OBJECTIVE

To evaluate the effects of small interference RNA protein kinase C-alpha (siRNA-PKCα) on experimental proliferative vitreoretinopathy (PVR) induced by dispase in mice.

METHODS

C57BL/6 mice PVR models (4-6 weeks old) were induced by intravitreal injection of dispase and then equally divided into six groups. After 1 week, the five treatment groups received 2 μL, intravitreal injections of siRNA-PKCα at a concentration of 250 nM, 500 nM, 750 nM, 1000 nM, and 1500 nM, respectively, while the negative control group received 2 μL of 500 nM no-silencing siRNA. SiRNA-PKCα was transfected by a square wave electroporator. Postoperative ophthalmic observations of lens clarity and the fundus of the eyes were performed periodically. The eyeballs of the mice were enucleated and imbedded in optimal cutting temperature to perform histological and immunofluorescence analysis at the end of a 4-week observation period.

RESULTS

Four weeks after the siRNA-PKCα injections, there are 100% lens dissolution and 100% PVR in the 250 nM group and 70%, 70%, 70%, and 50% PVR in the 500 nM, 750 nM, 1000 nM, and 1500 nM groups, respectively, which is significantly different from the negative group. Abnormalities in fundus appearance were related to the concentrations of siRNA-PKCα; a higher concentration of siRNA-PKCα resulted in a more normal fundus. Histological sections by hematoxylin-eosin staining of the eyes support the clinical observation. Immunofluorescence analysis showed that RPE65, glutamine synthase, glial acidic fibrillary protein, and α-smooth muscle actin were increasing in the retina with the decreasing concentration of siRNA-PKCα, indicating that intraocular siRNA-PKCα can partly inhibit changes of markers for glia cells, fibroblast cells, retinal pigment epithelium cells, and Müller cells in the process of PVR.

CONCLUSIONS

Gene therapy with siRNA-PKCα could effectively inhibit PVR in mice and provide us with a novel therapeutic target on PVR.

OBJECTIVE

To investigate the effect of the iron chelator deferiprone (DFP) on sodium iodate (NaIO3)-induced retinal degeneration and on the hereditary retinal degeneration caused by the rd6 mutation.

METHODS

Retinas from NaIO3-treated C57BL/6J mice, with or without DFP cotreatment, were analyzed by histology, immunofluorescence, and quantitative PCR to investigate the effect of DFP on retinal degeneration. To facilitate photoreceptor quantification, we developed a new function of MATLAB to perform this task in a semiautomated fashion. Additionally, rd6 mice treated with or without DFP were analyzed by histology to assess possible protection.

RESULTS

In NaIO3-treated mice, DFP protected against retinal degeneration and significantly decreased expression of the oxidative stress-related gene heme oxygenase-1 and the complement gene C3. DFP treatment partially protected against NaIO3-induced reduction in the levels of mRNAs encoded by visual cycle genes rhodopsin (Rho) and retinal pigment epithelium-specific 65 kDa protein (Rpe65), consistent with the morphological data indicating preservation of photoreceptors and RPE, respectively. DFP treatment also protected photoreceptors in rd6 mice.

CONCLUSIONS

The oral iron chelator DFP provides significant protection against retinal degeneration induced through different modalities. This suggests that iron chelation could be useful as a treatment for retinal degeneration even when the main etiology does not appear to be iron dysregulation.

UNASSIGNED

These data provide proof of principle that the oral iron chelator DFP can protect the retina against diverse insults. Further testing of DFP in additional animal retinal degeneration models at a range of doses is warranted.

RPE65 is the isomerase catalyzing conversion of all-trans-retinyl ester (atRE) into 11-cis-retinol in the retinal visual cycle. Crystal structures of RPE65 and site-directed mutagenesis reveal aspects of its catalytic mechanism, especially retinyl moiety isomerization, but other aspects remain to be determined. To investigate potential interactions between RPE65 and lipid metabolism enzymes, HEK293-F cells were transfected with expression vectors for visual cycle proteins and co-transfected with either fatty acyl:CoA ligases (ACSLs) 1, 3, or 6 or the SLC27A family fatty acyl-CoA synthase FATP2/SLCA27A2 to test their effect on isomerase activity. These experiments showed that RPE65 activity was reduced by co-expression of ACSLs or FATP2. Surprisingly, however, in attempting to relieve the ACSL-mediated inhibition, we discovered that triacsin C, an inhibitor of ACSLs, also potently inhibited RPE65 isomerase activity in cellulo. We found triacsin C to be a competitive inhibitor of RPE65 (IC50 = 500 nm). We confirmed that triacsin C competes directly with atRE by incubating membranes prepared from chicken RPE65-transfected cells with liposomes containing 0-1 μM atRE. Other inhibitors of ACSLs had modest inhibitory effects compared with triascin C. In conclusion, we have identified an inhibitor of ACSLs as a potent inhibitor of RPE65 that competes with the atRE substrate of RPE65 for binding. Triacsin C, with an alkenyl chain resembling but not identical to either acyl or retinyl chains, may compete with binding of the acyl moiety of atRE via the alkenyl moiety. Its inhibitory effect, however, may reside in its nitrosohydrazone/triazene moiety.

Spark Therapeutics recently reported positive phase III results for SPK-RPE65 targeting the treatment of visual impairment caused by RPE65 gene mutations (often referred to as Leber congenital amaurosis type 2, or LCA2, but may include other retinal disorders), marking an important inflection point for the field of gene therapy. The results highlight the ability to successfully design and execute a randomized trial of a gene therapy and also reinforce the potentially predictive nature of early preclinical and clinical data. The results are expected to pave the way for the first approved gene therapy product in the United States and should sustain investor interest and confidence in gene therapy for many approaches, including retina targeting and beyond.

Retinal pigment epithelium (RPE) is a major component of the eye. This highly specialized cell type facilitates maintenance of the visual system. Because RPE loss induces an irreversible visual impairment, RPE generation techniques have recently been investigated as a potential therapeutic approach to RPE degeneration. A microRNA-based technique is a new strategy for producing RPE cells from adult stem cell sources. Previously, we identified that antisense microRNA-410 (anti-miR-410) induces RPE differentiation from amniotic epithelial stem cells. In this study, we investigated RPE differentiation from umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) via anti-miR-410 treatment. We identified miR-410 as a RPE-relevant microRNA in UCB-MSCs from among 21 putative human RPE-depleted microRNAs. Inhibition of miR-410 induces overexpression of immature and mature RPE-specific factors, including MITF, LRAT, RPE65, Bestrophin, and EMMPRIN. The RPE-induced cells were able to phagocytize microbeads. Results of our microRNA-based strategy demonstrated proof-of-principle for RPE differentiation in UCB-MSCs by using anti-miR-410 treatment without the use of additional factors or exogenous transduction.

The visual cycle is a multi-step pathway to recycle 11-cis retinal, the chromophore for both rod and cone visual pigments. The isomerohydrolase RPE65, a membrane-associated enzyme, converts atRE (all-trans-retinyl ester) to 11-cis-retinol, a key step in the visual cycle. Previously, it has been shown that membrane association of RPE65 is essential for its catalytic activity. Using purified recombinant chicken RPE65 and an in vitro liposome-based floatation assay, we present evidence that the RPE65 membrane-binding affinity was significantly facilitated by incorporation of atRE, the substrate of RPE65, into liposomal membrane. Using tryptophan emission fluorescence quenching and CD spectroscopy, we showed that, upon membrane binding, RPE65 undergoes conformational changes at both the tertiary and secondary structural levels. Specifically, tryptophan fluorescence quenching showed that the tertiary RPE65 structure became more open towards the hydrophilic environment upon its association with the membrane. Simultaneously, a decrease in the α-helix content of RPE65 was revealed upon binding with the lipid membrane containing atRE. These results demonstrated that RPE65's functional activity depends on its conformational changes caused by its association with the membrane.

CONCLUSIONS

A prognostic model was obtained for LC. Several critical genes were unveiled. They could be potentially applied for LC recurrence prediction.

OBJECTIVE

Gene expression data of laryngeal cancer (LC) were analyzed to identify critical genes associated with recurrence.

METHODS

Two gene expression datasets were downloaded from the Gene Expression Omnibus. Dataset GSE27020 is used as the training set, containing 75 non-recurred LC cases and 34 recurred LC cases.

RESULTS

A total of 725 DEGs were identified from the training set. A total of 4126 gene pairs showed significant correlations in non-recurred LC only, corresponding to 533 genes. A total of 7235 gene pairs showed significant correlations in recurred LC only, corresponding to 608 genes. Besides, 1694 gene pairs showed significant correlations in both non-recurred and recurred LC, corresponding to 322 genes. Functional enrichment analysis was performed for the three groups of DEGs. Seven overlapping biological functions were revealed: positive regulation of chondrocyte differentiation, autoimmune thyroid disease, focal adhesion, linoleic acid metabolism, drug metabolism, organic cation transport, and ECM-receptor interaction. Eight feature genes (PDIA3, MYH11, PDK1, SDC3, RPE65, LAMC3, BTK, and UPK1B) were identified. Their prognostic effect was validated by independent test set as well as survival analysis.

UNASSIGNED

Oxidative stress, partly due to light, has an important role in many retinal diseases, including macular degeneration and retinal dystrophies. The Leu450Met variant of RPE65 is expressed in C57BL/6 and in many genetically modified mice. It confers significant resistance to light induced retinal degeneration (LIRD). Our goal was to develop an effective and efficient method to induce LIRD in resistant mice that would recapitulate mechanisms seen in known models of LIRD.

UNASSIGNED

The retinas of C57BL/6J mice were exposed to light using a murine fundus camera. Two protocols (with and without intraperitoneal fluorescein) were used. Optical coherence tomography (OCT) helped determine the location and extent of retinal damage. Histology, TUNEL assay, quantitative (q) PCR, and immunohistochemistry were performed.

UNASSIGNED

Both protocols consistently generated LIRD in C57BL/6J mice. Optical coherence tomography and histology demonstrated that retinal damage starts at the level of the photoreceptor/outer retina and is more prominent in the superior retina. Fundus camera-delivered light-induced retinal degeneration (FCD-LIRD) is associated with apoptosis, subretinal microglia/macrophages, increased expression of oxidative stress response genes, and C3d deposition.

UNASSIGNED

We characterize two new models of light-induced retinal degeneration that are effective in C57BL/6J mice, and can be modulated in terms of severity. We expect FCD-LIRD to be useful in exploring mechanisms of LIRD in resistant mice, which will be important in increasing our understanding of the retinal response to light damage and oxidative stress.

UNASSIGNED

To assess the immunohistochemical and histopathological changes in a subject with Autosomal Dominant Vitreoretinochoroidopathy (ADVIRC).

UNASSIGNED

Case study.

UNASSIGNED

Ninety two year-old Caucasian male with ADVIRC.

UNASSIGNED

The subject was documented clinically for 54 Years. The retina/choroid complex of the right eye was evaluated with cryosections stained with hematoxylin and eosin or periodic acid schiff reagent. Cryosections were also evaluated with immunofluorescence or alkaline phosphatase immunohistochemistry (IHC) using primary antibodies against bestrophin1, GFAP, PEDF, RPE65, TGFβ, VEGF, and vimentin. The left retina and choroid were evaluated as flat mounts using immunofluorescence. UEA lectin was used to stain viable vasculature.

UNASSIGNED

The immunohistochemical and histopathological changes in retina and choroid from a subject with ADVIRC.

UNASSIGNED

The subject had a heterozygous c.248G>A variant in exon 4 of the BEST1 gene. There was widespread chorioretinal degeneration and atrophy except for an island of spared RPE monolayer in the perimacula/macula OU. In this region, some photoreceptors were present, choriocapillaris was spared, and retinal pigment epithelial cells were in their normal disposition. There was a Muller cell periretinal membrane throughout much of the fundus. Bestrophin-1 was not detected or only minimally present by IHC in the ADVIRC RPE, even in the spared RPE area. Beyond the island of retained RPE monolayer on Bruch's membrane (BrMb), there was migration of RPE into the neuro-retina, often ensheathing blood vessels and producing excessive matrix within their perivascular aggregations.

UNASSIGNED

The primary defect in ADVIRC is in RPE, the only cells in the eye that express the BEST1 gene. The dysfunctional RPE cells may go through epithelial/mesenchymal transition as they migrate from BrMb to form papillary aggregations in the neuro-retina, often ensheathing blood vessels. This may be the reason for retinal blood vessel nonperfusion. Migration of RPE from BrMb was also associated with attenuation of the choriocapillaris.

Novel therapeutics for inherited retinal dystrophies (IRDs) have rapidly evolved since groundbreaking clinical trials for LCA due to RPE65 mutations led to the first FDA-approved in vivo gene therapy. Since then, advancements in viral vectors have led to more efficient AAV transduction and developed other viral vectors for gene augmentation therapy of large gene targets. Furthermore, significant developments in gene editing and RNA modulation technologies have introduced novel capabilities for treatment of autosomal dominant diseases, intronic mutations, and/or large genes otherwise unable to be treated with current viral vectors. We highlight strategies currently being evaluated in gene therapy clinical trials and promising preclinical developments for IRDs.

Keywords: RNA modulation; gene editing; gene therapy; inherited retinal dystrophies.

We have previously shown that immunization with RPE65 produces in rats of four strains a severe inflammatory eye disease, designated experimental autoimmune uveitis (EAU). Here, we examined the uveitogenicity of RPE65 in six strains of mice. Only one strain, C57Bl/6, was found to develop consistently moderate levels of EAU, whereas other strains (BALB/c, B10.A, B10.BR, B10.RIII, C57BL/10J) were found to be essentially resistant to disease induced by RPE65. Analysis of the expression of RPE65 mRNA in thymi of the six mouse strains revealed detectable levels of the transcript in all strains, but with remarkable quantitative differences, with the lowest levels seen in thymi of C57Bl/6 mice, the only strain susceptible to RPE65-induced EAU. Moreover, unlike the finding with the mice, no RPE65 mRNA was detected in thymi of any of the four rat strains (Lewis, BN, F344, SHR) all of which are susceptible to the disease. These data thus indicate that the susceptibility to RPE65-induced EAU is inversely related to the thymic expression of the molecule. The data also suggest that this disease can be induced only in mice in which thymic expression of RPE65 is sufficiently low to allow the escape from deletion of T-cells with the adequate capacity to initiate the pathogenic immune response.

Ocular gene therapy with recombinant adeno-associated virus (AAV) has shown vector-mediated gene augmentation to be safe and efficacious in the retina in one set of diseases (retinitis pigmentosa and Leber congenital amaurosis (LCA) caused by RPE65 deficiency), with excellent safety profiles to date and potential for efficacy in several additional diseases. However, size constraints imposed by the packaging capacity of the AAV genome restrict application to diseases with coding sequence lengths that are less than 5,000 nt. The most prevalent retinal diseases with monogenic inheritance are caused by mutations in genes that exceed this capacity. Here, we designed a spliceosome mediated pre-mRNA trans-splicing strategy to rescue expression of CEP290, which is associated with Leber congenital amaurosis type 10 (LCA10) and several syndromic diseases including Joubert syndrome. We used this reagent to demonstrate editing of CEP290 in cell lines in vitro and in vivo in a mini-gene mouse model. This study is the first to show broad editing of CEP290 transcripts and in vivo proof of concept for editing of CEP290 transcripts in photoreceptors and paves the way for future studies evaluating therapeutic effects.

Bone marrow stem and progenitor cells can differentiate into a range of non-hematopoietic cell types, including retinal pigment epithelium (RPE)-like cells. In this study, we programmed bone marrow-derived cells (BMDCs) ex vivo by inserting a stable RPE65 transgene using a lentiviral vector. We tested the efficacy of systemically administered RPE65-programmed BMDCs to prevent visual loss in the superoxide dismutase 2 knockdown (Sod2 KD) mouse model of age-related macular degeneration. Here, we present evidence that these RPE65-programmed BMDCs are recruited to the subretinal space, where they repopulate the RPE layer, preserve the photoreceptor layer, retain the thickness of the neural retina, reduce lipofuscin granule formation, and suppress microgliosis. Importantly, electroretinography and optokinetic response tests confirmed that visual function was significantly improved. Mice treated with non-modified BMDCs or BMDCs pre-programmed with LacZ did not exhibit significant improvement in visual deficit. RPE65-BMDC administration was most effective in early disease, when visual function and retinal morphology returned to near normal, and less effective in late-stage disease. This experimental paradigm offers a minimally invasive cellular therapy that can be given systemically overcoming the need for invasive ocular surgery and offering the potential to arrest progression in early AMD and other RPE-based diseases.

RPE65, an abundant membrane-associate protein in the retinal pigment epithelium (RPE), is a key retinoid isomerase of the visual cycle necessary for generating 11-cis-retinal that functions not only as a molecular switch for activating cone and rod visual pigments in response to light stimulation, but also as a chaperone for normal trafficking of cone opsins to the outer segments. Many mutations in RPE65 are associated with Leber congenital amaurosis (LCA). A R91W substitution, the most frequent LCA-associated mutation, results in a severe decrease in protein level and enzymatic activity of RPE65, causing cone opsin mislocalization and early cone degeneration in the mutation knock-in mouse model of LCA. Here we show that R91W RPE65 undergoes ubiquitination-dependent proteasomal degradation in the knock-in mouse RPE due to misfolding. The 26S proteasome non-ATPase regulatory subunit 13 mediated degradation specifically of misfolded R91W RPE65. The mutation disrupted membrane-association and colocalization of RPE65 with lecithin:retinol acyltransferase (LRAT) that provides the hydrophobic substrate for RPE65. Systemic administration of sodium 4-phenylbutyrate (PBA), a chemical chaperone, increased protein stability, enzymatic activity, membrane-association, and colocalization of R91W RPE65 with LRAT. This rescue effect increased synthesis of 11-cis-retinal and 9-cis-retinal, a functional iso-chromophore of the visual pigments, led to alleviation of S-opsin mislocalization and cone degeneration in the knock-in mice. Importantly, PBA-treatment also improved cone-mediated vision in the mutant mice. These results indicate that PBA, a U.S. Food and Drug Administration-approved safe oral medication, may provide a noninvasive therapeutic intervention that delays daylight vision loss in patients with RPE65 mutations.

LCA is a severe early onset retinal dystrophy. Recent clinical trials of gene therapy have implicated the need of an alternative or combination therapy to improve cone survival and function in patients with LCA caused by RPE65 mutations. Using a mouse model carrying the most frequent LCA-associated mutation (R91W), we found that the mutant RPE65 underwent ubiquitination-dependent proteasomal degradation due to misfolding. Treatment of the mice with a chemical chaperone partially corrected stability, enzymatic activity, and subcellular localization of R91W RPE65, which was also accompanied by improvement of cone survival and vision. These findings identify an in vivo molecular pathogenic mechanism for R91W mutation and provide a feasible pharmacological approach that can delay vision loss in patients with RPE65 mutations.

OBJECTIVE

To present detailed phenotypic and molecular findings in four patients from four families with atypical, mild, recessive RPE65-related retinal dystrophy and discuss potential implications for gene replacement therapy.

METHODS

Four patients from four families with early onset retinal dystrophy underwent clinical examination, retinal imaging and electrophysiological testing. Bidirectional Sanger sequencing of all exons and intron-exon boundaries of RPE65 was performed.

RESULTS

All patients presented with nyctalopia in early childhood but demonstrated a mild phenotype with good visual acuity until at least 19 years of age. All had generalised retinal dysfunction on electroretinography. Central macular thickness on optical coherence tomography was preserved in those patients with good visual acuity. One patient had extensive white dots throughout the retina reminiscent of fundus albipunctatus with electrophysiological evidence of partial recovery of rod function after prolonged dark adaptation. Sanger sequencing identified RPE65 mutations in all patients including three missense variants likely to represent hypomorphic alleles.

CONCLUSIONS

Hypomorphic mutations of RPE65 are associated with mild disease in childhood with preservation of good visual acuity into adulthood; they may in rare cases be associated with a flecked retina appearance similar to fundus albipunctatus. The presence of normal visual acuity in patients with hypomorphic mutations in RPE65 suggests that efficiency of transduction may not be the limiting factor in improving visual acuity in trials of gene replacement therapy. Rather, it suggests that for optimal recovery of visual acuity gene replacement therapy may need to be given much earlier in childhood.

BACKGROUND

The RPE65 gene was recently described to cause autosomal dominant retinitis pigmentosa (adRP), presenting with a phenotype resembling choroideremia. This study presents the 2-year progression of RPE65 adRP in a patient.

METHODS

This is an observational case report of one patient. The patient received a full ophthalmic examination during both visits, including diagnostic imaging such as spectral domain optical coherence tomography (SD-OCT), OCT-angiography (OCT-A), short-wave fundus autofluorescence (FAF), and fundus photography. Genetic characterization was obtained by DNA sequencing from peripheral blood lymphocytes obtained during the first visit.

RESULTS

RPE65 adRP phenocopied choroideremia at the initial fundoscopy. Upon the patient's return to our clinic 2 years later, DNA sequencing revealed a heterozygous mutation in the RPE65 gene. Diagnostic imaging by SD-OCT and FAF suggested disease progression. In conjunction with clinical examination and imaging, the diagnosis was revised to adRP caused by RPE65.

CONCLUSIONS

adRP due to a mutation in the gene encoding RPE65 phenocopied choroideremia. Based on our analysis of the 2-year disease progression in this patient, RPE65 adRP is mild and has a slow rate of disease progression.

Recessive mutations in RLBP1 cause a form of retinitis pigmentosa in which the retina, before its degeneration leads to blindness, abnormally slowly recovers sensitivity after exposure to light. To develop a potential gene therapy for this condition, we tested multiple recombinant adeno-associated vectors (rAAVs) composed of different promoters, capsid serotypes, and genome conformations. We generated rAAVs in which sequences from the promoters of the human RLBP1, RPE65, or BEST1 genes drove the expression of a reporter gene (green fluorescent protein). A promoter derived from the RLBP1 gene mediated expression in the retinal pigment epithelium and Müller cells (the intended target cell types) at qualitatively higher levels than in other retinal cell types in wild-type mice and monkeys. With this promoter upstream of the coding sequence of the human RLBP1 gene, we compared the potencies of vectors with an AAV2 versus an AAV8 capsid in transducing mouse retinas, and we compared vectors with a self-complementary versus a single-stranded genome. The optimal vector (scAAV8-pRLBP1-hRLBP1) had serotype 8 capsid and a self-complementary genome. Subretinal injection of scAAV8-pRLBP1-hRLBP1 in Rlbp1 nullizygous mice improved the rate of dark adaptation based on scotopic (rod-plus-cone) and photopic (cone) electroretinograms (ERGs). The effect was still present after 1 year.

Most of the genes causing autosomal recessive retinitis pigmentosa (AR-RP) are rare and cause 1% of all cases. Some of the genes, like PDE6 (PDE6A, PDE6B, PDE6G), RP25, and RPE65, have higher prevalence, about 2-5% of all cases. Overall, autosomal recessive RP accounts for about 15-20% of all cases of RP. Clinically, it shows all the classic features of RP, such as attenuated retinal blood vessels, intraretinal pigmentation, waxy pallor of the optic disc, and hyperfluorescent rings on fundus autofluorescence (FAF) (Figs. 25.1, 25.2 and 25.3). The ring is suggestive of increased metabolic burden of the corresponding retinal pigment epithelium (RPE).

The retinal pigment epithelium (RPE)-dependent visual cycle provides 11-cis-retinal to opsins in the photoreceptor outer segments to generate functional visual pigments that initiate phototransduction in response to light stimuli. Both RPE65 isomerase of the visual cycle and the rhodopsin visual pigment have recently been identified as critical players in mediating light-induced retinal degeneration. These findings suggest that the expression and function of RPE65 and rhodopsin need to be coordinately controlled to sustain normal vision and to protect the retina from photodamage. However, the mechanism controlling the development of the retinal visual system remains poorly understood. Here, we show that deficiency in ciliary neurotrophic factor (CNTF) up-regulates the levels of rod and cone opsins accompanied by an increase in the thickness of the outer nuclear layers and the lengths of cone and rod outer segments in the mouse retina. Moreover, retinoid isomerase activity, expression levels of RPE65 and lecithin:retinol acyltransferase (LRAT), which synthesizes the RPE65 substrate, were also significantly increased in the Cntf -/- RPE. Rod a-wave and cone b-wave amplitudes of electroretinograms were increased in Cntf -/- mice, but rod b-wave amplitudes were unchanged compared with those in WT mice. Up-regulated RPE65 and LRAT levels accelerated both the visual cycle rate and recovery rate of rod light sensitivity in Cntf -/- mice. Of note, rods and cones in Cntf -/- mice exhibited hypersusceptibility to light-induced degeneration. These results indicate that CNTF is a common extracellular factor that prevents excessive production of opsins, the photoreceptor outer segments, and 11-cis-retinal to protect rods and cones from photodamage.

OBJECTIVE

To investigate the pathogenic potential and sites of retinal pigment epithelium-specific 65-kDa protein (RPE65) for inducing experimental autoimmune uveitis (EAU) in Lewis rats.

METHODS

Twenty-six peptides were chemically synthesized based on the amino acid sequences of human RPE65. These peptides spanned the entire RPE65 sequence. Each peptide was injected into a footpad and the peritoneum of Lewis rats. The eyes were examined by slit-lamp biomicroscopy, and the findings were correlated with the histological findings. The serum antibody titer and lymphocyte reactivity against each peptide was also determined by enzyme-liked immunosorbent assay (ELISA) and lymphocyte proliferation assay, respectively.

RESULTS

Active immunization of rats resulted in the induction of EAU with 14 (3 severe and 11 mild) of the 26 peptides. The clinical course of the EAU was similar to that induced by the injection of retinal antigens such as S-antigen or inter-photoreceptor retinoid binding protein (IRBP). However, the histopathologic changes differed from the EAU induced by these retinal antigens. The inflammation was induced mainly from the retinal pigment epithelium (RPE) and the choroid, while the retina was relatively well-preserved except for some granulomatous changes adjacent to the RPE.

CONCLUSIONS

Active immunization with peptides making up RPE65 will induce EAU. RPE65 has multiple EAU-inducing sites for Lewis rats.

Pigment 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-MSCs^PEDF, PEDF+) using a nonviral gene delivery system and evaluated the characteristics of PD-MSCs^PEDF and their potential regenerative effects on RPE cells damaged by H₂O₂-induced OS. PD-MSCs^PEDF maintained their stemness, cell surface marker, and differentiation potential characteristics. Compared to naive cells, PD-MSCs^PEDF 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-MSCs^PEDF 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-MSCs^PEDF. Furthermore, OS-damaged RPE cells cocultured with PD-MSCs^PEDF 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-MSCs^PEDF alleviated proinflammatory cytokines and restored retinal structure and function by increasing PEDF expression and decreasing VEGF expression. Intravitreal administration of PD-MSCs^PEDF 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.