Centre for Eye Research Australia (CERA) - Research Publications

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    AAV-Mediated CRISPR/Cas Gene Editing of Retinal Cells In Vivo
    Hung, SSC ; Chrysostomou, V ; Li, F ; Lim, JKH ; Wang, J-H ; Powell, JE ; Tu, L ; Daniszewski, M ; Lo, C ; Wong, RC ; Crowston, JG ; Pebay, A ; King, AE ; Bui, BV ; Liu, G-S ; Hewitt, AW (ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2016-06-01)
    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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    Clinical Outcomes of a Treat and Extend Regimen with Intravitreal Aflibercept Injections in Patients with Diabetic Macular Edema: Experience in Clinical Practice
    Curry, BA ; Sanfilippo, PG ; Chan, S ; Hewitt, AW ; Verma, N (SPRINGER INTERNATIONAL PUBLISHING AG, 2019-11-21)
    INTRODUCTION: Treat-and-extend (T&E) and pro re nata (PRN; 'as needed') regimens of intravitreal anti-vascular endothelial growth factor (VEGF) treatment have been found to reduce the injection burden on patients and improve the cost effectiveness of the treatment of macular edema. The aim of this study was to assess the effectiveness of a T&E regimen of aflibercept, in a clinical setting, in patients with diabetic macular edema (DME) who were either intravitreal anti-VEGF therapy naive or with minimal exposure to anti-VEGF (≤ 6 treatments) in the previous 12 months. METHODS: This prospective, single arm, open label study recruited patients with DME (macular thickness of ≥ 300 µm) and best-corrected visual acuity (BCVA) between 28-78 ETDRS letters. Participants received five loading doses of intravitreal aflibercept at 4-weekly intervals. BCVA measurements and macular optical coherence tomography were performed at each visit. If no disease activity was detected, treatment intervals were increased by 2 weeks to a maximum of 12 weeks. Outcome measures included: changes in BCVA and retinal anatomical measures (central foveal thickness [CFT] and central macular volume within 6 mm of the fovea [CSVol]) between baseline and 2 years, patient treatment intervals; and adverse events. RESULTS: Of the 36 patients who provided informed consent to participate in the study and were screened, 26 patients (eyes) were eligible to participate in the study. After regression analysis, adjustment for repeated measures, and significant covariates, the mean BCVA increased by 3.8 letters (95% confidence interval [CI] 1.1, 6.4) and the CFT and CSVol decreased by 127.2 µm (95% CI 91.7, 162.5) and 1.6 mm3 (95% CI 1.2, 2.0), respectively, over the course of the study. In the second year, 16 of the 25 patients still participating had their treatment intervals extended to 12 weeks. There was no evidence of any new adverse events that would require changes to the aflibercept safety profile. CONCLUSION: For the majority of patients presenting with DME, a T&E regimen of aflibercept in the first 2 years of therapy is a practical alternative to PRN treatment with regular review. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry number, ACTRN12618000428268. FUNDING: This investigator-initiated study was supported by Bayer Australia Ltd. who provided the study treatment and some financial assistance.
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    Peeking into the molecular trove of discarded surgical specimens
    Hewitt, AW ; Cook, AL ; Pebay, A (WILEY-BLACKWELL, 2016-11-01)
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    Response: Cycloplegia in refraction: age and cycloplegics
    Sanfilippo, PG ; Chu, B-S ; Bigault, O ; Kearns, LS ; Boon, M-Y ; Young, TL ; Hammond, CJ ; Hewitt, AW ; Mackey, DA (WILEY-BLACKWELL, 2016-08-01)
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    A single-cell transcriptome atlas of the adult human retina
    Lukowski, SW ; Lo, CY ; Sharov, AA ; Nguyen, Q ; Fang, L ; Hung, SSC ; Zhu, L ; Zhang, T ; Grunert, U ; Nguyen, T ; Senabouth, A ; Jabbari, JS ; Welby, E ; Sowden, JC ; Waugh, HS ; Mackey, A ; Pollock, G ; Lamb, TD ; Wang, P-Y ; Hewitt, AW ; Gillies, MC ; Powell, JE ; Wong, RCB (WILEY, 2019-09-16)
    The retina is a specialized neural tissue that senses light and initiates image processing. Although the functional organization of specific retina cells has been well studied, the molecular profile of many cell types remains unclear in humans. To comprehensively profile the human retina, we performed single-cell RNA sequencing on 20,009 cells from three donors and compiled a reference transcriptome atlas. Using unsupervised clustering analysis, we identified 18 transcriptionally distinct cell populations representing all known neural retinal cells: rod photoreceptors, cone photoreceptors, Müller glia, bipolar cells, amacrine cells, retinal ganglion cells, horizontal cells, astrocytes, and microglia. Our data captured molecular profiles for healthy and putative early degenerating rod photoreceptors, and revealed the loss of MALAT1 expression with longer post-mortem time, which potentially suggested a novel role of MALAT1 in rod photoreceptor degeneration. We have demonstrated the use of this retina transcriptome atlas to benchmark pluripotent stem cell-derived cone photoreceptors and an adult Müller glia cell line. This work provides an important reference with unprecedented insights into the transcriptional landscape of human retinal cells, which is fundamental to understanding retinal biology and disease.
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    Seeing the impact of the Glaucoma Inheritance Study in Tasmania after 25 years
    Mackey, DA ; Craig, JE ; Hewitt, AW (WILEY, 2019-07-01)
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    Current state and future prospects of artificial intelligence in ophthalmology: a review
    Hogarty, DT ; Mackey, DA ; Hewitt, AW (WILEY, 2019-01-01)
    Artificial intelligence (AI) has emerged as a major frontier in computer science research. Although AI has broad application across many medical fields, it will have particular utility in ophthalmology and will dramatically change the diagnostic and treatment pathways for many eye conditions such as corneal ectasias, glaucoma, age-related macular degeneration and diabetic retinopathy. However, given that AI has primarily been driven as a computer science, its concepts and terminology are unfamiliar to many medical professionals. Important key terms such as machine learning and deep learning are often misunderstood and incorrectly used interchangeably. This article presents an overview of AI and new developments relevant to ophthalmology.
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    Plurality in multi-disciplinary research: multiple institutional affiliations are associated with increased citations
    Sanfilippo, P ; Hewitt, AW ; Mackey, DA (PEERJ INC, 2018-09-24)
    BACKGROUND: The institutional affiliations and associated collaborative networks that scientists foster during their research careers are salient in the production of high-quality science. The phenomenon of multiple institutional affiliations and its relationship to research output remains relatively unexplored in the literature. METHODS: We examined 27,612 scientific articles, modelling the normalized citation counts received against the number of authors and affiliations held. RESULTS: In agreement with previous research, we found that teamwork is an important factor in high impact papers, with average citations received increasing concordant with the number of co-authors listed. For articles with more than five co-authors, we noted an increase in average citations received when authors with more than one institutional affiliation contributed to the research. DISCUSSION: Multiple author affiliations may play a positive role in the production of high-impact science. This increased researcher mobility should be viewed by institutional boards as meritorious in the pursuit of scientific discovery.
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    Family-Based Genome-Wide Association Study of South Indian Pedigrees Supports WNT7B as a Central Corneal Thickness Locus
    Fan, BJ ; Chen, X ; Sondhi, N ; Sharmila, PF ; Soumittra, N ; Sripriya, S ; Sacikala, S ; Asokan, R ; Friedman, DS ; Pasquale, LR ; Gao, XR ; Vijaya, L ; Bailey, JC ; Vitart, V ; MacGregor, S ; Hammond, CJ ; Khor, CC ; Haines, JL ; George, R ; Wiggs, JL (ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2018-05-01)
    Purpose: To identify genetic risk factors contributing to central corneal thickness (CCT) in individuals from South India, a population with a high prevalence of ocular disorders. Methods: One hundred ninety-five individuals from 15 large South Indian pedigrees were genotyped using the Omni2.5 bead array. Family-based association for CCT was conducted using the score test in MERLIN. Results: Genome-wide association study (GWAS) identified strongest association for single nucleotide polymorphisms (SNPs) in the first intron of WNT7B and CCT (top SNP rs9330813; β = -0.57, 95% confidence interval [CI]: -0.78 to -0.36; P = 1.7 × 10-7). We further investigated rs9330813 in a Latino cohort and four independent European cohorts. A meta-analysis of these data sets demonstrated statistically significant association between rs9330813 and CCT (β = -3.94, 95% CI: -5.23 to -2.66; P = 1.7 × 10-9). WNT7B SNPs located in the same genomic region that includes rs9330813 have previously been associated with CCT in Latinos but with other ocular quantitative traits related to myopia (corneal curvature and axial length) in a Japanese population (rs10453441 and rs200329677). To evaluate the specificity of the observed WNT7B association with CCT in the South Indian families, we completed an ocular phenome-wide association study (PheWAS) for the top WNT7B SNPs using 45 ocular traits measured in these same families including corneal curvature and axial length. The ocular PheWAS results indicate that in the South Indian families WNT7B SNPs are primarily associated with CCT. Conclusions: The results indicate robust evidence for association between WNT7B SNPs and CCT in South Indian pedigrees, and suggest that WNT7B SNPs can have population-specific effects on ocular quantitative traits.
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    Genome-Wide Association Study Identifies a Susceptibility Locus for Comitant Esotropia and Suggests a Parent-of-Origin Effect
    Shaaban, S ; MacKinnon, S ; Andrews, C ; Staffieri, SE ; Maconachie, GDE ; Chan, W-M ; Whitman, MC ; Morton, SU ; Yazar, S ; MacGregor, S ; Elder, JE ; Traboulsi, EI ; Gottlob, I ; Hewitt, AW ; Hunter, DG ; Mackey, DA ; Engle, EC (ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2018-08-01)
    Purpose: To identify genetic variants conferring susceptibility to esotropia. Esotropia is the most common form of comitant strabismus, has its highest incidence in European ancestry populations, and is believed to be inherited as a complex trait. Methods: White European American discovery cohorts with nonaccommodative (826 cases and 2991 controls) or accommodative (224 cases and 749 controls) esotropia were investigated. White European Australian and United Kingdom cohorts with nonaccommodative (689 cases and 1448 controls) or accommodative (66 cases and 264 controls) esotropia were tested for replication. We performed a genome-wide case-control association study using a mixed linear additive model. Meta-analyses of discovery and replication cohorts were then conducted. Results: A significant association with nonaccommodative esotropia was discovered (odds ratio [OR] = 1.41, P = 2.84 × 10-09) and replicated (OR = 1.23, P = 0.01) at rs2244352 [T] located within intron 1 of the WRB (tryptophan rich basic protein) gene on chromosome 21 (meta-analysis OR = 1.33, P = 9.58 × 10-11). This single nucleotide polymorphism (SNP) is differentially methylated, and there is a statistically significant skew toward paternal inheritance in the discovery cohort. Meta-analysis of the accommodative discovery and replication cohorts identified an association with rs912759 [T] (OR = 0.59, P = 1.89 × 10-08), an intergenic SNP on chromosome 1p31.1. Conclusions: This is the first genome-wide association study (GWAS) to identify significant associations in esotropia and suggests a parent-of-origin effect. Additional cohorts will permit replication and extension of these findings. Future studies of rs2244352 and WRB should provide insight into pathophysiological mechanisms underlying comitant strabismus.