Invest Ophthalmol Vis Sci. 2024 Dec 2;65(14):15. doi: 10.1167/iovs.65.14.15.

ABSTRACT

PURPOSE: Choroidal inflammation, complement deposition, and accumulation of C-reactive protein (CRP) are involved in age-related macular degeneration (AMD) pathology. The pro-inflammatory signals that regulate immune cell recruitment in the choroid of patients with AMD remain to be determined. We performed cytokine profiling of human AMD and age-matched control donor tissue to identify inflammatory molecules upregulated in AMD tissue.

METHODS: Protein was isolated from 25 AMD and 21 control donor RPE/choroid macular punches. Total protein was quantified, and 50 µg assayed for expression of 40 cytokines using an inflammation array. We validated the elevated expression of granulocyte colony stimulating factor (G-CSF) protein by ELISA in a second cohort of 22 control and 26 AMD donors. To identify an AMD associated stressor responsible for upregulating G-CSF we assayed for changes in G-CSF protein secretion in RPE/choroid organ cultures treated with the monomeric (m)CRP, an inflammatory protein elevated in AMD.

RESULTS: Using a multiplex array, we identified elevated G-CSF protein in the choroid of AMD donors compared to age-matched non-AMD controls. Differential expression of G-CSF was confirmed via ELISA in an independent cohort of samples (P = 0.01). The mCRP, which is deposited in AMD choroids, increased G-CSF protein secretion in RPE/choroid organ cultures. Single nuclei RNA sequencing identified choroidal endothelial cells and fibroblasts as the primary cell types responsible for increased G-CSF secretion in response to mCRP. The G-CSF receptor is expressed primarily by choroidal macrophages and dendritic cells and anti-G-CSFR colocalizes with anti-CD45 and anti-CD68 in human donor choroid tissue.

CONCLUSIONS: Elevated G-CSF expression in AMD donor tissue as a result of increased levels of mCRP may be involved in immune cell recruitment in AMD contributing to inflammatory stress in the choroid.

PMID:39641748 | DOI:10.1167/iovs.65.14.15

Exp Eye Res. 2024 Oct 16;248:110128. doi: 10.1016/j.exer.2024.110128. Online ahead of print.

ABSTRACT

The choriocapillaris is a dense vascular bed in the inner choroid that supplies the photoreceptor cells and retinal pigment epithelium (RPE). While loss of choriocapillaris density has been described in association with age-related macular degeneration (AMD), whether these changes are primary or secondary to RPE degenerative changes in AMD has been debated. In this study we characterized choriocapillaris loss by quantifying "ghost" vessels in a series of 99 human donor maculae labeled with the UEA-I lectin, and found significant increases in early-intermediate AMD and a greater difference in geographic atrophy in areas with intact RPE. Eyes were genotyped at the CFH Tyr402His locus, and those homozygous for the His allele showed significantly more ghost vessels than those with other genotypes. When only non-AMD eyes were evaluated, His homozygotes had increased ghost vessel density but this trend did not reach statistical significance. These results support the notion that choriocapillaris death often precedes RPE degeneration in AMD and that this loss is an important therapeutic consideration for AMD.

PMID:39419369 | DOI:10.1016/j.exer.2024.110128

HGG Adv. 2024 Sep 30:100357. doi: 10.1016/j.xhgg.2024.100357. Online ahead of print.

ABSTRACT

Correct identification of the molecular consequences of pathogenic genetic variants is essential to the development of allele-specific therapies. However, such molecular effects may remain ambiguous following genetic sequence analysis alone. Here, we identify exonic codon-altering variants that are also predicted to disrupt normal RNA splicing in the context of inherited retinal disease. NR2E3 c.932G>A (p.Arg311Gln) is a variant commonly associated with Enhanced S Cone Syndrome (ESCS). Previous studies using mutagenized cDNA constructs have shown that the arginine to glutamine substitution at position 311 of NR2E3 does not meaningfully diminish function of the rod-specific transcription factor. Using retinal organoids, we explored the molecular consequences of NR2E3 c.932G>A when expressed endogenously during human rod photoreceptor cell development. Retinal organoids carrying the NR2E3 c.932G>A allele expressed a transcript containing a 186-nucleotide deletion of exon 6 within the ligand binding domain. This short transcript was not detected in control organoids or control human donor retina samples. A minigene containing exons 5 and 6 of NR2E3 showed sufficiency of the c.932G>A variant to cause the observed splicing defect. These results support the hypothesis that the pathogenic NR2E3 c.932G>A variant leads to photoreceptor disease by causing a splice defect and not through an amino acid substitution as previously supposed. They also explain the relatively mild effect of Arg311Gln on NR2E3 function in vitro. We also used in silico prediction tools to show that similar changes are likely to affect other inherited retinal disease variants in genes such as CEP290, ABCA4, and BEST1.

PMID:39354715 | DOI:10.1016/j.xhgg.2024.100357

J Clin Invest. 2024 Apr 23:e173892. doi: 10.1172/JCI173892. Online ahead of print.

ABSTRACT

While dysfunction and death of light-detecting photoreceptor cells underlie most inherited retinal dystrophies, knowledge of the species-specific details of human rod and cone photoreceptor cell development remains limited. Here, we generate retinal organoids carrying retinal disease-causing variants in NR2E3, as well as isogenic and unrelated controls. Organoids were sampled using single-cell RNA sequencing across the developmental window encompassing photoreceptor specification, emergence, and maturation. Using scRNAseq data, we reconstruct the rod photoreceptor developmental lineage and identify a branchpoint unique to the disease state. We show that the rod-specific transcription factor NR2E3 is required for the proper expression of genes involved in phototransduction, including rhodopsin, which is absent in divergent rods. NR2E3-null rods additionally misexpress several cone-specific phototransduction genes. Using joint multimodal single-cell sequencing, we further identify putative regulatory sites where rod-specific factors act to steer photoreceptor cell development. Finally, we show that rod-committed photoreceptor cells form and persist throughout life in a patient with NR2E3-associated disease. Importantly, these findings are strikingly different than those observed in Nr2e3 rodent models. Together, these data provide a roadmap of human photoreceptor development and leverage patient iPSCs to define the specific roles of rod transcription factors in photoreceptor cell emergence and maturation in health and disease.

PMID:38652563 | DOI:10.1172/JCI173892

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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