these cells led to the development of the International Society for Cellular Therapy criteria39, but problems continue to be reported despite this and may limit the degree to which MSCs can successfully be used to confer neuroprotection to retinal ganglion cells. 

There have been significant concerns regarding the unregulated use of stem cells in various centres around the world. Most ‘stem cell’ treatments for glaucoma administered worldwide are unregulated and currently most registered trials are patient funded, open-label studies. Kuriyan et al. reported severe bilateral visual loss in three patients who received intravitreal injections of autologous adipose tissue–derived stem cells for age-related macular degeneration40. Visual loss was related to ocular hypertension, haemorrhagic retinopathy, vitreous haemorrhage, combined traction and rhegmatogenous retinal detachment, or lens dislocation. The pre-injection visual acuity ranged from 20/30 to 20/200 and measured between 20/200  and no light perception one year post injection. 

There are currently at least four registered clinical trials evaluating the use of stem cells in glaucoma. The Intravitreal Mesenchymal Stem Cell Transplantation in Advanced Glaucoma study (NCT02330978) is a Phase 1 safety study in 10 patients who meet the legal definition of bilateral blindness, with intravitreal injection of autologous MSCs administered to the worst-affected eye. Secondary outcome measures include visual acuity, visual fields, optical coherence tomography and electroretinography. Stem Cell Ophthalmology Treatment Study (NCT01920867) and Stem Cell Ophthalmology Treatment Study II (NCT03011541) are non-randomised, open label efficacy studies evaluating the use of MSCs in multiple eye diseases including glaucoma delivered through either the sub-Tenon’s, retrobulbar, intravitreal or intravenous route. The study is patient funded and claims to be associated with a risk of potential complications of 0.0008% to 5% depending on the physician-selected treatment protocol chosen for individual patients. The study website declares that the investigators hope for visual improvement in the vast majority of individuals enrolled, but very little information is provided with regard to outcomes of those already treated. The Effectiveness and Safety of Adipose-Derived Regenerative Cells for Treatment of Glaucomatous Neurodegeneration study (NCT02144103) is an open-label safety and efficacy study in Russia. Stem cells are delivered via the sub-Tenon’s route with structural and functional endpoints and is a single-arm study with no control group.

It remains conceivable that stem cells do have the potential to provide a useful treatment strategy for glaucoma, either by a neuroprotective or neuroregenerative mechanism.  However, in order to assess such strategies we need randomised, masked, controlled clinical trials and further work on the optimal mode of delivery. Care needs to be taken to prevent the exploitation of vulnerable patients and false promise of success with unproven treatments. 

Neurotrophins
Ciliary Neurotrophic Factor
Ciliary neurotrophic factor (CNTF) has been shown to be neuroprotective in experimental glaucoma19. A randomised, sham-controlled, masked Phase II clinical trial by Neurotech Pharmaceuticals is currently underway to assess the effects of encapsulated cell-based delivery of ciliary neurotrophic factor (CNTF) in 54 glaucoma patients using the NT-501 device. Retinal pigment epithelial cells that have been engineered to release CNTF are encased in a semi-permeable membrane which permits the selective and sustained release of CNTF to retinal ganglion cells when implanted intravitreally. The NT-501 implant has also been shown to slow the progression of retinal photoreceptor degeneration and stabilise visual function in the patients with macular-telangiectasia type 2 (NCT01949324) 41. A Phase III study to further assess the effects of NT-501 in macular-telangiectasia type 2 is currently enrolling across the US, Australia and Europe. 

Recombinant human nerve growth factor
Eye drops containing recombinant human nerve growth factor (rhNGF) have received FDA approval to treat neurotrophic keratitis42 with encouraging safety and efficacy results in dry eye disease (NCT02101281) with data from further trials to follow (NCT03019627, NCT03035864). A Phase 1b randomised, double-masked study to assess the safety and tolerability of rhNGF eye drops in progressive POAG patients compared to vehicle has completed recruitment (NCT02855450). Secondary objectives include structural and functional assessments using changes in visual field, ERG and optical coherence tomography of the ganglion cell layer and retinal nerve fibre layer thickness. The investigators also plan functional assessments to determine whether any biological effect persists following discontinuation of the study treatment. 
Memantine
Oral administration of existing therapies that have been repurposed for glaucoma is an approach which can help overcome some issues with safety profile and regulatory agency approval meaning that an effective treatment can reach the clinic more quickly. Memantine is a non-competitive NMDA receptor antagonist8 which is used in the treatment of moderate to severe Alzheimer’s disease and showed promising results in a monkey model of glaucoma43. NMDA receptors are widely distributed throughout the central nervous system  and are essential for neurotransmission and the healthy function of neuronal cells. However, overstimulation in the presence of excessive glutamate can lead to Ca2+ - mediated neurotoxicity. The dysregulation of this cascade has been widely implicated in many chronic neurodegenerative conditions including glaucoma and led to the memantine glaucoma trials, the results of which were anticipated with great interest. However, large scale multicentre, randomised double-masked placebo controlled Phase III clinical trials (NCT00141882, NCT00168350) conducted to test the efficacy of oral memantine for glaucoma, showed there was no evidence of any statistical benefit compared to placebo in reducing visual field progression. The failure to demonstrate statistical significance relative to placebo in trials that took nearly 5 years at an estimated cost of over $100 million highlighted a need for better glaucoma clinical trial design and more effective use of relevant endpoints as required by regulatory agencies.

Nicotinamide (Vitamin B3)
A team from the Centre for Eye Research Australia led by Jonathan Crowston has recently completed patient recruitment for a neuroprotection pilot study investigating the short-term effect of taking the nicotinamide adenine dinucleotide (NAD) precursor nicotinamide (Vitamin B3) supplementation on patients with glaucoma (Universal Trial Number U1111-1197-0197). NAD is an essential co-factor in the redox reactions of the mitochondrial respiratory chain required to maintain sufficient levels of adenosine triphosphate (ATP). The intraocular portion of the retinal ganglion cell axon is unmyelinated and therefore has a particularly high energy requirement. Nicotinamide deficiency and NAD depletion therefore have the potential to disrupt mitochondrial and energy metabolism and ganglion cell function. Recent work by Nzoughet et al44 has demonstrated a significantly lower plasma nicotinamide concentration in POAG patients relative to controls which lends support to this hypothesis.
 
Furthermore, Williams et al. demonstrated that nicotinamide supplementation conferred significant neuroprotection both prophylactically and as an intervention in the DBA/2J chronic, age related open angle mouse model of glaucoma which confers a phenotype similar to pigment dispersion syndrome45. Treatment at the highest dose (2000 mg/kg/day) tested reduced the risk of developing glaucoma by 10-fold (equivalent human dose 162.6 mg/kg/day). The authors had also demonstrated an age-dependent reduction in retinal levels of NAD and postulated that this reduction led to mitochondrial dysfunction and rendered aging neurons more susceptible to raised IOP46. They analysed age- and IOP- dependent changes in transcripts of NAD producing enzymes within retinal ganglion cells and showed that nicotinamide treatment prevents these transcriptomic changes. Similar neuroprotective effects were also seen by overexpressing Nmnat1, an enzyme required for NAD production, using a gene therapy approach. The ability to confer neuroprotection by modulating mitochondrial vulnerability builds upon the significant body of work implicating age, metabolic stress and mitochondrial dysfunction in POAG47. 

Primary outcomes for the nicotinamide clinical trial include functional changes measured using visual fields and electroretinography and structural outcomes including hyperspectral imaging and OCT. Participants were randomly assigned to take oral nicotinamide or placebo daily for 12 weeks with functional and structural testing at baseline, 6 and 12 weeks post-intervention. A crossover design switched patients without a washout period to take placebo or nicotinamide for another 12 weeks with the same outcome measures repeated at 6 and 12 weeks post-intervention. 

The safety and efficacy of oral nicotinamide has already been demonstrated in a Phase III randomised clinical trial (UTN U1111-1131-4069) published in the New England Journal of Medicine, where the rate of new non-melanoma skin cancers and actinic keratoses in high-risk patients was reduced following oral supplementation at a dose of 500 mg of nicotinamide twice daily for 12 months compared to placebo48. The protective effects of nicotinamide in this trial were thought be mediated by minimising the damage from UV radiation. Radiation exposure coupled with a bioenergetic defect from mitochondrial dysfunction has also been implicated in POAG, rendering retinal ganglion cells susceptible to injury49.  

The promise from the pre-clinical data and other fields, together with the use of electroretinography and hyperspectral imaging to provide functional outcomes in a short period of time has the potential to make a significant impact and the results from this trial are anticipated with interest.  

Gene therapy
At present, no gene therapy for glaucoma neuroprotection has reached the clinical trial stage. Successes in gene therapy to treat other progressive retinal and optic nerve pathologies that were previously deemed untreatable have been encouraging, not only with regards to prospects for POAG treatment, but for the scientific and medical community as a whole. 

Those trials that have progressed the furthest are single gene replacement therapies for a specific genetic defect in people with inherited retinal diseases. The immediate comparison for glaucoma patients is perhaps for those with congenital or juvenile open angle glaucoma who have a specific genetic defect. Dominant gain of function mutations in myocilin have been shown to affect trabecular meshwork function and raise the IOP with subsequent development of glaucoma. Jain et al. disrupted the effects of the mutant myocilin gene using AAV-CRISPR/Cas9 (Ad5-cas9 and Ad5-crMYOC) in a mouse model of myocilin-associated POAG50. They were able to lower the IOP in treated eyes and prevent further glaucomatous damage. The authors also applied these constructs to human explants and demonstrated a reduction in trabecular meshwork myocilin mRNA. More recently, mutations in angiopoietin receptor TEK (tunica interna endothelial cell kinase, also known as Tie2) have been implicated in Schlemm’s canal development with congenital glaucoma phenotypes seen in mice that lack functional angiopoietin-TEK signalling51. Human mutations in TEK with variable expressivity have also recently been identified in a primary congenital glaucoma cohort that do not carry mutations in other known disease-causing genes52 and work is ongoing to assess the effects of TEK augmentation in these patients. 

Gene therapy for glaucoma patients who have a specific genetic defect is conceivable, but gene therapy could also be used to deliver protective factors in patients with disease not caused by single gene defects. Astellas Pharmaceuticals have recently acquired a gene therapy construct which overexpresses BDNF and its receptor TrkB53,54 with a view to taking it into Phase I/IIa trials in POAG patients in the near future. The construct is designed to bolster the natural neuroprotective levels of BDNF on retinal ganglion cell survival, which have been proposed to fall in glaucoma partly due to glaucoma-related pathophysiology of axonal transport55. The simultaneous dual expression of ligand and receptor is also designed to maintain beneficial effects of BDNF for prolonged periods by chronic activation of survival pathways. 

A new era of translational medicine: lessons learned from other gene therapy trials 
Below we summarise how lessons learned from trials for other diseases may be used to guide the development of neuroprotective gene therapies for glaucoma.

FDA approval for the first directly administered gene therapy product for any condition due to mutations in a specific gene was granted to Spark Therapeutics for Luxturna, (AAV2-hRPE65v2) a viral vector administered subretinally to augment function of the retinal pigment epithelial cells in Leber’s Congenital Amaurosis (LCA). LCA is an inherited retinal condition caused by a loss of function mutation in the RPE65 gene. This leads to excessive build-up of all-trans retinal, disruption of the photochemical conversion of light into electrical impulses and severe visual impairment from childhood which was previously deemed untreatable. Russell et al. demonstrated significant functional improvement in children with LCA up to 1 year following Luxturna administration in Phase III clinical trials (NCT00999609)56 and this effect has been reported to be maintained at 3 years. The primary efficacy endpoint was change in bilateral multiluminance mobility test (MLMT). The MLMT assesses the ability to navigate an obstacle course at varying light levels and was specifically designed for the trial to be a functional measure that would best capture the impact of treatment.  A change of one light level in passing was considered clinically meaningful although it is not fully clear as yet how the MLMT score translates to real world outcomes. At one year, treated eyes were associated with a difference of 1.8 in the MLMT change score in treated eyes versus 0.2 in control eyes. 
 
This trial was a landmark achievement which demonstrated the safety and efficacy of directly administered gene therapy for genetic conditions, particularly as early trials for patients with X-linked severe combined immunodeficiency induced leukaemia were associated with mortality in treated children. This subsequently halted the further development of gene therapy approaches for several years while risks were re-assessed and strategies were refined. Clinical trials using gene therapy to treat other debilitating genetic conditions such as spinal muscular atrophy57, which has also recently been granted FDA approval, and junctional epidermolysis bullosa58 are also showing considerable promise with good safety profiles and functional improvement. Although evaluation is ongoing and the longer term impact remains uncertain, with other trials for LCA (NCT00643747) reporting a decline in the degree of visual improvement59 and the subsequent development of an optimised AAV5 viral vector and transgene expression cassette to improve retinal transduction and yield higher levels of RPE6560, these advances all lend strong support to the use of directly administered gene therapies forming a core part of a clinician’s toolkit when treating intractable illnesses in the foreseeable future. 

There has been an economic burden associated with these significant scientific advances. The cost price for Luxturna is $850 000 for a single treatment (or $425 000 per eye). Following review by the Institute for Clinical and Economic Review and giving special weighting to the very rare nature of the disease, the price was considered to approach cost-effectiveness compared to current standard of care in a ‘best-case’ scenario, where a child aged 3 would receive a one off treatment that would last the duration of a lifetime. Spark Therapeutics have included refunds in the event of treatment failure and payment over several years as part of their overall strategy. 

Concerns about current cost are understandable, but we would argue should not preclude continued development of gene therapies. An international gene therapy trial involving over 100 patients across 9 countries in the EU and North America is now underway for choroideremia, an X-linked retinal degeneration resulting in progressive blindness due to mutations in Rab-escort protein 1 (REP1). The results of the initial pilot study (NCT01461213)  were reported in Nature Medicine where a significant improvement in visual acuity at 2 years following subretinal injection of AAV2.REP1 was seen in 14 choroideremia patients compared with controls (median 4.5 letter gain, versus 1.5 letter loss, p=0.04). 6 of the treated eyes gained more than one line of vision at 2 years61. The timescale for development of gene therapies continues to fall and the economics of providing gene therapies for common conditions such as POAG are likely to drive production costs down further over time. 

Significant advances are also being made for optic neuropathies and clinical trials are currently being conducted to evaluate the efficacy of GS010, an AAV2 construct containing the human wild-type NADH dehydrogenase subunit 4 (ND4) gene to treat patients with Leber’s Hereditary Optic Neuropathy due to the G11778A ND4 mitochondrial mutation62,63. Preliminary data from the Phase I/II trial showed promising results (NCT02064569) and provided the foundation for two Phase III clinical trials which are currently underway (NCT02652780, NCT02652767). Clinically meaningful functional improvement has recently been reported with 72 week data from the REVERSE Phase III clinical trial (NCT02652780) which demonstrates that 45.9% of treated eyes achieved a significant improvement of 0.3 LogCS or more compared to 24.9% improvement seen in sham treated eyes (p-0.0047)64. Treated eyes were estimated to be more likely to achieve a visual acuity of 20/200 or more (p=0.0012) and this was also associated with relative preservation of structural parameters including the ganglion cell layer volume, papillomacular bundle thickness and total macular thickness. Transport of the administered vector to the fellow eye, reorganisation of the visual cortex or a placebo effect may go some way to towards understanding the basis of the improvement in visual acuity seen in the sham injected eyes. The randomisation code for the study will be broken later this year and will enable individual patient responses to the therapy to be determined which should help refine the analysis. A separate arm of the study, the REFLECT study (NCT03293524), evaluating the efficacy and safety of bilateral intravitreal injection of GS010 in LHON patients with the ND4 mutation, is currently recruiting and will also help to assess the sham-treated improvement and help guide future clinical trial design for gene therapies for POAG. 

The most frequent treatment-emergent adverse effect (TEAE) seen in the initial Phase I/II clinical trial was intraocular inflammation, with 13 events reported in 11 out of 15 subjects. These were usually mild and treated topically but two patients required oral steroid administration to treat the inflammation. The greatest percentage of those affected was seen at the highest vector doses: 33% (1 of 3 patients) at 9x109 vector genomes/eye, 67% (2 of 3 patients) at 3 x 1010 vector genomes/eye, 83% (5 of 6 patients) at 9 x 1010 vector genomes/eye and 100% (3 of 3 patients) at 1.8 x 1011 vector genomes/eye. By week 96, no related TEAE required ongoing treatment and no structural or visual loss was reported as a result of vector administration or TEAE. A secondary analysis of the Phase I/II trial data calculated a composite ocular inflammation (OIS) score using the Standardization of Uveitis Nomenclature and correlated this with the viral titre and systemic immune response65. The maximum OIS was seen in a patient with a previous history of idiopathic uveitis. The authors did not find an association between the OIS, humoral immune response, immune status of the patient or viral titre administered and overall concluded there were no intraocular inflammatory safety concerns that would preclude further clinical trials. However, in view of the transient intraocular inflammation that was reported after vector administration, it would appear that AAV vectors have a degree of immunogenicity. The authors recommended the systematic use of prophylactic steroids in future trials to decrease the inflammatory responses and also pointed to the need for ongoing animal studies to help elucidate the local immune response to gene therapy. The AAV8.RS1 Phase I/II three-dose-escalation trial (NCT02317887) for X-linked retinoschisis administers a healthy copy of retinoschisin to target photoreceptors and showed a dose-related intraocular inflammation that resolved with topical and oral steroids66. Systemic antibodies against AAV8 also increased in a dose-related fashion, but no antibodies against RS1 were observed.

It has been suggested that innate immune responses induced by the toll-like receptor (TLR) pathway can affect the adaptive immune responses to AAV gene transfer with TLR2, TLR9, and MyD88 being implicated in particular67–71. A number of refinements are ongoing to limit activation of the immune response while achieving efficient transgene transfer following AAV administration. These include single cell transcriptome based development of AAV vectors and capsid modifications to alter the tropism and pattern of activity of viral efficacy72. This has been postulated to confer an increased ability to bypass structural barriers including the vitreous body while limiting the effect of neutralising antibodies. 

Exosomal AAVs, where viral particles are carried inside small membrane vesicles, are another promising approach and have been shown to yield a higher retinal transduction efficiency and are more resistant to neutralising anti-AAV antibodies73–75. This technology confers additional advantages over traditional AAV2 based approaches as the development of neutralising antibodies may preclude repeat injections in the event of treatment failure, reduced efficacy or the development of a separate disease process that may also be treated by gene therapy approaches. Scope therefore also exists to target multiple processes involved in glaucoma pathogenesis rather than taking a single ‘one-size-fits-all’ approach. It is unlikely that the immune response to viral vectors will be completely eliminated despite refinement. The degree of efficacy provided versus risk of inflammation should form part of the decision making prior to administering therapy. 

It is also of interest that the rAAV2/2-ND4 trial enrolled patients from the age of 15 years onwards with diagnosed ND4 LHON. The pharmacokinetics of the vitreous in younger patients may well be different from the often elderly population affected by POAG, and it remains to be seen whether the presence of vitreous syneresis or posterior vitreous detachment will lessen the structural barrier to viral transduction hence improving transgene expression. It is encouraging that efficacy has been demonstrated following intravitreal injection in the younger LHON cohort who typically have a thicker internal limiting membrane and denser vitreous composition. The AAV8-RS1 gene therapy trial for X-linked retinoschisis also uses an intravitreal approach to administer a healthy copy of retinoschisin to target photoreceptors. Cukras et al. postulated that the retinal degeneration associated with the X-linked retinoschisis also compromises the structural integrity of the internal limiting membrane, facilitating retinal penetration following intravitreal administration of the viral vector66.  Little is known about the structural integrity of the internal limiting membrane in glaucoma patients and how much of a barrier this will pose to viral transduction for future gene therapy trials. 

Optimising clinical trial endpoints
The failure to demonstrate efficacy in the oral memantine POAG neuroprotection trial highlighted the need to develop more relevant and appropriate clinical endpoints.  Slow progression of POAG in patients recruited to clinical trials of neuroprotection has suggested that lengthy trials would be required with the individual variability that occurs when performing functional tests necessitating large group sizes to determine whether there is evidence of any therapeutic effect. 

It is therefore of considerable interest that the UK Glaucoma Treatment Study (UKGTS)3 which evaluated vision preservation in patients taking the prostaglandin analogue Latanoprost for POAG in a randomised, multicentre, placebo-controlled trial demonstrated statistically significant differences between treatment groups within one year with approximately 250 patients per group. It is conceivable that recruitment of patients progressing at a pre-defined rate prior to trial entry as well as enhancement of functional endpoints by clustering of measurements at the beginning and the end of studies could help to shorten neuroprotection trials and reduce the group sizes required still further. 

De Moraes et al. proposed that decreasing the rate of visual field progression by 30% in a clinical trial of 12 to 18 month duration would yield clinically meaningful results and showed that a 30% decrease in rate of visual field progression had a significant effect on health-related quality of life76. It has also been suggested that the 10-2 visual field may be a more appropriate outcome measure as the 24-2 field can underestimate the degree of damage at the macular region involved in glaucomatous optic neuropathy77. The 10-2 visual field has also been shown to correlate with quality of life outcome measures78 and may reduce the time required to detect glaucomatous progression79. 

Wu et al. also indicate that lengthy and large trials are not essential, suggesting that the sample size required to assess the effect of any neuroprotective intervention depends on the baseline rate of progression, study duration, and quantification and outcome parameter80. They suggest that the sample size could be lowered by assessing progression using the average mean deviation (MD) change rather than event-based guided progression analysis (GPA) analysis. Quigley suggested that selecting rapid progressors would limit the generalisability of any study, increase the time taken for recruitment and that selecting patients with low variability in previous tests would be most efficient, and stressed a greater role for futility trial designs to determine promising candidates to take to larger clinical trials81.  

UKGTS was also one of the first major clinical trials to use optical coherence tomography and structural parameters as a secondary outcome. Garway-Heath et al. showed that combining structural and functional outcome measures yielded higher sensitivity but did not improve overall discrimination. However, the study used time-domain OCT and the higher resolution provided by spectral-domain or swept-source OCT may help to refine endpoints further in a clinically meaningful way. The use of probability maps rather than summary measures when analysing outcomes has also been suggested to improve sensitivity and earlier detection82,83. 

DARC Technology (Detection of Apoptosing Retinal Cells) is a technique currently being assessed in Phase II clinical trials which enables the direct visualisation of apoptosing retinal cells in real time following intravenous administration of ANX776 (annexin-5 labelled with a fluorescent dye DY-776). The proof-of-concept open-label Phase I study (ISRCTN10751859) demonstrated an increase in the ‘DARC count’ or total number of ANX776 spots in glaucoma patients relative to healthy controls, and the DARC count also corresponded with rates of structural or functional progression84. 

The DARC count does not identify retinal ganglion cells specifically and the correlation of apoptosis detection through annexin V labelling with the ‘health status’ of a human retinal ganglion cell undergoing glaucomatous injury remains to be determined. However, DARC Technology does offer the potential to monitor retinal disease activity in vivo and longitudinally at the level of an individual cell and may prove to be a useful adjunct to clinical endpoints when assessing treatment efficacy. 

An understanding of the compartmentalised degenerative process will not only help to identify new targeted approaches for neuroprotection but also help to refine outcome measures and timings for therapeutic interventions in clinical trials. Ou et al. correlated structural and functional parameters of subsets of retinal ganglion cells following laser induced ocular hypertension using biolistic labelling and multielectrode array recordings of individual retinal ganglion cells85. They found that  OFF-transient retinal ganglion cells were more susceptible to glaucomatous injury both structurally, with higher rates of cell death and reduction in dendrite area and synapse density, and functionally, with decreases in spontaneous activity and receptive field size. Interestingly, while the function of the OFF-sustained and ON-sustained retinal ganglion cells were preserved, there was a decrease in excitatory post-synaptic sites of these cells, indicating that synapse loss occurs prior to any observable functional loss. They also reported an increased vulnerability of the presynaptic ribbon in the OFF sublamina of the inner plexiform layer, which suggests that retinal ganglion cells with dendrites stratifying in the OFF sublamina may be damaged earlier in the degenerative process. Whether the presynaptic changes occur prior to postsynaptic changes and are a secondary or primary event remains to be determined, including whether synapses that receive different bipolar cell type inputs are lost uniformly. Early dendrite pruning with synapse loss occurring prior to axon or soma loss has also been reported elsewhere using the microbead model of induced ocular hypertension86 and the genetic DBA/2J model of chronic glaucoma87, with dendritic atrophy also occurring prior to functional loss through visual field testing in primates88. The authors did note preservation of the dendritic arbour in DBA/2J mice in eyes that had more significant ganglion cell loss and postulated that selective vulnerability of retinal ganglion cell types, along with possible functional recovery, may account for this finding.  

Several factors are implicated in the loss of dendrites, synaptic pruning, remodelling and neuronal maintenance following glaucomatous injury including the transport, number and distribution of mitochondria, retrograde blockade of neurotrophic support to the cell body and synapses, complement cascade dysregulation, microglial activation and changes in neuronal activity. Whether the latter influences early structural remodelling or is a consequence remains under active debate.  Impaired inner retinal function has been shown to precede defects in anterograde active axonal transport of cholera toxin B following intraocular pressure elevation89, with a redistribution in the size and location of the axon initial segment postulated to alter action potential propagation following glaucomatous injury. Visual stimulation and electrical activity has also been shown to influence axonal regeneration90. Little is yet known about the relationship between retrograde transport blockade or the transport of endogenous proteins and functional impairment. 

The degree of IOP elevation appears to influence whether the retinal ganglion cell body or axon undergoes preferential degeneration20. The mechanisms of degeneration are distinct and compartmentalised as are the functional requirements of retinal ganglion cells according to their distribution within the retina and between the myelinated, non-myelinated and laminar regions of the optic nerve. The Wlds allele, which slows Wallerian degeneration, has been shown to protect axons91 but not retinal ganglion cell somata following experimental glaucoma92. 

Functional recovery has previously been demonstrated in glaucoma patients following intervention, suggesting the possibility of reversal of glaucomatous dysfunction of retinal ganglion cells and their connections. Caprioli et al. demonstrated evidence of persistent improvement in visual function in the longer-term93. They assessed visual field progression before and up to 5 years following trabeculectomy in 74 eyes of 64 patients.  Fifty-seven percent of eyes improved in 10 or more test locations compared with pre-operative values and the number of improved visual field loci correlated with the IOP lowering. Casson et al. demonstrated a glucose-mediated neuroenhancement of retinal function, where topical glucose was applied to pseudophakic glaucoma patients and significantly improved contrast sensitivity94. Preclinical studies would indicate that ageing retinal ganglion cells recover more slowly95, and that exercise-mediated BDNF expression can enhance functional recovery of retinal ganglion cells following IOP elevation96. 

Refinements in our ability to assess the health and activity of retinal ganglion cells in these distinct compartments with continued development of modalities to identify retinal ganglion subtypes in humans as well as earlier measures of functional damage in glaucoma will help to assess disease activity and treatment efficacy. 

Towards precision medicine
Several neuroprotective approaches show significant promise but intrinsic regulatory mechanisms may determine their effectiveness in different individuals. In a similar vein, alternative compensatory pathways may limit the degree of efficacy which a neuroprotective approach can offer, reaching a plateau, without the modification of underlying pathophysiology. Scope therefore exists to continue to pinpoint specific mechanisms that are disrupted in POAG and the relative contribution of these in individuals to tailor treatment.
 
An effective treatment strategy may well require a multifaceted combinatorial approach to yield maximum therapeutic effect both to target pathological processes and compartmentalised degeneration. How we administer this combination approach is of interest and it is likely that neuroprotection strategies will serve as an adjunct to IOP-lowering therapies in the first instance. 

We have recently described a novel gene sequence to encode both a ligand (BDNF) and its TrkB receptor in a single transgene separated by a short viral-2A sequence53. The single transgene is processed intracellularly to produce the two proteins of interest, which are then independently transported to their final cellular locations: TrkB receptors to the cell membrane, and BDNF contained within vesicles to be secreted. This approach when delivered to retinal ganglion cells via an AAV2 vector yielded functional proteins which demonstrated neuroprotection in both optic nerve crush and experimental glaucoma models54. It is conceivable that combining two or more transgenes coding for proteins targeting multiple pathways related to glaucoma pathogenesis could provide an effective approach to deliver targeted, sustained and individualised treatment to maximise efficacy. We have also discussed the use of CRISPR/Cas9 as a gene editing tool for glaucoma elsewhere97 and other gene and post-translation modification approaches to modify ocular disease have been extensively reviewed98. 

Valuable lessons can be learned from the oncology community, where breast cancer molecular and genetic phenotyping are now used widely in clinical practice to tailor treatment to individuals99–101. These molecular subtypes have phenotypic diversity with regard to multiple clinical outcomes, including response to chemotherapy, disease-free survival, and overall survival. It is therefore conceivable that continued progress in the molecular profiling of adult-onset POAG patients will improve our understanding of relative contributions of the various disease processes we know are implicated in POAG in individual patients and determine how they should be treated according to their underlying pathophysiology.

The UK Biobank holds large-scale genomic and detailed phenotypic and health-related data for 500,000 people and has already proved a powerful tool in linking genetic and epidemiological factors to disease risk and an understanding of the mechanisms that underlie those associations102–104. The project, which benefits greatly from the infrastructure of the NHS, has an open access approach so that researchers from around the world are able to use the dataset. To date there have been at least 8,294 approved registrations, and 796 formally registered projects, the results of which have been communicated in over 500 publications in peer-reviewed journals.  Glaucoma parameters have also been included in the data collection process105 and we are starting to see the development of polygenic risk scores106 to predict glaucoma progression and the need for intervention. The UK Biobank data, combined with other biobank centres and initiatives such as the 100,000 Genomes project has the potential to significantly enhance our understanding of the relative contributions of pathological mechanisms to a disease process and enable us to predict individual risk to target screening programmes, enhance our diagnostic capability and tailor treatment according to the pathological process. 



REFERENCES 

1. Leske MC, Heijl A, Hyman L, Bengtsson B, Dong L, Yang Z. Predictors of Long-term Progression in the Early Manifest Glaucoma Trial. Ophthalmology. 2007;114(11):1965–1972. doi:10.1016/j.ophtha.2007.03.016
2. Friedman DS, Wilson MR, Liebmann JM, Fechtner RD, Weinreb RN. An evidence-based assessment of risk factors for the progression of ocular hypertension and glaucoma. Am. J. Ophthalmol. 2004;138(3 SUPPL.). doi:10.1016/j.ajo.2004.04.058
3. Garway-Heath DF, Crabb DP, Bunce C, Lascaratos G, Amalfitano F, Anand N, Azuara-Blanco A, Bourne RR, Broadway DC, Cunliffe IA, et al. Latanoprost for open-angle glaucoma (UKGTS): A randomised, multicentre, placebo-controlled trial. Lancet. 2015;385(9975):1295–1304. doi:10.1016/S0140-6736(14)62111-5
4. Malihi M, Moura Filho ER, Hodge DO, Sit AJ. Long-Term Trends in Glaucoma-Related Blindness in Olmsted County, Minnesota. Ophthalmology. 2014;121(1):134–141. doi:10.1016/j.ophtha.2013.09.003.Long-Term
5. Landers J, Martin K, Sarkies N, Bourne R, Watson P. A twenty-year follow-up study of trabeculectomy: Risk factors and outcomes. Ophthalmology. 2012;119(4):694–702. doi:10.1016/j.ophtha.2011.09.043
6. Parc CE, Johnson DH, Oliver JE, Hattenhauer MG, Hodge DO. The long-term outcome of glaucoma filtration surgery. Am. J. Ophthalmol. 2001;132(1):27–35. doi:10.1016/S0002-9394(01)00923-0
7. Quigley H, Broman AT. The number of people with glaucoma worldwide in 2010 and 2020. Br. J. Ophthalmol. 2006;90(3):262–267. doi:10.1136/bjo.2005.081224
8. Vorwerk CK, Lipton SA, Zurakowski D, Hyman BT, Sabel BA, Dreyer EB. Chronic low-dose glutamate is toxic to retinal ganglion cells: Toxicity blocked by memantine. Invest. Ophthalmol. Vis. Sci. 1996;37(8):1618–1624.
9. Russo R, Cavaliere F, Berliocchi L, Nucci C, Gliozzi M, Mazzei C, Tassorelli C, Corasaniti MT, Rotiroti D, Bagetta G, et al. Modulation of pro-survival and death-associated pathways under retinal ischemia/reperfusion: effects of NMDA receptor blockade. J. Neurochem. 2008;107(5):1347–1357. doi:10.1111/j.1471-4159.2008.05694.x
10. Hernández M, Urcola JH, Vecino E. Retinal ganglion cell neuroprotection in a rat model of glaucoma following brimonidine, latanoprost or combined treatments. Exp.Eye Res. 2008;86(5):798–806. doi:10.1016/j.exer.2008.02.008
11. Martin KRG, Quigley HA, Zack DJ, Levkovitch-Verbin H, Kielczewski J, Valenta D, Baumrind L, Pease ME, Klein RL, Hauswirth WW. Gene Therapy with Brain-Derived Neurotrophic Factor As a Protection: Retinal Ganglion Cells in a Rat Glaucoma Model. Invest. Ophthalmol. Vis. Sci. 2003;44:4357–4365.
12. Geyer O, Almog J, Lupu-Meiri M, Lazar M, Oron Y. Nitric oxide synthase inhibitors protect rat retina against ischemic injury. FEBS Lett. 1995;374(3):399–402. doi:10.1016/0014-5793(95)01147-7
13. Howell GR, MacNicoll KH, Braine CE, Soto I, Macalinao DG, Sousa GL, John SWM. Combinatorial targeting of early pathways profoundly inhibits neurodegeneration in a mouse model of glaucoma. Neurobiol. Dis. 2014;71:44–52. doi:10.1016/j.nbd.2014.07.016
14. Rosenthal R, Fromm M. Endothelin antagonism as an active principle for glaucoma therapy. Br. J. Pharmacol. 2011;162(4):806–816. doi:10.1111/j.1476-5381.2010.01103.x
15. Mozaffarieh M, Grieshaber MC, Orgül S, Flammer J. The Potential Value of Natural Antioxidative Treatment in Glaucoma. Surv. Ophthalmol. 2016;53(5):479–505. doi:10.1016/j.survophthal.2008.06.006
16. Cheung ZH, So K-F, Lu Q, Yip HK, Wu W, Shan JJ, Pang PKT, Chen C-F. Enhanced Survival and Regeneration of Axotomized Retinal Ganglion Cells by a Mixture of Herbal Extracts. J. Neurotrauma. 2002;19(3):369–378. doi:10.1089/089771502753594936
17. Kim S-Y, Kwak J-S, Shin J-P, Lee S-H. The Protection of the Retina from Ischemic Injury by the Free Radical Scavenger EGb 761 and Zinc in the Cat Retina. Ophthalmologica. 1998;212(4):268–274.
18. Gregory-Ksander MS, Fei F, Marshak-Rothstein A, Ksander B, Krishnan A. Soluble Fas ligand provides long-term protection in a chronic mouse model of glaucoma by inhibiting glial activation, inflammation, and apoptosis. Invest. Ophthalmol. Vis. Sci. 2016;57(12).
19. Pease ME, Zack DJ, Berlinicke C, Bloom K, Cone F, Wang Y, Klein RL, Hauswirth WW, Quigley HA. Effect of CNTF on retinal ganglion cell survival in experimental glaucoma. Invest. Ophthalmol. Vis. Sci. 2009;50(5):2194–200. doi:10.1167/iovs.08-3013
20. Park YH, Snook JD, Ostrin EJ, Kim S, Chen R, Frankfort BJ. Transcriptomic profiles of retinal ganglion cells are defined by the magnitude of intraocular pressure elevation in adult mice. Sci. Rep. 2019;9(1):1–12. doi:10.1038/s41598-019-39141-1
21. Crowston JG, Kong YXG, Trounce IA, Dang TM, Fahy ET, Bui B V., Morrison JC, Chrysostomou V. An acute intraocular pressure challenge to assess retinal ganglion cell injury and recovery in the mouse. Exp. Eye Res. 2014;141:3–8. doi:10.1016/j.exer.2015.03.006
22. Woldemussie E, Ruiz G, Wijono M, Wheeler LA. Neuroprotection of Retinal Ganglion Cells by Brimonidine in Rats with Laser-Induced Chronic Ocular Hypertension. Invest. Ophthalmol. Vis. Sci. 2001;42(12):2849–2855.
23. Gao H, Qiao X, LB C, WuDunn D. Upregulation of brain-derived neurotrophic factor expression by brimonidine in rat retinal ganglion cells. Arch. Ophthalmol. 2002;120(6):797–803.
24. Feke GT, Bex PJ, Taylor CP, Rhee DJ, Turalba A V, Chen TC, Wand M, Pasquale LR. Effect of Brimonidine on Retinal Vascular Autoregulation and Short-term Visual Function in Normal Tension Glaucoma. Am. J. Ophthalmol. 2014;158(1):105–112.e1. doi:10.1016/j.ajo.2014.03.015
25. Wheeler LA, Lai R, WoldeMussie E. From the Lab to the Clinic: Activation of an Alpha-2 Agonist Pathway is Neuroprotective in Models of Retinal and Optic Nerve Injury. Eur. J. Ophthalmol. 9(Suppl. 1):17. doi:10.5301/EJO.2008.3854
26. Dong CJ, Guo Y, Agey P, Wheeler L, Hare WA. Alpha2 adrenergic modulation of NMDA receptor function as a major mechanism of RGC protection in experimental glaucoma and retinal excitotoxicity. Invest. Ophthalmol. Vis. Sci. 2008;49(10):4515–4522. doi:10.1167/iovs.08-2078
27. Kent AR, Nussdorf JD, David R, Tyson F, Small D, Fellows D. Vitreous concentration of topically applied brimonidine tartrate 0.2%. Ophthalmology. 2001;108(4):784–787. doi:10.1016/S0161-6420(00)00654-0
28. Burke J, Schwartz M. Preclinical evaluation of brimonidine. Surv. Ophthalmol. 1996;41:S9–S18. doi:http://dx.doi.org/10.1016/S0039-6257(96)82027-3
29. Krupin T, Liebmann JM, Greenfield DS, Ritch R, Gardiner S. A randomized trial of brimonidine versus timolol in preserving visual function: Results from the low-pressure glaucoma treatment study. Am. J. Ophthalmol. 2011;151(4):671–681. doi:10.1016/j.ajo.2010.09.026
30. Comparison of glaucomatous progression between untreated patients with normal-tension glaucoma and patients with therapeutically reduced intraocular pressures. Collaborative Normal-Tension Glaucoma Study Group. Am. J. Ophthalmol. 1998;126(4):487—497.
31. Johnson T V., Bull ND, Hunt DP, Marina N, Tomarev SI, Martin KR. Neuroprotective effects of intravitreal mesenchymal stem cell transplantation in experimental glaucoma. Invest. Ophthalmol. Vis. Sci. 2010;51(4):2051–2059. doi:10.1167/iovs.09-4509
32. Emre E, Yüksel N, Duruksu G, Pirhan D, Subaşi C, Erman G, Karaöz E. Neuroprotective effects of intravitreally transplanted adipose tissue and bone marrow–derived mesenchymal stem cells in an experimental ocular hypertension model. Cytotherapy. 2015;17(5):543–559. doi:10.1016/j.jcyt.2014.12.005
33. Yu S, Tanabe T, Dezawa M, Ishikawa H, Yoshimura N. Effects of bone marrow stromal cell injection in an experimental glaucoma model. Biochem. Biophys. Res. Commun. 2006;344(4):1071–1079. doi:10.1016/j.bbrc.2006.03.231
34. Johnson T V., Dekorver NW, Levasseur VA, Osborne A, Tassoni A, Lorber B, Heller JP, Villasmil R, Bull ND, Martin KR, et al. Identification of retinal ganglion cell neuroprotection conferred by platelet-derived growth factor through analysis of the mesenchymal stem cell secretome. Brain. 2014;137(2):503–519. doi:10.1093/brain/awt292
35. Osborne A, Sanderson J, Martin KR. Neuroprotective Effects of Human Mesenchymal Stem Cells and Platelet-Derived Growth Factor on Human Retinal Ganglion Cells. Stem Cells. 2018;36(1):65–78. doi:10.1002/stem.2722
36. Tassoni A, Gutteridge A, Barber AC, Osborne A, Martin KR. Molecular Mechanisms Mediating Retinal Reactive Stem Cell Transplantation. Stem Cells. 2015;33:3006–3016. doi:10.1002/stem.2095
37. Tzameret A, Sher I, Belkin M, Treves AJ, Meir A, Nagler A, Levkovitch-Verbin H, Barshack I, Rosner M, Rotenstreich Y. Transplantation of human bone marrow mesenchymal stem cells as a thin subretinal layer ameliorates retinal degeneration in a rat model of retinal dystrophy. Exp. Eye Res. 2014;118:135–144. doi:10.1016/j.exer.2013.10.023
38. Kim JY, You YS, Kim SH, Kwon OW. Epiretinal membrane formation after intravitreal autologous stem cell implantation in a retinitis pigmentosa patient. Retin. Cases Brief Rep. 2016. doi:10.1097/ICB.0000000000000327
39. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating  a, Prockop D, Horwitz E. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 2006;8(4):315–7. doi:10.1080/14653240600855905
40. Kuriyan AE, Albini TA, Townsend JH, Rodriguez M, Pandya HK, Leonard RE, Parrott MB, Rosenfeld PJ, Flynn HW, Goldberg JL. Vision Loss after Intravitreal Injection of Autologous “Stem Cells” for AMD. N. Engl. J. Med. 2017;376(11):1047–1053. doi:10.1056/NEJMoa1609583
41. Chew EY, Clemons TE, Jaffe GJ, Johnson CA, Farsiu S, Lad EM, Guymer R, Rosenfeld P, Hubschman JP, Constable I, et al. Effect of Ciliary Neurotrophic Factor on Retinal Neurodegeneration in Patients with Macular Telangiectasia Type 2: A Randomized Clinical Trial. Ophthalmology. 2019;126(4):540–549. doi:10.1016/j.ophtha.2018.09.041
42. Lambiase A, Rama P, Bonini S, Caprioglio G, Aloe L. Topical Treatment with Nerve Growth Factor for Corneal Neurotrophic Ulcers. N. Engl. J. Med. 1998;338(17):1174–1180. doi:10.1056/NEJM199804233381702
43. Hare WA, WoldeMussie E, Lai RK, Ton H, Ruiz G, Chun T, Wheeler L. Efficacy and Safety of Memantine Treatment for Reduction of Changes Associated with Experimental Glaucoma in Monkey, I: Functional Measures. Invest. Ophthalmol. Vis. Sci. 2004;45(8):2625. doi:10.1167/iovs.03-0566
44. Kouassi Nzoughet J, Chao de la Barca JM, Guehlouz K, Leruez S, Coulbault L, Allouche S, Bocca C, Muller J, Amati-Bonneau P, Gohier P, et al. Nicotinamide Deficiency in Primary Open-Angle GlaucomaNicotinamide Deficiency in Glaucoma. Invest. Ophthalmol. Vis. Sci. 2019;60(7):2509–2514. doi:10.1167/iovs.19-27099
45. Williams PA, Harder JM, Foxworth NE, Cochran KE, Philip VM, Porciatti V, Smithies O, John SWM. Vitamin B3 modulates mitochondrial vulnerability and prevents glaucoma in aged mice. Science. 2017;760(February):756–760. doi:10.1120/science.aal0092
46. Williams PA, Harder JM, Cardozo BH, Foxworth NE, John SWM. Nicotinamide treatment robustly protects from inherited mouse glaucoma. Commun. Integr. Biol. 2018;11(1). doi:10.1080/19420889.2017.1356956
47. Williams PA, Harder JM, John SWM. Glaucoma as a Metabolic Optic Neuropathy: Making the Case for Nicotinamide Treatment in Glaucoma. J. Glaucoma. 2017;26(12):1161–1168. doi:10.1097/IJG.0000000000000767 
48. Chen AC, Martin AJ, Choy B, Fernández-Peñas P, Dalziell RA, McKenzie CA, Scolyer RA, Dhillon HM, Vardy JL, Kricker A, et al. A Phase 3 Randomized Trial of Nicotinamide for Skin-Cancer Chemoprevention. N. Engl. J. Med. 2015;373(17):1618–1626. doi:10.1056/NEJMoa1506197
49. Osborne NN, Lascaratos G, Bron AJ, Chidlow G, Wood JPM. A hypothesis to suggest that light is a risk factor in glaucoma and the mitochondrial optic neuropathies. Br. J.  Ophthalmol. 2006;90(2):237–241. doi:10.1136/bjo.2005.082230
50. Jain A, Zode G, Kasetti RB, Ran FA, Yan W, Sharma TP, Bugge K, Searby CC, Fingert JH, Zhang F, et al. CRISPR-Cas9–based treatment of myocilin-associated glaucoma. Proc. Natl. Acad. Sci. 2017;114(42):11199–11204. doi:10.1073/pnas.1706193114
51. Thomson BR, Heinen S, Jeansson M, Ghosh AK, Fatima A, Sung H-K, Onay T, Chen H, Yamaguchi S, Economides AN, et al. A lymphatic defect causes ocular hypertension and glaucoma in mice. J. Clin. Invest. 2014;124(10):4320–4324. doi:10.1172/JCI77162
52. Souma T, Tompson SW, Thomson BR, Siggs OM, Kizhatil K, Yamaguchi S, Feng L, Limviphuvadh V, Whisenhunt KN, Maurer-Stroh S et al. Angiopoietin receptor TEK mutations underlie primary congenital glaucoma with variable expressivity. J Clin Invest. 2016;126(7):2575–2587. doi:10.1172/JCI85830
53. Osborne A, Wang AXZ, Tassoni A, Widdowson PS, Martin KR. Design of a Novel Gene Therapy Construct to Achieve Sustained Brain-Derived Neurotrophic Factor Signaling in Neurons. Hum. Gene Ther. 2018;29(7):828–841. doi:10.1089/hum.2017.069
54. Osborne A, Khatib TZ, Songra L, Barber AC, Hall K, Kong GYX, Widdowson PS, Martin KR. Neuroprotection of retinal ganglion cells by a novel gene therapy construct that achieves sustained enhancement of brain-derived neurotrophic factor/tropomyosin-related kinase receptor-B signaling. Cell Death Dis. 2018;9(10). doi:10.1038/s41419-018-1041-8
55. Quigley HA, McKinnon SJ, Zack DJ, Pease ME, Kerrigan-Baumrind LA, Kerrigan DF, Mitchell RS. Retrograde axonal transport of BDNF in retinal ganglion cells is blocked by acute IOP elevation in rats. Invest. Ophthalmol. Vis. Sci. 2000;41(11):3460–3466.
56. Russell S, Bennett J, Wellman JA, Chung DC, Yu Z-F, Tillman A, Wittes J, Pappas J, Elci O, McCague S, et al. Efficacy and safety of voretigene neparvovec (AAV2-hRPE65v2) in patients with <em>RPE65</em>-mediated inherited retinal dystrophy: a randomised, controlled, open-label, phase 3 trial. Lancet. 2017;390(10097):849–860. doi:10.1016/S0140-6736(17)31868-8
57. Mendell JR, Al-Zaidy S, Shell R, Arnold WD, Rodino-Klapac LR, Prior TW, Lowes L, Alfano L, Berry K, Church K, et al. Single-Dose Gene-Replacement Therapy for Spinal Muscular Atrophy. N. Engl. J. Med. 2017;377(18):1713–1722. doi:10.1056/NEJMoa1706198
58. Hirsch T, Rothoeft T, Teig N, Bauer JW, Rosa L De, Scaglione D, Reichelt J, Klausegger A, Kneisz D, Romano O, et al. Regeneration of the entire human epidermis by transgenic stem cells. Nature 2017;551(7680):327-332. doi: 10.1038/nature24487
59. Bainbridge JWB, Mehat MS, Sundaram V, Robbie SJ, Barker SE, Ripamonti C, Georgiadis A, Mowat FM, Beattie SG, Gardner PJ, et al. Long-Term Effect of Gene Therapy on Leber’s Congenital Amaurosis. N. Engl. J. Med. 2015;372(20):1887–1897. doi:10.1056/NEJMoa1414221
60. Georgiadis A, Duran Y, Ribeiro J, Abelleira-Hervas L, Robbie SJ, Sünkel-Laing B, Fourali S, Gonzalez-Cordero A, Cristante E, Michaelides M, et al. Development of an optimized AAV2/5 gene therapy vector for Leber congenital amaurosis owing to defects in RPE65. Gene Ther. 2016;23:857.
61. Xue K, Jolly JK, Barnard AR, Rudenko A, Salvetti AP, Patrício MI, Edwards TL, Groppe M, Orlans HO, Tolmachova T, et al. Beneficial effects on vision in patients undergoing retinal gene therapy for choroideremia. Nat. Med. 2018;24(10):1507–1512. doi:10.1038/s41591-018-0185-5
62. Vignal C, Uretsky S, Fitoussi S, Galy A, Blouin L, Girmens J-F, Bidot S, Thomasson N, Bouquet C, Valero S, et al. Safety of rAAV2/2- ND4 Gene Therapy for Leber Hereditary Optic Neuropathy. Ophthalmology. 2018;125(6):945–947. doi:10.1016/j.ophtha.2017.12.036
63. Wan X, Pei H, Zhao M, Yang S, Hu W, He H, Ma S, Zhang G, Dong X, Chen C, et al. Efficacy and Safety of rAAV2-ND4 Treatment for Leber’s Hereditary Optic Neuropathy. Sci. Rep. 2016;6:21587. doi:10.1038/srep21587
64. Moster ML, Sadun A, Klopstock T, Vignal-Clermont C, Newman NJ, Carelli V, Yu-Wai-Man P, Sahel J-A, Katz B. rAAV2/2-ND4 for the Treatment of LHON: 72-week Data from the REVERSE Phase III Clinical Trial (Plen02.002). Neurology. 2019;92(15 Supplement):Plen02.002.
65. Bouquet C, Vignal Clermont C, Galy A, Fitoussi S, Blouin L, Munk MR, Valero S, Meunier S, Katz B, Sahel JA, et al. Immune Response and Intraocular Inflammation in Patients With Leber Hereditary Optic Neuropathy Treated With Intravitreal Injection of Recombinant Adeno-Associated Virus 2 Carrying the ND4 Gene: A Secondary Analysis of a Phase 1/2 Clinical Trial Outcomes JAMA Ophthalmol. 2019;137(4):399–406. doi:10.1001/jamaophthalmol.2018.6902
66. Cukras C, Wiley HE, Jeffrey BG, Sen HN, Turriff A, Zeng Y, Vijayasarathy C, Marangoni D, Ziccardi L, Kjellstrom S, et al. Retinal AAV8-<em>RS1</em> Gene Therapy for X-Linked Retinoschisis: Initial Findings from a Phase I/IIa Trial by Intravitreal Delivery. Mol. Ther. 2018;26(9):2282–2294. doi:10.1016/j.ymthe.2018.05.025
67. Rogers GL, Herzog RW. TLR9 and Dendritic Cells Are Required for CD8+ T Cell Responses to the AAV Capsid. Blood. 2014;124(21):552 LP-552.
68. Rogers GL, Suzuki M, Zolotukhin I, Markusic DM, Morel LM, Lee B, Ertl HCJ, Herzog RW. Unique Roles of TLR9- and MyD88-Dependent and -Independent Pathways in Adaptive Immune Responses to AAV-Mediated Gene Transfer. Journal Innate Immun. 2015;7(3):302–314. doi:10.1159/000369273
69. Hösel M, Broxtermann M, Janicki H, Esser K, Arzberger S, Hartmann P, Gillen S, Kleeff J, Stabenow D, Odenthal M, et al. Toll-like receptor 2–mediated innate immune response in human nonparenchymal liver cells toward adeno-associated viral vectors. Hepatology. 2012;55(1):287–297. doi:10.1002/hep.24625
70. Zhu J, Huang X, Yang Y. The TLR9-MyD88 pathway is critical for adaptive immune responses to adeno-associated virus gene therapy vectors in mice. J. Clin. Invest. 2009;119(8):2388–2398. doi:10.1172/JCI37607
71. Martino AT, Suzuki M, Markusic DM, Zolotukhin I, Ryals RC, Moghimi B, Ertl HCJ, Muruve DA, Lee B, Herzog RW. The genome of self-complementary adeno-associated viral vectors increases Toll-like receptor 9-dependent innate immune responses in the liver. Blood. 2011;117(24):6459–6468. doi:10.1182/blood-2010-10-314518
72. Bartel M, Schaffer D, Büning H. Enhancing the Clinical Potential of AAV Vectors by Capsid Engineering to Evade Pre-Existing Immunity. Front. Microbiol. 2011;2:204. doi:10.3389/fmicb.2011.00204
73. Carvalho LS, György B, Mu D, Shah S, Vandenberghe LH, Maguire CA. 152. Retinal Tropism of Exosome-Associated AAV Vector Via Intravitreal Delivery. Mol. Ther. 2015;23:S61. doi:10.1016/S1525-0016(16)33757-1
74. György B, Wassmer S, Carvalho L, Maguire C, Vandenberghe LH. 594. Exosome-Associated AAV Enhances Retinal Transduction Following Intravitreal Injection. Mol. Ther. 2016;24:S235. doi:10.1016/S1525-0016(16)33402-5
75. Wassmer SJ, Carvalho LS, György B, Vandenberghe LH, Maguire CA. Exosome-associated AAV2 vector mediates robust gene delivery into the murine retina upon intravitreal injection. Sci. Rep. 2017;7:45329.
76. De Moraes CG, Liebmann JM, Levin LA. Detection and measurement of clinically meaningful visual field progression in clinical trials for glaucoma. Prog. Retin. Eye Res. 2017;56:107–147. doi:10.1016/j.preteyeres.2016.10.001
77. De Moraes CG, Hood DC, Thenappan A, Girkin CA, Medeiros FA, Weinreb RN, Zangwill LM, Liebmann JM. 24-2 Visual Fields Miss Central Defects Shown on 10-2 Tests in Glaucoma Suspects, Ocular Hypertensives, and Early Glaucoma. Ophthalmology. 2017;124(10):1449–1456. doi:10.1016/j.ophtha.2017.04.021
78. Blumberg DM, De Moraes CG, Prager AJ, Yu Q, Al-Aswad L, Cioffi GA, Liebmann JM, Hood DC. Association Between Undetected 10-2 Visual Field Damage and Vision-Related Quality of Life in Patients With Glaucoma. JAMA Ophthalmol. 2017;135(7):742–747. doi:10.1001/jamaophthalmol.2017.1396
79. Wu Z, Medeiros FA, Weinreb RN, Girkin CA, Zangwill LM. Comparing 10-2 and 24-2 Visual Fields for Detecting Progressive Central Visual Loss in Glaucoma Eyes with Early Central Abnormalities. Ophthalmol. Glaucoma. 2019;2(2):95–102. doi:https://doi.org/10.1016/j.ogla.2019.01.003
80. Wu Z, Crabb DP, Chauhan BC, Crowston JG, Medeiros FA. Improving the Feasibility of Glaucoma Clinical Trials Using Trend-Based Visual Field Progression End Points. Ophthalmol. Glaucoma. 2019;2(2):72–77. doi:10.1016/j.ogla.2019.01.004
81. Quigley HA. Glaucoma Neuroprotection Trials Are Practical Using Visual Field Outcomes. Ophthalmol. Glaucoma. 2019;2(2):69–71. doi:10.1016/j.ogla.2019.01.009
82. Hood DC, Raza AS. Method for comparing visual field defects to local RNFL and RGC damage seen on frequency domain OCT in patients with glaucoma. Biomed. Opt. Express 2011;2(5):1097–1105. doi:10.1364/BOE.2.001097
83. Hood DC, Raza AS. On improving the use of OCT imaging for detecting glaucomatous damage. Br. J. Ophthalmol. 2014;98 Suppl 2(Suppl 2):ii1-ii9. doi:10.1136/bjophthalmol-2014-305156
84. Cordeiro MF, Normando EM, Cardoso MJ, Miodragovic S, Jeylani S, Davis BM, Guo L, Ourselin S, A’Hern R, Bloom PA. Real-time imaging of single neuronal cell apoptosis in patients with glaucoma. Brain. 2017;140(6):1757–1767. doi:10.1093/brain/awx088
85. Ou Y, Jo RE, Ullian EM, Wong ROL, Della Santina L. Selective Vulnerability of Specific Retinal Ganglion Cell Types and Synapses after Transient Ocular Hypertension. J. Neurosci. 2016;36(35):9240–9252. doi:10.1523/jneurosci.0940-16.2016
86. El-Danaf RN, Huberman AD. Characteristic Patterns of Dendritic Remodeling in Early-Stage Glaucoma: Evidence from Genetically Identified Retinal Ganglion Cell Types. J. Neurosci. 2015;35(6):2329–2343. doi:10.1523/jneurosci.1419-14.2015
87. Williams PA, Howell GR, Barbay JM, Braine CE, Sousa GL, John SWM, Morgan JE. Retinal ganglion cell dendritic atrophy in DBA/2J glaucoma. PLoS ONE. 2013;8(8):1–10. doi:10.1371/journal.pone.0072282
88. Weber AJ, Harman CD. Structure-function relations of parasol cells in the normal and glaucomatous primate retina. Invest. Ophthalmol. Vis. Sci. 2005;46(9):3197–3207. doi:10.1167/iovs.04-0834
89. Fahy ET, Chrysostomou V, Abbott CJ, van Wijngaarden P, Crowston JG. Axonal transport along retinal ganglion cells is grossly intact during reduced function post-injury. Exp. Eye Res. 2016;146:289–292. doi:10.1016/j.exer.2016.03.001
90. Lim J-HA, Stafford BK, Nguyen PL, Lien B V, Wang C, Zukor K, He Z, Huberman AD. Neural activity promotes long-distance, target-specific regeneration of adult retinal axons. Nat. Neurosci. 2016;19(July):1–14. doi:10.1038/nn.4340
91. Howell GR, Libby RT, Jakobs TC, Smith RS, Phalan FC, Barter JW, Barbay JM, Marchant JK, Mahesh N, Porciatti V, et al. Axons of retinal ganglion cells are insulted in the optic nerve early in DBA/2J glaucoma. J. Cell Biol. 2007;179(7):1523 LP-1537. doi:10.1083/jcb.200706181
92. Beirowski B, Babetto E, Coleman MP, Martin KR. The WldS gene delays axonal but not somatic degeneration in a rat glaucoma model. Eur. J. Neurosci. 2008;28(6):1166–1179. doi:10.1111/j.1460-9568.2008.06426.x
93. Caprioli J, De Leon JM, Azarbod P, Chen A, Morales E, Nouri-Mahdavi K, Coleman A, Yu F, Afifi A. Trabeculectomy Can Improve Long-Term Visual Function in Glaucoma. Ophthalmology. 2016;123(1):117–128. doi:10.1016/j.ophtha.2015.09.027
94. Casson RJ, Han G, Ebneter A, Chidlow G, Glihotra J, Newland H, Wood JPM. Glucose-induced temporary visual recovery in primary open-angle glaucoma: A double-blind, randomized study. Ophthalmology. 2014;121(6):1203–1211. doi:10.1016/j.ophtha.2013.12.011
95. Kong YXG, van Bergen N, Bui B V., Chrysostomou V, Vingrys AJ, Trounce IA, Crowston JG. Impact of aging and diet restriction on retinal function during and after acute intraocular pressure injury. Neurobiol. Aging. 2012;33(6):1126.e15-1126.e25. doi:10.1016/J.NEUROBIOLAGING.2011.11.026
96. Chrysostomou V, Kezic JM, Trounce IA, Crowston JG. Forced exercise protects the aged optic nerve against intraocular pressure injury. Neurobiol. of Aging. 2014;35(7):1722–1725. doi:10.1016/j.neurobiolaging.2014.01.019
97. Ratican SE, Osborne A, Martin KR. Progress in Gene Therapy to Prevent Retinal Ganglion Cell Loss in Glaucoma and Leber’s Hereditary Optic Neuropathy. Neural Plast. 2018;2018:1–11. doi:10.1155/2018/7108948
98. Lee JH, Wang J-H, Chen J, Li F, Edwards TL, Hewitt AW, Liu G-S. Gene therapy for visual loss: Opportunities and concerns. Prog. Retin. Eye Res. 2019;68:31–53. doi:https://doi.org/10.1016/j.preteyeres.2018.08.003
99. Sioshansi S, Huber KE, Wazer DE. The Implications of Breast Cancer Molecular Phenotype for Radiation Oncology. Front. Oncol. 2011;1(June):1–9. doi:10.3389/fonc.2011.00012
100. Cappelletti V, Iorio E, Miodini P, Silvestri M, Dugo M, Daidone MG. Metabolic Footprints and Molecular Subtypes in Breast Cancer. Dis. Markers. 2017;2017:1–19. doi:10.1155/2017/7687851
101. Dai X, Xiang L, Li T, Bai Z. Cancer hallmarks, biomarkers and breast cancer molecular subtypes. J. Cancer. 2016;7(10):1281–1294. doi:10.7150/jca.13141
102. Bycroft C, Freeman C, Petkova D, Band G, Elliott LT, Sharp K, Motyer A, Vukcevic D, Delaneau O, O’Connell J, et al. The UK Biobank resource with deep phenotyping and genomic data. Nature. 2018;562(7726):203–209. doi:10.1038/s41586-018-0579-z
103. Chua SYL, Thomas D, Allen N, Lotery A, Desai P, Patel P, Muthy Z, Sudlow C, Peto T, Khaw PT, et al. Cohort profile: Design and methods in the eye and vision consortium of UK Biobank. BMJ Open. 2019;9(2):1–13. doi:10.1136/bmjopen-2018-025077
104. Ollier W, Sprosen T, Peakman T. UK Biobank: from concept to reality. Pharmacogenomics. 2005;6(6):639–646. doi:10.2217/14622416.6.6.639
105. Chan MPY, Grossi CM, Khawaja AP, Yip JLY, Khaw KT, Patel PJ, Khaw PT, Morgan JE, Vernon SA, Foster PJ. Associations with intraocular pressure in a large cohort: Results from the UK Biobank. Ophthalmology. 2016;123(4):771–782. doi:10.1016/j.ophtha.2015.11.031
106. Gao XR, Huang H, Kim H. Polygenic Risk Score Is Associated With Intraocular Pressure and Improves Glaucoma Prediction in the UK Biobank Cohort. Transl. Vis. Sci.  Technol. 2019;8(2):10. doi:10.1167/tvst.8.2.10