Centre for Eye Research Australia (CERA) - Research Publications
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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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ItemA drug-tunable Flt23k gene therapy for controlled intervention in retinal neovascularization(SPRINGER, 2021-02)Gene therapies that chronically suppress vascular endothelial growth factor (VEGF) represent a new approach for managing retinal vascular leakage and neovascularization. However, constitutive suppression of VEGF in the eye may have deleterious side effects. Here, we developed a novel strategy to introduce Flt23k, a decoy receptor that binds intracellular VEGF, fused to the destabilizing domain (DD) of Escherichia coli dihydrofolate reductase (DHFR) into the retina. The expressed DHFR(DD)-Flt23k fusion protein is degraded unless "switched on" by administering a stabilizer; in this case, the antibiotic trimethoprim (TMP). Cells transfected with the DHFR(DD)-Flt23k construct expressed the fusion protein at levels correlated with the TMP dose. Stabilization of the DHFR(DD)-Flt23k fusion protein by TMP was able to inhibit intracellular VEGF in hypoxic cells. Intravitreal injection of self-complementary adeno-associated viral vector (scAAV)-DHFR(DD)-Flt23k and subsequent administration of TMP resulted in tunable suppression of ischemia-induced retinal neovascularization in a rat model of oxygen-induced retinopathy (OIR). Hence, our study suggests a promising novel approach for the treatment of retinal neovascularization. Schematic diagram of the tunable system utilizing the DHFR(DD)-Flt23k approach to reduce VEGF secretion. a The schematic shows normal VEGF secretion. b Without the ligand TMP, the DHFR(DD)-Flt23k protein is destabilized and degraded by the proteasome. c In the presence of the ligand TMP, DHFR(DD)-Flt23k is stabilized and sequestered in the ER, thereby conditionally inhibiting VEGF. Green lines indicate the intracellular and extracellular distributions of VEGF. Blue lines indicate proteasomal degradation of the DHFR(DD)-Flt23k protein. Orange lines indicate the uptake of cell-permeable TMP. TMP, trimethoprim; VEGF, vascular endothelial growth factor; ER, endoplasmic reticulum.
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ItemClinical Outcomes of a Treat and Extend Regimen with Intravitreal Aflibercept Injections in Patients with Diabetic Macular Edema: Experience in Clinical Practice(SPRINGER INTERNATIONAL PUBLISHING AG, 2020-03)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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ItemPeeking into the molecular trove of discarded surgical specimens(WILEY-BLACKWELL, 2016-11)
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ItemResponse: Cycloplegia in refraction: age and cycloplegics(WILEY-BLACKWELL, 2016-08)
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ItemA single-cell transcriptome atlas of the adult human retina(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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ItemCurrent state and future prospects of artificial intelligence in ophthalmology: a review(WILEY, 2019-01)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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ItemPlurality in multi-disciplinary research: multiple institutional affiliations are associated with increased citations(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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ItemGenomic locus modulating corneal thickness in the mouse identifies POU6F2 as a potential risk of developing glaucoma(PUBLIC LIBRARY SCIENCE, 2018-01)Central corneal thickness (CCT) is one of the most heritable ocular traits and it is also a phenotypic risk factor for primary open angle glaucoma (POAG). The present study uses the BXD Recombinant Inbred (RI) strains to identify novel quantitative trait loci (QTLs) modulating CCT in the mouse with the potential of identifying a molecular link between CCT and risk of developing POAG. The BXD RI strain set was used to define mammalian genomic loci modulating CCT, with a total of 818 corneas measured from 61 BXD RI strains (between 60-100 days of age). The mice were anesthetized and the eyes were positioned in front of the lens of the Phoenix Micron IV Image-Guided OCT system or the Bioptigen OCT system. CCT data for each strain was averaged and used to QTLs modulating this phenotype using the bioinformatics tools on GeneNetwork (www.genenetwork.org). The candidate genes and genomic loci identified in the mouse were then directly compared with the summary data from a human POAG genome wide association study (NEIGHBORHOOD) to determine if any genomic elements modulating mouse CCT are also risk factors for POAG.This analysis revealed one significant QTL on Chr 13 and a suggestive QTL on Chr 7. The significant locus on Chr 13 (13 to 19 Mb) was examined further to define candidate genes modulating this eye phenotype. For the Chr 13 QTL in the mouse, only one gene in the region (Pou6f2) contained nonsynonymous SNPs. Of these five nonsynonymous SNPs in Pou6f2, two resulted in changes in the amino acid proline which could result in altered secondary structure affecting protein function. The 7 Mb region under the mouse Chr 13 peak distributes over 2 chromosomes in the human: Chr 1 and Chr 7. These genomic loci were examined in the NEIGHBORHOOD database to determine if they are potential risk factors for human glaucoma identified using meta-data from human GWAS. The top 50 hits all resided within one gene (POU6F2), with the highest significance level of p = 10-6 for SNP rs76319873. POU6F2 is found in retinal ganglion cells and in corneal limbal stem cells. To test the effect of POU6F2 on CCT we examined the corneas of a Pou6f2-null mice and the corneas were thinner than those of wild-type littermates. In addition, these POU6F2 RGCs die early in the DBA/2J model of glaucoma than most RGCs. Using a mouse genetic reference panel, we identified a transcription factor, Pou6f2, that modulates CCT in the mouse. POU6F2 is also found in a subset of retinal ganglion cells and these RGCs are sensitive to injury.