Retinal Degeneration: From Pathophysiology to Therapeutic Approaches

Retinitis pigmentosa (RP) is a generic term for a group of genetic diseases characterized by loss of rod and cone photoreceptor cells. Although the genetic causes of RP frequently only affect the rod photoreceptor cells, cone photoreceptors become stressed in the absence of rods and undergo a secondary degeneration. Changes in the gene expression profile of cone photoreceptor cells are likely to occur prior to observable physiological changes. To this end, we sought to achieve greater understanding of the changes in cone photoreceptor cells early in the degeneration process of the Rho^−/− mouse model. To account for gene expression changes attributed to loss of cone photoreceptor cells, we normalized PCR in the remaining number of cones to a cone cell reporter (OPN1-GFP). Gene expression profiles of key components involved in the cone phototransduction cascade were correlated with tests of retinal cone function prior to cell loss. A significant downregulation of the photoreceptor transcription factor Crx was observed, which preceded a significant downregulation in cone opsin transcripts that coincided with declining cone function. Our data add to the growing understanding of molecular changes that occur prior to cone dysfunction in a model of rod-cone dystrophy. It is of interest that gene supplementation of CRX by adeno-associated viral vector delivery prior to cone cell loss did not prevent cone photoreceptor degeneration in this mouse model. Full article

Age-related macular degeneration (AMD) is a leading cause for visual impairment in aging populations with limited established therapeutic interventions available. Oxidative stress plays an essential role in the pathogenesis of AMD, damaging the retinal pigment epithelium (RPE), which is essential for the function and maintenance of the light-sensing photoreceptors. This study aimed to evaluate the effects of crocetin, one of the main components of Saffron, on an in vitro RPE model of tert-butyl hydroperoxide (TBHP) induced oxidative stress using ARPE19 cells. The effects of crocetin were assessed using lactate de-hydrogenase (LDH) and ATP assays, as well as immunocytochemistry for cell morphology, junctional integrity, and nuclear morphology. The mechanism of crocetin action was determined via assessment of energy production pathways, including mitochondrial respiration and glycolysis in real-time as well as investigation of extracellular signal-regulated kinase 1/2 (ERK1/2) activation and distribution. Our results show that crocetin pre-treatment protects ARPE19 cells from TBHP-induced LDH release, intracellular ATP depletion, nuclear condensation, and disturbance of junctional integrity and cytoskeleton. The protective effect of crocetin is mediated via the preservation of energy production pathways and activation of ERK1/2 in the first minutes of TBHP exposure to potentiate survival pathways. The combined data suggest that a natural antioxidant, such as crocetin, represents a promising candidate to prevent oxidative stress in RPE cells and might halt or delay disease progression in AMD. Full article

Retinitis pigmentosa (RP) is a category of inherited retinal dystrophies that is best prognosticated using electroretinography (ERG). In this retrospective cohort study of 25 patients with RP, we evaluated the correlation between 30 Hz flicker ERG and structural parameters in the retina. Internationally standardized 30 Hz flicker ERG recordings, short-wavelength autofluorescence (SW-AF), and spectral domain–optical coherence tomography (SD-OCT) were acquired at two visits at least one year apart. Vertical and horizontal hyperautofluorescent ring diameter measurements with SW-AF, as well as ellipsoid zone (EZ) line width measurements with SD-OCT, were used as structural parameters of disease progression. The 30 Hz flicker ERG amplitude decreased by 2.2 ± 0.8 µV/year (p = 0.011), while implicit times remained unchanged. For SD-OCT, the EZ line decreased by 204.1 ± 34.7 µm/year (p < 0.001). Horizontal and vertical hyperautofluorescent ring diameters decreased by 161.9 ± 25.6 µm/year and 146.9 ± 34.6 µm/year, respectively (p = 0.001), with SW-AF. A correlation was found between the progression rates of the 30 Hz flicker amplitude recorded with Burian–Allen electrodes and both the horizontal ring diameter (p = 0.020) and EZ line (p = 0.044). SW-AF and SD-OCT, two readily available imaging techniques, may be used to prognosticate disease progression because of the reliability of their measurements and correlation with functional outcome. Full article

The nervous system demands an adequate oxygen and metabolite exchange, making pericytes (PCs), the only vasoactive cells on the capillaries, essential to neural function. Loss of PCs is a hallmark of multiple diseases, including diabetes, Alzheimer’s, amyotrophic lateral sclerosis (ALS) and Parkinson’s. Platelet-derived growth factor receptors (PDGFRs) have been shown to be critical to PC function and survival. However, how PDGFR-mediated PC activity affects vascular homeostasis is not fully understood. Here, we tested the hypothesis that imatinib, a chemotherapeutic agent and a potent PDGFR inhibitor, alters PC distribution and thus induces vascular atrophy. We performed a morphometric analysis of the vascular elements in sham control and imatinib-treated NG2-DsRed mice. Vascular morphology and the integrity of the blood–retina barrier (BRB) were evaluated using blood albumin labeling. We found that imatinib decreased the number of PCs and blood vessel (BV) coverage in all retinal vascular layers; this was accompanied by a shrinkage of BV diameters. Surprisingly, the total length of capillaries was not altered, suggesting a preferential effect of imatinib on PCs. Furthermore, blood–retina barrier disruption was not evident. In conclusion, our data suggest that imatinib could help in treating neurovascular diseases and serve as a model for PC loss, without BRB disruption. Full article

Best vitelliform macular dystrophy (BD), autosomal dominant vitreoretinochoroidopathy (ADVIRC), and the autosomal recessive bestrophinopathy (ARB), together known as the bestrophinopathies, are caused by mutations in the bestrophin-1 (BEST1) gene affecting anion transport through the plasma membrane of the retinal pigment epithelium (RPE). To date, while no treatment exists a better understanding of BEST1-related pathogenesis may help to define therapeutic targets. Here, we systematically characterize functional consequences of mutant BEST1 in thirteen RPE patient cell lines differentiated from human induced pluripotent stem cells (hiPSCs). Both BD and ARB hiPSC-RPEs display a strong reduction of BEST1-mediated anion transport function compared to control, while ADVIRC mutations trigger an increased anion permeability suggesting a stabilized open state condition of channel gating. Furthermore, BD and ARB hiPSC-RPEs differ by the degree of mutant protein turnover and by the site of subcellular protein quality control with adverse effects on lysosomal pH only in the BD-related cell lines. The latter finding is consistent with an altered processing of catalytic enzymes in the lysosomes. The present study provides a deeper insight into distinct molecular mechanisms of the three bestrophinopathies facilitating functional categorization of the more than 300 known BEST1 mutations that result into the distinct retinal phenotypes. Full article

Mutations in more than 200 retina-specific genes have been associated with inherited retinal diseases. Genome editing represents a promising emerging field in the treatment of monogenic disorders, as it aims to correct disease-causing mutations within the genome. Genome editing relies on highly specific endonucleases and the capacity of the cells to repair double-strand breaks (DSBs). As DSB pathways are cell-cycle dependent, their activity in postmitotic retinal neurons, with a focus on photoreceptors, needs to be assessed in order to develop therapeutic in vivo genome editing. Three DSB-repair pathways are found in mammalian cells: Non-homologous end joining (NHEJ); microhomology-mediated end joining (MMEJ); and homology-directed repair (HDR). While NHEJ can be used to knock out mutant alleles in dominant disorders, HDR and MMEJ are better suited for precise genome editing, or for replacing entire mutation hotspots in genomic regions. Here, we analyzed transcriptomic in vivo and in vitro data and revealed that HDR is indeed downregulated in postmitotic neurons, whereas MMEJ and NHEJ are active. Using single-cell RNA sequencing analysis, we characterized the dynamics of DSB repair pathways in the transition from dividing cells to postmitotic retinal cells. Time-course bulk RNA-seq data confirmed DSB repair gene expression in both in vivo and in vitro samples. Transcriptomic DSB repair pathway profiles are very similar in adult human, macaque, and mouse retinas, but not in ground squirrel retinas. Moreover, human-induced pluripotent stem-cell-derived neurons and retinal organoids can serve as well suited in vitro testbeds for developing genomic engineering approaches in photoreceptors. Our study provides additional support for designing precise in vivo genome-editing approaches via MMEJ, which is active in mature photoreceptors. Full article

Human induced pluripotent stem cells (hiPSCs) promise a great number of future applications to investigate retinal development, pathophysiology and cell therapies for retinal degenerative diseases. Specific approaches to genetically modulate hiPSC would be valuable for all of these applications. Vectors based on adeno-associated virus (AAV) have shown the ability for gene delivery to retinal organoids derived from hiPSCs. Thus far, little work has been carried out to investigate mechanisms of AAV-mediated gene delivery and the potential advantages of engineered AAVs to genetically modify retinal organoids. In this study, we compared the early transduction efficiency of several recombinant and engineered AAVs in hiPSC-derived RPE cells and retinal organoids in relation to the availability of their cell-surface receptors and as a function of time. The genetic variant AAV2-7m8 had a superior transduction efficiency when applied at day 44 of differentiation on retinal organoids and provided long-lasting expressions for at least 4 weeks after infection without compromising cell viability. All of the capsids we tested transduced the hiPSC-RPE cells, with the AAV2-7m8 variant being the most efficient. Transduction efficiency was correlated with the presence of primary cell-surface receptors on the hiPS-derived organoids. Our study explores some of the mechanisms of cell attachment of AAVs and reports long-term gene expression resulting from gene delivery in retinal organoids. Full article

Aquaporins (AQPs) are involved in hypoxia-induced angiogenesis and retinal damage. Bumetanide is a diuretic agent, Na⁺/K⁺/Cl⁻ cotransporter (NKCC1), and AQP 1–4 inhibitor. We tested the hypothesis that early postnatal treatment with bumetanide suppresses biomarkers of angiogenesis and decreases severe retinopathy oxygen-induced retinopathy (OIR). Neonatal rats were exposed at birth (P0) to either (1) room air (RA); (2) hyperoxia (50% O₂); or (3) intermittent hypoxia (IH) consisting of 50% O₂ with brief, clustered episodes of 12% O₂ from P0 to postnatal day 14 (P14), during which they were treated intraperitoneally (IP) with bumetanide (0.1 mg/kg/day) or an equivalent volume of saline, on P0–P2. Pups were examined at P14 or allowed to recover in RA from P14–P21. Retinal angiogenesis, morphometry, pathology, AQPs, and angiogenesis biomarkers were determined at P14 and P21. Bumetanide reduced vascular abnormalities associated with severe OIR. This was associated with reductions in AQP-4 and VEGF. Bumetanide suppressed sVEGFR-1 in the serum and vitreous fluid, but levels were increased in the ocular tissues during recovery. Similar responses were noted for IGF-I. In this model, early systemic bumetanide administration reduces severe OIR, the benefits of which appear to be mediated via suppression of AQP-4 and VEGF. Further studies are needed to determine whether bumetanide at the right doses may be considered a potential pharmacologic agent to treat retinal neovascularization. Full article

This study describes the clinical, genetic, and histopathological features in patients with RPGR-associated retinal dystrophies. Nine male patients from eight unrelated families underwent a comprehensive ophthalmic examination. Additionally, the histopathology of the right eye from a patient with an end-stage cone-rod-dystrophy (CRD)/sector retinitis pigmentosa (RP) phenotype was examined. All RPGR mutations causing a CRD phenotype were situated in exon ORF15. The mean best-corrected visual acuity (BCVA, decimals) was 0.58 (standard deviation (SD)): 0.34; range: 0.05–1.13); and the mean spherical refractive error was −4.1 D (SD: 2.11; range: −1.38 to −8.19). Hyperautofluorescent rings were observed in six patients. Full-field electroretinography responses were absent in all patients. The visual field defects ranged from peripheral constriction to central islands. The mean macular sensitivity on microperimetry was 11.6 dB (SD: 7.8; range: 1.6–24.4) and correlated significantly with BCVA (r = 0.907; p = 0.001). A histological examination of the donor eye showed disruption of retinal topology and stratification, with a more severe loss found in the peripheral regions. Reactive gliosis was seen in the inner layers of all regions. Our study demonstrates the highly variable phenotype found in RPGR-associated retinal dystrophies. Therapies should be applied at the earliest signs of photoreceptor degeneration, prior to the remodeling of the inner retina. Full article

Ocular inflammation contributes to the pathogenesis of blind-causing retinal degenerative diseases, such as age-related macular degeneration (AMD) or photic maculopathy. Here, we report on inflammatory mechanisms that are associated with retinal degeneration induced by bright visible light, which were revealed while using a rabbit model. Histologically and electrophysiologically noticeable degeneration of the retina is preceded and accompanied by oxidative stress and inflammation, as evidenced by granulocyte infiltration and edema in this tissue, as well as the upregulation of total protein, pro-inflammatory cytokines, and oxidative stress markers in aqueous humor (AH). Consistently, quantitative lipidomic studies of AH elucidated increase in the concentration of arachidonic (AA) and docosahexaenoic (DHA) acids and lyso-platelet activating factor (lyso-PAF), together with pronounced oxidative and inflammatory alterations in content of lipid mediators oxylipins. These alterations include long-term elevation of prostaglandins, which are synthesized from AA via cyclooxygenase-dependent pathways, as well as a short burst of linoleic acid derivatives that can be produced by both enzymatic and non-enzymatic free radical-dependent mechanisms. The upregulation of all oxylipins is inhibited by the premedication of the eyes while using mitochondria-targeted antioxidant SkQ1, whereas the accumulation of prostaglandins and lyso-PAF can be specifically suppressed by topical treatment with cyclooxygenase inhibitor Nepafenac. Interestingly, the most prominent antioxidant and anti-inflammatory benefits and overall retinal protective effects are achieved by simultaneous administrating of both drugs indicating their synergistic action. Taken together, these findings provide a rationale for using a combination of mitochondria-targeted antioxidant and cyclooxygenase inhibitor for the treatment of inflammatory components of retinal degenerative diseases. Full article

Biblical references aside, restoring vision to the blind has proven to be a major technical challenge. In recent years, considerable advances have been made towards this end, especially when retinal degeneration underlies the vision loss such as occurs with retinitis pigmentosa. Under these conditions, optogenetic therapies are a particularly promising line of inquiry where remaining retinal cells are made into “artificial photoreceptors”. However, this strategy is not without its challenges and a model system using human retinal explants would aid its continued development and refinement. Here, we cultured post-mortem human retinas and show that explants remain viable for around 7 days. Within this period, the cones lose their outer segments and thus their light sensitivity but remain electrophysiologically intact, displaying all the major ionic conductances one would expect for a vertebrate cone. We optogenetically restored light responses to these quiescent cones using a lentivirus vector constructed to express enhanced halorhodopsin under the control of the human arrestin promotor. In these ‘reactivated’ retinas, we show a light-induced horizontal cell to cone feedback signal in cones, indicating that transduced cones were able to transmit their light response across the synapse to horizontal cells, which generated a large enough response to send a signal back to the cones. Furthermore, we show ganglion cell light responses, suggesting the cultured explant’s condition is still good enough to support transmission of the transduced cone signal over the intermediate retinal layers to the final retinal output level. Together, these results show that cultured human retinas are an appropriate model system to test optogenetic vision restoration approaches and that cones which have lost their outer segment, a condition occurring during the early stages of retinitis pigmentosa, are appropriate targets for optogenetic vision restoration therapies. Full article

Gene therapy for adRP due to RHO mutations was recently shown to prevent photoreceptor death in a canine model of Class B disease. Among translational steps to be taken, one is to determine a method to detect efficacy in a human clinical trial. The relatively slow progression of adRP becomes a difficulty for clinical trials requiring an answer to whether there is slowed progression of degeneration in response to therapy. We performed a single-center, retrospective observational study of cross-sectional and longitudinal data. The study was prompted by our identification of a pericentral disease distribution in Class B RHO-adRP. Ultrawide optical coherence tomography (OCT) scans were used. Inferior retinal pericentral defects was an early disease feature. Degeneration further inferior in the retina merged with the pericentral defect, which extended into superior retina. In about 70% of patients, there was an asymmetric island of structure with significantly greater superior than inferior ellipsoid zone (EZ) extent. Serial measures of photoreceptor structure by OCT indicated constriction in superior retinal extent within a two-year interval. We conclude that these results should allow early-phase trials of therapy in RHO-adRP to move forward by inclusion of patients with an asymmetric extent of photoreceptor structure and by monitoring therapeutic effects over two years in the superior retina, a reasonable target for subretinal injection. Full article

Background: Age-related macular degeneration (AMD) is the leading cause of blindness and can be classified into two types called atrophic AMD (dry AMD) and neovascular AMD (wet AMD). Dry AMD is characterized by cellular degeneration of the retinal pigment epithelium, choriocapillaris, and photoreceptors. Wet AMD is characterized by the invasion of abnormal vessels from the choroid. Although anti-vascular endothelial growth factor (VEGF) therapy has a potent therapeutic effect against the disease, there is a possibility of chorio-retinal atrophy and adverse systemic events due to long-term robust VEGF antagonism. We focused on hypoxia-inducible factor (HIF) regulation of VEGF transcription, and report the suppressive effects of HIF inhibition against ocular phenotypes in animal models. Many of the known HIF inhibitors are categorized as anti-cancer drugs, and their systemic side effects are cause for concern in clinical use. In this study, we explored food ingredients that have HIF inhibitory effects and verified their effects in an animal model of AMD. Methods: Food ingredients were screened using a luciferase assay. C57BL6/J mice were administered the Garcinia cambogia extract (Garcinia extract) and hydroxycitric acid (HCA). Choroidal neovascularization (CNV) was induced by laser irradiation. Results: Garcinia extract and HCA showed inhibitory effects on HIF in the luciferase assay. The laser CNV model mice showed significant reduction of CNV volume by administering Garcinia extract and HCA. Conclusions: Garcinia extract and HCA showed therapeutic effects in a murine AMD model. Full article

Age-related macular degeneration (AMD) is one of the main causes of vision impairment in the elderly. Autophagy is the process of delivery of cytoplasmic components into lysosomes for cleavage; its age-related malfunction may contribute to AMD. Here we showed that the development of AMD-like retinopathy in OXYS rats is accompanied by retinal transcriptome changes affecting genes involved in autophagy. These genes are associated with kinase activity, immune processes, and FoxO, mTOR, PI3K-AKT, MAPK, AMPK, and neurotrophin pathways at preclinical and manifestation stages, as well as vesicle transport and processes in lysosomes at the progression stage. We demonstrated a reduced response to autophagy modulation (inhibition or induction) in the OXYS retina at age 16 months: expression of genes Atg5, Atg7, Becn1, Nbr1, Map1lc3b, p62, and Gabarapl1 differed between OXYS and Wistar (control) rats. The impaired reactivity of autophagy was confirmed by a decreased number of autophagosomes under the conditions of blocked autophagosome–lysosomal fusion according to immunohistochemical analysis and transmission electron microscopy. Thus, the development of AMD signs occurs against the background of changes in the expression of autophagy-related genes and a decrease in autophagy reactivity: the ability to enhance autophagic flux in response to stress. Full article

Νeuronal and glial cells play an important role in the development of vasculature in the retina. In this study, we investigated whether re-vascularization occurs in retinal neurodegenerative injury models. To induce retinal injury, N-methyl-D-aspartic acid (NMDA, 200 nmol) or kainic acid (KA, 20 nmol) was injected into the vitreous chamber of the eye on postnatal day (P)7. Morphological changes in retinal neurons and vasculature were assessed on P14, P21, and P35. Prevention of vascular growth and regression of some capillaries were observed on P14 in retinas of NMDA- and KA-treated eyes. However, vascular growth and re-vascularization started on P21, and the retinal vascular network was established by P35 in retinas with neurodegenerative injuries. The re-vascularization was suppressed by a two-day treatment with KRN633, an inhibitor of VEGF receptor tyrosine kinase, on P21 and P22. Astrocytes and Müller cells expressed vascular endothelial growth factor (VEGF), and the distribution pattern of VEGF was almost the same between the control and the NMDA-induced retinal neurodegenerative injury model, except for the difference in the thickness of the inner retinal layer. During re-vascularization, angiogenic sprouts from pre-existing blood vessels were present along the network of fibronectins formed by astrocytes. These results suggest that glial cells contribute to angiogenesis in neonatal rat models of retinal neurodegeneration. Full article

Variations in the Crumbs homolog-1 (CRB1) gene are associated with a wide variety of autosomal recessive retinal dystrophies, including early onset retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA). CRB1 belongs to the Crumbs family, which in mammals includes CRB2 and CRB3. Here, we studied the specific roles of CRB2 in rod photoreceptor cells and whether ablation of CRB2 in rods exacerbates the Crb1-disease. Therefore, we assessed the morphological, retinal, and visual functional consequences of specific ablation of CRB2 from rods with or without concomitant loss of CRB1. Our data demonstrated that loss of CRB2 in mature rods resulted in RP. The retina showed gliosis and disruption of the subapical region and adherens junctions at the outer limiting membrane. Rods were lost at the peripheral and central superior retina, while gross retinal lamination was preserved. Rod function as measured by electroretinography was impaired in adult mice. Additional loss of CRB1 exacerbated the retinal phenotype leading to an early reduction of the dark-adapted rod photoreceptor a-wave and reduced contrast sensitivity from 3-months-of-age, as measured by optokinetic tracking reflex (OKT) behavior testing. The data suggest that CRB2 present in rods is required to prevent photoreceptor degeneration and vision loss. Full article

To unravel the mechanisms behind the higher resistance to light damage of juvenile (JR) versus adult (AR) rats, Sprague Dawley rats were exposed to a bright luminous environment of 10, 000 lux. The light-induced retinopathy (LIR) was assessed with histology, electroretinography and immunohistochemistry (IHC). In JR, 2 days of exposure induced the typical LIR, while >3 days added little LIR. IHC revealed a subtle migration of microglia (Iba1 marker) from the inner to the outer retina after 3 days of exposure in JR contrasting with the stronger reaction seen after 1 day in AR. Similarly, in JR, the Müller cells expressed less intense GFAP, CNTF and FGF2 staining compared to AR. Our results suggest that in JR the degree of retinal damage is not proportional to the duration of light exposure (i.e., dose-independent retinopathy), contrasting with the dose-dependent LIR reported in AR. The immature immune system in JR may explain the delayed and/or weaker inflammatory response compared to AR, a finding that would also point to the devastating contribution of the immune system in generating the LIR phenotype, a claim also advanced to explain the pathophysiology of other retinal degenerative disorders such as Age-related Macular Degeneration, Diabetic Retinopathy and Retinitis Pigmentosa. Full article

Recessively-inherited NR2E3 gene mutations cause an unusual retinopathy with abnormally-increased short-wavelength sensitive cone (S-cone) function, in addition to reduced rod and long/middle-wavelength sensitive cone (L/M-cone) function. Progress toward clinical trials to treat patients with this otherwise incurable retinal degeneration prompted the need to determine efficacy outcome measures. Comparisons were made between three computerized perimeters available in the clinic. These perimeters could deliver short-wavelength stimuli on longer-wavelength adapting backgrounds to measure whether S-cone vision can be quantified. Results from a cohort of normal subjects were compared across the three perimeters to determine S-cone isolation and test-retest variability. S-cone perimetry data from NR2E3-ESCS (enhanced S-cone syndrome) patients were examined and determined to have five stages of disease severity. Using these stages, strategies were proposed for monitoring efficacy of either a focal or retina-wide intervention. This work sets the stage for clinical trials. Full article

Inherited retinal dystrophies and optic neuropathies cause chronic disabling loss of visual function. The development of recombinant adeno-associated viral vectors (rAAV) gene therapies in all disease fields have been promising, but the translation to the clinic has been slow. The safety and efficacy profiles of rAAV are linked to the dose of applied vectors. DNA changes in the rAAV gene cassette affect potency, the expression pattern (cell-specificity), and the production yield. Here, we present a library of rAAV vectors and elements that provide a workflow to design novel vectors. We first performed a meta-analysis on recombinant rAAV elements in clinical trials (2007–2020) for ocular gene therapies. We analyzed 33 unique rAAV gene cassettes used in 57 ocular clinical trials. The rAAV gene therapy vectors used six unique capsid variants, 16 different promoters, and six unique polyadenylation sequences. Further, we compiled a list of promoters, enhancers, and other sequences used in current rAAV gene cassettes in preclinical studies. Then, we give an update on pro-viral plasmid backbones used to produce the gene therapy vectors, inverted terminal repeats, production yield, and rAAV safety considerations. Finally, we assess rAAV transgene and bioactivity assays applied to cells or organoids in vitro, explants ex vivo, and clinical studies. Full article

The retinal pigment epithelium (RPE) and the adjacent light-sensitive photoreceptors form a single functional unit lining the back of the eye. Both cell layers are essential for normal vision. RPE degeneration is usually followed by photoreceptor degeneration and vice versa. There are currently almost no effective therapies available for RPE disorders such as Stargardt disease, specific types of retinitis pigmentosa, and age-related macular degeneration. RPE replacement for these disorders, especially in later stages of the disease, may be one of the most promising future therapies. There is, however, no consensus regarding the optimal RPE source, delivery strategy, or the optimal experimental host in which to test RPE replacement therapy. Multiple RPE sources, delivery methods, and recipient animal models have been investigated, with variable results. So far, a systematic evaluation of the (variables influencing) efficacy of experimental RPE replacement parameters is lacking. Here we investigate the effect of RPE transplantation on vision and vision-based behavior in animal models of retinal degenerated diseases. In addition, we aim to explore the effect of RPE source used for transplantation, the method of intervention, and the animal model which is used. Methods: In this study, we systematically identified all publications concerning transplantation of RPE in experimental animal models targeting the improvement of vision (e.g., outcome measurements related to the morphology or function of the eye). A variety of characteristics, such as species, gender, and age of the animals but also cell type, number of cells, and other intervention characteristics were extracted from all studies. A risk of bias analysis was performed as well. Subsequently, all references describing one of the following outcomes were analyzed in depth in this systematic review: a-, b-, and c-wave amplitudes, vision-based, thickness analyses based on optical coherence tomography (OCT) data, and transplant survival based on scanning laser ophthalmoscopy (SLO) data. Meta-analyses were performed on the a- and b-wave amplitudes from electroretinography (ERG) data as well as data from vision-based behavioral assays. Results: original research articles met the inclusion criteria after two screening rounds. Overall, most studies were categorized as unclear regarding the risk of bias, because many experimental details were poorly reported. Twenty-three studies reporting one or more of the outcome measures of interest were eligible for either descriptive (thickness analyses based on OCT data; n = 2) or meta-analyses. RPE transplantation significantly increased ERG a-wave (Hedges’ g 1.181 (0.471–1.892), n = 6) and b-wave (Hedges’ g 1.734 (1.295–2.172), n = 42) amplitudes and improved vision-based behavior (Hedges’ g 1.018 (0.826–1.209), n = 96). Subgroup analyses revealed a significantly increased effect of the use of young and adolescent animals compared to adult animals. Moreover, transplanting more cells (in the range of 10⁵ versus in the range of 10⁴) resulted in a significantly increased effect on vision-based behavior as well. The origin of cells mattered as well. A significantly increased effect was found on vision-based behavior when using ARPE-19 and OpRegen^® RPE. Conclusions: This systematic review shows that RPE transplantation in animal models for retinal degeneration significantly increases a- and b- wave amplitudes and improves vision-related behavior. These effects appear to be more pronounced in young animals, when the number of transplanted cells is larger and when ARPE-19 and OpRegen^® RPE cells are used. We further emphasize that there is an urgent need for improving the reporting and methodological quality of animal experiments, to make such studies more comparable. Full article

The treatment of dominantly inherited retinal diseases requires silencing of the pathogenic allele. RNA interference to suppress gene expression suffers from wide-spread off-target effects, while CRISPR-mediated gene disruption creates permanent changes in the genome. CRISPR interference uses a catalytically inactive ‘dead’ Cas9 directed by a guide RNA to block transcription of chosen genes without disrupting the DNA. It is highly specific and potentially reversible, increasing its safety profile as a therapy. Pre-clinical studies have demonstrated the versatility of CRISPR interference for gene silencing both in vivo and in ex vivo modification of iPSCs for transplantation. Applying CRISPR interference techniques for the treatment of autosomal dominant inherited retinal diseases is promising but there are few in vivo studies to date. This review details how CRISPR interference might be used to treat retinal diseases and addresses potential challenges for clinical translation. Full article

The loss of cone photoreceptor function in retinitis pigmentosa (RP) severely impacts the central and daily vision and quality of life of patients affected by this disease. The loss of cones follows the degeneration of rods, in a manner independent of the causing mutations in numerous genes associated with RP. We have explored this phenomenon and proposed that the loss of rods triggers a reduction in the expression of rod-derived cone viability factor (RdCVF) encoded by the nucleoredoxin-like 1 (NXNL1) gene which interrupts the metabolic and redox signaling between rods and cones. After providing scientific evidence supporting this mechanism, we propose a way to restore this lost signaling and prevent the cone vision loss in animal models of RP. We also explain how we could restore this signaling to prevent cone vision loss in animal models of the disease and how we plan to apply this therapeutic strategy by the administration of both products of NXNL1 encoding the trophic factor RdCVF and the thioredoxin enzyme RdCVFL using an adeno-associated viral vector. We describe in detail all the steps of this translational program, from the design of the drug, its production, biological validation, and analytical and preclinical qualification required for a future clinical trial that would, if successful, provide a treatment for this incurable disease. Full article

Diabetic retinopathy (DR) is an ocular complication of diabetes mellitus (DM). International Diabetic Federations (IDF) estimates up to 629 million people with DM by the year 2045 worldwide. Nearly 50% of DM patients will show evidence of diabetic-related eye problems. Therapeutic interventions for DR are limited and mostly involve surgical intervention at the late-stages of the disease. The lack of early-stage diagnostic tools and therapies, especially in DR, demands a better understanding of the biological processes involved in the etiology of disease progression. The recent surge in literature associated with NOD-like receptors (NLRs) has gained massive attraction due to their involvement in mediating the innate immune response and perpetuating inflammatory pathways, a central phenomenon found in the pathogenesis of ocular diseases including DR. The NLR family of receptors are expressed in different eye tissues during pathological conditions suggesting their potential roles in dry eye, ocular infection, retinal ischemia, cataract, glaucoma, age-related macular degeneration (AMD), diabetic macular edema (DME) and DR. Our group is interested in studying the critical early components involved in the immune cell infiltration and inflammatory pathways involved in the progression of DR. Recently, we reported that NLRP3 inflammasome might play a pivotal role in the pathogenesis of DR. This comprehensive review summarizes the findings of NLRs expression in the ocular tissues with special emphasis on its presence in the retinal microglia and DR pathogenesis. Full article

RNA editing aims to treat genetic disease through altering gene expression at the transcript level. Pairing site-directed RNA-targeting mechanisms with engineered deaminase enzymes allows for the programmable correction of G>A and T>C mutations in RNA. This offers a promising therapeutic approach for a range of genetic diseases. For inherited retinal degenerations caused by point mutations in large genes not amenable to single-adeno-associated viral (AAV) gene therapy such as USH2A and ABCA4, correcting RNA offers an alternative to gene replacement. Genome editing of RNA rather than DNA may offer an improved safety profile, due to the transient and potentially reversible nature of edits made to RNA. This review considers the current site-directing RNA editing systems, and the potential to translate these to the clinic for the treatment of inherited retinal degeneration. Full article

Inherited retinal dystrophies (IRDs) are a group of clinically and genetically heterogeneous degenerative disorders. To date, mutations have been associated with IRDs in over 270 disease genes, but molecular diagnosis still remains elusive in about a third of cases. The methodologic developments in genome sequencing techniques that we have witnessed in this last decade have represented a turning point not only in diagnosis and prognosis but, above all, in the identification of new therapeutic perspectives. The discovery of new disease genes and pathogenetic mechanisms underlying IRDs has laid the groundwork for gene therapy approaches. Several clinical trials are ongoing, and the recent approval of Luxturna, the first gene therapy product for Leber congenital amaurosis, marks the beginning of a new era. Due to its anatomical and functional characteristics, the retina is the organ of choice for gene therapy, although there are quite a few difficulties in the translational approaches from preclinical models to humans. In the first part of this review, an overview of the current knowledge on methodological issues and future perspectives of gene therapy applied to IRDs is discussed; in the second part, the state of the art of clinical trials on the gene therapy approach in IRDs is illustrated. Full article

Diabetic retinopathy (DR), a sight-threatening neurovasculopathy, is the leading cause of irreversible blindness in the developed world. DR arises as the result of prolonged hyperglycemia and is characterized by leaky retinal vasculature, retinal ischemia, retinal inflammation, angiogenesis, and neovascularization. The number of DR patients is growing with an increase in the elderly population, and therapeutic approaches are limited, therefore, new therapies to prevent retinal injury and enhance repair are a critical unmet need. Besides vascular endothelial growth factor (VEGF)-induced vascular proliferation, several other mechanisms are important in the pathogenesis of diabetic retinopathy, including vascular inflammation. Thus, combining anti-VEGF therapy with other new therapies targeting these pathophysiological pathways of DR may further optimize treatment outcomes. Technological advancements have allowed for high-throughput proteomic studies examining biofluids such as aqueous humor, vitreous humor, tear, and serum. Many DR biomarkers have been identified, especially proteins involved in retinal inflammatory processes. This review attempts to summarize the proteomic biomarkers of DR-associated retinal inflammation identified over the last several years. Full article