Patient-Specific iPSC-Derived RPE for Modeling of Retinal Diseases

Inherited retinal diseases, such as age-related macular degeneration and retinitis pigmentosa, are the leading cause of blindness in the developed world. Currently, treatments for these conditions are limited. Recently, considerable attention has been given to the possibility of using patient-specific induced pluripotent stem cells (iPSCs) as a treatment for these conditions. iPSCs reprogrammed from adult somatic cells offer the possibility of generating patient-specific cell lines in vitro. In this review, we will discuss the current literature pertaining to iPSC modeling of retinal disease, gene therapy of iPSC-derived retinal pigmented epithelium (RPE) cells, and retinal transplantation. We will focus on the use of iPSCs created from patients with inherited eye diseases for testing the efficacy of gene or drug-based therapies, elucidating previously unknown mechanisms and pathways of disease, and as a source of autologous cells for cell replacement

Late-stage rescue of visually guided behavior in the context of a significantly remodeled retinitis pigmentosa mouse model

Patients with progressive neurodegenerative disorder retinitis pigmentosa (RP) are diagnosed in the midst of ongoing retinal degeneration and remodeling. Here, we used a Pde6b-deficient RP gene therapy mouse model to test whether treatment at late disease stages can halt photoreceptor degeneration and degradative remodeling, while sustaining constructive remodeling and restoring function. We demonstrated that when fewer than 13% of rods remain, our genetic rescue halts photoreceptor degeneration, electroretinography (ERG) functional decline and inner retinal remodeling. In addition, in a water maze test, the performance of mice treated at 16 weeks of age or earlier was indistinguishable from wild type. In contrast, no efficacy was apparent in mice treated at 24 weeks of age, suggesting the photoreceptors had reached a point of no return. Further, remodeling in the retinal pigment epithelium (RPE) and retinal vasculature was not halted at 16 or 24 weeks of age, although there appeared to be some slowing of blood vessel degradation. These data suggest a novel working model in which restoration of clinically significant visual function requires only modest threshold numbers of resilient photoreceptors, halting of destructive remodeling and sustained constructive remodeling. These novel findings define the potential and limitations of RP treatment and suggest possible nonphotoreceptor targets for gene therapy optimization

Erythropoetin receptor expression in the human diabetic retina

<p>Abstract</p> <p>Background</p> <p>Recent evidence suggests erythropoietin (<it>EPO</it>) and the erythropoietin receptor (<it>EPOR</it>) may play a direct role in the pathogenesis of diabetic retinopathy. Better characterization of the <it>EPO-EPOR </it>signaling system in the ischemic retina may offer a new therapeutic modality for ischemic ophthalmic diseases. This study was performed to identify <it>EPOR </it>mRNA expression in the human diabetic eye.</p> <p>Findings</p> <p><it>EPOR </it>antisense RNA probes were validated on human pancreas tissue. In the normal eye, <it>EPOR </it>was expressed in the retinal ganglion cell layer. Minimal expression was observed in the inner and outer nuclear layer. Under conditions of diabetic retinopathy, <it>EPOR </it>expression shifted to photoreceptor cells. Increased expression was also observed in the peripheral retina.</p> <p>Conclusion</p> <p><it>EPOR </it>expression may be a biomarker or contribute to disease mechanisms in diabetic retinopathy.</p

Unilateral electronegative ERG in a presumed central retinal artery occlusion

A unilateral electronegative electroretinogram (ERG) was seen in a 94-year-old man with presumed central retinal artery occlusion. Goldmann perimetry revealed central scotoma in the right eye and no abnormalities in the left eye. Full-field ERG in the right eye described a reduction of the b-wave with a relative preservation of the a-wave which is characteristic of electronegative ERG. Hence, our case illustrates that ERG testing is essential for the work-up of individuals with suspected retinal vascular disorders

Nutrigenetic reprogramming of oxidative stress

Retinal disorders such as retinitis pigmentosa, age-related retinal degeneration, oxygen-induced retinopathy, and ischemia-reperfusion injury cause debilitating and irreversible vision loss. While the exact mechanisms underlying these conditions remain unclear, there has been a growing body of evidence demonstrating the pathological contributions of oxidative stress across different cell types within the eye. Nuclear factor erythroid-2-related factor (Nrf2), a transcriptional activator of antioxidative genes, and its regulator Kelch-like ECH-associated protein 1 (Keap1) have emerged as promising therapeutic targets. The purpose of this review is to understand the protective role of the Nrf2-Keap1 pathway in different retinal tissues and shed light on the complex mechanisms underlying these processes. In the photoreceptors, we highlight that Nrf2 preserves their survival and function by maintaining oxidation homeostasis. In the retinal pigment epithelium, Nrf2 similarly plays a critical role in oxidative stabilization but also maintains mitochondrial motility and autophagy-related lipid metabolic processes. In endothelial cells, Nrf2 seems to promote proper vascularization and revascularization through concurrent activation of antioxidative and angiogenic factors as well as inhibition of inflammatory cytokines. Finally, Nrf2 protects retinal ganglion cells against apoptotic cell death. Importantly, we show that Nrf2-mediated protection of the various retinal tissues corresponds to a preservation of functional vision. Altogether, this review underscores the potential of the Nrf2-Keap1 pathway as a powerful tool against retinal degeneration. Key insights into this elegant oxidative defense mechanism may ultimately pave the path toward a universal therapy for various inherited and environmental retinal disorders

Precision Medicine: Genetic Repair of Retinitis Pigmentosa in PatientDerived Stem Cells

Induced pluripotent stem cells (iPSCs) generated from patient fibroblasts could potentially be used as a source of autologous cells for transplantation in retinal disease. Patient-derived iPSCs, however, would still harbor disease-causing mutations. To generate healthy patient-derived cells, mutations might be repaired with new gene-editing technology based on the bacterial system of clustered regularly interspersed short palindromic repeats (CRISPR)/Cas9, thereby yielding grafts that require no patient immunosuppression. We tested whether CRISPR/Cas9 could be used in patient-specific iPSCs to precisely repair an RPGR point mutation that causes X-linked retinitis pigmentosa (XLRP). Fibroblasts cultured from a skin-punch biopsy of an XLRP patient were transduced to produce iPSCs carrying the patient’s c.3070G>T mutation. The iPSCs were transduced with CRISPR guide RNAs, Cas9 endonuclease, and a donor homology template. Despite the gene’s repetitive and GC-rich sequences, 13% of RPGR gene copies showed mutation correction and conversion to the wild-type allele. This is the first report using CRISPR to correct a pathogenic mutation in iPSCs derived from a patient with photoreceptor degeneration. This important proof-of-concept finding supports the development of personalized iPSC-based transplantation therapies for retinal disease

Inhibition of Ca 2+ channel surface expression by mutant bestrophin‐1 in RPE cells

The BEST1 gene product bestrophin-1, a Ca2+-dependent anion channel, interacts with CaV1.3 Ca2+ channels in the retinal pigment epithelium (RPE). BEST1 mutations lead to Best vitelliform macular dystrophy. A common functional defect of these mutations is reduced trafficking of bestrophin-1 into the plasma membrane. We hypothesized that this defect affects the interaction partner CaV1.3 channel affecting Ca2+ signaling and altered RPE function. Thus, we investigated the protein interaction between CaV1.3 channels and bestrophin-1 by immunoprecipitation, CaV1.3 activity in the presence of mutant bestrophin-1 and intracellular trafficking of the interaction partners in confluent RPE monolayers. We selected four BEST1 mutations, each representing one mutational hotspot of the disease: T6P, F80L, R218C, and F305S. Heterologously expressed L-type channels and mutant bestrophin-1 showed reduced interaction, reduced CaV1.3 channel activity, and changes in surface expression. Transfection of polarized RPE (porcine primary cells, iPSC-RPE) that endogenously express CaV1.3 and wild-type bestrophin-1, with mutant bestrophin-1 confirmed reduction of CaV1.3 surface expression. For the four selected BEST1 mutations, presence of mutant bestrophin-1 led to reduced CaV1.3 activity by modulating pore-function or decreasing surface expression. Reduced CaV1.3 activity might open new ways to understand symptoms of Best vitelliform macular dystrophy such as reduced electro-oculogram, lipofuscin accumulation, and vision impairment

Stem Cell Therapies in Retinal Disorders

Stem cell therapy has long been considered a promising mode of treatment for retinal conditions. While human embryonic stem cells (ESCs) have provided the precedent for regenerative medicine, the development of induced pluripotent stem cells (iPSCs) revolutionized this field. iPSCs allow for the development of many types of retinal cells, including those of the retinal pigment epithelium, photoreceptors, and ganglion cells, and can model polygenic diseases such as age-related macular degeneration. Cellular programming and reprogramming technology is especially useful in retinal diseases, as it allows for the study of living cells that have genetic variants that are specific to patients’ diseases. Since iPSCs are a self-renewing resource, scientists can experiment with an unlimited number of pluripotent cells to perfect the process of targeted differentiation, transplantation, and more, for personalized medicine. Challenges in the use of stem cells are present from the scientific, ethical, and political realms. These include transplant complications leading to anatomically incorrect placement, concern for tumorigenesis, and incomplete targeting of differentiation leading to contamination by different types of cells. Despite these limitations, human ESCs and iPSCs specific to individual patients can revolutionize the study of retinal disease and may be effective therapies for conditions currently considered incurable

Functional Analysis of Retinal Flecks in Stargardt Disease

To evaluate visual function of flecked areas in a series of patients with Stargardt disease (STGD) and compare them with adjacent non flecked areas