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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ItemAAV-Mediated CRISPR/Cas Gene Editing of Retinal Cells In Vivo(ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2016-06)PURPOSE: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) has recently been adapted to enable efficient editing of the mammalian genome, opening novel avenues for therapeutic intervention of inherited diseases. In seeking to disrupt yellow fluorescent protein (YFP) in a Thy1-YFP transgenic mouse, we assessed the feasibility of utilizing the adeno-associated virus 2 (AAV2) to deliver CRISPR/Cas for gene modification of retinal cells in vivo. METHODS: Single guide RNA (sgRNA) plasmids were designed to target YFP, and after in vitro validation, selected guides were cloned into a dual AAV system. One AAV2 construct was used to deliver Streptococcus pyogenes Cas9 (SpCas9), and the other delivered sgRNA against YFP or LacZ (control) in the presence of mCherry. Five weeks after intravitreal injection, retinal function was determined using electroretinography, and CRISPR/Cas-mediated gene modifications were quantified in retinal flat mounts. RESULTS: Adeno-associated virus 2-mediated in vivo delivery of SpCas9 with sgRNA targeting YFP significantly reduced the number of YFP fluorescent cells of the inner retina of our transgenic mouse model. Overall, we found an 84.0% (95% confidence interval [CI]: 81.8-86.9) reduction of YFP-positive cells in YFP-sgRNA-infected retinal cells compared to eyes treated with LacZ-sgRNA. Electroretinography profiling found no significant alteration in retinal function following AAV2-mediated delivery of CRISPR/Cas components compared to contralateral untreated eyes. CONCLUSIONS: Thy1-YFP transgenic mice were used as a rapid quantifiable means to assess the efficacy of CRISPR/Cas-based retinal gene modification in vivo. We demonstrate that genomic modification of cells in the adult retina can be readily achieved by viral-mediated delivery of CRISPR/Cas.
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ItemUtility of Self-Destructing CRISPR/Cas Constructs for Targeted Gene Editing in the Retina(MARY ANN LIEBERT, INC, 2019-11-01)Safe delivery of CRISPR/Cas endonucleases remains one of the major barriers to the widespread application of in vivo genome editing. We previously reported the utility of adeno-associated virus (AAV)-mediated CRISPR/Cas genome editing in the retina; however, with this type of viral delivery system, active endonucleases will remain in the retina for an extended period, making genotoxicity a significant consideration in clinical applications. To address this issue, we have designed a self-destructing "kamikaze" CRISPR/Cas system that disrupts the Cas enzyme itself following expression. Four guide RNAs (sgRNAs) were initially designed to target Streptococcus pyogenes Cas9 (SpCas9) and after in situ validation, the selected sgRNAs were cloned into a dual AAV vector. One construct was used to deliver SpCas9 and the other delivered sgRNAs directed against SpCas9 and the target locus (yellow fluorescent protein [YFP]), in the presence of mCherry. Both constructs were packaged into AAV2 vectors and intravitreally administered in C57BL/6 and Thy1-YFP transgenic mice. After 8 weeks, the expression of SpCas9 and the efficacy of YFP gene disruption were quantified. A reduction of SpCas9 mRNA was found in retinas treated with AAV2-mediated YFP/SpCas9 targeting CRISPR/Cas compared with those treated with YFP targeting CRISPR/Cas alone. We also show that AAV2-mediated delivery of YFP/SpCas9 targeting CRISPR/Cas significantly reduced the number of YFP fluorescent cells among mCherry-expressing cells (∼85.5% reduction compared with LacZ/SpCas9 targeting CRISPR/Cas) in the transfected retina of Thy1-YFP transgenic mice. In conclusion, our data suggest that a self-destructive "kamikaze" CRISPR/Cas system can be used as a robust tool for genome editing in the retina, without compromising on-target efficiency.
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ItemLongitudinal expression profiling of CD4+and CD8+cells in patients with active to quiescent giant cell arteritis(BMC, 2018-07-23)BACKGROUND: Giant cell arteritis (GCA) is the most common form of vasculitis affecting elderly people. It is one of the few true ophthalmic emergencies but symptoms and signs are variable thereby making it a challenging disease to diagnose. A temporal artery biopsy is the gold standard to confirm GCA, but there are currently no specific biochemical markers to aid diagnosis. We aimed to identify a less invasive method to confirm the diagnosis of GCA, as well as to ascertain clinically relevant predictive biomarkers by studying the transcriptome of purified peripheral CD4+ and CD8+ T lymphocytes in patients with GCA. METHODS: We recruited 16 patients with histological evidence of GCA at the Royal Victorian Eye and Ear Hospital, Melbourne, Australia, and aimed to collect blood samples at six time points: acute phase, 2-3 weeks, 6-8 weeks, 3 months, 6 months and 12 months after clinical diagnosis. CD4+ and CD8+ T-cells were positively selected at each time point through magnetic-assisted cell sorting. RNA was extracted from all 195 collected samples for subsequent RNA sequencing. The expression profiles of patients were compared to those of 16 age-matched controls. RESULTS: Over the 12-month study period, polynomial modelling analyses identified 179 and 4 statistically significant transcripts with altered expression profiles (FDR < 0.05) between cases and controls in CD4+ and CD8+ populations, respectively. In CD8+ cells, two transcripts remained differentially expressed after 12 months; SGTB, associated with neuronal apoptosis, and FCGR3A, associatied with Takayasu arteritis. We detected genes that correlate with both symptoms and biochemical markers used for predicting long-term prognosis. 15 genes were shared across 3 phenotypes in CD4 and 16 across CD8 cells. In CD8, IL32 was common to 5 phenotypes including Polymyalgia Rheumatica, bilateral blindness and death within 12 months. CONCLUSIONS: This is the first longitudinal gene expression study undertaken to identify robust transcriptomic biomarkers of GCA. Our results show cell type-specific transcript expression profiles, novel gene-phenotype associations, and uncover important biological pathways for this disease. In the acute phase, the gene-phenotype relationships we have identified could provide insight to potential disease severity and as such guide in initiating appropriate patient management.
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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.
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ItemPSEN1ΔE9, APPswe, and APOE4 Confer Disparate Phenotypes in Human iPSC-Derived Microglia(CELL PRESS, 2019-10-08)Here we elucidate the effect of Alzheimer disease (AD)-predisposing genetic backgrounds, APOE4, PSEN1ΔE9, and APPswe, on functionality of human microglia-like cells (iMGLs). We present a physiologically relevant high-yield protocol for producing iMGLs from induced pluripotent stem cells. Differentiation is directed with small molecules through primitive erythromyeloid progenitors to re-create microglial ontogeny from yolk sac. The iMGLs express microglial signature genes and respond to ADP with intracellular Ca2+ release distinguishing them from macrophages. Using 16 iPSC lines from healthy donors, AD patients and isogenic controls, we reveal that the APOE4 genotype has a profound impact on several aspects of microglial functionality, whereas PSEN1ΔE9 and APPswe mutations trigger minor alterations. The APOE4 genotype impairs phagocytosis, migration, and metabolic activity of iMGLs but exacerbates their cytokine secretion. This indicates that APOE4 iMGLs are fundamentally unable to mount normal microglial functionality in AD.
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ItemSingle-Cell Profiling Identifies Key Pathways Expressed by iPSCs Cultured in Different Commercial Media(CELL PRESS, 2018-09-28)We assessed the pluripotency of human induced pluripotent stem cells (iPSCs) maintained on an automated platform using StemFlex and TeSR-E8 media. Analysis of transcriptome of single cells revealed similar expression of core pluripotency genes, as well as genes associated with naive and primed states of pluripotency. Analysis of individual cells from four samples consisting of two different iPSC lines each grown in the two culture media revealed a shared subpopulation structure with three main subpopulations different in pluripotency states. By implementing a machine learning approach, we estimated that most cells within each subpopulation are very similar between all four samples. The single-cell RNA sequencing analysis of iPSC lines grown in both media reports the molecular signature in StemFlex medium and how it compares to that observed in the TeSR-E8 medium.