Centre for Eye Research Australia (CERA) - Research Publications
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ItemAAV capsid bioengineering in primary human retina models(NATURE PORTFOLIO, 2023-12-11)Adeno-associated viral (AAV) vector-mediated retinal gene therapy is an active field of both pre-clinical as well as clinical research. As with other gene therapy clinical targets, novel bioengineered AAV variants developed by directed evolution or rational design to possess unique desirable properties, are entering retinal gene therapy translational programs. However, it is becoming increasingly evident that predictive preclinical models are required to develop and functionally validate these novel AAVs prior to clinical studies. To investigate if, and to what extent, primary retinal explant culture could be used for AAV capsid development, this study performed a large high-throughput screen of 51 existing AAV capsids in primary human retina explants and other models of the human retina. Furthermore, we applied transgene expression-based directed evolution to develop novel capsids for more efficient transduction of primary human retina cells and compared the top variants to the strongest existing benchmarks identified in the screening described above. A direct side-by-side comparison of the newly developed capsids in four different in vitro and ex vivo model systems of the human retina allowed us to identify novel AAV variants capable of high transgene expression in primary human retina cells.
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ItemNo Preview AvailableA village in a dish model system for population-scale hiPSC studies(NATURE PORTFOLIO, 2023-06-09)The mechanisms by which DNA alleles contribute to disease risk, drug response, and other human phenotypes are highly context-specific, varying across cell types and different conditions. Human induced pluripotent stem cells are uniquely suited to study these context-dependent effects but cell lines from hundreds or thousands of individuals are required. Village cultures, where multiple induced pluripotent stem lines are cultured and differentiated in a single dish, provide an elegant solution for scaling induced pluripotent stem experiments to the necessary sample sizes required for population-scale studies. Here, we show the utility of village models, demonstrating how cells can be assigned to an induced pluripotent stem line using single-cell sequencing and illustrating that the genetic, epigenetic or induced pluripotent stem line-specific effects explain a large percentage of gene expression variation for many genes. We demonstrate that village methods can effectively detect induced pluripotent stem line-specific effects, including sensitive dynamics of cell states.
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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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ItemNo Preview AvailableGenome-Wide Meta-analysis Identifies Risk Loci and Improves Disease Prediction of Age-Related Macular Degeneration(ELSEVIER SCIENCE INC, 2024-01)PURPOSE: To identify age-related macular degeneration (AMD) risk loci and to establish a polygenic prediction model. DESIGN: Genome-wide association study (GWAS) and polygenic risk score (PRS) construction. PARTICIPANTS: We included 64 885 European patients with AMD and 568 740 control participants (with overlapped samples) in the UK Biobank, Genetic Epidemiology Research on Aging (GERA), International AMD Consortium, FinnGen, and published early AMD GWASs in meta-analyses, as well as 733 European patients with AMD and 20 487 control participants from the Canadian Longitudinal Study on Aging (CLSA) and non-Europeans from the UK Biobank and GERA for polygenic risk score validation. METHODS: A multitrait meta-analysis of GWASs comprised 64 885 patients with AMD and 568 740 control participants; the multitrait approach accounted for sample overlap. We constructed a PRS for AMD based on both previously reported as well as unreported AMD loci. We applied the PRS to nonoverlapping data from the CLSA. MAIN OUTCOME MEASURES: We identified several single nucleotide polymorphisms associated with AMD and established a PRS for AMD risk prediction. RESULTS: We identified 63 AMD risk loci alongside the well-established AMD loci CFH and ARMS2, including 9 loci that were not reported in previous GWASs, some of which previously were linked to other eye diseases such as glaucoma (e.g., HIC1). We applied our PRS to nonoverlapping data from the CLSA. A new PRS was constructed using the PRS method, PRS-CS, and significantly improved the prediction accuracy of AMD risk compared with PRSs from previously published datasets. We further showed that even people who carry all the well-known AMD risk alleles at CFH and ARMS2 vary considerably in their AMD risk (ranging from close to 0 in individuals with low PRS to > 50% in individuals with high PRS). Although our PRS was derived in individuals of European ancestry, the PRS shows potential for predicting risk in people of East Asian, South Asian, and Latino ancestry. CONCLUSIONS: Our findings improve the knowledge of the genetic architecture of AMD and help achieve better accuracy in AMD prediction. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.
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ItemRNA-targeting strategies as a platform for ocular gene therapy(Elsevier, 2023-01)Genetic medicine is offering hope as new therapies are emerging for many previously untreatable diseases. The eye is at the forefront of these advances, as exemplified by the approval of Luxturna® by the United States Food and Drug Administration (US FDA) in 2017 for the treatment of one form of Leber Congenital Amaurosis (LCA), an inherited blindness. Luxturna® was also the first in vivo human gene therapy to gain US FDA approval. Numerous gene therapy clinical trials are ongoing for other eye diseases, and novel delivery systems, discovery of new drug targets and emerging technologies are currently driving the field forward. Targeting RNA, in particular, is an attractive therapeutic strategy for genetic disease that may have safety advantages over alternative approaches by avoiding permanent changes in the genome. In this regard, antisense oligonucleotides (ASO) and RNA interference (RNAi) are the currently popular strategies for developing RNA-targeted therapeutics. Enthusiasm has been further fuelled by the emergence of clustered regularly interspersed short palindromic repeats (CRISPR)-CRISPR associated (Cas) systems that allow targeted manipulation of nucleic acids. RNA-targeting CRISPR-Cas systems now provide a novel way to develop RNA-targeted therapeutics and may provide superior efficiency and specificity to existing technologies. In addition, RNA base editing technologies using CRISPR-Cas and other modalities also enable precise alteration of single nucleotides. In this review, we showcase advances made by RNA-targeting systems for ocular disease, discuss applications of ASO and RNAi technologies, highlight emerging CRISPR-Cas systems and consider the implications of RNA-targeting therapeutics in the development of future drugs to treat eye disease.
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ItemDiagnostic yield of candidate genes in an Australian corneal dystrophy cohort(WILEY, 2022-10)Corneal dystrophies describe a clinically and genetically heterogeneous group of inherited disorders. The International Classification of Corneal Dystrophies (IC3D) lists 22 types of corneal dystrophy, 17 of which have been demonstrated to result from pathogenic variants in 19 identified genes. In this study, we investigated the diagnostic yield of genetic testing in a well-characterised cohort of 58 individuals from 44 families with different types of corneal dystrophy. Individuals diagnosed solely with Fuchs endothelial corneal dystrophy were excluded. Clinical details were obtained from the treating ophthalmologist. Participants and their family members were tested using a gene candidate and exome sequencing approach. We identified a likely molecular diagnosis in 70.5% families (31/44). The detection rate was significantly higher among probands with a family history of corneal dystrophy (15/16, 93.8%) than those without (16/28, 57.1%, p = .015), and among those who had undergone corneal graft surgery (9/9, 100.0%) compared to those who had not (22/35, 62.9%, p = .041). We identified eight novel variants in five genes and identified five families with syndromes associated with corneal dystrophies. Our findings highlight the genetic heterogeneity of corneal dystrophies and the clinical utility of genetic testing in reaching an accurate clinical diagnosis.
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ItemAttitudes Toward Glaucoma Genetic Risk Assessment in Unaffected Individuals(ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2022-10)PURPOSE: Integrating polygenic risk scores (PRS) into healthcare has the potential to stratify an individual's risk of glaucoma across a broad population. Glaucoma is the most common cause of irreversible blindness worldwide, therefore effective screening for glaucoma endorsed by the population is highly important. This study assessed the attitude of unaffected individuals toward PRS testing for glaucoma, and sought to identify factors associated with interest in testing. METHODS: We surveyed 418 unaffected individuals including 193 with a first-degree relative with glaucoma, 117 who had a recent eye examination, and 108 general members of the community. RESULTS: Overall, 71.3% of the individuals indicated an interest in taking a polygenic risk test for glaucoma. Interest was more likely in those who believed glaucoma to be a severe medical condition (odds ratio [OR] = 14.58, 95% confidence interval [CI] = 1.15-185.50, P = 0.039), those concerned about developing glaucoma (OR = 4.37, 95% CI = 2.32-8.25, P < 0.001), those with an intention to take appropriate measures regarding eye health (OR = 2.39, 95% CI = 1.16-4.95, P = 0.019), and those preferring to know if considered to be at-risk or not (OR = 4.52, 95% CI = 2.32-8.83, P < 0.001). CONCLUSIONS: Our results show strong interest in genetic risk assessment for glaucoma among unaffected individuals in Australia. TRANSLATIONAL RELEVANCE: These findings represent a valuable assessment of interest in glaucoma polygenic risk testing among potential target populations, which will be integral to the implementation and uptake of novel PRS-based tests into clinical practice.
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ItemFundoscopy use in neurology departments and the utility of smartphone photography: a prospective prevalence and crossover diagnostic accuracy study amongst neurology inpatients(WILEY, 2022-08)BACKGROUND AND PURPOSE: Although fundoscopy is a crucial part of the neurological examination, it is challenging, under-utilized and unreliably performed. The aim was to determine the prevalence of fundus pathology amongst neurology inpatients and the diagnostic accuracy of current fundoscopy practice compared with systematic screening with smartphone fundoscopy (SF) and portable non-mydriatic fundus photography (NMFP). METHODS: This was a prospective cross-sectional surveillance and diagnostic accuracy study on adult patients admitted under neurology in an Australian hospital. Inpatients were randomized to initial NMFP (RetinaVue 100, Welch Allyn) or SF (D-EYE) followed by a crossover to the alternative modality. Images were graded by neurology doctors, using telemedicine consensus neuro-ophthalmology NMFP grading as the reference standard. Feasibility parameters included ease, comfort and speed. RESULTS: Of 79 enrolled patients, 14.1% had neurologically relevant pathology (seven, disc pallor; one, hypertensive retinopathy; three, disc swelling). The neurology team performed direct ophthalmoscopy in 6.6% of cases and missed all abnormalities. SF had a sensitivity of 30%-40% compared with NMFP (45.5%); however, it had a lower rate of screening failure (1% vs. 13%, p < 0.001), a shorter examination time (1.10 vs. 2.25 min, p < 0.001) and a slightly higher patient comfort rating (9.2 vs. 8/10, p < 0.001). CONCLUSION: Our study demonstrates a clinically significant prevalence of fundus pathology amongst neurology inpatients which was missed by current fundoscopy practices. Portable NMFP screening appears more accurate than SF, whilst both are diagnostically superior to routine fundoscopic practice, feasible and well tolerated by patients.
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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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ItemA saturated map of common genetic variants associated with human height(NATURE PORTFOLIO, 2022-10-27)Common single-nucleotide polymorphisms (SNPs) are predicted to collectively explain 40-50% of phenotypic variation in human height, but identifying the specific variants and associated regions requires huge sample sizes1. Here, using data from a genome-wide association study of 5.4 million individuals of diverse ancestries, we show that 12,111 independent SNPs that are significantly associated with height account for nearly all of the common SNP-based heritability. These SNPs are clustered within 7,209 non-overlapping genomic segments with a mean size of around 90 kb, covering about 21% of the genome. The density of independent associations varies across the genome and the regions of increased density are enriched for biologically relevant genes. In out-of-sample estimation and prediction, the 12,111 SNPs (or all SNPs in the HapMap 3 panel2) account for 40% (45%) of phenotypic variance in populations of European ancestry but only around 10-20% (14-24%) in populations of other ancestries. Effect sizes, associated regions and gene prioritization are similar across ancestries, indicating that reduced prediction accuracy is likely to be explained by linkage disequilibrium and differences in allele frequency within associated regions. Finally, we show that the relevant biological pathways are detectable with smaller sample sizes than are needed to implicate causal genes and variants. Overall, this study provides a comprehensive map of specific genomic regions that contain the vast majority of common height-associated variants. Although this map is saturated for populations of European ancestry, further research is needed to achieve equivalent saturation in other ancestries.