Ophthalmology (Eye & Ear Hospital) - Research Publications
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ItemNo Preview AvailableA semi-automated pipeline for quantifying drusen-like deposits in human induced pluripotent stem cell-derived retinal pigment epithelium cells.(Elsevier BV, 2023-08-30)Age-Related Macular Degeneration (AMD) is a highly prevalent form of retinal disease amongst Western communities over 50 years of age. A hallmark of AMD pathogenesis is the accumulation of drusen underneath the retinal pigment epithelium (RPE), a biological process also observable in vitro. The accumulation of drusen has been shown to predict the progression to advanced AMD, making accurate characterisation of drusen in vitro models valuable in disease modelling and drug development. More recently, deposits above the RPE in the subretinal space, called reticular pseudodrusen (RPD) have been recognized as a sub-phenotype of AMD. While in vitro imaging techniques allow for the immunostaining of drusen-like deposits, quantification of these deposits often requires slow, low throughput manual counting of images. This further lends itself to issues including sampling biases, while ignoring critical data parameters including volume and precise localization. To overcome these issues, we developed a semi-automated pipeline for quantifying the presence of drusen-like deposits in vitro, using RPE cultures derived from patient-specific induced pluripotent stem cells (iPSCs). Using high-throughput confocal microscopy, together with three-dimensional reconstruction, we developed an imaging and analysis pipeline that quantifies the number of drusen-like deposits, and accurately and reproducibly provides the location and composition of these deposits. Extending its utility, this pipeline can determine whether the drusen-like deposits locate to the apical or basal surface of RPE cells. Here, we validate the utility of this pipeline in the quantification of drusen-like deposits in six iPSCs lines derived from patients with AMD, following their differentiation into RPE cells. This pipeline provides a valuable tool for the in vitro modelling of AMD and other retinal disease, and is amenable to mid and high throughput screenings.
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ItemRetinal ganglion cell-specific genetic regulation in primary open-angle glaucoma(ELSEVIER, 2022-06-08)To assess the transcriptomic profile of disease-specific cell populations, fibroblasts from patients with primary open-angle glaucoma (POAG) were reprogrammed into induced pluripotent stem cells (iPSCs) before being differentiated into retinal organoids and compared with those from healthy individuals. We performed single-cell RNA sequencing of a total of 247,520 cells and identified cluster-specific molecular signatures. Comparing the gene expression profile between cases and controls, we identified novel genetic associations for this blinding disease. Expression quantitative trait mapping identified a total of 4,443 significant loci across all cell types, 312 of which are specific to the retinal ganglion cell subpopulations, which ultimately degenerate in POAG. Transcriptome-wide association analysis identified genes at loci previously associated with POAG, and analysis, conditional on disease status, implicated 97 statistically significant retinal ganglion cell-specific expression quantitative trait loci. This work highlights the power of large-scale iPSC studies to uncover context-specific profiles for a genetically complex disease.
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ItemTranscriptomic and proteomic retinal pigment epithelium signatures of age-related macular degeneration(NATURE PORTFOLIO, 2022-07-26)There are currently no treatments for geographic atrophy, the advanced form of age-related macular degeneration. Hence, innovative studies are needed to model this condition and prevent or delay its progression. Induced pluripotent stem cells generated from patients with geographic atrophy and healthy individuals were differentiated to retinal pigment epithelium. Integrating transcriptional profiles of 127,659 retinal pigment epithelium cells generated from 43 individuals with geographic atrophy and 36 controls with genotype data, we identify 445 expression quantitative trait loci in cis that are asssociated with disease status and specific to retinal pigment epithelium subpopulations. Transcriptomics and proteomics approaches identify molecular pathways significantly upregulated in geographic atrophy, including in mitochondrial functions, metabolic pathways and extracellular cellular matrix reorganization. Five significant protein quantitative trait loci that regulate protein expression in the retinal pigment epithelium and in geographic atrophy are identified - two of which share variants with cis- expression quantitative trait loci, including proteins involved in mitochondrial biology and neurodegeneration. Investigation of mitochondrial metabolism confirms mitochondrial dysfunction as a core constitutive difference of the retinal pigment epithelium from patients with geographic atrophy. This study uncovers important differences in retinal pigment epithelium homeostasis associated with geographic atrophy.
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ItemAAV2-mediated gene therapy for Bietti crystalline dystrophy provides functional CYP4V2 in multiple relevant cell models(NATURE PORTFOLIO, 2022-06-09)Bietti crystalline dystrophy (BCD) is an inherited retinal disease (IRD) caused by mutations in the CYP4V2 gene. It is a relatively common cause of IRD in east Asia. A number of features of this disease make it highly amenable to gene supplementation therapy. This study aims to validate a series of essential precursor in vitro experiments prior to developing a clinical gene therapy for BCD. We demonstrated that HEK293, ARPE19, and patient induced pluripotent stem cell (iPSC)-derived RPE cells transduced with AAV2 vectors encoding codon optimization of CYP4V2 (AAV2.coCYP4V2) resulted in elevated protein expression levels of CYP4V2 compared to those transduced with AAV2 vectors encoding wild type CYP4V2 (AAV2.wtCYP4V2), as assessed by immunocytochemistry and western blot. Similarly, we observed significantly increased CYP4V2 enzyme activity in cells transduced with AAV2.coCYP4V2 compared to those transduced with AAV2.wtCYP4V2. We also showed CYP4V2 expression in human RPE/choroid explants transduced with AAV2.coCYP4V2 compared to those transduced with AAV2.wtCYP4V2. These preclinical data support the further development of a gene supplementation therapy for a currently untreatable blinding condition-BCD. Codon-optimized CYP4V2 transgene was superior to wild type in terms of protein expression and enzyme activity. Ex vivo culture of human RPE cells provided an effective approach to test AAV-mediated transgene delivery.
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ItemComparison of CRISPR/Cas Endonucleases forin vivoRetinal Gene Editing(FRONTIERS MEDIA SA, 2020-09-10)CRISPR/Cas has opened the prospect of direct gene correction therapy for some inherited retinal diseases. Previous work has demonstrated the utility of adeno-associated virus (AAV) mediated delivery to retinal cells in vivo; however, with the expanding repertoire of CRISPR/Cas endonucleases, it is not clear which of these are most efficacious for retinal editing in vivo. We sought to compare CRISPR/Cas endonuclease activity using both single and dual AAV delivery strategies for gene editing in retinal cells. Plasmids of a dual vector system with SpCas9, SaCas9, Cas12a, CjCas9 and a sgRNA targeting YFP, as well as a single vector system with SaCas9/YFP sgRNA were generated and validated in YFP-expressing HEK293A cell by flow cytometry and the T7E1 assay. Paired CRISPR/Cas endonuclease and its best performing sgRNA was then packaged into an AAV2 capsid derivative, AAV7m8, and injected intravitreally into CMV-Cre:Rosa26-YFP mice. SpCas9 and Cas12a achieved better knockout efficiency than SaCas9 and CjCas9. Moreover, no significant difference in YFP gene editing was found between single and dual CRISPR/SaCas9 vector systems. With a marked reduction of YFP-positive retinal cells, AAV7m8 delivered SpCas9 was found to have the highest knockout efficacy among all investigated endonucleases. We demonstrate that the AAV7m8-mediated delivery of CRISPR/SpCas9 construct achieves the most efficient gene modification in neurosensory retinal cells in vivo.