Previous studies have tested gene replacement therapy in RPE65 deficient dogs using recombinant adeno-associated virus 2/2 (rAAV2/2), -2/1 or -2/5 mediated delivery of the RPE65 gene. They all documented restoration of dark- and light-adapted ERG responses and improved psychophysical outcomes. Use of a specific RPE65 promoter and a rAAV vector that targets transgene expression specifically to the retinal pigmented epithelium (RPE) may, however, provide a safer setting for the long-term therapeutic expression of RPE65. Subretinal injection of rAAV2 pseudotyped with serotype 4 (rAAV2/4) specifically targets the RPE. The purpose of our study was to evaluate a rAAV2/4 vector carrying a human RPE65cDNA driven by a human RPE65 promoter, for the ability to restore vision in RPE65-/- purebred Briard dogs. Recombinant rAAV2/4 and rAAV2/2 vectors containing similar human RPE65 promoter and cDNA cassettes were generated and administered subretinally in 8 affected dogs, ages 8 to 30 months (n = 6 with rAAV2/4, n = 2 with rAAV2/2). Although fluorescein angiography and OCT examinations displayed retinal abnormalities in treated retinas, electrophysiological analysis demonstrated that restoration of rod and cone photoreceptor function started as soon as 15 days post-injection, reaching maximal function at 3 months post-injection, and remaining stable thereafter in all animals treated at 8 to 11 months of age. As assessed by the ability of these animals to avoid obstacles in both dim and normal light, functional vision was restored in the treated eye, while the untreated contralateral eye served as an internal control. The dog treated at a later age (30 months) did not recover retinal function or vision, suggesting that there might be a therapeutic window for the successful treatment of RPE65 -/- dogs by gene replacement therapy.

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Inherited retinal diseases are the leading cause of sight impairment in people of working age in England and Wales, and the second commonest in childhood. Gene therapy offers the potential for benefit.

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Pubmed and clinicaltrials.gov.

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Gene therapy can improve vision in RPE65-associated Leber Congenital Amaurosis (RPE65-LCA). Potential benefit depends on efficient gene transfer and is limited by the extent of retinal degeneration.

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The magnitude of vision improvement from RPE65-LCA gene therapy is suboptimal, and its durability may be limited by progressive retinal degeneration.

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The safety and potential benefit of gene therapy for inherited and acquired retinal diseases is being explored in a rapidly expanding number of trials.

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Developments in vector design and delivery will enable greater efficiency and safety of gene transfer. Optimization of trial design will accelerate reliable assessment of outcomes.

As our understanding of the genetic basis for inherited retinal disease has expanded, gene therapy has advanced into clinical development. When the gene mutations associated with inherited retinal dystrophies were identified, it became possible to create animal models in which individual gene were altered to match the human mutations. The retina of these animals were then characterized to assess whether the mutated genes produced retinal phenotypes characteristic of disease-affected patients. Following the identification of a subpopulation of patients with the affected gene and the development of techniques for the viral gene transduction of retinal cells, it has become possible to deliver a copy of the normal gene into the retinal sites of the mutated genes. When this was performed in animal models of monogenic diseases, at an early stage of retinal degeneration when the affected cells remained viable, successful gene augmentation corrected the structural and functional lesions characteristic of the specific diseases in the areas of the retina that were successfully transduced. These studies provided the essential proof-of-concept needed to advance monogenic gene therapies into clinic development; these therapies include treatments for: Leber's congenital amaurosis type 2, caused by mutations to RPE65, retinoid isomerohydrolase; choroideremia, caused by mutations to REP1, Rab escort protein 1; autosomal recessive Stargardt disease, caused by mutations to ABCA4, the photoreceptor-specific ATP-binding transporter; Usher 1B disease caused by mutations to MYO7A, myosin heavy chain 7; X-linked juvenile retinoschisis caused by mutations to RS1, retinoschisin; autosomal recessive retinitis pigmentosa caused by mutations to MERTK, the proto-oncogene tyrosine-protein kinase MER; Leber's hereditary optic neuropathy caused by mutations to ND4, mitochondrial nicotinamide adenine dinucleotide ubiquinone oxidoreductase (complex I) subunit 4 and achromatopsia, caused by mutations to CNGA3, cyclic nucleotide-gated channel alpha 3 and CNGB3, cyclic nucleotide-gated channel beta 3. This review includes a tabulated summary of treatments for these monogenic retinal dystrophies that have entered into clinical development, as well as a brief summary of the preclinical data that supported their advancement into clinical development.

To characterize the course of sodium iodate (SI)-induced retinal degeneration in young adult albino and pigmented mice.

Single intraperitoneal (IP) injections of SI (25, 50, and 100 mg/kg) were performed in 7- to 8-week-old BALB/c and C57Bl/6J mice. Retinal function and structure was assessed at baseline, 24 hours, 3 days, 1, 2, 3, and 4 weeks postinjection by optokinetic tracking response, ERG, optical coherence tomography (OCT), and histologic and immunohistochemical techniques.

The 50 mg/kg SI dosage was selected after dose ranging due to consistent retinal effects and lack of systemic toxicity. Time-dependent deterioration in retinal function and morphology was consistently observed between 1 and 4 weeks in all measured parameters. These include reduction of ERG responses, thinning of retinal layers as observed by OCT and histology, and loss of RPE nuclei. Immunohistochemistry revealed rapid RPE disorganization with loss of tight junctions and markedly reduced expression of RPE65 and rod opsin, accompanied by mislocalization of cone opsins. Earlier time points displayed variable results, including partial recovery of visual acuity at 1 week and supranormal ERG cone responses at 24 hours, suggesting possible limitations of early intervention and assessment in the SI model.

A single IP injection of 50 mg/kg SI leads to severe RPE injury followed by vision impairment, dysfunction, and loss of photoreceptors in both BALB/c and C57Bl/6J mice. This easily induced and reproducible noninherited model may serve as a useful tool for seeking and evaluating novel therapeutic modalities for the treatment of retinal degenerations caused by primary failure of the RPE.

Geographic atrophy (GA) is the late stage of non-neovascular age-related macular degeneration. A lack of animal models for GA has hampered treatment efforts. Presented herein is a rat model for GA using subretinal injection of sodium iodate (NaIO3).

Rats were given subretinal injections of NaIO3 (5 μg/μL) using a pico-injector. Fundus photographs and spectral domain optical coherent tomography scans were collected at 1, 3, 7, 14, and 28 days after injection, at which time rats were euthanized and eyes were enucleated. Eyes were either cryopreserved or dissected into retinal and choroidal flatmounts. Fluorescence immunohistochemistry was performed for retinal glial fibrillary acidic protein (activated Müller cells and astrocytes) and vimentin (Müller cells), as well as peanut agglutin lectin (photoreceptors) labeling. RPE/choroids were labeled for RPE65 and CD34. Images were collected on Zeiss confocal microscopes.

Fundus photos, spectral domain optical coherent tomography, and RPE65 staining revealed well-demarcated areas with focal loss of RPE and photoreceptors in NaIO3-treated rats. At 1 day after injection, RPE cells appeared normal. By 3 days, there was patchy RPE and photoreceptor loss in the injected area. RPE and photoreceptors were completely degenerated in the injected area by 7 days. A large subretinal glial membrane occupied the degenerated area. Choriocapillaris was highly attenuated in the injected area at 14 and 28 days.

The rat model reported herein mimics the photoreceptor cell loss, RPE atrophy, glial membrane formation, and choriocapillaris degeneration seen in GA. This model will be valuable for developing and testing drugs and progenitor cell regenerative therapies for GA.

Biallelic mutations in the RPE65 gene are associated with inherited retinal degenerations/dystrophies (IRD) and disrupt the visual cycle, leading to loss of vision. A new adenoviral vector-based gene therapy surgically delivered to retinal cells provides normal human RPE65 protein that can restore the visual cycle and some vision. To view this Bench to Bedside, open or download the PDF.

OBJECTIVE

C57/Bl6, Cpfl1(-/-) (cone photoreceptors function loss 1; pure rod function), Gnat1a(-/-) (rod α-transducin; pure cone function), and Rpe65(-/-);Rho(-/-) double-knockout mice were studied to distinguish the respective contributions of the different photoreceptor (PR) systems that enable light perception and mediate a visual reflex in adult Rpe65(-/-) mice, with a simple behavioral procedure.

METHODS

Visual function was estimated using a rotating automated optomotor drum covered with vertical black-and-white stripes at spatial frequencies of 0.025 to 0.5 cycles per degree (cyc/deg) in both photopic and scotopic conditions. Mouse strains with different luminances were tested to evaluate the contribution and the light-intensity threshold of each PR system.

RESULTS

Stripe rotation elicited head movements in the wild-type (WT) animals in photopic and scotopic conditions, depending on the spatial frequency, whereas the Cpfl1(-/-) mice show a reduced activity in the photopic condition and the Gnat1a(-/-) mice an almost absent response in the scotopic condition. A robust visual response was obtained with Rpe65(-/-) knockout mice at 0.075 and 0.1 cyc/deg in the photopic condition. The residual rod function in the Rpe65(-/-) animals was demonstrated by testing Rpe65(-/-);Rho(-/-) mice that present no response in photopic conditions.

CONCLUSIONS

The optomotor test is a simple method of estimating the visual function and evaluating the respective contributions of rod and cone systems. This test was used to demonstrate that in Rpe65(-/-) mice, devoid of functional cones and of detectable 11-cis-retinal protein, the rods mimic cone function in part, by mediating vision in photopic conditions.

The protein RPE65 is essential for the generation of the native chromophore, 11-cis retinal, of visual pigments. However, the Rpe65 knockout (Rpe65-/-) mouse shows a minimal visual response due to the presence of a pigment, isorhodopsin, formed with 9-cis retinal. Isorhodopsin accumulates linearly with prolonged dark-rearing of the animals. The majority of Rpe65-/- mice have an agouti coat color. A tan coat color subset of Rpe65-/- mice was found to have an enhanced visual response as measured by electroretinograms. The enhanced response was found to be due to increased levels of 9-cis retinal and isorhodopsin pigment levels. Animals of both coat colors reared in cyclic light have minimal levels of regenerated pigment and show photoreceptor degeneration. On dark-rearing, pigment accumulates and photoreceptor degeneration is decreased. In the tan Rpe65-/- mice, the level of photoreceptor degeneration is less than in the agouti animals, which have an increased pigment and decreased free opsin level. Therefore, photoreceptor damage correlates with the amount of the apoprotein present, supporting findings that the activity from unregenerated opsin can lead to photoreceptor degeneration.

Rpe65(-/-) mice are unable to produce 11-cis-retinal, the chromophore of visual pigments. Consequently, the pigment is present as the apoprotein opsin with a minute level of pigment containing 9-cis-retinal as chromophore. Notably, a 10-20% fraction of this opsin is mono-phosphorylated independently of light conditions. To determine the role of rhodopsin kinase (GRK1) in phosphorylating this opsin and to test whether eliminating this phosphorylation would accelerate photoreceptor degeneration, we generated the Rpe65(-/-)Grk1(-/-) mouse. The retinae of Rpe65(-/-)Grk1(-/-) mice had negligible opsin phosphorylation, extensive degeneration with decreased opsin levels, and diminished light-evoked rod responses relative to Rpe65(-/-) mice. These data show that opsin phosphorylation in the Rpe65(-/-) mouse is due to the action of GRK1 and is neuroprotective. However, despite the higher activity of unphosphorylated opsin, the severe loss of opsin in the rapidly degenerating Rpe65(-/-)Grk1(-/-) mice resulted in lower overall opsin activity and in higher rod sensitivity compared with Rpe65(-/-) mice. In Rpe65(-/-)Grk1(-/-)Gnat1(-/-) mice where transduction activation was blocked, degeneration was only partially prevented. Therefore, increased opsin activity in the absence of phosphorylation was not the only mechanism for the accelerated retinal degeneration. Finally, the deletion of GRK1 triggered retinal degeneration in Grk1(-/-) mice after 1 month, even in the absence of apo-opsin. This degeneration was independent of light conditions and occurred even in the absence of transducin in Grk1(-/-)Gnat1(-/-) mice. Taken together, our results demonstrate a light-independent mechanism for retinal degeneration in the absence of GRK1, suggesting a second, not previously recognized role for that kinase.

OBJECTIVE

Tests of vision for mice remain limited and the visual phenotype of some retinal disorders in mice remain poorly understood. A novel assay of vision was used to determine how the form and extent of retinal disease affects visual phenotype in mice.

METHODS

Retinal histology, the suppression of locomotion by light and visual guidance of locomotion, were assessed in mice with progressive photoreceptor degeneration (rd/rd) or visual cycle dysfunction (Rpe65 rd12).

RESULTS

In wild-type mice, there was visual guidance of locomotor activity in dim light and suppression of activity (negative masking) in bright light. In rd/rd mice, vision was sufficient to guide locomotion at postnatal day (P)34 but was lost from P46 onward. In bright light rd/rd mice had enhanced negative masking. Although Rpe65 rd12 mice had no dim light response, with high illumination, vision was sufficient to guide locomotion at all ages tested.

CONCLUSIONS

A major concern for gene and cell replacement therapies is the development of visual pathways through which restored retinal function can connect to visual centers of the brain. The residual retinal response to high illumination in Rpe65 rd12 mice translates into useful vision, and visual pathways remain functional--a prerequisite for restoring vision in disorders of the retina. Similarly, useful vision in young rd/rd mice shows that there is visual pathway function before photoreceptor degeneration and suggests the potential for early therapy. Together, these findings recommend observation of masking responses in the assessment of gene and cell replacement therapies.

In HEK293S cells expressing opsin, rhodopsin regenerates on addition of all-trans retinol. This study was to determine if key proteins in the retinal pigment epithelium (RPE) are present in these cells. Cellular retinoid binding protein, cellular retinoic-acid binding protein, RPE65, caveolin-1-alpha- and -beta-isoforms, interphotoreceptor retinoid binding protein, and 11-cis retinol dehydrogenase, but not lecithin:retinol acyltransferase (LRAT), were identified by Western blot analysis. LRAT transcripts were found by RT-PCR and Southern blot analysis. Small interference RNA specific to LRAT reduced ester formation, confirming that the enzyme is present. Therefore, HEK293S cells contain the functional components of the retinoid cycle found in the RPE.

Primary cultures of human retinal pigment epithelium (RPE) requires young human donors with short post-mortem time and no known retinal diseases. The use of an established human RPE cell line, like ARPE-19, would be a welcomed alternative to primary cultures. This cell line retains many of the characteristics of RPE cells, including cell morphology, functional tight junctions and expression of CRALBP and RPE65. This study was conducted in order to investigate integrin alpha5 expression at both the gene and protein level in the ARPE-19 cell line and compare the results with those obtained with primary cultures of RPE cells. The potential use of this cell line as a substitute for primary cultures of RPE cells was also considered. Integrin alpha5 protein was detected on RPE and ARPE-19 cultures at different confluencies by immunofluorescence and immunoprecipitation analyses. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to study alpha5 mRNA levels. Transient transfections were performed in order to compare alpha5 promoter strength in both types of cells. Immunofluorescence studies showed that both primary RPE and ARPE-19 cells yielded similar alpha5 staining patterns at all cell confluencies. Both immunoprecipitation and RT-PCR analyses provided evidence that sub-confluent and confluent RPE and ARPE-19 cells have similar cell surface alpha5 protein and mRNA levels whereas post-confluent cells had a marked decrease in both protein and transcript levels. ARPE-19 cells show a large increase in promoter strength compared to primary cultures. When compared to primary cultures, the cell line exhibited major differences in the way the alpha5 promoter is regulated, even if both cell types are cultured under identical conditions. This study demonstrates that primary cultures of human RPE and ARPE-19 cells show reductions in both the alpha5 protein and the mRNA when cells reach post-confluency. However, major differences have been observed in the strength of the alpha5 promoter between both cell types. We also show that culturing ARPE-19 cells in a different growth medium alters the transcriptional activity directed by the alpha5 promoter.

Disturbance in vitamin A metabolism seems to be an important attribute of cancer cells. Retinoids, particularly retinoic acid, have critical regulatory functions and appear to modulate tumor development and progression. The key step of vitamin A metabolism is the esterification of all-trans retinol, catalyzed by lecithin/retinol acyltransferase (LRAT). In this work, we show that malignant melanoma cells are able to esterify all-trans retinol and subsequently isomerize all-trans retinyl esters (RE) into 11-cis retinol, whereas their benign counterparts-melanocytes are not able to catalyze these reactions. Besides, melanoma cell lines express lecithin/retinol acyltranseferase both at the mRNA and protein levels. In contrast, melanocytes do not express this enzyme at the protein level, but mRNA of lecithin/retinol acyltransefrase could still be present at mRNA level. RPE65 is expressed in both melanocytic counterparts, and could be involved in the subsequent isomerization of RE produced by lecithin/retinol acyltransefrase to 11-cis retinol. Cellular retinol-binding protein 2 does not appear to be involved in the regulation of all-trans retinol esterification in these cells. Expression of LRAT and RPE65 can be modulated by retinoids. We propose that the post-transcriptional regulation of lecithin/retinol acyltransefrase could be involved in the differential expression of this enzyme. Besides, activities of LRAT and RPE65 may be important for removal of all-trans retinal which is the substrate for retinoic acid production in skin cells. Consequently, the decreasing cellular amount of retinoic acid and its precursor molecules could result in a change of gene regulation.

Leber congenital amaurosis (LCA) is a clinically and genetically heterogeneous severe retinal dystrophy presenting in infancy. To explain the phenotypical variability observed in two affected siblings of a consanguineous pedigree diagnosed with LCA and establish a genotype-phenotype correlation, we screened GUCY2D, RPE65, CRX, AIPL1, and RPGRIP1 for mutations. The more severely affected sibling carried a heterozygous missense mutation in the GUCY2D gene (Ile539Val), which did not segregate with the disease phenotype. Subsequently, a homozygous nonsense mutation (Glu102STOP) in the RPE65 gene was identified in both affected siblings, thus identifying the causative gene. This data provides evidence for the presence of genetic modulation in LCA. It appears that the heterozygous GUCY2D mutation further disrupts the already compromised photoreceptor function resulting in more severe retinal dysfunction in the older sibling. We suggest that the unusual phenotypic variability in these two siblings with LCA is caused by the modifying effect of a heterozygous GUCY2D mutation observed against the disease background of a homozygous RPE65 mutation.

OBJECTIVE

The C57BL/6-c(2J) (c2J) mouse strain has been shown in earlier studies to be highly resistant to light damage. Subsequent studies related this resistance to an amino acid substitution (leu450met) in a pigment epithelial enzyme (RPE65), which slowed the rate of rhodopsin regeneration. The present study was conducted to examine patterns of photoreceptor death, electrophysiological function (the ERG) and trophic factor expression over the life of the C57BL/6-c(2J) retina.

METHODS

Observations were made on two C57BL/6J-c(2J) substrains, one albino (Tyr/Tyr) and one pigmented (Tyr/(+)), and two nondegenerative strains, one albino (BALB/cJ) and one pigmented (C57BL/6J). Mice were raised in dim cyclic light (12 hours at 5 lux, 12 hours in the dark), and a developmental series of retinas of each strain was taken between postnatal day (P)4 and (P365(+)). Retinas were examined for cell death by using the TUNEL technique, stress-induced protein expression (FGF-2 and GFAP), and measures of retinal thickness. The dark-adapted ERG was recorded in dark-adapted conditions in early adulthood (13-15 weeks) and late adulthood (>1 year).

RESULTS

In both C57BL/6-c(2J) substrains, the retina showed marked degenerative features when compared with two control strains, BALB/cJ (leucine at codon 450 in RPE65) and C57BL/6J (methionine). During development and into young adulthood, photoreceptor death rates were abnormally high, levels of two stress-inducible proteins (FGF-2 and GFAP) were abnormally high, and the ERG (electroretinogram) was significantly reduced in amplitude (<50% of values in BALB/cJ or C57BL/6J). The rate of photoreceptor death remained abnormally high into young adulthood (2-3 months) but decreased to control levels by 1 year. Accordingly, the thickness of the outer nuclear layer and the ERG were stable over the same period.

CONCLUSIONS

Results suggest that a still-unidentified stress increases photoreceptor death in the C57BL/6-c(2J) retina during the critical period of photoreceptor development and into young adulthood, upregulates stress-inducible factors, and markedly limits the amplitude of the ERG. These degenerative changes do not continue after early adulthood, the retina remaining stable in structure and function into late adulthood. The degenerative changes were apparent in both albino and pigmented C57BL/6-c(2J) substrains. Their genetic cause remains unknown.

OBJECTIVE

To identify retinoids and retinoid processing proteins in the ocular ciliary epithelium (CE), and to compare in cultured ciliary epithelial cell lines promoter activities of the cellular retinaldehyde binding protein (CRALBP) and interphotoreceptor retinoid binding protein (IRBP).

METHODS

Retinoid processing proteins were detected by RT-PCR, western analysis and immunocytochemistry. PCR products were verified by DNA sequence analysis. Retinoids were measured by normal phase HPLC and UV visible spectroscopy. Reporter product from CRALBP and IRBP promoter fragments was measured following transient transfection in bovine pigmented and nonpigmented CE cells.

RESULTS

CRALBP, IRBP, cellular retinol binding protein (CRBP), 11-cis-retinol dehydrogenase (11-cis-RDH), lecithin:retinol acyltransferase (LRAT), and ATP binding cassette receptor (ABCR) were detected in human CE tissue by RT-PCR. Retinal pigment epithelium specific protein 65 kDa (RPE65) mRNA and protein were also detected. CRALBP and IRBP were detected by western analysis in tissue extracts from bovine CE and were localized to the PE and NPE cell layers, respectively, by immunocytochemistry. IRBP immunoreactivity was also detected in aqueous humor. Retinoids identified in the bovine CE include retinyl esters (7.4+/-3.5 pMol/mg of protein) and all-trans-retinol (14.9+/-1.1 pMol/mg of protein). Betacarotene was also tentatively identified. 11-cis-Retinoids were not detected. In CE cell cultures, the CRALBP p2.1-kb promoter construct exhibited reporter activity 15-30 fold above basal level, with 2 fold more activity in pigmented than nonpigmented CE cells. IRBP promoter constructs exhibited low level reporter activities in vitro in both CE cell layers.

CONCLUSIONS

The ocular CE expresses genes encoding components of the rod visual cycle. The differential localization of CRALBP and IRBP along the bilayer of the CE suggests a potential role in retinoid transport and/or retinoid metabolism. However, the absence of 11-cis-retinoids suggests that the function of retinoid processing proteins in the CE differs from that of the retina.

Compared with many induced pluripotent stem cell (iPSC) lines generated using retrovirus and other non-integrating methods, the utilization of human protein-induced iPSC (piPSC) lines may provide a safer alternative for the generation of retinal pigment epithelial (RPE) cells for transplantation in retinal degenerative diseases. Here we assess the ability of piPSCs to differentiate into RPE cells, and to perform native RPE cell behavior. piPSCs were seeded in 6-well low-attachment plates to allow embryoid body formation, and then analyzed for pluripotent stem cell markers NANOG, SSEA4 and TRA-1-60 by immunofluorescence. Following colony formation, piPSCs were assessed for confirmation of RPE cell differentiation by staining for zonula occludens (ZO-1), bestrophin, microphthalmia-associated transcription factor (MITF) and retinal pigment epithelium specific protein-65 (RPE65). To evaluate piPSC-RPE cell phagocytic ability, adult bovine photoreceptor rod outer segments (ROS) were fed to piPSC-RPE cells, which were analyzed by fluorescent microscopy and flow cytometry. Undifferentiated piPSCs expressed all pluripotent markers assessed and formed embryoid body aggregates after 7 days. Differentiated piPSC-RPE cells expressed ZO-1, bestrophin, MITF and RPE65, typical RPE cell markers. Flow cytometry revealed robust ingestion of fluorescently-labeled ROS by piPSC-RPE cells, which was over four-times greater than that of undifferentiated piPSCs and comparable to that of an immortalized RPE cell line. Phagocytosis activity by piPSC-RPE cells was significantly reduced after the addition of anti-integrin αVβ5. In conclusion, piPSCs can be differentiated toward an RPE cell fate, expressing RPE cell markers and resembling native RPE cells in behavior. These results demonstrate that piPSCs can be differentiated into RPE-like cells using a method that has an increased safety profile, a critical consideration for the development of better treatments for retinal degenerative diseases such as age-related macular degeneration (AMD).

The retinal pigment epithelium (RPE) is a highly specialized cell type located between the choroid and neural retina of the eye. RPE degeneration causes irreversible visual impairment, extending to blindness. Cell therapy has recently emerged as a potential therapeutic approach for retinal degeneration. MicroRNA-based differentiation of stem cells is a new strategy for producing tissue-specific cell types. In this study, we developed a novel microRNA-based strategy for RPE induction from human amniotic epithelial stem cells (AESCs). We identified microRNAs involved in RPE development in AESCs. Of 29 putative human RPE-relevant microRNAs, microRNA-410 (miR-410) was predicted to target multiple RPE development-relevant genes. Inhibition of miR-410 induces overexpression of immature and mature RPE-specific factors, including OTX2, RPE65, Bestrophin and EMMPRIN. These RPE-like cells were morphologically altered toward a cobblestone-like shape and were able to phagocytize microbeads. We showed that miR-410 directly regulates predicted target genes OTX2 and RPE65. Our microRNA-based strategy demonstrated RPE differentiation in AESCs by treatment of an antisense microRNA-410 (anti-miR-410), without the use of additional factors or exogenous transduction. These findings suggest that miR-410 inhibition can be a useful tool for directed cell differentiation and an attractive method for cell therapy in human retinal degenerative diseases.

OBJECTIVE

The use of canine models of retinal disease in the development of therapeutic strategies for inherited retinal disorders is a growing area of research. To evaluate accurately the success of potential vision-enhancing treatments, reliable methods for objectively assessing visual function in canine models is necessary.

METHODS

A simple vision-testing device was constructed that consisted of a junction box with four exit tunnels. Dogs were placed in the junction box and given one vision-based choice for exit. The first-choice tunnel and time to exit were recorded and analyzed. Two canine models of retinal disease with distinct molecular defects, a null mutation in the gene encoding the alpha subunit of rod cyclic GMP phosphodiesterase (PDE6A), and a null mutation in the gene encoding a retinal pigment epithelium-specific protein (RPE65) were tested and compared to those in unaffected dogs.

RESULTS

With the use of bright light versus dim red light, the test differentiated between unaffected dogs and dogs affected with either mutation with a high degree of certainty. The white-light intensity series showed a significantly different performance between the unaffected and affected dogs. A significant difference in performance was detected between the dogs with each mutation.

CONCLUSIONS

The results indicate that this novel canine vision-testing method is an accurate and sensitive means of distinguishing between unaffected dogs and dogs affected with two different forms of inherited retinal disease and should be useful as a means of assessing response to therapy in future studies.

OBJECTIVE

Due to the low abundance of cone photoreceptors in the mouse retina and the scarcity of alternative animal models, little is known about mechanisms of cone degeneration. Nrl knockout mice develop exclusively the cone-type of photoreceptors. However, the cone photoreceptor layer in Nrl(-/-) mice displays an irregular morphology with severe rosette formation. Retinas of Rpe65(-/-);Nrl(-/-) mice have no rosettes due to the lack of 11-cis-retinal, but also are not functional. To develop a model with a functional all-cone retina that is morphologically well structured, we generated R91W;Nrl(-/-) double-mutant mice, which express a hypomorphic Rpe65 allele (R91W).

METHODS

The following analyses were used to characterize the R91W;Nrl(-/-)mice: morphology by light and electron microscopy, protein distribution by immunofluorescence, cone function by electroretinography and optomotor response, RNA levels by RT-PCR, and chromophore levels by HPLC. Cone degeneration was assessed in R91W;Nrl(-/-) mice treated with MNU, and in triple R91W;Nrl(-/-);Cpfl1 and quadruple R91W;Nrl(-/-);Cpfl1;rd10 mutant mice.

RESULTS

The all-cone retina of R91W;Nrl(-/-) mice is functional and relatively stable with only very slow age-related degeneration. Using triple and quadruple mutant mice, or a chemical treatment, we demonstrated that cone degeneration could be induced and analyzed in these mice.

CONCLUSIONS

The reduced levels of visual chromophore prevented rosette formation and sustained function in the R91W;Nrl(-/-) retina. Thus, the R91W;Nrl(-/-) mouse allows study of the etiology of diseases related to cone degeneration in a "morphologically intact" and functional all-cone photoreceptor retina.

OBJECTIVE

Quantifying phenotypic variation at the level of protein expression (variegation) within populations of retinal pigment epithelium (RPE) cells may be important in the study of pathologies associated with this variation. The lack of quantitative methods for examining single cells, however, and the variable presence of pigment and/or lipofuscin complicate this experimental goal. We have applied the technique of laser scanning cytometry (LSC) to paraffin sections of mouse and human eyes to evaluate the utility of LSC for these measurements.

METHODS

Mouse eyes were perfusion fixed in 4% paraformaldehyde and embedded in paraffin. Postmortem human eyes were fixed and dissected to obtain a 9-mm punch, which was then embedded in paraffin. A laser scanning cytometer equipped with violet, argon, and helium-neon lasers and the detectors for blue, green, and long red were used to record the fluorescence of each individual cell at all three wavelengths. Raw data were recorded and processed using the WinCyte software. Individual nuclei were identified by the fluorescence of the 4',6-diamidino-2-phenylindole (DAPI) nuclear counterstain. Next, RPE cells were uniquely identified in the green channel using an anti-retinal pigment epithelium-specific protein 65 kDa (anti-RPE65) monoclonal antibody with an Alexa Fluor 488-labeled secondary antibody. Mn-superoxide dismutase (MnSOD) was quantified in the long-red channel using an anti-MnSOD antibody and an Alexa Fluor 647-labeled secondary antibody. MnSOD(+) and RPE65(+) cells exhibited peaks in the plot of fluorescence intensity versus cell number, which could be characterized by the mean fluorescence intensity (MFI), the coefficient of variation (CV), and the percentage of total RPE cells that were also labeled for MnSOD.

RESULTS

RPE cells can be uniquely identified in human and mouse paraffin sections by immunolabeling with anti-RPE65 antibody. A second antigen, such as MnSOD, can then be probed only within this set of RPE. Results are plotted primarily with the population frequency diagram, which can be subdivided into multiple regions. The data collected for each region include the MFI, the CV, and the number of cells that are immunolabeled in that region. Background interference from pigment or autofluorescent material can be successfully overcome by elevating the concentrations of fluorescent secondary antibodies. In the human and mouse eyes, age-related changes in MFI, CV, and percent RPE cells immunolabeled for MnSOD were observed.

CONCLUSIONS

The extent of the variability of gene expression in RPE cells at the protein level can be quantified by LSC. Relative changes in the MFI, the CV, and/or percentage of RPE cells double labeled for a second antigen quantify the changes observed. The analysis of these data also suggest whether the effects observed are related to local changes in transcription (alterations of CV) or major changes of protein expression (MFI), which are likely to be due to changes in the chromatin structure. The changes of these variables with age suggest that the observed age-related variegation is primarily due to changes in the chromatin structure in individual cells.

BACKGROUND

To evaluate the knockdown of placental growth factor (PlGF) gene expression in human retinal pigment epithelium (RPE) cells and its effect on cell proliferation, apoptosis and angiogenic potential of RPE cells.

METHODS

Human RPE cells were isolated by dispase I solution and cultured in DMEM/F12 supplemented with 10% fetal calf serum (FCS). A small interfering RNA (siRNA) corresponding to PlGF mRNA and a scrambled siRNA (scRNA) were introduced into the cells. Cell proliferation and cell death were examined by ELISA. PlGF mRNA and protein were quantified by real-time polymerase chain reaction (PCR) and western blot. The levels of gene expression for human retinal pigment epithelium-specific protein 65 kDa (RPE65), cellular retinaldehyde-binding protein (CRALBP) and tyrosinase were examined by real-time PCR. The angiogenic activity of RPE cell-derived conditioned media was assayed by a tube formation assay using human umbilical vein endothelial cells (HUVECs).

RESULTS

At a final siRNA concentration of 20 pmol/ml, the transfection efficiency was about 80%. The amount of PlGF transcripts was reduced to 10% after 36 h of incubation, and the amount of PlGF protein in culture supernatant was significantly decreased. Suppression of PlGF gene had no effect on RPE cell proliferation and survival, and there were no notable changes in the transcript levels of RPE65, CRALBP or tyrosinase for the cultures treated by siRNA cognate to PlGF. Vascular tube formation was efficiently reduced in HUVECs.

CONCLUSIONS

Our findings present PlGF as a key modulator of angiogenic potential in RPE cells of the human retina.

OBJECTIVE

The rd12 mouse was reported as a recessively inherited Rpe65 mutation. We asked if the rd12 mutation resides in Rpe65 and how the mutation manifests itself.

METHODS

A complementation test was performed by mating Rpe65(KO) (KO/KO) and rd12 mice together to determine if the rd12 mutation is in the Rpe65 gene. Visual function of wild-type (+/+), KO/+, rd12/+, KO/KO, rd12/rd12, and KO/rd12 mice was measured by optokinetic tracking (OKT) and ERG. Morphology was assessed by retinal cross section. qRT-PCR quantified Rpe65 mRNA levels. Immunoblotting measured the size and level of RPE65 protein. Rpe65 mRNA localization was visualized with RNA fluorescence in situ hybridization (FISH). Fractions of Rpe65 mRNA-bound proteins were separated by linear sucrose gradient fractionation.

RESULTS

The KO and rd12 alleles did not complement. The rd12 allele induced a negative semidominant effect on visual function; OKT responses became undetectable 120 days earlier in rd12/rd12 mice compared with KO/KO mice. rd12/+ mice lost approximately 21% visual acuity by P210. rd12/rd12 mice had fewer cone photoreceptor nuclei than KO/KO mice at P60. rd12/rd12 mice expressed 71% +/+ levels of Rpe65 mRNA, but protein was undetectable. Mutant mRNA was appropriately spliced, exported to the cytoplasm, trafficked, and contained no other coding mutation aside from the known nonsense mutation. Mutant mRNA was enriched on ribosome-free messenger ribonucleoproteins (mRNPs), whereas wild-type mRNA was enriched on actively translating polyribosomes.

CONCLUSIONS

The rd12 lesion is in Rpe65. The rd12 mutant phenotype inherits in a semidominant manner. The effects of the mutant mRNA on visual function may result from inefficient binding to ribosomes for translation.