OBJECTIVE

To identify the functional promoter region and cis-acting elements that regulate the expression of RPE65, the retinal pigment epithelium (RPE)-specific gene responsible for certain forms of autosomal recessive childhood-onset severe retinal dystrophy.

METHODS

A human genomic DNA clone containing the 5'-flanking region of RPE65 was isolated and, 4.0 kb proximal to the transcription start site, was sequenced and analyzed for the presence of transcription factor-binding sites. Promoter activity was assayed by transient transfection of luciferase reporter constructs containing nested deletions of the upstream sequence in the human RPE cell lines ARPE19 and D407, as well as in the SK-Mel-28 and HeLa cell lines. Specific DNA protein-binding sites present in the 340 bp upstream of the transcription start site were identified by DNase I footprint analysis.

RESULTS

Sequence analysis places the polymorphic marker, D1S2803, within the RPE65 upstream region and identifies a number of sequences homologous to the gene encoding the cellular retinaldehyde-binding protein. Functional analysis indicates that basal promoter activity is conferred by the sequence from -83 to +39 and is approximately equivalent in all cell lines tested, with no other control elements detected in 3.6 kb of the upstream sequence. At least eight protected regions are identified in DNase I footprint assays, including sequences corresponding to the predicted TATA box, AP-4, and nuclear factor-1 DNA protein-binding sites.

CONCLUSIONS

These findings localize the basal promoter activity of RPE65, identify potential cis-acting elements that act as positive regulators of gene expression, and suggest that additional regulatory elements are likely to be involved in restricting gene expression to the retinal pigment epithelium. Identification of promoter elements and genetic markers in the upstream sequence will enable the screening of patients with retinal degeneration for possible mutations that affect RPE65 expression.

Inherited eye disorders are a significant cause of vision loss. Genetic testing can be particularly helpful for patients with inherited retinal dystrophies because of genetic heterogeneity and overlapping phenotypes. The need to identify a molecular diagnosis for retinal dystrophies is particularly important in the era of developing novel gene therapy-based treatments, such as the RPE65 gene-based clinical trials and others on the horizon, as well as recent advances in reproductive options. The introduction of massively parallel sequencing technologies has significantly advanced the identification of novel gene candidates and has expanded the landscape of genetic testing. In a relatively short time clinical medicine has progressed from limited testing options to a plethora of choices ranging from single-gene testing to whole-exome sequencing. This article outlines currently available genetic testing and factors to consider when selecting appropriate testing for patients with inherited retinal dystrophies.

The suitability of the rat derived SV-40T immortalized RPE-J cell line for identifying proteome changes associated with RPE differentiation was evaluated by surveying changes in protein expression levels. Rat RPE-J cells were induced to undergo differentiation in culture by growth at the nonpermissive temperature of 40 degrees C in the presence of retinoic acid. Total proteins were extracted from cells grown under proliferating or differentiating conditions and separated by 1D and 2D gel electrophoresis. Gel spots were excised, digested in situ with trypsin, and analysed by mass spectrometry to identify proteins. Computer assisted image analysis was used to align gel patterns and quantify spot intensities. Neither proliferating nor differentiating RPE-J cell cultures exhibited detectable levels of cellular retinaldehyde-binding protein, RPE65, 11- cis -retinol dehydrogenase or lecithin retinol acyl transferase, suggesting that RPE-J cells are not appropriate for visual cycle studies. About 18% of the 61 identified proteins appear to change expression levels with the cell growth conditions. Seven proteins appeared to be up-regulated and four proteins down-regulated when the cells were changed from proliferating to differentiating culture conditions. The majority of the apparent changes in protein expression levels were associated with stress response genes. Significant changes in the apparent mass and charge properties of proteins were also observed and for select proteins, the modifications appeared to be correlated with cell growth conditions. The results demonstrate that proteome differences in RPE-J cells associated with growth conditions can be identified and support the suitability of RPE-J cells for more targeted and/or more global proteome analysis of RPE differentiation.

Over 70 different missense mutations, including a dominant mutation, in RPE65 retinoid isomerase are associated with distinct forms of retinal degeneration; however, the disease mechanisms for most of these mutations have not been studied. Although some mutations have been shown to abolish enzyme activity, the molecular mechanisms leading to the loss of enzymatic function and retinal degeneration remain poorly understood. Here we show that the 26 S proteasome non-ATPase regulatory subunit 13 (PSMD13), a newly identified negative regulator of RPE65, plays a critical role in regulating pathogenicity of three mutations (L22P, T101I, and L408P) by mediating rapid degradation of mutated RPE65s via a ubiquitination- and proteasome-dependent non-lysosomal pathway. These mutant RPE65s were misfolded and formed aggregates or high molecular complexes via disulfide bonds. Interaction of PSMD13 with mutant RPE65s promoted degradation of misfolded but not properly folded mutant RPE65s. Many mutations, including L22P, T101I, and L408P, were mapped on non-active sites. Although their activities were very low, these mutant RPE65s were catalytically active and could be significantly rescued at low temperature, whereas mutant RPE65s with a distinct active site mutation could not be rescued under the same conditions. Sodium 4-phenylbutyrate and glycerol displayed a significant synergistic effect on the low temperature rescue of the mutant RPE65s by promoting proper folding, reducing aggregation, and increasing membrane association. Our results suggest that a low temperature eye mask and sodium 4-phenylbutyrate, a United States Food and Drug Administration-approved oral medicine, may provide a promising "protein repair therapy" that can enhance the efficacy of gene therapy by reducing the cytotoxic effect of misfolded mutant RPE65s.

OBJECTIVE

To perform ocular motility recordings of infantile nystagmus (IN) in RPE65-deficient canines and determine whether they can be used as a motor indicator of restored retinal function to investigate the effects of gene therapy.

METHODS

Treated and untreated canines were comfortably suspended in a custom-built sling and encouraged to fixate on distant targets at gaze angles varying between +/-15 degrees horizontally and +/-10 degrees vertically. Ocular motility recordings were made, using two distinct methods-infrared reflection and high-speed video. The resultant recordings from three untreated, four treated, and three pre- and post-treatment dogs were analyzed for using the eXpanded Nystagmus Acuity Function (NAFX), which yields an objective assessment of best potential visual acuity, based on the duration and repeatable accuracy of foveation and centralisation.

RESULTS

During fixation, the untreated dogs exhibited large-amplitude, classic IN waveforms, including pendular and jerk in both the horizontal and vertical planes, which prevented them from keeping the targets within the area centralis (the region of highest receptor density, spanning +/-3 degrees horizontally by +/-1.5 degrees vertically, analogous to the fovea). Some untreated dogs also had small-amplitude (0.5-1 degrees), high-frequency (6-9 Hz) oscillations. Under the same conditions, successfully treated canines no longer exhibited clinically detectable IN. Their IN was converted to waveforms with very low amplitudes that yielded higher NAFX values and allowed target images to remain well within the area centralis. Of note, uniocular treatment appeared to damp the IN in both eyes. Behaviorally, the treated dogs were able to successfully navigate through obstacles more easily without inadvertent contact, a task beyond the untreated dogs' ability.

CONCLUSIONS

Gene therapy that successfully restored retinal function also reduced the accompanying IN to such a great extent that it was not clinically detectable approximately 90% of the time in many of the dogs. IN improvement, as quantified by the NAFX, is an objective motor indicator of visual improvement due to gene therapy.

Spontaneous canine models exist for several inherited retinal dystrophies. This review will summarize the models and indicate where they have been used in translational gene therapy trials. The RPE65 gene therapy trials to treat childhood blindness are a good example of how studies in dogs have contributed to therapy development. Outcomes in human clinical trials are compared and contrasted with the result of the preclinical dog trials.

Cone photoreceptors mediate visual acuity under daylight conditions, so loss of cone-mediated central vision of course dramatically affects the quality of life of patients suffering from retinal degeneration. Therefore, promoting cone survival has become the goal of many ocular therapies and defining the stage of degeneration that still allows cell rescue is of prime importance. Using the Rpe65(R91W/R91W) mouse, which carries a mutation in the Rpe65 gene leading to progressive photoreceptor degeneration in both patients and mice, we defined stages of retinal degeneration that still allow cone rescue. We evaluated the therapeutic window within which cones can be rescued, using a subretinal injection of a lentiviral vector driving expression of RPE65 in the Rpe65(R91W/R91W) mice. Surprisingly, when applied to adult mice (1 month) this treatment not only stalls or slows cone degeneration but, actually, induces cone-specific protein expression that was previously absent. Before the intervention only part of the cones (40% of the number found in wild-type animals) in the Rpe65(R91W/R91W) mice expressed cone transducin (GNAT2); this fraction increased to 64% after treatment. Correct S-opsin localization is also recovered in the transduced region. In consequence these results represent an extended therapeutic window compared to the Rpe65(-/-) mice, implying that patients suffering from missense mutations might also benefit from a prolonged therapeutic window. Moreover, cones are not only rescued during the course of the degeneration, but can actually recover their initial status, meaning that a proportion of altered cones in chromophore deficiency-related disease can be rehabilitated even though they are severely affected.

Carotenoids are plant-derived pigment molecules that vertebrates cannot synthesize de novo that protect the fovea of the primate retina from oxidative stress and light damage. meso-Zeaxanthin is an ocular-specific carotenoid for which there are no common dietary sources. It is one of the three major carotenoids present at the foveal center, but the mechanism by which it is produced in the eye is unknown. An isomerase enzyme is thought to be responsible for the transformation of lutein to meso-zeaxanthin by a double-bond shift mechanism, but its identity has been elusive. We previously found that meso-zeaxanthin is produced in a developmentally regulated manner in chicken embryonic retinal pigment epithelium (RPE)/choroid in the absence of light. In the present study, we show that RPE65, the isomerohydrolase enzyme of the vertebrate visual cycle that catalyzes the isomerization of all-trans-retinyl esters to 11-cis-retinol, is also the isomerase enzyme responsible for the production of meso-zeaxanthin in vertebrates. Its RNA is up-regulated 23-fold at the time of meso-zeaxanthin production during chicken eye development, and we present evidence that overexpression of either chicken or human RPE65 in cell culture leads to the production of meso-zeaxanthin from lutein. Pharmacologic inhibition of RPE65 function resulted in significant inhibition of meso-zeaxanthin biosynthesis during chicken eye development. Structural docking experiments revealed that the epsilon ring of lutein fits into the active site of RPE65 close to the nonheme iron center. This report describes a previously unrecognized additional activity of RPE65 in ocular carotenoid metabolism.

Retinal dystrophies are inherited disorders of photoreceptor and retinal pigment epithelial function that may result in severe visual impairment. Advances in molecular genetics have helped identify many of the gene defects responsible, and progress in gene transfer technology has enabled therapeutic strategies to be developed and applied. The first human clinical trials of gene therapy for RPE65 associated retinal dystrophy have shown promising initial results and have helped prepare the way for further trials of gene therapy for inherited retinal disorders. The results of these trials will provide further insight into the safety and efficacy of gene therapy for a range of currently untreatable and debilitating eye disorders.

Emixustat is a visual cycle modulator that has entered clinical trials as a treatment for age-related macular degeneration (AMD). This molecule has been proposed to inhibit the visual cycle isomerase RPE65, thereby slowing regeneration of 11-cis-retinal and reducing production of retinaldehyde condensation byproducts that may be involved in AMD pathology. Previously, we reported that all-trans-retinal (atRAL) is directly cytotoxic and that certain primary amine compounds that transiently sequester atRAL via Schiff base formation ameliorate retinal degeneration. Here, we have shown that emixustat stereoselectively inhibits RPE65 by direct active site binding. However, we detected the presence of emixustat-atRAL Schiff base conjugates, indicating that emixustat also acts as a retinal scavenger, which may contribute to its therapeutic effects. Using agents that lack either RPE65 inhibitory activity or the capacity to sequester atRAL, we assessed the relative importance of these 2 modes of action in protection against retinal phototoxicity in mice. The atRAL sequestrant QEA-B-001-NH2 conferred protection against phototoxicity without inhibiting RPE65, whereas an emixustat derivative incapable of atRAL sequestration was minimally protective, despite direct inhibition of RPE65. These data indicate that atRAL sequestration is an essential mechanism underlying the protective effects of emixustat and related compounds against retinal phototoxicity. Moreover, atRAL sequestration should be considered in the design of next-generation visual cycle modulators.

Detection of light in the eye underlies image-forming vision, but also regulates adaptive responses in physiology and behavior. Typically these adaptive responses do not involve image-forming vision, but depend on a relatively absolute measure of brightness (nonimage-forming irradiance detection). The goal of this study was to further understand how image-forming vision and nonimage-forming irradiance detection contribute to the effects of light on behavior. Three light dependent behaviors were assessed in wild-type, Rpe65-/- and rd1 mice. In Rpe65-/- mice, nonimage-forming irradiance detection is severely attenuated, but rod based visual acuity is relatively preserved. In rd1 mice visual acuity is nonrecordable, but nonimage-forming responses are less severely attenuated than Rpe65-/-. Positive masking, an image-forming vision dependent increase in wheel running, was absent in rd1 and restricted to higher irradiances in Rpe65-/-. Negative masking, a suppression of wheel running sensitivity with nonimage-forming irradiance detection input, was increased in rd1, but reduced in Rpe65-/- mice. By contrast, light aversion, an avoidance of brightly lit areas, was abolished in both Rpe65-/- and rd1. This shows that image-forming vision is not sufficient for light aversion, suggesting nonimage-forming irradiance detection motivates this behavior. Further, the differing effects of disease suggest that negative masking and light aversion are distinct responses with specialized nonimage-forming irradiance detection pathways.

RPE65 is a potential retinoid-processing protein expressed in the retinal pigment epithelium. Mutations in the RPE65 gene have been shown to cause certain inherited retinal dystrophies. Previous studies have shown that salamander cone photoreceptor cells have a unique retinoid processing mechanism which is distinct from that of rods. To determine whether RPE65 is expressed in photoreceptors, the RPE65 cDNA was cloned from a salamander retinal cDNA library. The deduced protein consists of 533 amino acids and is 85% identical to human and bovine RPE65. The RPE65 mRNA was detected in all of the single cone cells isolated from the salamander retina, as well as in the retinal pigment epithelium by RT-PCR, but not in the isolated rods. The RT-PCR products have been confirmed to be RPE65 by DNA sequencing. The results indicate that this potential retinoid processing protein is expressed in the cone photoreceptor cells but not in rods. Therefore, this protein may contribute to the unique retinoid processing capabilities in salamander cones.

In vivo gene transfer in a large group of RPE65 null mutation dogs have been recently performed. The present study was aimed at determining, through visual behavioral and electroretinographic (ERG) testing, if there is a volume effect of the gene construct administered. Eleven Beagle-Briard dogs homozygous for the RPE65 null mutation and two unaffected control dogs were included. Affected animals were unilaterally treated with either a high (70-100 micro1; N = 6) or a low volume (30-60 microl; N = 5) of subretinally injected rAAV.RPE65 construct, at the age of 4 months to 2.5 years. Fellow eyes were treated with a subretinal injection of rAAV.GFP or sham operated and used as internal controls. Retinal function was measured pre- and 10-12 weeks post-surgically, using simultaneous bilateral full-field flash ERGs. A significant improvement in all ERG responses studied was identified for the high volume treated group compared to pre-surgical parameters. A significant improvement for the high intensity scotopic a-wave response for the low volume rAAV.RPE65 treated group was also found. Objective and subjective dim and day light visual maze testing, in eight of the affected treated animals, and the two control dogs, revealed better vision in daylight than in dim light for all animals. Vision in dogs treated with the high volume of gene construct was significantly better in day light than in dim light. No significant difference was noted between day and dim light testing for the control group or those animals treated with a low volume of the gene construct. Significantly better vision was noted in the control group when compared with the low volume group under dim light conditions, and the high volume group under day light conditions. No significant difference in functional vision could be identified between the high volume treated animals and control animals in day light conditions. These findings support the hypothesis that functional vision is improved by subretinal rAAV.RPE65 injection in a volume-dependent manner.

OBJECTIVE

To develop an hypoxia-regulated retinal pigment epithelium (RPE)-specific adeno-associated virus (AAV) gene transfer platform that exploits hypoxia as a physiologic trigger for an early antiangiogenic treatment strategy directed at arresting neovascularization in the eye.

METHODS

Tissue-specific and hypoxia-regulated expression vectors were constructed with tandem combinations of hypoxia responsive elements and aerobically silenced elements (HRSE) that together induce gene expression in hypoxia and suppress it in normoxia. For RPE-specific expression, the HRSE and a (6x) HRE (hypoxia responsive element) oligomer were ligated upstream of the Rpe65 promoter in a pGL3 firefly luciferase plasmid (pGL3-HRSE-6xHRE-Rpe65). The cell specificity of this novel hybrid promoter was tested in human RPE (ARPE-19), human glioblastoma, rat C6 glioma, mouse hippocampal neurons, and human embryonic kidney cell lines. Expression of all cell types in normoxia, and following 40 h hypoxia, was analyzed by dual luciferase assay. After confirmation of its tissue-specificity and hypoxia-inducibility, the hybrid promoter construct was integrated into a replication-deficient AAV delivery system and tested for cell-selectivity and hypoxia-inducible green fluorescent protein (GFP) reporter expression.

RESULTS

The HRSE-6xHRE-Rpe65 promoter was highly selective for RPE cells, strongly induced in hypoxia, and similar in activation strength to the cytomegalovirus (CMV) promoter. The AAV.HRSE.6xHRE.Rpe65 vector induced robust GFP expression in hypoxic ARPE-19 cells, but elicited no GFP expression in hypoxia in other cell types or in normoxic ARPE-19 cells.

CONCLUSIONS

The hypoxia-regulated, aerobically-silenced RPE-targeting vector forms a platform for focal autoregulated delivery of antiangiogenic agents in hypoxic regions of the RPE. Such autoinitiated therapy provides a means for early intervention in choroidal neovascularization, when it is most sensitive to inhibition.

The retinal pigment epithelium (RPE)-specific protein RPE65 is the major protein of the RPE microsomal membrane fraction. Though RPE65 lacks transmembrane domains or signal peptide, detergents are required for its maximally effective solubilization in isotonic buffers. However, in 0.75-1.0 M KCl, RPE65 is as soluble without detergent, indicating a peripheral membrane association. We wished to understand why this non-membrane-inserted protein was so closely associated with RPE microsomal membranes. To explore the possible involvement of interactions with phospholipids, an isotonic salt-soluble extract of RPE was incubated with phosphatidylcholine (PC)/phosphatidylserine (PS)/phosphatidylinositol liposomes and centrifuged to sediment the liposomes. RPE65 cosedimented with the liposome pellet. RPE65 also cosedimented with synthetic dipalmitoyl-, 1-palmitoyl, 2-docosahexaenoyl-PC or dipalmitoyl-PS liposomes. Incubation with 1 mM Ca2+ or 1 mM EGTA had no effect, indicating a Ca2+-independent association. A spectrophotometric assay showed that this interaction of RPE65 with phospholipid vesicles resulted in increased light scattering, consistent with phospholipid vesicle aggregation. Resonance energy transfer experiments showed that any putative aggregation occurred without subsequent vesicle fusion. This PC affinity was further confirmed by incubation of RPE extract with dimyristoyl-PC-immobilized artificial membrane (IAM.PC) matrix. The RPE65 selectively bound and was elutable with 2% detergent. This RPE65-phospholipid liposome association may explain the solubilization characteristics of RPE65 and may be related to the function of RPE65 and to its physical association with the RPE smooth endoplasmic reticulum.

Retinal pigment epithelium-specific 65 kDa (RPE65)-associated Leber congenital amaurosis is an autosomal recessive disease that results in reduced visual acuity and night blindness beginning at birth. It is one of the few retinal degenerative disorders for which promising clinical gene transfer trials are currently underway. However, the ability to enroll patients in a gene augmentation trial is dependent on the identification of 2 bona fide disease-causing mutations, and there are some patients with the phenotype of RPE65-associated disease who might benefit from gene transfer but are ineligible because 2 disease-causing genetic variations have not yet been identified. Some such patients have novel mutations in RPE65 for which pathogenicity is difficult to confirm. The goal of this study was to determine if an intronic mutation identified in a 2-year-old patient with presumed RPE65-associated disease was truly pathogenic and grounds for inclusion in a clinical gene augmentation trial. Sequencing of the RPE65 gene revealed 2 mutations: (1) a previously identified disease-causing exonic leucine-to-proline mutation (L408P) and (2) a novel single point mutation in intron 3 (IVS3-11) resulting in an A>G change. RT-PCR analysis using RNA extracted from control human donor eye-derived primary RPE, control iPSC-RPE cells, and proband iPSC-RPE cells revealed that the identified IVS3-11 variation caused a splicing defect that resulted in a frameshift and insertion of a premature stop codon. In this study, we demonstrate how patient-specific iPSCs can be used to confirm pathogenicity of unknown mutations, which can enable positive clinical outcomes.

Leber congenital amaurosis (LCA) is a group of severe autosomal recessive retinal dystrophies unified by the onset of blindness at birth and absence of ERG signals. Mutations in fourteen genes are currently associated with LCA, accounting for approximately 60% of patients. LCA genes encode retinal proteins that participate in a variety of significant retinal pathways ranging from photoreceptor development to replenishing vitamin A in the retinoid cycle. In some of the genetic subtypes of LCA (e.g. RPE65), viable photoreceptors and a relatively intact retina have been discovered. Consequently, successful photoreceptor and visual rescue have been achieved in mice and canine models of LCA. Two research groups, one in Philadelphia, USA (Maguire et al.) another in London, England (Bainbridge et al.) recently tested RPE65 replacement in two human trials with a total of six LCA patients. Remarkably, visual improvements were documented by ETDRS visual acuity measurements, pupillometry, nystagmus frequency, visual field measures, perimetry and an obstacle course. There were no local, or systemic side effects, nor improvements in ERG measures. These spectacular results will now stimulate further investigations of younger patients, higher dosages, and clinically or genetically related retinal disorders.

Transplanted retinal pigment epithelium (RPE) cells hold promise for treatment of age-related macular degeneration (AMD) and Stargardt disease (SD), but it is conceivable that the degenerated host Bruch's membrane (BM) as a natural substrate for RPE might not optimally support transplanted cell survival with correct cellular organization. We fabricated novel ultrathin three-dimensional (3-D) nanofibrous membranes from collagen type I and poly(lactic-co-glycolic acid) (PLGA) by an advanced clinical-grade needle-free electrospinning process. The nanofibrillar 3-D networks closely mimicked the fibrillar architecture of the native inner collagenous layer of human BM. Human RPE cells grown on our nanofibrous membranes bore a striking resemblance to native human RPE. They exhibited a correctly orientated monolayer with a polygonal cell shape and abundant sheet-like microvilli on their apical surfaces. RPE cells built tight junctions and expressed RPE65 protein. Flat 2-D PLGA film and cover glass as controls delivered inferior RPE layers. Our nanofibrous membranes may imitate the natural BM to such extent that they allow for the engineering of an in vivo-like human RPE monolayer that maintains the natural biofunctional characteristics. Such ultrathin membranes may provide a promising vehicle for a functional RPE cell monolayer implantation in the subretinal space in patients with AMD or SD.

No treatments exist to effectively treat many retinal diseases. Retinal pigmented epithelium (RPE) and neural retina can be generated from human embryonic stem cells/induced pluripotent stem cells (hESCs/hiPSCs). The efficacy of current protocols is, however, limited. It was hypothesised that generation of laminated neural retina and/or RPE from hiPSCs/hESCs could be enhanced by three dimensional (3D) culture in hydrogels. hiPSC- and hESC-derived embryoid bodies (EBs) were encapsulated in 0.5% RGD-alginate; 1% RGD-alginate; hyaluronic acid (HA) or HA/gelatin hydrogels and maintained until day 45. Compared with controls (no gel), 0.5% RGD-alginate increased: the percentage of EBs with pigmented RPE foci; the percentage EBs with optic vesicles (OVs) and pigmented RPE simultaneously; the area covered by RPE; frequency of RPE cells (CRALBP+); expression of RPE markers (TYR and RPE65) and the retinal ganglion cell marker, MATH5. Furthermore, 0.5% RGD-alginate hydrogel encapsulation did not adversely affect the expression of other neural retina markers (PROX1, CRX, RCVRN, AP2α or VSX2) as determined by qRT-PCR, or the percentage of VSX2 positive cells as determined by flow cytometry. 1% RGD-alginate increased the percentage of EBs with OVs and/or RPE, but did not significantly influence any other measures of retinal differentiation. HA-based hydrogels had no significant effect on retinal tissue development. The results indicated that derivation of retinal tissue from hESCs/hiPSCs can be enhanced by culture in 0.5% RGD-alginate hydrogel. This RGD-alginate scaffold may be useful for derivation, transport and transplantation of neural retina and RPE, and may also enhance formation of other pigmented, neural or epithelial tissue.

The burden of retinal disease is ever growing with the increasing age of the world-wide population. Transplantation of retinal tissue derived from human pluripotent stem cells (PSCs) is considered a promising treatment. However, derivation of retinal tissue from PSCs using defined media is a lengthy process and often variable between different cell lines. This study indicated that alginate hydrogels enhanced retinal tissue development from PSCs, whereas hyaluronic acid-based hydrogels did not. This is the first study to show that 3D culture with a biomaterial scaffold can improve retinal tissue derivation from PSCs. These findings indicate potential for the clinical application of alginate hydrogels for the derivation and subsequent transplantation retinal tissue. This work may also have implications for the derivation of other pigmented, neural or epithelial tissue.

Recent studies have suggested that bone marrow-derived mesenchymal stem cells (BMMSCs) are capable of retinal tissue-specific differentiation but not retinal pigment epithelium (RPE) cell-specific differentiation. Photoreceptor outer segments (POS) contribute to RPE development and maturation. However, there has been no standard culture system that fosters the differentiation of BMMSCs into mature RPE cells in vitro. In this study, we investigated if the soluble factors from RPE cells and POS could differentiate BMMSCs into cells having a phenotype characteristic of RPE cells. Rat BMMSCs were separately co-cultured with RPE cells, or they were exposed to either control medium, RPE cell-conditioned medium (RPECM), POS, or a combination of RPECM and POS (RPECM-POS). After 7 days, the cells were analyzed for morphology and the expression of RPE markers (cytokeratin 8, CRALBP, and RPE65) to assess the RPE differentiation. Significantly higher pigment accumulation and increased protein expression of the three markers were seen in cells cultured in RPECM-POS than in other treated cultures. Furthermore, the RPECM-POS-treated cultures displayed ultrastructural features typical of RPE cells, expressed RPE cell functional proteins, and had the capability to phagocytose POS. Together, theses results suggest the combination of RPECM and POS stimulate BMMSCs differentiation toward a functional RPE phenotype. Our results provide the foundation for a new route to RPE regenerative therapy involving BMMSCs. Future work isolating the active agent in RPECM and POS would be useful in therapies for RPE diseases or in developing appropriately pre-differentiated BMMSCs for tissue-engineered RPE reconstruction.

The advances in gene therapy hold significant promise for the treatment of ophthalmic conditions. Several studies using animal models have been published. Animal models on retinitis pigmentosa, Leber's Congenital Amaurosis (LCA), and Stargardt disease have involved the use of adeno-associated virus (AAV) to deliver functional genes into mice and canines. Mice models have been used to show that a mutation in cGMP phosphodiesterase that results in retinitis pigmentosa can be corrected using rAAV vectors. Additionally, rAAV vectors have been successfully used to deliver ribozyme into mice with a subsequent improvement in autosomal dominant retinitis pigmentosa. By using dog models, researchers have made progress in studying X-linked retinitis pigmentosa which results from a RPGR gene mutation. Mouse and canine models have also been used in the study of LCA. The widely studied form of LCA is LCA2, resulting from a mutation in the gene RPE65. Mice and canines that were injected with normal copies of RPE65 gene showed signs such as improved retinal pigment epithelium transduction, visual acuity, and functional recovery. Studies on Stargardt disease have shown that mutations in the ABCA4 gene can be corrected with AAV vectors, or nanoparticles. Gene therapy for the treatment of red-green color blindness was successful in squirrel monkeys. Plans are at an advanced stage to begin clinical trials. Researchers have also proved that CD59 can be used with AMD. Gene therapy is also able to treat primary open angle glaucoma (POAG) in animal models, and studies show it is economically viable.

Gene and cell therapies have the potential to prevent, halt, or reverse diseases of the retina in patients with currently incurable blinding conditions. Over the past 2 decades, major advances in our understanding of the pathobiologic basis of retinal diseases, coupled with growth of gene transfer and cell transplantation biotechnologies, have created optimism that previously blinding retinal conditions may be treatable. It is now possible to deliver cloned genes safely and stably to specific retinal cell types in humans. Preliminary results testing gene augmentation strategies in human recessive diseases suggest promising safety and efficacy profiles, including improved visual function outcomes over extended periods. Additional gene-based strategies under development include approaches to autosomal dominant disease ("gain of function"), attempts to deliver genes encoding therapeutic proteins with proven mechanisms of action interfering with specific disease pathways, and approaches that could be used to render retinal cells other than atrophied photoreceptors light sensitive. In the programs that are the furthest along-pivotal regulatory safety and efficacy trials studying individuals with retinal degeneration resulting from RPE65 mutations-initial results reveal a robust safety profile and clinically significant improvements in visual function, thereby making this program a frontrunner for the first approved gene therapy product in the United States. Similar to gene therapy, progress in regenerative or stem cell-based transplantation strategies has been substantial. It is now possible to deliver safely stem cell-derived, terminally differentiated, biologically and genetically defined retinal pigment epithelium (RPE) to the diseased human eye. Although demonstration of clinical efficacy is still well behind the gene therapy field, multiple programs investigating regenerative strategies in RPE disease are beginning to enroll subjects, and initial results suggest possible signs of efficacy. Stem cells capable of becoming other retinal cell types, such as photoreceptors, are on the cusp of clinical trials. Stem cell-derived transplants can be delivered to precise target locations in the eye, and their ability to ameliorate, reverse, regenerate, or neuroprotect against disease processes can be assessed. Results from these studies will provide foundational knowledge that may lead to clinically significant therapies for currently untreatable retinal disease.

Leber congenital amaurosis (LCA) represents the most severe form of inherited retinal dystrophies with an onset during the first year of life. Currently, 21 genes are known to be associated with LCA and recurrent mutations have been observed in AIPL1, CEP290, CRB1 and GUCY2D. In addition, sequence analysis of LRAT and RPE65 may be important in view of treatments that are emerging for patients carrying variants in these genes. Screening of the aforementioned variants and genes was performed in 64 Danish LCA probands. Upon the identification of heterozygous variants, Sanger sequencing was performed of the relevant genes to identify the second allele. In combination with prior arrayed primer extension analysis, this led to the identification of two variants in 42 of 86 cases (49%). Remarkably, biallelic RPE65 variants were identified in 16% of the cases, and one novel variant, p.(D110G), was found in seven RPE65 alleles. We also collected all previously published RPE65 variants, identified in 914 alleles of 539 patients with LCA or early-onset retinitis pigmentosa, and deposited them in the RPE65 Leiden Open Variation Database (LOVD). The in silico pathogenicity assessment of the missense and noncanonical splice site variants, as well as an analysis of their frequency in ~60 000 control individuals, rendered 864 of the alleles to affect function or probably affect function. This comprehensive database can now be used to select patients eligible for gene augmentation or retinoid supplementation therapies.