Ophthalmology (Eye & Ear Hospital) - 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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ItemAAV-mediated gene delivery of the calreticulin anti-angiogenic domain inhibits ocular neovascularization(SPRINGER, 2018-02)Ocular neovascularization is a common pathological feature in diabetic retinopathy and neovascular age-related macular degeneration that can lead to severe vision loss. We evaluated the therapeutic efficacy of a novel endogenous inhibitor of angiogenesis, the calreticulin anti-angiogenic domain (CAD180), and its functional 112-residue fragment, CAD-like peptide 112 (CAD112), delivered using a self-complementary adeno-associated virus serotype 2 (scAAV2) in rodent models of oxygen-induced retinopathy and laser-induced choroidal neovascularization. The expression of CAD180 and CAD112 was elevated in human umbilical vein endothelial cells transduced with scAAV2-CAD180 or scAAV2-CAD112, respectively, and both inhibited angiogenic activity in vitro. Intravitreal gene delivery of scAAV2-CAD180 or scAAV2-CAD112 significantly inhibited ischemia-induced retinal neovascularization in rat eyes (CAD180: 52.7% reduction; CAD112: 49.2% reduction) compared to scAAV2-mCherry, as measured in retinal flatmounts stained with isolectin B4. Moreover, the retinal structure and function were unaffected by scAAV2-CAD180 or scAAV2-CAD112, as measured by optical coherence tomography and electroretinography. Moreover, subretinal delivery of scAAV2-CAD180 or scAAV2-CAD112 significantly attenuated laser-induced choroidal neovascularization in mouse eyes compared to scAAV2-mCherry, as measured by fundus fluorescein angiography (CAD180: 62.4% reduction; CAD112: 57.5% reduction) and choroidal flatmounts (CAD180: 40.21% reduction; CAD112: 43.03% reduction). Gene delivery using scAAV2-CAD180 or scAAV2-CAD112 has significant potential as a therapeutic option for the management of ocular neovascularization.
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ItemNo Preview AvailableMethods for In Vivo CRISPR/Cas Editing of the Adult Murine Retina(HUMANA PRESS INC, 2018)Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) is used by some bacteria and most archaea to protect against viral phage intrusion and has recently been adapted to allow for efficient editing of the mammalian genome. Whilst CRISPR/Cas-based technology has been used to modify genes in mammalian cells in vitro, delivery of CRISPR/Cas system into mammalian tissue and/or organs is more difficult and often requires additional vectors. With the use of adeno-associated virus (AAV) gene delivery system, active CRISPR/Cas enzyme can be maintained for an extended period of time and enable efficient editing of genome in the retina in vivo. Herein we outline the method to edit the genome in mouse retina using a dual AAV vector -mediated CRISPR/Cas9 system.
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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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ItemMicroRNA-143 plays a protective role in ischemia-induced retinal neovascularization( 2019-02-13)Retinal neovascularization is a severe complication of proliferative diabetic retinopathy. MicroRNAs (miRNAs) are master regulators of gene expression that play important roles in retinal neovascularization. Here, we investigated the retinal miRNA expression profile in a rat model of oxygen-induced retinopathy (OIR) through miRNA-Seq. We found that miR-143-3p, miR-126-3p, miR-150-5p and miR-145-5p were significantly down-regulated in the retina of OIR rats, and directly involved in the development of retinal neovascularization. Of these identified miRNAs, miR-143 is enriched in retina and was first reported being associated with pathological retinal angiogenesis. Our RNA-Seq data further suggested that miR-143 alleviates retinal neovascularization by mediating the inflammation/stress pathways via Fos. Moreover, the computational analysis indicated that Transforming Growth Factor-beta Activated Kinase 1 (TAK1) is involved in several key pathways associated with the dysregulated miRNAs. The pharmacological inhibition of TAK1 suppressed angiogenesis in vitro and retinal neovascularization in vivo. Our data highlight the utility of next-generation sequencing in the development of therapeutics for ocular neovascularization and further suggest that therapeutic targeting the dysregulated miRNAs or TAK1 may be a feasible adjunct therapeutic approach in patients with retinal neovascularization.
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ItemNo Preview AvailableThe effect of ageing on the recovery of retinal function and structure following intraocular pressure elevation in mice(Association for Research in Vision and Ophthalmology, 2019-07-01)Purpose : To investigate the effect of ageing on the capacity of the eye to cope with acute intraocular pressure (IOP) elevation in mice Methods : IOP was elevated to 50 mmHg for 30 minutes in anaesthetised (ketamine/xylazine) 3- and 12-month old (3mo and 12mo) C57Bl/6 mice by infusing Hanks’ Balance Salt Solution through a glass micropipette (~50μm tip) inserted into the anterior chamber of one randomly selected eye. The contralateral eye served as an untreated control. Retinal function was assessed using electroretinogram to provide an index of the health of the major cell classes in the eye. Retinal structure was assessed using optical coherence tomography (OCT) which returns thickness for a range of retinal layers. Responses were collected one week prior to and at 3 (n=13 3mo, n=11 12mo), 7 (n=13 3mo, n=10 12mo), 14 (n=10 3mo, n=11 12mo) or 28 (n=11 3mo, n=11 12mo) days after IOP elevation. Responses in the high IOP eye were expressed relative (%) to their contralateral control eye (mean±SEM). As retinal ganglion cell (RGC) responses are influenced by input from the outer retina, we expressed the functional recovery of RGC as the % difference between relative RGC (output cells) and photoreceptor (input cells) function. The effect of age on RGC functional recovery and retinal structural changes at the various recovery time points was analysed using two-way ANOVA. Results : In 3-month old eyes, 3 days after IOP elevation, RGC function was -37.3±7.0% worse than expected from photoreceptoral input. By 7 days after IOP elevation, RGC responses were similar to photoreceptor responses (-5.7±7.2%) and remained so at 14 (-9.7±6.0%) and 28 (15.6±16.4%) days of recovery. In contrast, 12-month old eyes showed slower recovery. RGC responses were worse than expected from photoreceptoral responses at 3 (-58.1±6.1%) and 7 (-34.8±10.5%) days. Only at 14 (-9.4±10.0%) and 28 (1.9±13.1%) days had RGC responses returned to levels comparable with photoreceptoral responses in 12-month old eyes. Two-way ANOVA confirmed a significant age effect in the functional recovery (p<0.05). There was, however, no significant differences in retinal layers measured using OCT with age. Conclusions : RGC function was more affected by acute IOP elevation than photoreceptoral responses. Ageing slowed down the functional recovery of RGC following an acute IOP stressor but appears to have little effect on retinal structure.
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ItemNo Preview AvailableA retinal imaging biomarker of Alzheimer's disease(Wiley, 2019-11-01)Background: Amyloid-beta (Ab) deposition in the brain is a diagnostic marker for Alzheimer's disease (AD), but current tests are costly and not widely available. Evidence from transgenic rodent models and post-mortem human tissues suggest that retinal accumulation of Ab may serve as a surrogate marker of brain Ab levels. As Ab has a wavelength-dependent effect on light scatter, we investigated the potential for in vivo retinal hyperspectral imaging to serve as a biomarker of brain Ab. Purpose: To develop and validate a retinal imaging biomarker of Alzheimer's disease. Methods: We performed human retinal hyperspectral imaging on individuals with high Ab burden on brain PET imaging and mild cognitive impairment (cases; n = 15), and age-matched PET-negative controls (n = 20). Image analysis methods were developed and validated on a second group of participants with and with (n = 4) and without (n = 13) moderate-to-high brain Ab burden and on transgenic mice (5xFAD) known to accumulate retinal Ab. Results: We show significant differences in retinal reflectance spectra between cases and controls in both cohorts (AUC ROC = 0.82, P = 0.001, 95% CI: 0.67-0.97). There was a moderate positive linear correlation between retinal imaging scores and brain Abburden (r = 0.46, 95%CI: 0.13-0.69, P = 0.008).The technique also enabled discrimination of AD-model mice from wild-type controls. Conclusion: We have developed a novel retinal imaging method to distinguish people with moderate-high brain Ab load from those without. This approach may have value for the diagnostic confirmation of AD.
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ItemNo Preview AvailableA tractable preclinical model of optic nerve demyelination(Association for Research in Vision and Ophthalmology, 2019-07-01)Purpose : Progress in the development of therapies to enhance remyelination in demyelinating diseases has been hampered by a lack of appropriate preclinical models - functional measures are often lacking or variable. We sought to develop a tractable and reproducible model of optic nerve demyelination with precise structural and functional measures. Methods : Oligodendrocytes of MBP-DTR 100a transgenic mice express diphtheria toxin receptor (DTR) and systemic diphtheria toxin (DT) administration induces diffuse demyelination of the central nervous system. In the present study we used retrobulbar DT injection to induce focal demyelination of the optic nerves of 3-month-old MBP-DTR 100a mice. Dose optimisation: anaesthetised mice underwent unilateral retrobulbar DT injection with 5, 10 or 15ng/kg DT (n=7 per dose, 1 µL per injection). Tissues were harvested three weeks after injection. Time-course study: Following baseline visual evoked potential (VEP) recording, electroretinogram (ERG) and optical coherence tomography (OCT), mice underwent retrobulbar DT injection with 15ng/kg DT or 1µL PBS. Follow-up measurements were taken at 2 (n=5 DT, 5 PBS), 4 (n=6 DT, 6 PBS), 8 (n=9 DT, 9 PBS) or 12-weeks (n=7 DT, 7 PBS). Animals were culled at each timepoint for tissue analysis. Tissue analysis: Optic nerves were resin embedded, sectioned (1µm) and stained with toluidine blue for myelin analysis, or cryosectioned for immunofluorescence, and retinas were flat-mounted for ganglion cell counts. Results : 3 weeks after injection with 15ng/kg DT, optic nerves showed colocalisation of activated caspase 3 & olig2, consistent with the apoptosis of oligodendroglia. Gliosis and axonal degeneration were evident.
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ItemNon-invasive in vivo hyperspectral imaging of the retina for potential biomarker use in Alzheimer's disease(NATURE PUBLISHING GROUP, 2019-09-17)Studies of rodent models of Alzheimer's disease (AD) and of human tissues suggest that the retinal changes that occur in AD, including the accumulation of amyloid beta (Aβ), may serve as surrogate markers of brain Aβ levels. As Aβ has a wavelength-dependent effect on light scatter, we investigate the potential for in vivo retinal hyperspectral imaging to serve as a biomarker of brain Aβ. Significant differences in the retinal reflectance spectra are found between individuals with high Aβ burden on brain PET imaging and mild cognitive impairment (n = 15), and age-matched PET-negative controls (n = 20). Retinal imaging scores are correlated with brain Aβ loads. The findings are validated in an independent cohort, using a second hyperspectral camera. A similar spectral difference is found between control and 5xFAD transgenic mice that accumulate Aβ in the brain and retina. These findings indicate that retinal hyperspectral imaging may predict brain Aβ load.
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ItemGene Therapy with Endogenous Inhibitors of Angiogenesis for Neovascular Age-Related Macular Degeneration: Beyond Anti-VEGF Therapy(HINDAWI LTD, 2015)Age-related macular degeneration (AMD) is the leading cause of substantial and irreversible vision loss amongst elderly populations in industrialized countries. The advanced neovascular (or "wet") form of the disease is responsible for severe and aggressive loss of central vision. Current treatments aim to seal off leaky blood vessels via laser therapy or to suppress vessel leakage and neovascular growth through intraocular injections of antibodies that target vascular endothelial growth factor (VEGF). However, the long-term success of anti-VEGF therapy can be hampered by limitations such as low or variable efficacy, high frequency of administration (usually monthly), potentially serious side effects, and, most importantly, loss of efficacy with prolonged treatment. Gene transfer of endogenous antiangiogenic proteins is an alternative approach that has the potential to provide long-term suppression of neovascularization and/or excessive vascular leakage in the eye. Preclinical studies of gene transfer in a large animal model have provided impressive preliminary results with a number of transgenes. In addition, a clinical trial in patients suffering from advanced neovascular AMD has provided proof-of-concept for successful gene transfer. In this mini review, we summarize current theories pertaining to the application of gene therapy for neovascular AMD and the potential benefits when used in conjunction with endogenous antiangiogenic proteins.