Treatment Of Retinal Degenerative Diseases Research Articles

Human primitive mesenchymal stem cell-derived retinal progenitor cells improved neuroprotection, neurogenesis, and vision in rd12 mouse model of retinitis pigmentosa

BackgroundCurrently, there is no treatment for retinal degenerative diseases (RDD) such as retinitis pigmentosa (RP). Stem cell-based therapies could provide promising opportunities to repair the damaged retina and restore vision. Thus far, primarily adult mesenchymal stem cells (MSCs) have been investigated in preclinical and clinical studies, and the results have not been convincing. We applied a new approach in which primitive (p) MSC-derived retinal progenitor cells (RPCs) were examined to treat retinal degeneration in an rd12 mouse model of RP.MethodsWell-characterized pMSCs and RPCs labeled with PKH26 were intravitreally injected into rd12 mice. The vision and retinal function of transplanted animals were analyzed using electroretinography. Animals were killed 4 and 8 weeks after cell transplantation for histological, immunological, molecular, and transcriptomic analyses of the retina.ResultsTransplanted RPCs significantly improved vision and retinal thickness as well as function in rd12 mice. pMSCs and RPCs homed to distinct retinal layers. pMSCs homed to the retinal pigment epithelium, and RPCs migrated to the neural layers of the retina, where they improved the thickness of the respective layers and expressed cell-specific markers. RPCs induced anti-inflammatory and neuroprotective responses as well as upregulated the expression of genes involved in neurogenesis. The transcriptomic analysis showed that RPCs promoted neurogenesis and functional recovery of the retina through inhibition of BMP and activation of JAK/STAT and MAPK signaling pathways.ConclusionsOur study demonstrated that RPCs countered inflammation, provided retinal protection, and promoted neurogenesis resulting in improved retinal structure and physiological function in rd12 mice.

HMGB2 causes photoreceptor death via down-regulating Nrf2/HO-1 and up-regulating NF-κB/NLRP3 signaling pathways in light-induced retinal degeneration model

In the pathogenesis of retinal degenerative diseases, oxidative stress is a key driver leading to photoreceptor death and eventually vision loss. Currently, there are no effective therapies available to rescue photoreceptors in these diseases. High-mobility group box 2 (HMGB2), a pro-inflammatory factor and damage-associated molecular patterns (DAMPs), has been proven to mediate various inflammatory diseases, but its role in retinal degenerative diseases, especially in retinal inflammation and photoreceptor degeneration, still remains unknown. In this study, we assessed the localization and function of HMGB2 under oxidative stress and explored the underlying mechanisms in a mouse model of light-induced retinal damage (LIRD). The results showed that increased oxidative stress, the photoreceptors death, as well as the pyroptosis-related proteins were evidenced in mice retina after light exposure. HMGB2 protein was predominantly expressed in the outer nuclear layer (ONL), which was translocated to the cytoplasm and released after injury. The mechanistic effect of HMGB2 was studied in the 661w cell line treated with H2O2, showing that exogenous recombinant HMGB2 protein reduced the expressions of the antioxidant protein nuclear erythroid factor 2-related factor 2 (Nrf2) and its downstream target heme oxygenase-1 (HO-1), and induced NF-κB/NLRP3 signaling pathway. HMGB2 knockdown increased cell viability, up-regulated the expressions of Nrf2 and HO-1, down-regulated the expressions of pyroptosis-related proteins in H2O2-treated 661w cells; and also prevented photoreceptors loss and maintained ONL in mice model of LIRD. The present study proposed HMGB2 as a potential therapeutic target for treatment of retinal degenerative diseases.

Polyphenols and Visual Health: Potential Effects on Degenerative Retinal Diseases.

Dietary polyphenols are a group of natural compounds that have been proposed to have beneficial effects on human health. They were first known for their antioxidant properties, but several studies over the years have shown that these compounds can exert protective effects against chronic diseases. Nonetheless, the mechanisms underlying these potential benefits are still uncertain and contradictory effects have been reported. In this review, we analyze the potential effects of polyphenol compounds on some visual diseases, with a special focus on retinal degenerative diseases. Current effective therapies for the treatment of such retinal diseases are lacking and new strategies need to be developed. For this reason, there is currently a renewed interest in finding novel ligands (or known ligands with previously unexpected features) that could bind to retinal photoreceptors and modulate their molecular properties. Some polyphenols, especially flavonoids (e.g., quercetin and tannic acid), could attenuate light-induced receptor damage and promote visual health benefits. Recent evidence suggests that certain flavonoids could help stabilize the correctly folded conformation of the visual photoreceptor protein rhodopsin and offset the deleterious effect of retinitis pigmentosa mutations. In this regard, certain polyphenols, like the flavonoids mentioned before, have been shown to improve the stability, expression, regeneration and folding of rhodopsin mutants in experimental in vitro studies. Moreover, these compounds appear to improve the integration of the receptor into the cell membrane while acting against oxidative stress at the same time. We anticipate that polyphenol compounds can be used to target visual photoreceptor proteins, such as rhodopsin, in a way that has only been recently proposed and that these can be used in novel approaches for the treatment of retinal degenerative diseases like retinitis pigmentosa; however, studies in this field are limited and further research is needed in order to properly characterize the effects of these compounds on retinal degenerative diseases through the proposed mechanisms.

PEDF-Mediated Mitophagy Triggers the Visual Cycle by Enhancing Mitochondrial Functions in a H2O2-Injured Rat Model.

Retinal degenerative diseases result from oxidative stress and mitochondrial dysfunction, leading to the loss of visual acuity. Damaged retinal pigment epithelial (RPE) and photoreceptor cells undergo mitophagy. Pigment epithelium-derived factor (PEDF) protects from oxidative stress in RPE and improves mitochondrial functions. Overexpression of PEDF in placenta-derived mesenchymal stem cells (PD-MSCs; PD-MSCsPEDF) provides therapeutic effects in retinal degenerative diseases. Here, we investigated whether PD-MSCsPEDF restored the visual cycle through a mitophagic mechanism in RPE cells in hydrogen peroxide (H2O2)-injured rat retinas. Compared with naïve PD-MSCs, PD-MSCsPEDF augmented mitochondrial biogenesis and translation markers as well as mitochondrial respiratory states. In the H2O2-injured rat model, intravitreal administration of PD-MSCsPEDF restored total retinal layer thickness compared to that of naïve PD-MSCs. In particular, PTEN-induced kinase 1 (PINK1), which is the major mitophagy marker, exhibited increased expression in retinal layers and RPE cells after PD-MSCPEDF transplantation. Similarly, expression of the visual cycle enzyme retinol dehydrogenase 11 (RDH11) showed the same patterns as PINK1 levels, resulting in improved visual activity. Taken together, these findings suggest that PD-MSCsPEDF facilitate mitophagy and restore the loss of visual cycles in H2O2-injured rat retinas and RPE cells. These data indicate a new strategy for next-generation MSC-based treatment of retinal degenerative diseases.

BrdU Positive Cells Induced in a Genetic Mouse Model of Glaucoma

Previous studies have shown that eye drop application of the selective α7 nicotinic acetylcholine receptor agonist, PNU-282987, induces neurogenesis of RGCs in adult wild-type rodents. This study was designed to test the hypothesis that PNU-282987 reverses the loss of RGCs associated with glaucoma. A DBA/2J mouse model that auto-induces a glaucoma-like condition in adulthood was used for these studies. Short-term effects using PNU-282987 and BrdU eye drop treatments were examined, as well as the effects of early treatment and the effects in a chronic early treatment group in DBA/2J mice aged 3, 6 and 10 months. With and without treatment, retinas were removed, fixed, immunostained and RGC counts were assessed. IOP measurements were obtained weekly using a Tonolab tonometer. Results showed an average typical loss of BrdU positive RGCs by 29% by 10 months of age in this DBA/2J colony corresponding with a significant increase in IOP. However, the two-week short term application of PNU-282987 and BrdU induced a significant 21% increase in RGCs for DBA/2J mice at all ages. Chronic early PNU-282987 treatment produced a similarly significant increase in RGCs, while acute early treatment had no effect on RGC numbers. IOP measurements were not affected with PNU-282987 treatment. These studies demonstrated that 2-week treatment with PNU-282987, as well as chronic long-term treatment, induced a significant increase in the number of RGCs in the DBA/2J retina, counteracting the effects of the DBA/2J genetic glaucoma-like condition. These results suggest a potential future treatment of degenerative retinal diseases with PNU-282987.

Peptide Derivatives of Retinylamine Prevent Retinal Degeneration with Minimal Side Effects on Vision in Mice.

Safe and effective molecular therapeutics for prophylactic treatment of retinal degenerative diseases are greatly needed. Disruptions in the clearance of all-trans-retinal (atRAL) by the visual (retinoid) cycle of the retina can lead to the accumulation of atRAL and its condensation products known to initiate progressive retinal dystrophy. Retinylamine (Ret-NH2) and its analogues are known to be effective in lowering the concentration of atRAL within the eye and thus preventing retinal degeneration in mouse models of human retinopathies. Here, we chemically modified Ret-NH2 with amino acids and peptides to improve the stability and ocular bioavailability of the resulting derivatives and to minimize their side effects. Fourteen Ret-NH2 derivatives were synthesized and tested in vitro and in vivo. These derivatives exhibited structure-dependent therapeutic efficacy in preventing light-induced retinal degeneration in Abca4-/-Rdh8-/- double-knockout mice, with the compounds containing glycine and/or L-valine generally exhibiting greater protective effects than Ret-NH2 or other tested amino acid derivatives of Ret-NH2. Ret-NH2-L-valylglycine amide (RVG) exhibited good stability in storage; and effective uptake and prolonged retention in mouse eyes. RVG readily formed a Schiff base with atRAL and did not inhibit RPE65 enzymatic activity. Administered by oral gavage, this retinoid also provided effective protection against light-induced retinal degeneration in Abca4-/-Rdh8-/- mice. Notably, the treatment with RVG had minimal effects on the regeneration of 11-cis-retinal and recovery of retinal function. RVG holds promise as a lead therapy for effective and safe treatment of human retinal degenerative diseases.

Establishment of Cattle Encephalon Glycoside and Ignotin Sustained-Release Drug Loading Nanostructured Lipid Carriers and Effect on the Repair Improving of Retinal Cells with Optic Nerve Injury.

The research aims to explore the preparation and physicochemical properties of cattle encephalon glycoside and ignotin (CEGI) sustained-release drug loading nanostructured lipid carriers (NLC) and to study the enhanced therapeutic efficacy of CEGI-NLC in treatment of retinal degenerative diseases, thereby providing an objective basis and guidance for clinical medication. The results showed that the particle size of drug-loaded microspheres was 333.2 nm and the potential was 24.9 MV, with an entrapment efficiency of (79±5.6)% and a drug loading efficiency of (7.76±2.3)%. The microspheres were spherical and evenly dispersed. The observations showed that drug-loaded microspheres had specific external sustained release characteristics compared with those of CEGI alone. CEGI-NLC can promote the activity of retinal cells. From the 7th to 28th day, the number of cell layers in the outer and inner nuclear layer retina decreased gradually. Electron microscopy observation showed that the apoptotic nuclear was deformed and the internal segment and cilia were broken down. After 14 and 28 days of CEGI-NLC treatment, the number of layers in the retina was thicker and the number of apoptotic cells was lower than those in the control group. The studies proved that CEGI-NLC has a positive effect on promoting the growth and the development of retinal cells as well as alleviating the process of retinal degeneration in RDS mice.

Thermo-Responsive PLGA-PEG-PLGA Hydrogels as Novel Injectable Platforms for Neuroprotective Combined Therapies in the Treatment of Retinal Degenerative Diseases.

The present study aims to develop a thermo-responsive-injectable hydrogel (HyG) based on PLGA-PEG-PLGA (PLGA = poly-(DL-lactic acid co-glycolic acid); PEG = polyethylene glycol) to deliver neuroprotective agents to the retina over time. Two PLGA-PEG PLGA copolymers with different PEG:LA:GA ratios (1:1.54:23.1 and 1:2.25:22.5) for HyG-1 and HyG-2 development respectively were synthetized and characterized by different techniques (gel permeation chromatography (GPC), nuclear magnetic resonance (NMR), dynamic light scattering (DLS), critical micelle concentration (CMC), gelation and rheological behaviour). According to the physicochemical characterization, HyG-1 was selected for further studies and loaded with anti-inflammatory drugs: dexamethasone (0.2%), and ketorolac (0.5%), alone or in combination with the antioxidants idebenone (1 µM) and D-α-Tocopherol polyethylene glycol 1000 succinate (TPGS) (0.002%). In vitro drug release and cytotoxicity studies were performed for the active substances and hydrogels (loaded and drug-free). A cellular model based on oxidative stress was optimized for anti-inflammatory and antioxidant screening of the formulations by using retinal-pigmented epithelial cell line hTERT (RPE-1). The copolymer 1, used to prepare thermo-responsive HyG-1, showed low polydispersity (PDI = 1.22) and a strong gel behaviour at 25% (w/v) in an isotonic buffer solution close to the vitreous temperature (31–34 °C). Sustained release of dexamethasone and ketorolac was achieved between 47 and 62 days, depending on the composition. HyG-1 was well tolerated (84.5 ± 3.2%) in retinal cells, with values near 100% when the anti-inflammatory and antioxidant agents were included. The combination of idebenone and dexamethasone promoted high oxidative protection in the cells exposed to H2O2, with viability values of 86.2 ± 14.7%. Ketorolac and dexamethasone-based formulations ameliorated the production of TNF-α, showing significant results (p ≤ 0.0001). The hydrogels developed in the present study entail a novel biodegradable tool to treat neurodegenerative processes of the retina overtime.

A Review on Mesenchymal Stem Cells for Treatment of Retinal Diseases.

Mesenchymal Stem Cells (MSCs) have been studied extensively for the treatment of several retinal diseases. The therapeutic potential of MSCs lies in its ability to differentiate into multiple lineages and secretome enriched with immunomodulatory, anti-angiogenic and neurotrophic factors. Several studies have reported the role of MSCs in repair and regeneration of the damaged retina where the secreted factors from MSCs prevent retinal degeneration, improve retinal morphology and function. MSCs also donate mitochondria to rescue the function of retinal cells and exosomes secreted by MSCs were found to have anti-apoptotic and anti-inflammatory effects. Based on several promising results obtained from the preclinical studies, several clinical trials were initiated to explore the potential advantages of MSCs for the treatment of retinal diseases. This review summarizes the various properties of MSCs that help to repair and restore the damaged retinal cells and its potential for the treatment of retinal degenerative diseases.

Overexpression of pigment epithelium-derived factor in placenta-derived mesenchymal stem cells promotes mitochondrial biogenesis in retinal cells

AbstractPigment epithelium-derived factor (PEDF) plays a role in protecting retinal pigment epithelial (RPE) cells from oxidative stress (OS), a causative factor of RPE cell death. Genetically modified mesenchymal stem cells (MSCs) can be used to treat critical and incurable retinal diseases. Here, we overexpressed PEDF in placenta-derived MSCs (PD-MSCsPEDF, PEDF+) using a nonviral gene delivery system and evaluated the characteristics of PD-MSCsPEDF and their potential regenerative effects on RPE cells damaged by H2O2-induced OS. PD-MSCsPEDF maintained their stemness, cell surface marker, and differentiation potential characteristics. Compared to naive cells, PD-MSCsPEDF promoted mitochondrial respiration by enhancing biogenesis regulators (e.g., NRF1, PPARGC1A, and TFAM) as well as antioxidant enzymes (e.g., HMOXs, SODs, and GPX1). Compared to OS-damaged RPE cells cocultured with naive cells, OS-damaged RPE cells cocultured with PD-MSCsPEDF showed PEDF upregulation and VEGF downregulation. The expression levels of antioxidant genes and RPE-specific genes, such as RPE65, RGR, and RRH, were significantly increased in RPE cells cocultured with PD-MSCsPEDF. Furthermore, OS-damaged RPE cells cocultured with PD-MSCsPEDF had dramatically enhanced mitochondrial functions, and antiapoptotic effects improved due to cell survival signaling pathways. In the H2O2-induced retinal degeneration rat model, compared to administration of the naive counterpart, intravitreal administration of PD-MSCsPEDF alleviated proinflammatory cytokines and restored retinal structure and function by increasing PEDF expression and decreasing VEGF expression. Intravitreal administration of PD-MSCsPEDF also protected retinal degeneration against OS by increasing antioxidant gene expression and regulating the mitochondrial ROS levels and biogenesis. Taken together, PEDF overexpression in PD-MSCs improved the mitochondrial activities and induced OS-damaged RPE cell regeneration by regulating the oxidative status and mitochondrial biogenesis in vitro and in vivo. These data suggest that genetic modification of PEDF in PD-MSCs might be a new cell therapy for the treatment of retinal degenerative diseases.Overexpression of pigment epithelium-derived factor (PEDF) in placenta-derived mesenchymal stem cells (PD-MSCs) improved the mitochondrial activities, and induced regeneration of oxidative stress-damaged RPE through regulating oxidative status and mitochondrial biogenesis. Therefore, genetic modification of PD-MSCs with PEDF might be a new cell therapy for treatment of retinal degenerative diseases.

Non-invasive electrical stimulation as a potential treatment for retinal degenerative diseases.

Non-invasive electrical stimulation as a potential treatment for retinal degenerative diseases.

Stem-cell-based therapies for retinal degenerative diseases: Current challenges in the establishment of new treatment strategies.

Various advances have been made in the treatment of retinal diseases, including new treatment strategies and innovations in surgical devices. However, the treatment of degenerative retinal diseases, such as retinitis pigmentosa (RP) and age‐related macular degeneration (AMD), continues to pose a significant challenge. In this review, we focus on the use of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to treat retinal diseases by harnessing the ability of stem cells to differentiate into different body tissues. The retina is a tissue specialized for light sensing, and its degradation leads to vision loss. As part of the central nervous system, the retina has very low regenerative capability, and therefore, treatment options are limited once it degenerates. Nevertheless, innovations in methods to induce the generation of retinal cells and tissues from ESCs/iPSCs enable the development of novel approaches for these irreversible diseases. Here we review some historical background and current clinical trials involving the use of stem‐cell‐derived retinal pigment epithelial cells for AMD treatment and stem cell‐derived retinal cells/tissues for RP therapy. Finally, we discuss our future vision of regenerative treatment for retinal diseases with a partial focus on our studies and introduce other interesting approaches for restoring vision.

An Overview of Scaffolds for Retinal Pigment Epithelium Research

The reduced capabilities of retinal pigment epithelium (RPE) marks early retinal degenerative diseases and leads to blindness. Regenerating a functional RPE monolayer in vitro becomes significant and promise for developing new therapies. Many research activities have been done to develop new materials, structures and fabrication technologies to mimic native RPE structure for cell attachment and subsequent tissue development. Scaffold-based tissue engineering approaches attract intensive investigation, due to their capability and potential in building preclinical models and developing possible treatment for retinal degenerative diseases. Scaffolds in facilitating regeneration of RPE tissue in vitro has been proved by recent studies. Various materials have been applied, such as synthetic polymers, plant or animal extracted proteins, and composite materials. This review outlines the most recent fabrication progresses using these materials for RPE scaffolds, including both in vitro and in vivo trials, and the challenges in building a whole tissue model construct and transplantation. Additive manufacturing technologies at micrometer- and nanometer-scale are proposed as promising methods to build biomimetic RPE scaffolds in the current studies.

Does Stem Cell Implantation Have an Effect on Severity of Retinitis Pigmentosa: Evaluation with a Classification System?

Background: Cell replacement therapies have been evaluated in recent years as an alternative for various retinal pathologies to evaluate the therapeutic efficacy of cell therapy, it is important to measure the severity of the disease. The aim of this study was to evaluate the effect of umbilical cord derived Mesenchymal Stem Cell (UC-MSC) implantation on severity of Retinitis Pigmentosa (RP). Methods: This single-center, clinical study included data of 138 eyes of 92 patients who had a confirmed diagnosis of RP and received stem cell implantation to the suprachoroidal area with a surgical procedure. Patients were evaluated before and 1 year after the surgery regarding to the outcome measures of Best Corrected Visual Acuity (BCVA), Optical Coherence Tomography (OCT) and Visual Field (VF) tests. BCVA, VF width and ellipsoid zone (EZ) width on OCT were recorded for each patient and a scoring criterion was established for each variable varying from 0 to 5 depending on its distribution. The cumulative score (from 0 to 15) was used to classify disease severity from grade 0 to 5. Results: All of the patients completed 12-month follow-up period. The median age of the patients was 40.8 years, 46% were female, 77% had been diagnosed within 10 years and 41% had a family history. 79% of the patients with family history had autosomal recessive inheritance pattern. There were statistically significant improvements in the mean BCVA and VF scores during the study (p < 0.05). The mean score and the mean grade of the disease also improved after the treatment (p < 0.05). There was a negative correlation between BCVA improvement and scoring and grading of the disease. Conclusions: This study demonstrated beneficial effect of suprachoroidally applied UC-MSCs on BCVA, VF and the severity score and grade of the disease during 12-month follow-up period. Cell mediated therapy based on the secretion of Growth Factors (GFs) seems to be an effective and safe option for the treatment of degenerative retinal diseases. This classification is simple, produces objective measure of disease severity and gives opportunity to compare the results of different treatment modalities.

Optimal timing for activation of sigma 1 receptor in the Pde6brd10/J (rd10) mouse model of retinitis pigmentosa

Sigma 1 Receptor (Sig1R), a pluripotent modulator of cell survival, is a promising target for treatment of retinal degenerative diseases. Previously, we reported that administration of the high-affinity, high-specificity Sig1R ligand (+)-pentazocine, ((+)-PTZ) beginning at post-natal day 14 (P14) and continuing every other day improves visual acuity and delays loss of photoreceptor cells (PRCs) in the Pde6βrd10/J (rd10) mouse model of retinitis pigmentosa. Whether administration of (+)-PTZ, at time points concomitant with (P18) or following (P21, P24) onset of PRC death, would prove neuroprotective was investigated in this study. Rd10 mice were administered (+)-PTZ intraperitoneally [0.5 mg/kg], starting at either P14, P18, P21 or P24. Injections continued every other day through P42. Visual acuity was assessed using the optokinetic tracking response (OKR). Rd10 mice treated with (+)-PTZ beginning at P14 retained visual acuity for the duration of the study (~0.33 c/d at P21, ~0.38 c/d at P28, ~0.32 c/d at P35, ~0.32 c/d at P42), whereas mice injected beginning at P18, P21, P24 showed a decline in acuity when tested at P35 and P42. Their acuity was only slightly better than rd10-non-treated mice. Electrophysiologic function was assessed using scotopic and photopic electroretinography (ERG) to assess rod and cone function, respectively. Photopic a- and b-wave amplitudes were significantly greater in rd10 mice treated with (+)-PTZ beginning at P14 compared with non-treated mice and those in the later-onset (+)-PTZ injection groups. Retinal architecture was visualized in living mice using spectral domain-optical coherence tomography (SD-OCT) allowing measurement of the total retinal thickness, the inner retina and the outer retina (the area most affected in rd10 mice). The outer retina measured ~35 μm in rd10 mice treated with (+)-PTZ beginning at P14, which was significantly greater than mice in the later-onset (+)-PTZ injection groups (~25 μm) and non-treated rd10 mice (~25 μm). Following the visual function studies performed in the living mice, eyes were harvested at P42 for histologic analysis. While the inner retina was largely intact in all (+)-PTZ-injection groups, there was a marked reduction in the outer retina of non-treated rd10 mice (e.g. in the outer nuclear layer there were ~10 PRCs/100 μm retinal length). The rd10 mice treated with (+)-PTZ beginning at P14 had ~20 PRCs/100 μm retinal length, whereas the mice in groups beginning P18, P21 and P24 had ~16 PRCs/100 μm retinal length. In conclusion, the data indicate that delaying (+)-PTZ injection past the onset of PRC death in rd10 mice – even by a few days – can negatively impact the long-term preservation of retinal function. Our findings suggest that optimizing the administration of Sig1R ligands is critical for retinal neuroprotection.

Photobiomodulation preserves mitochondrial redox state and is retinoprotective in a rodent model of retinitis pigmentosa

Photobiomodulation (PBM) by far-red (FR) to near-infrared (NIR) light has been demonstrated to restore the function of damaged mitochondria, increase the production of cytoprotective factors and prevent cell death. Our laboratory has shown that FR PBM improves functional and structural outcomes in animal models of retinal injury and retinal degenerative disease. The current study tested the hypothesis that a brief course of NIR (830 nm) PBM would preserve mitochondrial metabolic state and attenuate photoreceptor loss in a model of retinitis pigmentosa, the P23H transgenic rat. P23H rat pups were treated with 830 nm light (180 s; 25 mW/cm2; 4.5 J/cm2) using a light-emitting diode array (Quantum Devices, Barneveld, WI) from postnatal day (p) 10 to p25. Sham-treated rats were restrained, but not treated with 830 nm light. Retinal metabolic state, function and morphology were assessed at p30 by measurement of mitochondrial redox (NADH/FAD) state by 3D optical cryo-imaging, electroretinography (ERG), spectral-domain optical coherence tomography (SD-OCT), and histomorphometry. PBM preserved retinal metabolic state, retinal function, and retinal morphology in PBM-treated animals compared to the sham-treated group. PBM protected against the disruption of the oxidation state of the mitochondrial respiratory chain observed in sham-treated animals. Scotopic ERG responses over a range of flash intensities were significantly greater in PBM-treated rats compared to sham controls. SD-OCT studies and histological assessment showed that PBM preserved the structural integrity of the retina. These findings demonstrate for the first time a direct effect of NIR PBM on retinal mitochondrial redox status in a well-established model of retinal disease. They show that chronic proteotoxic stress disrupts retinal bioenergetics resulting in mitochondrial dysfunction, and retinal degeneration and that therapies normalizing mitochondrial metabolism have considerable potential for the treatment of retinal degenerative disease.

Novel Therapeutic Approaches for the Treatment of Retinal Degenerative Diseases: Focus on CRISPR/Cas-Based Gene Editing.

Inherited retinal diseases encompass a highly heterogenous group of disorders caused by a wide range of genetic variants and with diverse clinical symptoms that converge in the common trait of retinal degeneration. Indeed, mutations in over 270 genes have been associated with some form of retinal degenerative phenotype. Given the immune privileged status of the eye, cell replacement and gene augmentation therapies have been envisioned. While some of these approaches, such as delivery of genes through recombinant adeno-associated viral vectors, have been successfully tested in clinical trials, not all patients will benefit from current advancements due to their underlying genotype or phenotypic traits. Gene editing arises as an alternative therapeutic strategy seeking to correct mutations at the endogenous locus and rescue normal gene expression. Hence, gene editing technologies can in principle be tailored for treating retinal degeneration. Here we provide an overview of the different gene editing strategies that are being developed to overcome the challenges imposed by the post-mitotic nature of retinal cell types. We further discuss their advantages and drawbacks as well as the hurdles for their implementation in treating retinal diseases, which include the broad range of mutations and, in some instances, the size of the affected genes. Although therapeutic gene editing is at an early stage of development, it has the potential of enriching the portfolio of personalized molecular medicines directed at treating genetic diseases.

Soy Protein Nanofiber Scaffolds for Uniform Maturation of Human Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium.

Retinal pigment epithelium (RPE) differentiated from human induced pluripotent stem cells, called induced retinal pigment epithelium (iRPE), is being explored as a cell-based therapy for the treatment of retinal degenerative diseases, especially age-related macular degeneration. The success of RPE implantation is linked to the use of biomimetic scaffolds that simulate Bruch's membrane and promote RPE maturation and integration as a functional tissue. Due to difficulties associated with animal protein-derived scaffolds, including sterility and pro-inflammatory responses, current practices favor the use of synthetic polymers, such as polycaprolactone (PCL), for generating nanofibrous scaffolds. In this study, we tested the hypothesis that plant protein-derived fibrous scaffolds can provide favorable conditions permissive for the maturation of RPE tissue sheets in vitro. Our natural, soy protein-derived nanofibrous scaffolds exhibited a J-shaped stress-strain curve that more closely resembled the mechanical properties of native tissues than PCL with significantly higher hydrophilicity of the natural scaffolds, favoring in vivo implantation. We then demonstrate that iRPE sheets growing on these soy protein scaffolds are equivalent to iRPE monolayers cultured on synthetic PCL nanofibrous scaffolds. Immunohistochemistry demonstrated RPE-like morphology and functionality with appropriate localization of RPE markers RPE65, PMEL17, Ezrin, and ZO1 and with anticipated histotypic polarization of vascular endothelial growth factor and pigment epithelium-derived growth factor as indicated by enzyme-linked immunosorbent assay. Scanning electron microscopy revealed dense microvilli on the cell surface and homogeneous tight junctional contacts between the cells. Finally, comparative transcriptome analysis in conjunction with principal component analysis demonstrated that iRPE on nanofibrous scaffolds, either natural or synthetic, matured more consistently than on nonfibrous substrates. Taken together, our studies suggest that the maturation of cultured iRPE sheets for subsequent clinical applications might benefit from the use of nanofibrous scaffolds generated from natural proteins. Impact statement Induced retinal pigment epithelium (iRPE) from patient-derived induced pluripotent stem cells (iPSCs) may yield powerful treatments of retinal diseases, including age-related macular degeneration. Recent studies, including early human clinical trials, demonstrate the importance of selecting appropriate biomaterial scaffolds to support tissue-engineered iRPE sheets during implantation. Electrospun scaffolds show particular promise due to their similarity to the structure of the native Bruch's membrane. In this study, we describe the use of electroprocessed nanofibrous soy protein scaffolds to generate polarized sheets of human iPSC-derived iRPE sheets. Our evaluation, including RNA-seq transcriptomics, indicates that these scaffolds are viable alternatives to scaffolds electrospun from synthetic polymers.

Development of chiral fluorinated alkyl derivatives of emixustat as drug candidates for the treatment of retinal degenerative diseases

The discovery of how a photon is converted into a chemical signal is one of the most important achievements in the field of vision. A key molecule in this process is the visual chromophore retinal. Several eye diseases are attributed to the abnormal metabolism of retinal in the retina and the retinal pigment epithelium. Also, the accumulation of two toxic retinal derivatives, N-retinylidene-N-retinylethanolamine and the retinal dimer, can damage the retina leading to blindness. RPE65 (Retinal pigment epithelium-specific 65 kDa protein) is one of the central enzymes that regulates the metabolism of retinal and the formation of its toxic metabolites. Its inhibition might decrease the rate of the retina’s degeneration by limiting the amount of retinal and its toxic byproducts. Two RPE65 inhibitors, (R)-emixustat and (R)-MB001, were recently developed for this purpose.

A 3D Printed Self-Sustainable Cell-Encapsulation Drug Delivery Device for Periocular Transplant-Based Treatment of Retinal Degenerative Diseases.

Self-sustainable release of brain-derived neurotrophic factor (BDNF) to the retina using minimally invasive cell-encapsulation devices is a promising approach to treat retinal degenerative diseases (RDD). Herein, we describe such a self-sustainable drug delivery device with human retinal pigment epithelial (ARPE-19) cells (cultured on collagen coated polystyrene (PS) sheets) enclosed inside a 3D printed semi-porous capsule. The capsule was 3D printed with two photo curable polymers: triethylene glycol dimethacrylate (TEGDM) and polyethylene glycol dimethylacrylate (PEGDM). The capsule’s semi-porous membrane (PEGDM) could serve three functions: protecting the cells from body’s immune system by limiting diffusion (5.97 ± 0.11%) of large molecules like immunoglobin G (IgG)(150 kDa); helping the cells to survive inside the capsule by allowing diffusion (43.20 ± 2.16%) of small molecules (40 kDa) like oxygen and necessary nutrients; and helping in the treatment of RDD by allowing diffusion of cell-secreted BDNF to the outside environment. In vitro results showed a continuous BDNF secretion from the device for at least 16 days, demonstrating future potential of the cell-encapsulation device for the treatment of RDD in a minimally invasive and self-sustainable way through a periocular transplant.