The dry (nonneovascular) form of age-related macular degeneration (AMD), a leading cause of blindness in the elderly, has few, if any, treatment options at present. It is characterized by early accumulation of cellular waste products in the retinal pigmented epithelium (RPE); rejuvenating impaired lysosome function in RPE is a well-justified target for treatment. It is now clear that amino acids and vacuolar-type H+ -ATPase (V-ATPase) regulate the mechanistic target of rapamycin, complex 1 (mTORC1) signaling in lysosomes. Here, we provide evidence for the first time that the amino acid transporter SLC36A4/proton-dependent amino acid transporter (PAT4) regulates the amino acid pool in the lysosomes of RPE. In Cryba1 (gene encoding βA3/A1-crystallin) KO (knockout) mice, where PAT4 and amino acid levels are increased in the RPE, the transcription factors EB (TFEB) and E3 (TFE3) are retained in the cytoplasm, even after 24 h of fasting. Consequently, genes in the coordinated lysosomal expression and regulation (CLEAR) network are not activated, and lysosomal function remains low. As these mice age, expression of RPE65 and lecithin retinol acyltransferase (LRAT), two vital visual cycle proteins, decreases in the RPE. A defective visual cycle would possibly slow down the regeneration of new photoreceptor outer segments (POS). Further, photoreceptor degeneration also becomes obvious during aging, reminiscent of human dry AMD disease. Electron microscopy shows basal laminar deposits in Bruch's membrane, a hallmark of development of AMD. For dry AMD patients, targeting PAT4/V-ATPase in the lysosomes of RPE cells may be an effective means of preventing or delaying disease progression.

In vertebrates, the absorption of light by rhodopsin leads to the isomerization of 11-cis-retinal chromophore to its all-trans form. In the visual cycle, all-trans retinal is converted back to 11-cis retinal. Mammalian visual cycle takes place in photoreceptor cells and retinal pigment epithelial (RPE) cells, while that of cephalopods is completed within a photoreceptor cell. To identify visual cycle system in the primitive chordate ascidians, we studied the localization of the ascidian visual cycle genes and proteins by in situ hybridization and whole-mount immunohistochemistry, respectively. We identified four genes encoding putative visual cycle proteins, homologs of retinal G protein-coupled receptor (Ci-opsin3), cellular retinaldehyde-binding protein (Ci-CRALBP), beta-carotene 15,15'monooxygenase (Ci-BCO) and RPE-specific 65 kDa protein (Ci-RPE65) in the ascidian, Ciona intestinalis. In contrast to Ci-BCO, which is predominantly localized in ocellus photoreceptor cells of the larva, Ci-RPE65 is not significantly expressed in the ocellus and brain vesicle of the larva. Ci-RPE65 is expressed in the neural complex, a photoreceptor organ of the adult ascidian, at a level comparable with that of Ci-opsin3 and Ci-CRALBP. Proteins of Ci-opsin3, Ci-CRALBP and Ci-BCO are localized in photoreceptor cells. These results suggest that the larval visual cycle uses Ci-opsin3 as a photoisomerase, while the visual cycle of the adult photoreceptors is RPE65-dependent. The colocalization of visual cycle proteins in the photoreceptor cells suggest that ascidian visual cycle takes place in a photoreceptor cell as seen in the cephalopod visual cycle.

Diabetes-associated visual cycle impairment has been implicated in diabetic retinopathy, and chronic hyperglycemia causes detrimental effects on visual function. Chrysin, a naturally occurring flavonoid found in various herbs, has anti-inflammatory, antioxidant, and neuroprotective properties. The goal of the current study was to identify the retinoprotective role of chrysin in maintaining robust retinoid visual cycle-related components. The in vitro study employed human retinal pigment epithelial (RPE) cells exposed to 33 mM of glucose or advanced glycation end products (AGEs) in the presence of 1⁻20 μM chrysin for three days. In the in vivo study, 10 mg/kg of chrysin was orally administrated to db/db mice. Treating chrysin reversed the glucose-induced production of vascular endothelial growth factor, insulin-like growth factor-1, and pigment epithelium-derived factor (PEDF) in RPE cells. The outer nuclear layer thickness of chrysin-exposed retina was enhanced. The oral gavage of chrysin augmented the levels of the visual cycle enzymes of RPE65, lecithin retinol acyltransferase (LRAT), retinol dehydrogenase 5 (RDH5), and rhodopsin diminished in db/db mouse retina. The diabetic tissue levels of the retinoid binding proteins and the receptor of the cellular retinol-binding protein, cellular retinaldehyde-binding protein-1, interphotoreceptor retinoid-binding protein and stimulated by retinoic acid 6 were restored to those of normal mouse retina. The presence of chrysin demoted AGE secretion and AGE receptor (RAGE) induction in glucose-exposed RPE cells and diabetic eyes. Chrysin inhibited the reduction of PEDF, RPE 65, LRAT, and RDH5 in 100 μg/mL of AGE-bovine serum albumin-exposed RPE cells. The treatment of RPE cells with chrysin reduced the activation of endoplasmic reticulum (ER) stress. Chrysin inhibited the impairment of the retinoid visual cycle through blocking ER stress via the AGE-RAGE activation in glucose-stimulated RPE cells and diabetic eyes. This is the first study demonstrating the protective effects of chrysin on the diabetes-associated malfunctioned visual cycle.

OBJECTIVE

Leber congenital amaurosis (LCA) is a group of childhood-onset retinal diseases characterized by severe visual impairment or blindness. One form is caused by mutations in the RPE65 gene, which encodes the retinal pigment epithelium (RPE) isomerase. In this study, the retinal structure and expression of molecular markers for different retinal cell types were characterized, and differences between control and RPE65 mutant dogs during the temporal evolution of the disease were analyzed.

METHODS

Retinas from normal and mutant dogs of different ages were examined by immunofluorescence with a panel of 16 different antibodies.

RESULTS

Cones and rods were preserved in the mutant retinas, and the number of cones was normal. However, there was altered expression of cone arrestin and delocalization of rod opsin. The ON bipolar cells showed sprouting of the dendritic arbors toward the outer nuclear layer (ONL) and retraction of their axons in the inner nuclear layer (INL). A decreased expression of GABA, and an increased expression of intermediate filament glial markers was also found in the mutant retinas. These changes were more evident in the adult than the young mutant retinas.

CONCLUSIONS

The structure of the retina is well preserved in the mutant retina, but several molecular changes take place in photoreceptors and in bipolar and amacrine cells. Some of these changes are structural, whereas others reflect a change in localization of the examined proteins. This study provides new information that can be applied to the interpretation of outcomes of retinal gene therapy in animal models and humans.

OBJECTIVE

To use ocular motility recordings to determine the changes over time of infantile nystagmus syndrome (INS) in RPE65-deficient canines with Leber Congenital Amaurosis (LCA) and assess the time course of the recalibration of the ocular motor system (OMS).

METHODS

Nine dogs were treated bilaterally with AAV-RPE65. A second cohort of four dogs was treated with AAV2.RPE65, an optimized vector. Their fixation eye movements were recorded before treatment and at 4-week intervals for 3 months, by using high-speed (500 Hz) digital videography. The dogs were suspended in a sling and encouraged to fixate on distant (57 inches) targets at gaze angles varying between +/-15 degrees horizontally and +/-10 degrees vertically. The records for each eye were examined for qualitative changes in waveform and for quantitative changes in centralisation with the expanded nystagmus acuity function (NAFX) and compared with ERG results for restoration of receptor function.

RESULTS

First group: Before treatment, five of the dogs had clinically apparent INS with jerk, pendular, or both waveforms and with peak-to-peak amplitudes as great as 15 degrees . One dog had intermittent nystagmus. At the 1- and 2-month examinations, no change in nystagmus waveform or NAFX was observed in any of the initial dogs, while at 10 weeks, one dog treated bilaterally with the standard dosage showed reduced nystagmus in only one eye. The other eye did not respond to treatment, as confirmed by ERG. This result was unexpected since it was previously documented that unilateral treatment leads to bilateral reduction of INS. The other dog treated with the standard dosage showed no reduction of its small-amplitude, high-frequency pendular nystagmus despite positive ERG responses. Second group: Only one dog of the four had clinically detectable INS, similar in characteristics to that seen in the affected dogs of the first group. Unlike any previous dog studied, this one showed a damping of the nystagmus within the first 4 weeks after treatment.

CONCLUSIONS

In all but one of the cases in which OMS recalibration occurred, as measured by the clinical appearance of nystagmus and by quantitative measurement using the NAFX, the improvement was apparent no sooner than 10 weeks after treatment. Longer term, dose-related studies are needed to determine the minimum necessary degree of restored receptor functionality, the duration after rescue for recalibration of the OMS, and the conditions under which recalibration information can successfully affect the contralateral eye.

OBJECTIVE

In a previous constant light-induced retinal damage (CLD) quantitative genetics study between the albino C57BL/6J-c2J (B6al) and BALB/c mouse strains, we identified a very strong and highly significant quantitative trait locus (QTL) on distal Chr 3 that we associated with a variant of the Rpe65 gene. The B6al strain carries the MET450 variant of RPE65 and is resistant to CLD while the BALB/c strain carries the LEU450 variant and is sensitive. Since then, we have discovered that the NZW/LacJ (NZW) albino mouse strain is sensitive to CLD but carries the MET450 variant of RPE65. The purpose of this study was to determine if the NZW mouse disproves the hypothesis that the MET450 variant of RPE65 protects the mouse retina against constant light-induced retinal damage.

METHODS

F2 progeny were bred from an intercross between the NZW/LacJ and B6al mouse strains. After a prolonged exposure to moderate constant light, F2 mice were phenotyped for retinal outer nuclear layer thickness as the quantitative trait. A subset of 156 of the 201 F2 mice was genotyped for a set of markers spanning the genome, and any marker with a significant association with the quantitative trait was genotyped in the remaining 45 F2s. Data were analyzed for QTL by the Map Manager QTX software.

RESULTS

No QTL was identified at distal Chr 3, although several QTL on Chrs 1 (two), 10, 13, 14, 16, and X were detected. One QTL on middle Chr 1 (LOD 5.22) mapped to the same location of a QTL (LOD 6.8) in a previous intense, short exposure light-induced retinal damage study conducted with an intercross between the 129S1/SvImJ and BALB/c strains. QTL on Chrs 1 (distal), 10, and 14 also appeared in other retinal damage quantitative genetics studies. Three pairs of genes exhibited significant epistatic effects. Two of the pairs involved synergistic interactions between NZW and B6al alleles, and the third between two B6al alleles.

CONCLUSIONS

If another gene besides Rpe65 was responsible for the QTL in the original BALB/c x B6al study, and the NZW mouse carried a light sensitive allele of this gene, a QTL should have been present in this study. Since a QTL on Chr 3 was not found, the hypothesis that RPE65-MET450 protects the retina from constant light-induced damage is left intact. The explanation for the NZW mouse being sensitive to constant light while carrying the RPE65-MET450 variant is that other light sensitive QTL (gene alleles) negate the protective effect.

This paper reviews the published histopathologic findings of patients with retinitis pigmentosa (RP) or an allied disease in whom the responsible gene defect was identified, including 10 cases with dominant RP (cases with mutations in RHO, PRPC8, and RP1), three with dominant spinocerebellar ataxia (SCA7), three X-linked RP carrier females (RPGR), two with congenital retinal blindness (AIPL1 and RPE65), two with mitochondrial encephalomyopathy overlap syndrome (MTTL1), and one case each with dominant cone degeneration (GCAP1), X-linked cone degeneration (RCP), enhanced S-cone syndrome (NR2E3), and dominant late-onset retinal degeneration (CTRP5). No histopathologic descriptions were found of the vast majority of genetically defined forms of retinal degeneration.

OBJECTIVE

To evaluate the occurrence and inheritance of various types of pigmentary retinopathy in patients followed at the outpatient clinic in the university hospital, Montpellier, France. To characterize genes and mutations causing these conditions.

METHODS

Ophthalmic examination and various visual tests were performed. Mutations were sought from genomic DNA by PCR amplification of exons associated with single-strand conformation analysis and/or direct sequencing.

RESULTS

Among 315 patients over an 8-year period, cases of retinitis pigmentosa (63.2%), Usher's syndrome (10.2%), Stargardt's disease (5.4%), choroideremia (3.2%), Leber's congenital amaurosis (3.2%), congenital stationary night blindness (2.9%), cone dystrophy (2.5%), dominant optic atrophy (1.9%), X-linked juvenile retinoschisis (1.6%), Best's disease (1.6%), and others (4.3%) were diagnosed. In retinitis pigmentosa, inheritance could be determined in 54.2% of the cases including dominant autosomic (26.6%), recessive autosomic (22.6%), and X-linked cases (5%) while it could not be confirmed in 45.7% of the cases (simplex cases in the majority). For the 6 examined genes, mutations were found in 22 out of 182 propositus (12.1%). Analysis of phenotype-genotype correlations indicates that in retinitis pigmentosa, RDS is more frequently associated with macular involvement and retinal flecks, RHO with regional disease, and RPE65 with the great severity of the disease with some cases of Leber's congenital amaurosis.

CONCLUSIONS

Identification of genes may help in diagnosis and in genetic counseling, especially in simplex cases with retinitis pigmentosa. In this latter condition, molecular diagnosis will be necessary to rationalize future treatments.

Mutations in RPE65 protein is characterized by the loss of photoreceptors, although the molecular pathways triggering retinal cell death remain largely unresolved. The role of the Bcl-2 family of proteins in retinal degeneration is still controversial. However, alteration in Bcl-2-related proteins has been observed in several models of retinal injury. In particular, Bax has been suggested to play a crucial role in apoptotic pathways in murine glaucoma model as well as in retinal detachment-associated cell death. We demonstrated that Bcl-2-related signaling pathway is involved in Rpe65-dependent apoptosis of photoreceptors during development of the disease. Pro-apoptotic Bax alpha and beta isoforms were upregulated in diseased retina. This was associated with a progressive reduction of anti-apoptotic Bcl-2, reflecting imbalanced Bcl-2/Bax ratio as the disease progresses. Moreover, specific translocation of Bax beta from cytosol to mitochondria was observed in Rpe65-deficient retina. This correlated with the initiation of photoreceptor cell loss at 4 months of age, and further increased during disease development. Altogether, these data suggest that Bcl-2-apoptotic pathway plays a crucial role in Leber's congenital amaurosis disease. They further highlight a new regulatory mechanism of Bax-dependent apoptosis based on regulated expression and activation of specific isoforms of this protein.

Lipofuscin is a fluorescent material with significant phototoxic potential that accumulates with age in the retinal pigment epithelium (RPE) of the eye. It is thought to be a factor in retinal degeneration diseases. The most extensively characterized lipofuscin component, N-retinylidene-N-retinylethanolamine (A2E), has been proposed to be a byproduct of reactions involving the visual pigment chromophore. To examine the impact of the visual pigment and photoreceptor cell type on lipofuscin accumulation, we analyzed the RPE from Nrl(-/-) mice of various ages for lipofuscin fluorescence and A2E levels. The photoreceptor cells of the Nrl(-/-) retina contain only cone-like pigments, and produce cone-like responses to photostimulation. The cone-like nature of these cells was confirmed by the presence of RPE65. Lipofuscin was measured with fluorescence imaging, whereas A2E was quantified by UV/VIS absorbance spectroscopy coupled to HPLC. The identity of A2E was corroborated with tandem mass spectrometry. Lipofuscin and A2E accumulated with age, albeit to lower levels compared with wild type mice. The emission spectra of RPE lipofuscin granules from Nrl(-/-) mice were similar to those from wild type mice, with λ(max) ca 610 nm. These results demonstrate that cone visual pigments can contribute to the production of lipofuscin and A2E.

Mutations in RPE65 or lecithin-retinol acyltransferase (LRAT) disrupt 11-cis-retinal synthesis and cause Leber congenital amaurosis (LCA), a severe hereditary blindness occurring in early childhood. The pathology is attributed to a combination of 11-cis-retinal deficiency and photoreceptor degeneration. The mistrafficking of cone membrane-associated proteins including cone opsins (M- and S-opsins), cone transducin (Gαt2), G-protein-coupled receptor kinase 1 (GRK1) and guanylate cyclase 1 (GC1) has been suggested to play a role in cone degeneration. However, their precise role in cone degeneration is unclear. Here we investigated the role of S-opsin (Opn1sw) in cone degeneration in Lrat(-) (/-), a murine model for LCA, by genetic ablation of S-opsin. We show that deletion of just one allele of S-opsin from Lrat(-) (/-) mice is sufficient to prevent the rapid cone degeneration for at least 1 month. Deletion of both alleles of S-opsin prevents cone degeneration for an extended period (at least 12 months). This genetic prevention is accompanied by a reduction of endoplasmic reticulum (ER) stress in Lrat(-) (/-) photoreceptors. Despite cone survival in Opn1sw(-/-)Lrat(-) (/-) mice, cone membrane-associated proteins (e.g. Gαt2, GRK1 and GC1) continue to have trafficking problems. Our results suggest that cone opsins are the 'culprit' linking 11-cis-retinal deficiency to cone degeneration in LCA. This result has important implications for the current gene therapy strategy that emphasizes the need for a combinatorial therapy to both improve vision and slow photoreceptor degeneration.

The retinal pigment epithelium (RPE) plays a key role in the maintenance of the normal functions of the retina. Tissue engineering using amniotic membrane as a substrate to culture RPE cells may provide a promising new strategy to replace damaged RPE. We established a method of culturing RPE cells over the amniotic membrane as a support for their growth and transplantation. The transcription of specific genes involved in cellular function of native RPE, including RPE65, CRALBP, VEGF, CD68, and tyrosinase, were then measured using quantitative real-time PCR. Data showed a considerable increase in transcription of RPE65, CD68, and VEGF in RPE cells cultured on amniotic membrane. The amounts of CRALBP and tyrosinase transcripts were not affected. This may simply indicate that amniotic membrane restricted dedifferentiation of RPE cells in culture. The results suggest that amniotic membrane may be considered as an elective biological substrate for RPE cell culture.

All-trans-retinal, a retinoid metabolite naturally produced upon photoreceptor light activation, is cytotoxic when present at elevated levels in the retina. To lower its toxicity, two experimentally validated methods have been developed involving inhibition of the retinoid cycle and sequestration of excess of all-trans-retinal by drugs containing a primary amine group. We identified the first-in-class drug candidates that transiently sequester this metabolite or slow down its production by inhibiting regeneration of the visual chromophore, 11-cis-retinal. Two enzymes are critical for retinoid recycling in the eye. Lecithin:retinol acyltransferase (LRAT) is the enzyme that traps vitamin A (all-trans-retinol) from the circulation and photoreceptor cells to produce the esterified substrate for retinoid isomerase (RPE65), which converts all-trans-retinyl ester into 11-cis-retinol. Here we investigated retinylamine and its derivatives to assess their inhibitor/substrate specificities for RPE65 and LRAT, mechanisms of action, potency, retention in the eye, and protection against acute light-induced retinal degeneration in mice. We correlated levels of visual cycle inhibition with retinal protective effects and outlined chemical boundaries for LRAT substrates and RPE65 inhibitors to obtain critical insights into therapeutic properties needed for retinal preservation.

Many different processes take place at the cell membrane interface. Indeed, for instance, ligands bind membrane proteins which in turn activate peripheral membrane proteins, some of which are enzymes whose action is also located at the membrane interface. Native cell membranes are difficult to use to gain information on the activity of individual proteins at the membrane interface because of the large number of different proteins involved in membranous processes. Model membrane systems, such as monolayers at the air-water interface, have thus been extensively used during the last 50 years to reconstitute proteins and to gain information on their organization, structure and activity in membranes. In the present paper, we review the recent work we have performed with membrane and peripheral proteins as well as enzymes in monolayers at the air-water interface. We show that the structure and orientation of gramicidin has been determined by combining different methods. Furthermore, we demonstrate that the secondary structure of rhodopsin and bacteriorhodopsin is indistinguishable from that in native membranes when appropriate conditions are used. We also show that the kinetics and extent of monolayer binding of myristoylated recoverin is much faster than that of the nonmyristoylated form and that this binding is highly favored by the presence polyunsaturated phospholipids. Moreover, we show that the use of fragments of RPE65 allow determine which region of this protein is most likely involved in membrane binding. Monomolecular films were also used to further understand the hydrolysis of organized phospholipids by phospholipases A2 and C.

The retinal pigment epithelium (RPE) is essential for maintaining the health of the neural retina. RPE cell dysfunction plays a critical role in many common blinding diseases including age-related macular degeneration (AMD), diabetic retinopathy, retinal dystrophies. Mouse models of ocular disease are commonly used to study these blinding diseases. Since isolating the RPE from the choroid has been challenging, most techniques separate the RPE from the retina, but not the choroid. As a result, the protein signature actually represents a heterogeneous population of cells that may not accurately represent the RPE response. Herein, we describe a method for separating proteins from the RPE that is free from retinal and choroidal contamination. After removing the anterior segment and retina from enucleated mouse eyes, protein from the RPE was extracted separately from the choroid by incubating the posterior eyecup with a protein lysis buffer for 10 min. Western blot analysis identified RPE65, an RPE specific protein in the RPE lysates, but not in choroidal lysates. The RPE lysates were devoid of rhodopsin and collagen VI, which are abundant in the retina and choroid, respectively. This technique will be very helpful for measuring the protein signal from the RPE without retinal or choroidal contamination.

Non-exudative age-related macular degeneration (NE-AMD) represents the leading cause of blindness in the elderly. Currently, there are no available treatments for NE-AMD. We have developed a NE-AMD model induced by superior cervical ganglionectomy (SCGx) in C57BL/6J mice, which reproduces the disease hallmarks. Several lines of evidence strongly support the involvement of oxidative stress in NE-AMD-induced retinal pigment epithelium (RPE) and outer retina damage. Melatonin is a proven and safe antioxidant. Our aim was analyzing the effect of melatonin in the RPE/outer retina damage within experimental NE-AMD. The treatment with melatonin starting 48 h after SCGx, which had no effect on the ubiquitous choriocapillaris widening, protected visual functions, and avoided Bruch's membrane thickening, RPE melanin content, melanosome number loss, retinoid isomerohydrolase (RPE65)-immunoreactivity decrease, and RPE and photoreceptor ultrastructural damage induced within experimental NE-AMD exclusively located at the central temporal (but not nasal) region. Melatonin also prevented the increase in outer retina/RPE oxidative stress markers, and a decrease in mitochondrial mass at 6 weeks post-SCGx. Moreover, when the treatment with melatonin started at 4 weeks post-SCGx, it restored visual functions and reversed the decrease in RPE melanin content and RPE65-immunoreactivity. These findings suggest that melatonin could become a promising safe therapeutic strategy for NE-AMD.

BACKGROUND

Retinitis pigmentosa (RP) and age related macular degeneration (AMD) are two retinal diseases that progress by photoreceptor cells death. In retinal transplantation studies, stem and progenitor cells inject into the sub retinal space or vitreous and then these cells can be migrate to the site of retinal degeneration and locate in the host retina and restitute vision.

OBJECTIVE

Our hypothesis suggests that using human conjunctiva stem cells (as the source for increasing the number of human stem cells progenitor cells in retina dysfunction diseases) with fibrin gel and also assessing its relating in vitro (cellular and molecular processes) and in vivo (vision tests and pathology) could be a promising strategy for treatment of AMD and RP disorders.

METHODS

In this idea, we describe a novel approach for retina tissue engineering with differentiation of conjunctiva mesenchymal stem cells (CJMSCs) into photoreceptor-like cells in fibrin gel with induction medium contain taurine. For assessment of differentiation, immunocytochemistry and real time PCR are used for the expression of Rhodopsin, RPE65, Nestin as differentiated photoreceptor cell markers in 2D and 3D culture. The results show that fibrin gel will offer a proper 3D scaffold for CJMSCs derived photoreceptor cell-like cells.

CONCLUSIONS

Application of immune-privileged, readily available sources of adult stem cells like human conjunctiva stem cells with fibrin gel would be a promising strategy to increase the number of photoreceptor progenitor cells and promote involuntary angiogenesis needed in retina layer repair and regeneration.

BACKGROUND

The retinal pigment epithelium (RPE), an important tissue monolayer of retina, sustains visual function and retinal homeostasis. In disease conditions such as Retinitis Pigmentosa (RP) and Age related Macular degeneration (AMD), the integrity and functional capacity of RPE monolayer is compromised. Human embryonic stem cells derived RPE (hESC-RPE) is ideal for cell based therapy because of their ability to morphologically and functionally mimic native fetal and adult RPE. However protocols for optimum culture of hESC-RPE are not well established.

OBJECTIVE

To describe a simplified protocol for differentiating human embryonic stem cells (hESC) into retinal pigment epithelial cells.

METHODS

hESC (WA09-DL-11) cell lines were grown with standard stem cell culture protocol. After cell colonies were established, basic fibroblast growth factor (bFGF) was deprived (day 0). hESC colonies expressing pigmentation were characterized for expression of RPE65 and Zonular occludens--1 (ZO-1) with immunocytochemistry on days 0, 36, 42, 56 and 70 and western blot analysis on days 0, 40, 48, 53 and 63. In addition, morphological assessment was conducted on transformed cells longitudinally.

RESULTS

Pigmented cells were noted 36 days after deprivation of bFGF from growth media. Immunofluorescence demonstrated progressive up regulation of RPE specific proteins (ZO-1 & RPE 65). Immunofluorescence of ZO-1 (in pixels) was (3.08 ± 0.31) on day 42, (5.33 ± 0.89, p = 0.0001) on day 56 and (4.87 ± 0.57, p = 0.0011) on day 70. Similarly expression of RPE 65 was (2.44 ± 0.31) on day 42, which continued to increase (4.23 ± 0.60, p = 0.0011) on day 56 and (5.59 ± 0.36, p < 0.0001) on day 70. Protein expression patterns using western blot confirmed the trends seen in immunofluorescence. Western blot analysis of ZO-1 expression (in optical density unit) was 272.57 ± 31.75 on day 40, 4212.20 ± 911.31 (p = 0.0004) on day 48, 5182.43 ± 1230.38 (p = 0.030) on day 53 and 5848.76 ± 241.04 (p < 0.0001) on day 63. Protein expression of RPE 65 was 1607.64 ± 247.76 on day 40, 2448.07 ± 152.66 on day 48 and (2341.15 ± 52.84) on day 63. hESC-RPE cells displayed a series of specific morphological changes (cytoplasmic, nuclear pigmentary and cell shape) over the course of time frame. By day 70, cells with hexagonal pattern, dark dense nucleus and uniform cytoplasm were noted in densely pigmented RPE colonies.

CONCLUSIONS

bFGF deprivation leads to successful differentiation of hESC into RPE cells. Longitudinal transformative changes were confirmed with measurement of ZO-1 and RPE 65, specific markers of RPE.

Age-related macular degeneration as well as some forms of Retinitis Pigmentosa (RP) are characterized by a retinal degeneration involving the retinal pigment epithelium (RPE). Various strategies were proposed to cure these disorders including the replacement of RPE cells using human pluripotent stem cells (hPSCs), an unlimited source material to generate in vitro RPE cells. The formulation strategy of the cell therapy (either a reconstructed sheet or a cell suspension) is crucial to achieve an efficient and long lasting therapeutic effect. We previously developed a hPSC-RPE sheet disposed on human amniotic membrane that sustained the vision of rodents with retinal degeneration compared to the same cells injected as a suspension. However, the transplantation strategy was difficult to implement in large animals. Herein we developed two medical devices for the preparation, conservation and implantation of the hPSC-RPE sheet in nonhuman primates. The surgery was safe and well tolerated during the 7-week follow up. The graft integrity was preserved in primates. Moreover, the hPSC-RPE sheet did not induce teratoma or grafted cell dispersion to other organs in rodent models. This work clears the way for the first cell therapy for RP patients carrying RPE gene mutations (LRAT, RPE65 and MERTK).

Previously, results at 2 years after subretinal injection of a recombinant adeno-associated virus vector expressing RPE65 (rAAV2-CB-hRPE65) in eight adults and four children with retinal degeneration caused by RPE65 mutations were reported. Now, results at 5 years after treatment in 11 of these subjects are reported. Subjects received a subretinal injection of rAAV2-CB-hRPE65 in the poorer-seeing eye, at either of two dose levels, and were followed for 5 years after treatment. The primary safety outcomes were ocular and non-ocular adverse events. Efficacy outcomes included changes in best corrected visual acuity, static perimetry hill of vision measurements for the central 30° (V30), and total (VTOT) visual field and kinetic perimetry visual field area. The only adverse events reported during years 3, 4, and 5 were minor intercurrent illnesses. Pediatric subjects had improvement in visual acuity and static perimetry in the treated eye, sometimes with a smaller improvement in the untreated eye, during the first 2 years of the study that persisted during years 3-5, with no consistent changes in kinetic perimetry during the study. Most adult subjects had no consistent changes in visual acuity or static perimetry during the study. Three adult subjects with markedly abnormal baseline kinetic visual field area had improvement in the treated eye during the first 1-2 years after treatment, but the absolute magnitude of the improvement was small and was not sustained at subsequent visits. There were no clinically significant adverse events. Visual acuity and static perimetry testing results suggest that treating patients at a younger age is associated with better visual function outcomes during 5 years after treatment.

OBJECTIVE

To investigate the role of the myocyte enhancer factor 2 (Mef2) transcription factor family in retinal diseases, Mef2c expression was assessed during retinal degeneration in the Rpe65(-/-) mouse model of Leber's congenital amaurosis (LCA). Mef2c-dependent expression of photoreceptor-specific genes was further addressed.

METHODS

Expression of Mef2 members was analyzed by oligonucleotide microarray, quantitative PCR (qPCR), and in situ hybridization. Mef2c-dependent transcriptional activity was assayed by luciferase assay in HEK293T cells.

RESULTS

Mef2c was the only Mef2 member markedly downregulated during retinal degeneration in Rpe65(-/-) mice. Mef2c mRNA level was decreased by more than 2-fold at 2 and 4 months and by 3.5-fold at 6 months in retinas of Rpe65(-/-) mice. Downregulation of Mef2c at the protein level was confirmed in Rpe65(-/-) retinas. The decrease in Mef2c mRNA levels in the developing Rpe65(-/-) retinas from postnatal day (P) 13 onward was concomitant with the decreased expression of the rod-specific transcription factors Nrl and Nr2e3. Nrl was further shown to drive Mef2c transcriptional activity, supporting a physiological role for Mef2c in the retina. In addition, Mef2c appeared to act as a transcriptional repressor of its own expression and the expression of the retina-specific retinal G-protein coupled receptor (Rgr), rhodopsin, and M-opsin genes.

CONCLUSIONS

These findings highlight the early altered regulation of the rod-specific transcriptional network in Rpe65-related disease. They also indicate that Mef2c may act as a novel transcription factor involved in the development and the maintenance of photoreceptor cells.

Cytosine base editors and adenine base editors (ABEs) can correct point mutations predictably and independent of Cas9-induced double-stranded DNA breaks (which causes substantial indel formation) and homology-directed repair (which typically leads to low editing efficiency). Here, we show, in adult mice, that a subretinal injection of an adeno-associated virus expressing an ABE and a single-guide RNA targeting a de novo nonsense mutation in the Rpe65 gene corrects the pathogenic mutation with up to 29% efficiency and with minimal formation of indel and off-target mutations, despite the absence of the canonical NGG sequence as a protospacer-adjacent motif. The ABE-treated mice displayed restored RPE65 expression and retinoid isomerase activity, and near-normal levels of retinal and visual functions. Our findings motivate the further testing of ABEs for the treatment of inherited retinal diseases and for the correction of pathological mutations with non-canonical protospacer-adjacent motifs.