Retinal Degenerations

Retinitis pigmentosa is a genetically heterogeneous disorder with an estimated prevalence of one in 4,000 that is classically characterized by the progressive constriction of peripheral vision and a later deterioration of visual acuity. Central vision can be compromised earlier in disease, however, in the approximately 25% of patients that have cystoid macular edema. This poorly understood problem can thus significantly impair patient quality of life, particularly as available treatments have limited efficacy. We will review clinical features of retinitis pigmentosa-associated cystoid macular edema, potential causative mechanisms, and finally, evidence supporting currently employed therapies with emphasis upon which management strategies require further evidence-based evaluation.

PURPOSE: Inherited retinal dystrophies are a significant cause of vision loss and are characterized by the loss of photoreceptors and the retinal pigment epithelium (RPE). Mutations in approximately 250 genes cause inherited retinal degenerations with a high degree of genetic heterogeneity. New techniques in next-generation sequencing are allowing the comprehensive analysis of all retinal disease genes thus changing the approach to the molecular diagnosis of inherited retinal dystrophies. This review serves to analyze clinical progress in genetic diagnostic testing and implications for retinal gene therapy. METHODS: A literature search of PubMed and OMIM was conducted to relevant articles in inherited retinal dystrophies. RESULTS: Next-generation genetic sequencing allows the simultaneous analysis of all the approximately 250 genes that cause inherited retinal dystrophies. Reported diagnostic rates range are high and range from 51% to 57%. These new sequencing tools are highly accurate with sensitivities of 97.9% and specificities of 100%. Retinal gene therapy clinical trials are underway for multiple genes including RPE65, ABCA4, CHM, RS1, MYO7A, CNGA3, CNGB3, ND4, and MERTK for which a molecular diagnosis may be beneficial for patients. CONCLUSION: Comprehensive next-generation genetic sequencing of all retinal dystrophy genes is changing the paradigm for how retinal specialists perform genetic testing for inherited retinal degenerations. Not only are high diagnostic yields obtained, but mutations in genes with novel clinical phenotypes are also identified. In the era of retinal gene therapy clinical trials, identifying specific genetic defects will increasingly be of use to identify patients who may enroll in clinical studies and benefit from novel therapies.

The G309 allele of SNPs in the mouse double minute (MDM2) promoter locus is associated with a higher risk of cancer and proliferative vitreoretinopathy (PVR), but whether SNP G309 contributes to the pathogenesis of PVR is to date unknown. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease (Cas) 9 from Streptococcus pyogenes (SpCas9) can be harnessed to manipulate a single or multiple nucleotides in mammalian cells. Here we delivered SpCas9 and guide RNAs using dual adeno-associated virus-derived vectors to target the MDM2 genomic locus together with a homologous repair template for creating the mutation of MDM2 T309G in human primary retinal pigment epithelial (hPRPE) cells whose genotype is MDM2 T309T. The next-generation sequencing results indicated that there was 42.51% MDM2 G309 in the edited hPRPE cells using adeno-associated viral CRISPR/Cas9. Our data showed that vitreous induced an increase in MDM2 and subsequent attenuation of p53 expression in MDM2 T309G hPRPE cells. Furthermore, our experimental results demonstrated that MDM2 T309G in hPRPE cells enhanced vitreous-induced cell proliferation and survival, suggesting that this SNP contributes to the pathogenesis of PVR.

Retinal degenerative diseases such as age-related macular degeneration, retinitis pigmentosa, and glaucoma result in permanent loss of retinal neurons and vision. Stem cell therapy could be a novel treatment strategy to restore visual function. In an ideal situation, a homogenous population of stem cell-derived retinal neurons with high purity is used for replacement therapy. Thus, it is crucial to elucidate the molecular mechanisms that regulate the development of retinal progenitor cells and subsequent generation of specific retinal neurons. Here, recent findings concerning the intrinsic and extrinsic factors that regulate retinal progenitor cell maintenance and differentiation are summarized, especially transcriptional factors and extrinsic signals. Understanding these mechanisms is indispensable because they have potential clinical applications, chiefly the generation of specific retinal cells such as retinal ganglion cells to treat glaucoma and other optic neuropathy diseases.

PURPOSE: Previous studies have shown that vitreous stimulates degradation of the tumor suppressor protein p53 and that knockdown of phosphatidylinositol 5-phosphate 4-kinases (PI5P4Kα and -β) abrogates proliferation of p53-deficient cells. The purpose of this study was to determine whether vitreous stimulated expression of PI5P4Kα and -β and whether suppression of PI5P4Kα and -β would inhibit vitreous-induced cellular responses and experimental proliferative vitreoretinopathy (PVR). METHODS: PI5P4Kα and -β encoded by PIP4K2A and 2B, respectively, in human ARPE-19 cells were knocked down by stably expressing short hairpin (sh)RNA directed at human PIP4K2A and -2B. In addition, we rescued expression of PI5P4Kα and -β by re-expressing mouse PIP4K2A and -2B in the PI5P4Kα and -β knocked-down ARPE-19 cells. Expression of PI5P4Kα and -β was determined by Western blot and immunofluorescence. The following cellular responses were monitored: cell proliferation, survival, migration, and contraction. Moreover, the cell potential of inducing PVR was examined in a rabbit model of PVR effected by intravitreal cell injection. RESULTS: We found that vitreous enhanced expression of PI5P4Kα and -β in RPE cells and that knocking down PI5P4Kα and -β abrogated vitreous-stimulated cell proliferation, survival, migration, and contraction. Re-expression of mouse PIP4Kα and -β in the human PI5P4Kα and -β knocked-down cells recovered the loss of vitreous-induced cell contraction. Importantly, suppression of PI5P4Kα and -β abrogated the pathogenesis of PVR induced by intravitreal cell injection in rabbits. Moreover, we revealed that expression of PI5P4Kα and -β was abundant in epiretinal membranes from PVR grade C patients. CONCLUSIONS: The findings from this study indicate that PI5P4Kα and -β could be novel therapeutic targets for the treatment of PVR.

PURPOSE: Marked attenuation of the single-flash electroretinographic (ERG) b-wave in the presence of a normal-amplitude or less-attenuated a-wave is commonly referred to as the "negative ERG." The purpose of this study was to investigate whether the disparate origins of the negative ERG in three murine models can be discriminated using flickering stimuli. METHODS: Three models were selected: (1) the Nyx (nob) mouse model of complete congenital stationary night blindness, (2) the oxygen-induced retinopathy (OIR) rat model of retinopathy of prematurity (ROP), and (3) the Rs1 knockout (KO) mouse model of X-linked juvenile retinoschisis. Directly after a dark-adapted, single-flash ERG luminance series, a flicker ERG frequency series (0.5-30 Hz) was performed at a fixed luminance of 0.5 log cd s/m(2). This series includes frequency ranges that are dominated by activity in (A) the rod pathways (below 5 Hz), (B) the cone ON-pathway (5-15 Hz), and (C) the cone OFF-pathway (above 15 Hz). RESULTS: All three models produced markedly attenuated single-flash ERG b-waves. In the Nyx (nob) mouse, which features postsynaptic deficits in the ON-pathways, the a-wave was normal and flicker responses were attenuated in ranges A and B, but not C. The ROP rat is characterized by inner-retinal ischemia which putatively affects both ON- and OFF-bipolar cell activity; flicker responses were reduced in all ranges (A-C). Notably, the choroid supplies the photoreceptors and is thought to be relatively intact in OIR, an idea supported by the nearly normal a-wave. Finally, in the Rs1 KO mouse, which has documented abnormality of the photoreceptor-bipolar synapse affecting both ON- and OFF-pathways, the flicker responses were attenuated in all ranges (A-C). The a-wave was also attenuated, likely as a consequence to schisms in the photoreceptor layer. CONCLUSION: Consideration of both single-flash and flickering ERG responses can discriminate the functional pathology of the negative ERG in these animal models of human disease.

The X-linked RP3 gene codes for the ciliary protein RPGR and accounts for over 10% of inherited retinal degenerations. The critical RPGR-ORF15 splice variant contains a highly repetitive purine-rich linker region that renders it unstable and difficult to adapt for gene therapy. To test the hypothesis that the precise length of the linker region is not critical for function, we evaluated whether adeno-associated virus-mediated replacement gene therapy with a human ORF15 variant containing in-frame shortening of the linker region could reconstitute RPGR function in vivo. We delivered human RPGR-ORF15 replacement genes with deletion of most (314 codons, 'short form') or 1/3 (126 codons, 'long form') of the linker region to Rpgr null mice. Human RPGR-ORF15 expression was detected post treatment with both forms of ORF15 transgenes. However, only the long form correctly localized to the connecting cilia and led to significant functional and morphological rescue of rods and cones. Thus the highly repetitive region of RPGR is functionally important but that moderate shortening of its length, which confers the advantage of added stability, preserves its function. These findings provide a theoretical basis for optimizing replacement gene design in clinical trials for X-linked RP3.

PURPOSE: To report swept-source optical coherence tomography findings of sarcoid choroidal granulomas in the posttreatment convalescent stage of disease. PATIENTS/METHODS: The authors retrospectively reviewed charts from patients with sarcoid-related choroidal granulomas and recorded pertinent examination and imaging findings. Swept-source optical coherence tomography was performed using the DRI 3D-OCT-1 Atlantis (Topcon) over the areas of previous choroidal granulomas after the patients had been treated. RESULTS: Three patients with sarcoid choroidal granulomas were imaged with swept-source optical coherence tomography. Findings included loss or alteration of choroidal architecture, subretinal fibrosis, and outer retinal tubulations in the areas of the sarcoid granulomas after treatment. In one case with an associated choroidal neovascular membrane, there was also disruption of Bruch membrane and loss of normal choroidal vascular network in the area of the lesion. CONCLUSION: Swept-source optical coherence tomography demonstrated significant anatomical sequelae that persisted after treatment of sarcoid granulomas. To the best of the authors' knowledge, this is the first report of outer retinal tubulations over healed sarcoid granulomas.

PURPOSE: Retinitis pigmentosa (RP) is a group of inherited retinal degenerations leading to blindness due to photoreceptor loss. Retinitis pigmentosa is a rare disease, affecting only approximately 100 000 people in the United States. There is no cure and no approved medical therapy to slow or reverse RP. The purpose of this clinical trial was to evaluate the safety, reliability, and benefit of the Argus II Retinal Prosthesis System (Second Sight Medical Products, Inc, Sylmar, CA) in restoring some visual function to subjects completely blind from RP. We report clinical trial results at 1 and 3 years after implantation. DESIGN: The study is a multicenter, single-arm, prospective clinical trial. PARTICIPANTS: There were 30 subjects in 10 centers in the United States and Europe. Subjects served as their own controls, that is, implanted eye versus fellow eye, and system on versus system off (native residual vision). METHODS: The Argus II System was implanted on and in a single eye (typically the worse-seeing eye) of blind subjects. Subjects wore glasses mounted with a small camera and a video processor that converted images into stimulation patterns sent to the electrode array on the retina. MAIN OUTCOME MEASURES: The primary outcome measures were safety (the number, seriousness, and relatedness of adverse events) and visual function, as measured by 3 computer-based, objective tests. RESULTS: A total of 29 of 30 subjects had functioning Argus II Systems implants 3 years after implantation. Eleven subjects experienced a total of 23 serious device- or surgery-related adverse events. All were treated with standard ophthalmic care. As a group, subjects performed significantly better with the system on than off on all visual function tests and functional vision assessments. CONCLUSIONS: The 3-year results of the Argus II trial support the long-term safety profile and benefit of the Argus II System for patients blind from RP. Earlier results from this trial were used to gain approval of the Argus II by the Food and Drug Administration and a CE mark in Europe. The Argus II System is the first and only retinal implant to have both approvals.

PURPOSE: To assess whether quantitative fundus autofluorescence (qAF), a measure of RPE lipofuscin, and spectral-domain optical coherence tomography (SD-OCT) can aid in the differentiation of patients with fundus features that could either be related to ABCA4 mutations or be part of the phenotypic spectrum of pattern dystrophies. METHODS: Autofluorescence images (30°, 488-nm excitation) from 39 patients (67 eyes) were acquired with a confocal scanning laser ophthalmoscope equipped with an internal fluorescent reference and were quantified as previously described. In addition, horizontal SD-OCT images through the fovea were obtained. Patients were screened for ABCA4 and PRPH2/RDS mutations. RESULTS: ABCA4 mutations were identified in 19 patients (mean age, 37 ± 12 years) and PRPH2/RDS mutations in 8 patients (mean age, 48 ± 13 years); no known ABCA4 or PRPH2/RDS mutations were found in 12 patients (mean age, 48 ± 9 years). Differentiation of the groups using phenotypic SD-OCT and AF features (e.g., peripapillary sparing, foveal sparing) was not reliable. However, patients with ABCA4 mutations could be discriminated reasonably well from other patients when qAF values were corrected for age and race. In general, ABCA4 patients had higher qAF values than PRPH2/RDS patients, while most patients without mutations in PRPH2/RDS or ABCA4 had qAF levels within the normal range. CONCLUSIONS: The high qAF levels of ABCA4-positive patients are a hallmark of ABCA4-related disease. The reason for high qAF among many PRPH2/RDS-positive patients is not known; higher RPE lipofuscin accumulation may be a primary or secondary effect of the PRPH2/RDS mutation.

PURPOSE: There is a need for automated retinal optical coherence tomography (OCT) image analysis tools for quantitative measurements in small animals. Some image processing techniques for retinal layer analysis have been developed, but reports about how useful those techniques are in actual animal studies are rare. This paper presents the use of a retinal layer detection method we developed in an actual mouse study that involves wild type and mutated mice carrying photoreceptor degeneration. METHODS: Spectral domain OCT scanning was performed by four experimenters over 12 months on 45 mouse eyes that were wild-type, deficient for ephrin-A2 and ephrin-A3, deficient for rhodopsin, or deficient for rhodopsin, ephrin-A2 and ephrin-A3. The thickness of photoreceptor complex between the outer plexiform layer and retinal pigment epithelium was measured on two sides of the optic disc as the biomarker of retinal degeneration. All the layer detection results were visually confirmed. RESULTS: Overall, 96% (8519 out of 9000) of the half-side images were successfully processed using our technique in a semi-automatic manner. There was no significant difference in success rate between mouse lines (p = 0.91). Based on a human observer's rating of image quality for images successfully and unsuccessfully processed, the odds ratios for 'easily visible' images and 'not clear' images to be successfully processed is 62 and 4, respectively, against 'indistinguishable' images. Thickness of photoreceptor complex was significantly different across the quadrants compared (p < 0.001). It was also found that the average thickness based on 4-point sparse sampling was not significantly different from the full analysis, while the range of differences between the two methods could be up to about 6 μm or 16% for individual eyes. Differences between mouse lines and progressive thickness reduction were revealed by both sampling measures. CONCLUSIONS: Although the thickness of the photoreceptor complex layer is not even, manual sparse sampling may be as sufficiently accurate as full analysis in some studies such as ours, where the error of sparse sampling was much smaller than the effect size of rhodopsin deficiency. It is also suggested that the image processing method can be useful in actual animal studies. Even for images poorly visible to human eyes the image processing method still has a good chance to extract the complex layer.