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A Retrospective Longitudinal Study of 460 Patients with ABCA4-Associated Retinal Disease

Sat, 2024-02-03 05:00

Ophthalmology. 2024 Feb 1:S0161-6420(24)00096-4. doi: 10.1016/j.ophtha.2024.01.035. Online ahead of print.

ABSTRACT

PURPOSE: To investigate the distribution of genotypes and natural history of ABCA4-associated retinal disease in a large cohort of patients seen at a single institution.

DESIGN: Retrospective, single institution, cohort review.

SUBJECTS: Patients seen at the University of Iowa between November 1986 and August 2022 clinically suspected to have disease caused by sequence variations in ABCA4.

METHODS: DNA samples from participants were subjected to a tiered testing strategy progressing from allele-specific screening to whole genome sequencing. Charts were reviewed and clinical data were tabulated. The pathogenic severity of the most common alleles was estimated by studying groups of patients that shared one allele. Groups of patients with shared genotypes were reviewed for evidence of modifying factor effects.

MAIN OUTCOME MEASURES: Age of first uncorrectable vision loss, best corrected visual acuity, and the area of the I2e isopter of the Goldmann visual field.

RESULTS: A total of 460 patients from 390 families had convincing clinical features of ABCA4-associated retinal disease. Complete genotypes were identified in 399 patients and partial genotypes in 61. The median age of first vision loss was 16 years (range 4-76 years). 265 of the families (68%) had a unique genotype and no more than 10 patients shared any single genotype. Review of the patients with shared genotypes revealed evidence of modifying factors that in several cases resulted in a greater than 15-year difference in age of first vision loss. 241 different alleles were identified among the members of this cohort and 161 of these (67%) were found in only a single individual.

CONCLUSIONS: ABCA4-associated retinal disease ranges from a very severe photoreceptor disease with an onset before age 5 years to a late-onset RPE-based condition resembling pattern dystrophy. Modifying factors frequently impact the ABCA4 disease phenotype to a degree that is similar in magnitude to the detectable ABCA4 alleles themselves. It is likely that the majority of patients in any cohort will have a unique genotype. The latter observations taken together suggest that patients' clinical findings will in most cases be more useful for predicting their clinical course than their genotype.

PMID:38309476 | DOI:10.1016/j.ophtha.2024.01.035

CRISPRi-Mediated Treatment of Dominant Rhodopsin-Associated Retinitis Pigmentosa

Mon, 2023-12-18 05:00

CRISPR J. 2023 Dec;6(6):502-513. doi: 10.1089/crispr.2023.0039.

ABSTRACT

Rhodopsin (RHO) mutations such as Pro23His are the leading cause of dominantly inherited retinitis pigmentosa in North America. As with other dominant retinal dystrophies, these mutations lead to production of a toxic protein product, and treatment will require knockdown of the mutant allele. The purpose of this study was to develop a CRISPR-Cas9-mediated transcriptional repression strategy using catalytically inactive Staphylococcus aureus Cas9 (dCas9) fused to the Krüppel-associated box (KRAB) transcriptional repressor domain. Using a reporter construct carrying green fluorescent protein (GFP) cloned downstream of the RHO promoter fragment (nucleotides -1403 to +73), we demonstrate a ∼74-84% reduction in RHO promoter activity in RHOpCRISPRi-treated versus plasmid-only controls. After subretinal transduction of human retinal explants and transgenic Pro23His mutant pigs, significant knockdown of rhodopsin protein was achieved. Suppression of mutant transgene in vivo was associated with a reduction in endoplasmic reticulum (ER) stress and apoptosis markers and preservation of photoreceptor cell layer thickness.

PMID:38108516 | DOI:10.1089/crispr.2023.0039

Demonstration of the pathogenicity of a common non-exomic mutation in ABCA4 using iPSC-derived retinal organoids and retrospective clinical data

Mon, 2023-11-06 05:00

Hum Mol Genet. 2023 Oct 31:ddad176. doi: 10.1093/hmg/ddad176. Online ahead of print.

ABSTRACT

Mutations in ABCA4 are the most common cause of Mendelian retinal disease. Clinical evaluation of this gene is challenging because of its extreme allelic diversity, the large fraction of non-exomic mutations, and the wide range of associated disease. We used patient-derived retinal organoids as well as DNA samples and clinical data from a large cohort of patients with ABCA4-associated retinal disease to investigate the pathogenicity of a variant in ABCA4 (IVS30 + 1321 A > G) that occurs heterozygously in 2% of Europeans. We found that this variant causes mis-splicing of the gene in photoreceptor cells such that the resulting protein contains 36 incorrect amino acids followed by a premature stop. We also investigated the phenotype of 10 patients with compound genotypes that included this mutation. Their median age of first vision loss was 39 years, which is in the mildest quintile of a large cohort of patients with ABCA4 disease. We conclude that the IVS30 + 1321 A > G variant can cause disease when paired with a sufficiently deleterious opposing allele in a sufficiently permissive genetic background.

PMID:37930186 | DOI:10.1093/hmg/ddad176

Gene Expression Within a Human Choroidal Neovascular Membrane Using Spatial Transcriptomics

Wed, 2023-10-25 05:00

Invest Ophthalmol Vis Sci. 2023 Oct 3;64(13):40. doi: 10.1167/iovs.64.13.40.

ABSTRACT

PURPOSE: Macular neovascularization is a relatively common and potentially visually devastating complication of age-related macular degeneration. In macular neovascularization, pathologic angiogenesis can originate from either the choroid or the retina, but we have limited understanding of how different cell types become dysregulated in this dynamic process.

METHODS: To study how gene expression is altered in focal areas of pathology, we performed spatial RNA sequencing on a human donor eye with macular neovascularization as well as a healthy control donor. We performed differential expression to identify genes enriched within the area of macular neovascularization and used deconvolution algorithms to predict the originating cell type of these dysregulated genes.

RESULTS: Within the area of neovascularization, endothelial cells demonstrated increased expression of genes related to Rho family GTPase signaling and integrin signaling. Likewise, VEGF and TGFB1 were identified as potential upstream regulators that could drive the observed gene expression changes produced by endothelial and retinal pigment epithelium cells in the macular neovascularization donor. These spatial gene expression profiles were compared to previous single-cell gene expression experiments in human age-related macular degeneration as well as a model of laser-induced neovascularization in mice. As a secondary aim, we investigated regional gene expression patterns within the macular neural retina and between the macular and peripheral choroid.

CONCLUSIONS: Overall, this study spatially analyzes gene expression across the retina, retinal pigment epithelium, and choroid in health and describes a set of candidate molecules that become dysregulated in macular neovascularization.

PMID:37878301 | DOI:10.1167/iovs.64.13.40

CGMP compliant microfluidic transfection of induced pluripotent stem cells for CRISPR-mediated genome editing

Sat, 2023-08-26 05:00

Stem Cells. 2023 Aug 26:sxad063. doi: 10.1093/stmcls/sxad063. Online ahead of print.

ABSTRACT

Inherited retinal degeneration is a term used to describe heritable disorders that result from the death of light sensing photoreceptor cells. Although we and others believe it will be possible to use gene therapy to halt disease progression early in its course, photoreceptor cell replacement will likely be required for patients who have already lost their sight. While advances in autologous photoreceptor cell manufacturing have been encouraging, development of technologies capable of efficiently delivering genome editing reagents to stem cells using current good manufacturing practices (cGMP) are needed. Gene editing reagents were delivered to induced pluripotent stem cells (iPSCs) using a Zephyr microfluidic transfection platform (CellFE). CRISPR-mediated cutting was quantified using an endonuclease assay. CRISPR correction was confirmed via digital PCR and Sanger sequencing. The resulting corrected cells were also karyotyped and differentiated into retinal organoids. We describe use of a novel microfluidic transfection platform to correct, via CRISPR-mediated homology dependent repair (HDR), a disease-causing NR2E3 mutation in patient-derived iPSCs using cGMP compatible reagents and approaches. We show that the resulting cell lines have a corrected genotype, exhibit no off-target cutting, retain pluripotency and a normal karyotype and can be differentiated into retinal tissue suitable for transplantation. The ability to codeliver CRISPR/Cas9 and HDR templates to patient-derived iPSCs without using proprietary transfection reagents will streamline manufacturing protocols, increase the safety of resulting cell therapies, and greatly reduce the regulatory burden of clinical trials.

PMID:37632456 | DOI:10.1093/stmcls/sxad063

Automated human induced pluripotent stem cell colony segmentation for use in cell culture automation applications

Sun, 2023-07-16 05:00

SLAS Technol. 2023 Jul 14:S2472-6303(23)00047-X. doi: 10.1016/j.slast.2023.07.004. Online ahead of print.

ABSTRACT

Human induced pluripotent stem cells (hiPSCs) have demonstrated great promise for a variety of applications that include cell therapy and regenerative medicine. Production of clinical grade hiPSCs requires reproducible manufacturing methods with stringent quality-controls such as those provided by image-controlled robotic processing systems. In this paper we present an automated image analysis method for identifying and picking hiPSC colonies for clonal expansion using the CellXTM robotic cell processing system. This method couples a light weight deep learning segmentation approach based on the U-Net architecture to automatically segment the hiPSC colonies in full field of view (FOV) high resolution phase contrast images with a standardized approach for suggesting pick locations. The utility of this method is demonstrated using images and data obtained from the CellXTM system where clinical grade hiPSCs were reprogrammed, clonally expanded, and differentiated into retinal organoids for use in treatment of patients with inherited retinal degenerative blindness.

PMID:37454765 | DOI:10.1016/j.slast.2023.07.004

GENE EXPRESSION WITHIN A HUMAN CHOROIDAL NEOVASCULAR MEMBRANE USING SPATIAL TRANSCRIPTOMICS

Mon, 2023-07-03 05:00

bioRxiv. 2023 Jun 17:2023.06.16.544770. doi: 10.1101/2023.06.16.544770. Preprint.

ABSTRACT

Macular neovascularization is a relatively common and potentially visually devastating complication of age-related macular degeneration. In macular neovascularization, pathologic angiogenesis can originate from either the choroid or the retina, but we have limited understanding of how different cell types become dysregulated in this dynamic process. In this study, we performed spatial RNA sequencing on a human donor eye with macular neovascularization as well as a healthy control donor. We identified genes enriched within the area of macular neovascularization and used deconvolution algorithms to predict the originating cell type of these dysregulated genes. Within the area of neovascularization, endothelial cells were predicted to increase expression of genes related to Rho family GTPase signaling and integrin signaling. Likewise, VEGF and TGFB1 were identified as potential upstream regulators that could drive the observed gene expression changes produced by endothelial and retinal pigment epithelium cells in the macular neovascularization donor. These spatial gene expression profiles were compared to previous single-cell gene expression experiments in human age-related macular degeneration as well as a model of laser-induced neovascularization in mice. As a secondary aim, we also investigated spatial gene expression patterns within the macular neural retina and between the macular and peripheral choroid. We recapitulated previously described regional-specific gene expression patterns across both tissues. Overall, this study spatially analyzes gene expression across the retina, retinal pigment epithelium, and choroid in health and describes a set of candidate molecules that become dysregulated in macular neovascularization.

PMID:37398429 | PMC:PMC10312719 | DOI:10.1101/2023.06.16.544770

Multimodal single-cell analysis of non-random heteroplasmy distribution in human retinal mitochondrial disease

Thu, 2023-06-08 05:00

JCI Insight. 2023 Jun 8:e165937. doi: 10.1172/jci.insight.165937. Online ahead of print.

ABSTRACT

Variants within the high copy number mitochondrial genome (mtDNA) can disrupt organelle function and lead to severe multi-system disease. The wide range of manifestations observed in mitochondrial disease patients results from varying fractions of abnormal mtDNA molecules in different cells and tissues, a phenomenon termed heteroplasmy. However, the landscape of heteroplasmy across cell types within tissues and its influence on phenotype expression in affected patients remains largely unexplored. Here, we identify non-random distribution of a pathogenic mtDNA variant across a complex tissue using single-cell RNA sequencing, mitochondrial single-cell ATAC sequencing, and multimodal single-cell sequencing. We profile the transcriptome, chromatin accessibility state, and heteroplasmy in cells from the eyes of a patient with mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) and healthy control donors. Utilizing the retina as a model for complex multi-lineage tissues, we found that the proportion of the pathogenic m.3243A>G allele was neither evenly nor randomly distributed across diverse cell types. All neuroectoderm-derived neural cells exhibited a high percentage of the mutant variant. However, a subset of mesoderm-derived lineage, namely the vasculature of the choroid, was near homoplasmic for the wildtype allele. Gene expression and chromatin accessibility profiles of cell types with high and low proportions of m.3243A>G implicate mTOR signaling in the cellular response to heteroplasmy. We further found by multimodal single-cell sequencing of retinal pigment epithelial cells that a high proportion of the pathogenic mtDNA variant was associated with transcriptionally and morphologically abnormal cells. Together, these findings show the non-random nature of mitochondrial variant partitioning in human mitochondrial disease and underscore its implications for mitochondrial disease pathogenesis and treatment.

PMID:37289546 | DOI:10.1172/jci.insight.165937

Propensity of Patient-Derived iPSCs for Retinal Differentiation: Implications for Autologous Cell Replacement

Tue, 2023-05-23 05:00

Stem Cells Transl Med. 2023 May 23:szad028. doi: 10.1093/stcltm/szad028. Online ahead of print.

ABSTRACT

Prior to use, newly generated induced pluripotent stem cells (iPSC) should be thoroughly validated. While excellent validation and release testing assays designed to evaluate potency, genetic integrity, and sterility exist, they do not have the ability to predict cell type-specific differentiation capacity. Selection of iPSC lines that have limited capacity to produce high-quality transplantable cells, places significant strain on valuable clinical manufacturing resources. The purpose of this study was to determine the degree and root cause of variability in retinal differentiation capacity between cGMP-derived patient iPSC lines. In turn, our goal was to develop a release testing assay that could be used to augment the widely used ScoreCard panel. IPSCs were generated from 15 patients (14-76 years old), differentiated into retinal organoids, and scored based on their retinal differentiation capacity. Despite significant differences in retinal differentiation propensity, RNA-sequencing revealed remarkable similarity between patient-derived iPSC lines prior to differentiation. At 7 days of differentiation, significant differences in gene expression could be detected. Ingenuity pathway analysis revealed perturbations in pathways associated with pluripotency and early cell fate commitment. For example, good and poor producers had noticeably different expressions of OCT4 and SOX2 effector genes. QPCR assays targeting genes identified via RNA sequencing were developed and validated in a masked fashion using iPSCs from 8 independent patients. A subset of 14 genes, which include the retinal cell fate markers RAX, LHX2, VSX2, and SIX6 (all elevated in the good producers), were found to be predictive of retinal differentiation propensity.

PMID:37221451 | DOI:10.1093/stcltm/szad028

Stress resilience-enhancing drugs preserve tissue structure and function in degenerating retina via phosphodiesterase inhibition

Mon, 2023-05-01 05:00

Proc Natl Acad Sci U S A. 2023 May 9;120(19):e2221045120. doi: 10.1073/pnas.2221045120. Epub 2023 May 1.

ABSTRACT

Chronic, progressive retinal diseases, such as age-related macular degeneration (AMD), diabetic retinopathy, and retinitis pigmentosa, arise from genetic and environmental perturbations of cellular and tissue homeostasis. These disruptions accumulate with repeated exposures to stress over time, leading to progressive visual impairment and, in many cases, legal blindness. Despite decades of research, therapeutic options for the millions of patients suffering from these disorders remain severely limited, especially for treating earlier stages of pathogenesis when the opportunity to preserve the retinal structure and visual function is greatest. To address this urgent, unmet medical need, we employed a systems pharmacology platform for therapeutic development. Through integrative single-cell transcriptomics, proteomics, and phosphoproteomics, we identified universal molecular mechanisms across distinct models of age-related and inherited retinal degenerations, characterized by impaired physiological resilience to stress. Here, we report that selective, targeted pharmacological inhibition of cyclic nucleotide phosphodiesterases (PDEs), which serve as critical regulatory nodes that modulate intracellular second messenger signaling pathways, stabilized the transcriptome, proteome, and phosphoproteome through downstream activation of protective mechanisms coupled with synergistic inhibition of degenerative processes. This therapeutic intervention enhanced resilience to acute and chronic forms of stress in the degenerating retina, thus preserving tissue structure and function across various models of age-related and inherited retinal disease. Taken together, these findings exemplify a systems pharmacology approach to drug discovery and development, revealing a new class of therapeutics with potential clinical utility in the treatment or prevention of the most common causes of blindness.

PMID:37126699 | DOI:10.1073/pnas.2221045120

The role of personal-use negative pressure wound therapy with enhanced functionality in achieving wound-related treatment goals: a small prospective study

Tue, 2023-03-14 05:00

Wounds. 2023 Mar;35(3):53-58.

ABSTRACT

BACKGROUND: NPWT is widely used to manage hard-to-heal wounds, and many different devices are available. Personal-use NPWT systems are becoming more popular, although current options have limited functionality.

PURPOSE: The primary objective was to determine acceptable progress of wounds towards a predefined goal of therapy for a variety of open wounds being treated with a novel NPWT personal-use system with enhanced functionality.

METHODS: In this prospective, nonrandomized, interventional study, patients were treated with a personal-use NPWT system over 4 weeks, initially in a wound care clinic setting, and were discharged home with the device. Clinician satisfaction with the device was also evaluated.

RESULTS: Ten patients were evaluated. Acceptable progress towards all predetermined goals was reached for all patients; a median reduction in wound volume of 84.6% and improved granulation was achieved within the 4-week treatment period. No device-related deficiencies were reported. In general, clinicians were satisfied with the device's ease of use and mobility.

CONCLUSION: Personal-use NPWT is easy to use, has positive effects on healing on a variety of wound types, and is well accepted by clinicians.

PMID:36917784

Automating iPSC generation to enable autologous photoreceptor cell replacement therapy

Wed, 2023-03-01 05:00

J Transl Med. 2023 Feb 28;21(1):161. doi: 10.1186/s12967-023-03966-2.

ABSTRACT

BACKGROUND: Inherited retinal degeneration is a leading cause of incurable vision loss in the developed world. While autologous iPSC mediated photoreceptor cell replacement is theoretically possible, the lack of commercially available technologies designed to enable high throughput parallel production of patient specific therapeutics has hindered clinical translation.

METHODS: In this study, we describe the use of the Cell X precision robotic cell culture platform to enable parallel production of clinical grade patient specific iPSCs. The Cell X is housed within an ISO Class 5 cGMP compliant closed aseptic isolator (Biospherix XVivo X2), where all procedures from fibroblast culture to iPSC generation, clonal expansion and retinal differentiation were performed.

RESULTS: Patient iPSCs generated using the Cell X platform were determined to be pluripotent via score card analysis and genetically stable via karyotyping. As determined via immunostaining and confocal microscopy, iPSCs generated using the Cell X platform gave rise to retinal organoids that were indistinguishable from organoids derived from manually generated iPSCs. In addition, at 120 days post-differentiation, single-cell RNA sequencing analysis revealed that cells generated using the Cell X platform were comparable to those generated under manual conditions in a separate laboratory.

CONCLUSION: We have successfully developed a robotic iPSC generation platform and standard operating procedures for production of high-quality photoreceptor precursor cells that are compatible with current good manufacturing practices. This system will enable clinical grade production of iPSCs for autologous retinal cell replacement.

PMID:36855199 | DOI:10.1186/s12967-023-03966-2

The degree of AAV-induced retinal inflammation varies based on serotype and route of delivery: intravitreal, subretinal or suprachoroidal

Thu, 2023-02-16 05:00

Hum Gene Ther. 2023 Feb 15. doi: 10.1089/hum.2022.222. Online ahead of print.

ABSTRACT

Adeno-associated virus (AAV) mediated gene therapy has great potential for treating a wide range of retinal degenerative diseases. However, some initial enthusiasm for gene therapy has been tempered by emerging evidence of AAV-associated inflammation, which in several instances has contributed to clinical trial discontinuation. Currently, there is a paucity of data describing the variable immune responses to different AAV serotypes, and similarly, little is known regarding how these responses differ depending on route of ocular delivery, including in animal models of disease. In this study, we characterize the severity and retinal distribution of AAV-associated inflammation in rats triggered by delivery of 5 different AAV vectors (AAV1, AAV2, AAV6, AAV8, AAV9), each of which contained eGFP driven under control of the constitutively active CMV promoter. We further compare the inflammation across 3 different potential routes (intravitreal, subretinal, suprachoroidal) of ocular delivery. Compared to buffer-injected controls for each route of delivery, AAV2 and AAV6 induced the most inflammation across all routes of delivery of the vectors tested, with AAV6 inducing the highest levels of inflammation when delivered suprachoroidally. AAV1-induced inflammation was highest when delivered suprachoroidally, whereas minimal inflammation was seen with intravitreal delivery. AAV8 and AAV9 induced minimal inflammation across all routes of delivery. Importantly, the degree of inflammation was not correlated with vector-mediated transduction and expression of eGFP. These data emphasize the importance of considering ocular inflammation when selecting AAV serotypes and ocular delivery routes for the development of gene therapy strategies.

PMID:36793189 | DOI:10.1089/hum.2022.222

Transcriptomic and chromatin accessibility analysis of the human macular and peripheral retinal pigment epithelium at the single cell level

Sun, 2023-02-12 05:00

Am J Pathol. 2023 Feb 10:S0002-9440(23)00045-7. doi: 10.1016/j.ajpath.2023.01.012. Online ahead of print.

ABSTRACT

Human retinal diseases are frequently characterized by pathology that is restricted to specific cell types and to specific regions of the eye. Several disease entities either selectively affect or spare the macula, the retinal region at the center of the posterior pole. Photoreceptor cells of the macula are responsible for high acuity vision and require metabolic support from non-neuronal cell types. Macular diseases often involve an epithelial cell layer known as the retinal pigment epithelium (RPE) that has several essential metabolic support functions for the overlying photoreceptors. In the current study, we probed how the RPE confers region-specific disease susceptibility by examining heterogeneity within human donor RPE. We profiled RPE nuclei from the macular and peripheral retina using joint single-cell RNA and ATAC sequencing. We found that the expression of several genes varies between macular and peripheral RPE. We further found region-specific ATAC peaks implying regulatory elements used exclusively by macular or peripheral RPE. Across anatomical regions, we identify subpopulations of RPE that appear to have differential expression levels of visual cycle genes. Finally, we examined loci connected to age related macular degeneration to better understand RPE-specific disease phenotypes. Together, these data show how gene expression regulation in the human RPE varies by region and subpopulation and provide a resource for better understanding the molecular basis of macular disease.

PMID:36775060 | DOI:10.1016/j.ajpath.2023.01.012

Retinal Pigment Epithelium-Secreted VEGF-A Induces Alpha-2-Macroglobulin Expression in Endothelial Cells

Fri, 2022-10-14 05:00

Cells. 2022 Sep 24;11(19):2975. doi: 10.3390/cells11192975.

ABSTRACT

Alpha-2-macroglobulin (A2M) is a protease inhibitor that regulates extracellular matrix (ECM) stability and turnover. Here, we show that A2M is expressed by endothelial cells (ECs) from human eye choroid. We demonstrate that retinal pigment epithelium (RPE)-conditioned medium induces A2M expression specifically in ECs. Experiments using chemical inhibitors, blocking antibodies, and recombinant proteins revealed a key role of VEGF-A in RPE-mediated A2M induction in ECs. Furthermore, incubation of ECs with RPE-conditioned medium reduces matrix metalloproteinase-2 gelatinase activity of culture supernatants, which is partially restored after A2M knockdown in ECs. We propose that dysfunctional RPE or choroidal blood vessels, as observed in retinal diseases such as age-related macular degeneration, may disrupt the crosstalk mechanism we describe here leading to alterations in the homeostasis of choroidal ECM, Bruch's membrane and visual function.

PMID:36230937 | PMC:PMC9564307 | DOI:10.3390/cells11192975

Characterization of a novel Pde6b-deficient rat model of retinal degeneration and treatment with adeno-associated virus (AAV) gene therapy

Thu, 2022-09-29 05:00

Gene Ther. 2022 Sep 29. doi: 10.1038/s41434-022-00365-y. Online ahead of print.

ABSTRACT

In humans, mutations in the beta subunit of cGMP-phosphodiesterase type 6 (PDE6B) cause autosomal recessive retinitis pigmentosa (RP), which typically has an aggressive clinical course of early-onset severe vision loss due to rapid photoreceptor degeneration. In this study, we describe the generation of a novel Pde6b-deficient rat model using CRISPR-Cas9 genome editing. We characterize the model at multiple time points using clinical imaging modalities as well as histology with immunohistochemistry to show rapid photoreceptor degeneration compared to wild-type and heterozygous animals. We describe the manufacture of two different adeno-associated viral (AAV) vectors (AAV2/1, AAV2/5) under current Good Manufacturing Practices (cGMP) and demonstrate their ability to drive human PDE6B expression in vivo. We further demonstrate the ability of AAV-mediated subretinal gene therapy to delay photoreceptor loss in Pde6b-deficient rats compared to untreated controls. However, severe progressive photoreceptor loss was noted even in treated eyes, likely due to the aggressive nature of the disease. These data provide useful preclinical data to guide the development of potential human gene therapy for PDE6B-associated RP. In addition, the rapid photoreceptor degeneration of the Pde6b-deficient rat with intact inner retina may provide a useful model for the study of cell replacement strategies.

PMID:36175490 | DOI:10.1038/s41434-022-00365-y

Vascular Findings in the Choriocapillaris in a Case of Radiation Retinopathy Secondary to Choroidal Melanoma

Mon, 2022-09-26 05:00

Case Rep Ophthalmol. 2022 Aug 15;13(2):589-598. doi: 10.1159/000525568. eCollection 2022 May-Aug.

ABSTRACT

The effects of radiation retinopathy on the retinal vasculature have been well established; however, the literature describing the pathologic changes in the choriocapillaris is relatively lacking. In this report, we describe the histologic findings of a donor eye with a choroidal melanoma with special attention to the choriocapillaris. Clinical and histological findings, including immunohistochemistry and transmission electron microscopy, are described for the retina and choroid of a donor eye affected by radiation retinopathy secondary to treatment of choroidal melanoma. Cells within the tumor exhibited an epithelioid structure and balloon melanosomes. Notable infiltration of macrophages with elongated morphology was also observed. Atrophy of photoreceptors, retinal pigmented epithelium, and choriocapillaris was observed on the inferior edge of the lesion and extending past the tumor. The choriocapillaris endothelium showed more severe dropout at the periphery of the lesion where loss of fenestration, thickened cytosol, and degenerated pericytes were observed. Morphologic analysis revealed choriocapillaris loss with pronounced degeneration of choroidal pericytes. Understanding the differences in sensitivity to radiation injury between different cell types and different patients will provide better insight into radiation retinopathy.

PMID:36160486 | PMC:PMC9459633 | DOI:10.1159/000525568

Systems genomics in age-related macular degeneration

Thu, 2022-09-15 05:00

Exp Eye Res. 2022 Dec;225:109248. doi: 10.1016/j.exer.2022.109248. Epub 2022 Sep 13.

ABSTRACT

Genomic studies in age-related macular degeneration (AMD) have identified genetic variants that account for the majority of AMD risk. An important next step is to understand the functional consequences and downstream effects of the identified AMD-associated genetic variants. Instrumental for this next step are 'omics' technologies, which enable high-throughput characterization and quantification of biological molecules, and subsequent integration of genomics with these omics datasets, a field referred to as systems genomics. Single cell sequencing studies of the retina and choroid demonstrated that the majority of candidate AMD genes identified through genomic studies are expressed in non-neuronal cells, such as the retinal pigment epithelium (RPE), glia, myeloid and choroidal cells, highlighting that many different retinal and choroidal cell types contribute to the pathogenesis of AMD. Expression quantitative trait locus (eQTL) studies in retinal tissue have identified putative causal genes by demonstrating a genetic overlap between gene regulation and AMD risk. Linking genetic data to complement measurements in the systemic circulation has aided in understanding the effect of AMD-associated genetic variants in the complement system, and supports that protein QTL (pQTL) studies in plasma or serum samples may aid in understanding the effect of genetic variants and pinpointing causal genes in AMD. A recent epigenomic study fine-mapped AMD causal variants by determing regulatory regions in RPE cells differentiated from induced pluripotent stem cells (iPSC-RPE). Another approach that is being employed to pinpoint causal AMD genes is to produce synthetic DNA assemblons representing risk and protective haplotypes, which are then delivered to cellular or animal model systems. Pinpointing causal genes and understanding disease mechanisms is crucial for the next step towards clinical translation. Clinical trials targeting proteins encoded by the AMD-associated genomic loci C3, CFB, CFI, CFH, and ARMS2/HTRA1 are currently ongoing, and a phase III clinical trial for C3 inhibition recently showed a modest reduction of lesion growth in geographic atrophy. The EYERISK consortium recently developed a genetic test for AMD that allows genotyping of common and rare variants in AMD-associated genes. Polygenic risk scores (PRS) were applied to quantify AMD genetic risk, and may aid in predicting AMD progression. In conclusion, genomic studies represent a turning point in our exploration of AMD. The results of those studies now serve as a driving force for several clinical trials. Expanding to omics and systems genomics will further decipher function and causality from the associations that have been reported, and will enable the development of therapies that will lessen the burden of AMD.

PMID:36108770 | DOI:10.1016/j.exer.2022.109248

The Essential Role of the Choriocapillaris in Vision: Novel Insights from Imaging and Molecular Biology

Thu, 2022-09-15 05:00

Annu Rev Vis Sci. 2022 Sep 15;8:33-52. doi: 10.1146/annurev-vision-100820-085958.

ABSTRACT

The choriocapillaris, a dense capillary network located at the posterior pole of the eye, is essential for supporting normal vision, supplying nutrients, and removing waste products from photoreceptor cells and the retinal pigment epithelium. The anatomical location, heterogeneity, and homeostatic interactions with surrounding cell types make the choroid complex to study both in vivo and in vitro. Recent advances in single-cell RNA sequencing, in vivo imaging, and in vitro cell modeling are vastly improving our knowledge of the choroid and its role in normal health and in age-related macular degeneration (AMD). Histologically, loss of endothelial cells (ECs) of the choriocapillaris occurs early in AMD concomitant with elevated formation of the membrane attack complex of complement. Advanced imaging has allowed us to visualize early choroidal blood flow changes in AMD in living patients, supporting histological findings of loss of choroidal ECs. Single-cell RNA sequencing is being used to characterize choroidal cell types transcriptionally and discover their altered patterns of gene expression in aging and disease. Advances in induced pluripotent stem cell protocols and 3D cultures will allow us to closely mimic the in vivo microenvironment of the choroid in vitro to better understand the mechanism leading to choriocapillaris loss in AMD.

PMID:36108103 | PMC:PMC9668353 | DOI:10.1146/annurev-vision-100820-085958

Biocompatibility of Human Induced Pluripotent Stem Cell-Derived Retinal Progenitor Cell Grafts in Immunocompromised Rats

Mon, 2022-06-27 05:00

Cell Transplant. 2022 Jan-Dec;31:9636897221104451. doi: 10.1177/09636897221104451.

ABSTRACT

Loss of photoreceptor cells is a primary feature of inherited retinal degenerative disorders including age-related macular degeneration and retinitis pigmentosa. To restore vision in affected patients, photoreceptor cell replacement will be required. The ideal donor cells for this application are induced pluripotent stem cells (iPSCs) because they can be derived from and transplanted into the same patient obviating the need for long-term immunosuppression. A major limitation for retinal cell replacement therapy is donor cell loss associated with simple methods of cell delivery such as subretinal injections of bolus cell suspensions. Transplantation with supportive biomaterials can help maintain cellular integrity, increase cell survival, and encourage proper cellular alignment and improve integration with the host retina. Using a pig model of retinal degeneration, we recently demonstrated that polycaprolactone (PCL) scaffolds fabricated with two photon lithography have excellent local and systemic tolerability. In this study, we describe rapid photopolymerization-mediated production of PCL-based bioabsorbable scaffolds, a technique for loading iPSC-derived retinal progenitor cells onto the scaffold, methods of surgical transplantation in an immunocompromised rat model and tolerability of the subretinal grafts at 1, 3, and 6 months of follow-up (n = 150). We observed no local or systemic toxicity, nor did we observe any tumor formation despite extensive clinical evaluation, clinical chemistry, hematology, gross tissue examination and detailed histopathology. Demonstrating the local and systemic compatibility of biodegradable scaffolds carrying human iPSC-derived retinal progenitor cells is an important step toward clinical safety trials of this approach in humans.

PMID:35758274 | PMC:PMC9247396 | DOI:10.1177/09636897221104451

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