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)
    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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    Comparison of CRISPR/Cas Endonucleases forin vivoRetinal Gene Editing
    Li, F ; Wing, K ; Wang, J-H ; Luu, CD ; Bender, JA ; Chen, J ; Wang, Q ; Lu, Q ; Nguyen Tran, MT ; Young, KM ; Wong, RCB ; Pebay, A ; Cook, AL ; Hung, SSC ; Liu, G-S ; Hewitt, AW (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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    Defined Medium Conditions for the Induction and Expansion of Human Pluripotent Stem Cell-Derived Retinal Pigment Epithelium
    Lidgerwood, GE ; Lim, SY ; Crombie, DE ; Ali, R ; Gill, KP ; Hernandez, D ; Kie, J ; Conquest, A ; Waugh, HS ; Wong, RCB ; Liang, HH ; Hewitt, AW ; Davidson, KC ; Pebay, A (SPRINGER, 2016-04)
    We demonstrate that a combination of Noggin, Dickkopf-1, Insulin Growth Factor 1 and basic Fibroblast Growth Factor, promotes the differentiation of human pluripotent stem cells into retinal pigment epithelium (RPE) cells. We describe an efficient one-step approach that allows the generation of RPE cells from both human embryonic stem cells and human induced pluripotent stem cells within 40-60 days without the need for manual excision, floating aggregates or imbedded cysts. Compared to methods that rely on spontaneous differentiation, our protocol results in faster differentiation into RPE cells. This pro-retinal culture medium promotes the growth of functional RPE cells that exhibit key characteristics of the RPE including pigmentation, polygonal morphology, expression of mature RPE markers, electrophysiological membrane potential and the ability to phagocytose photoreceptor outer segments. This protocol can be adapted for feeder, feeder-free and serum-free conditions. This method thereby provides a rapid and simplified production of RPE cells for downstream applications such as disease modelling and drug screening.
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    Enriched retinal ganglion cells derived from human embryonic stem cells
    Gill, KP ; Hung, SSC ; Sharov, A ; Lo, CY ; Needham, K ; Lidgerwood, GE ; Jackson, S ; Crombie, DE ; Nayagam, BA ; Cook, AL ; Hewitt, AW ; Pebay, A ; Wong, RCB (NATURE PORTFOLIO, 2016-08-10)
    Optic neuropathies are characterised by a loss of retinal ganglion cells (RGCs) that lead to vision impairment. Development of cell therapy requires a better understanding of the signals that direct stem cells into RGCs. Human embryonic stem cells (hESCs) represent an unlimited cellular source for generation of human RGCs in vitro. In this study, we present a 45-day protocol that utilises magnetic activated cell sorting to generate enriched population of RGCs via stepwise retinal differentiation using hESCs. We performed an extensive characterization of these stem cell-derived RGCs by examining the gene and protein expressions of a panel of neural/RGC markers. Furthermore, whole transcriptome analysis demonstrated similarity of the hESC-derived RGCs to human adult RGCs. The enriched hESC-RGCs possess long axons, functional electrophysiological profiles and axonal transport of mitochondria, suggestive of maturity. In summary, this RGC differentiation protocol can generate an enriched population of functional RGCs from hESCs, allowing future studies on disease modeling of optic neuropathies and development of cell therapies.
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    Study of mitochondrial respiratory defects on reprogramming to human induced pluripotent stem cells
    Hung, SSC ; Van Bergen, NJ ; Jackson, S ; Liang, H ; Mackey, DA ; Hernandez, D ; Lim, SY ; Hewitt, AW ; Trounce, I ; Pebay, A ; Wong, RCB (IMPACT JOURNALS LLC, 2016-05)
    Reprogramming of somatic cells into a pluripotent state is known to be accompanied by extensive restructuring of mitochondria and switch in metabolic requirements. Here we utilized Leber's hereditary optic neuropathy (LHON) as a mitochondrial disease model to study the effects of homoplasmic mtDNA mutations and subsequent oxidative phosphorylation (OXPHOS) defects in reprogramming. We obtained fibroblasts from a total of 6 LHON patients and control subjects, and showed a significant defect in complex I respiration in LHON fibroblasts by high-resolution respiratory analysis. Using episomal vector reprogramming, our results indicated that human induced pluripotent stem cell (hiPSC) generation is feasible in LHON fibroblasts. In particular, LHON-specific OXPHOS defects in fibroblasts only caused a mild reduction and did not significantly affect reprogramming efficiency, suggesting that hiPSC reprogramming can tolerate a certain degree of OXPHOS defects. Our results highlighted the induction of genes involved in mitochondrial biogenesis (TFAM, NRF1), mitochondrial fusion (MFN1, MFN2) and glycine production (GCAT) during reprogramming. However, LHON-associated OXPHOS defects did not alter the kinetics or expression levels of these genes during reprogramming. Together, our study provides new insights into the effects of mtDNA mutation and OXPHOS defects in reprogramming and genes associated with various aspects of mitochondrial biology.
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    Mitochondrial replacement in an iPSC model of Leber's hereditary optic neuropathy
    Wong, RCB ; Lim, SY ; Hung, SSC ; Jackson, S ; Khan, S ; Van Bergen, NJ ; De Smit, E ; Liang, HH ; Kearns, LS ; Clarke, L ; Mackey, DA ; Hewitt, AW ; Trounce, IA ; Pebay, A (IMPACT JOURNALS LLC, 2017-04)
    Cybrid technology was used to replace Leber hereditary optic neuropathy (LHON) causing mitochondrial DNA (mtDNA) mutations from patient-specific fibroblasts with wildtype mtDNA, and mutation-free induced pluripotent stem cells (iPSCs) were generated subsequently. Retinal ganglion cell (RGC) differentiation demonstrates increased cell death in LHON-RGCs and can be rescued in cybrid corrected RGCs.
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    Generation of a human induced pluripotent stem cell line CERAi001-A-6 using episomal vectors
    Wong, RCB ; Hung, SS ; Jackson, S ; Singh, V ; Khan, S ; Liang, HH ; Kearns, LS ; Nguyen, T ; Conquest, A ; Daniszewski, M ; Hewitt, AW ; Pebay, A (ELSEVIER, 2017-07)
    We report the generation of the hiPSC line CERAi001-A-6 from primary human dermal fibroblasts. Reprogramming was performed using episomal vector delivery of OCT4, SOX2, KLF4, L-MYC, LIN28 and shRNA for p53.
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    Friedreich's ataxia induced pluripotent stem cell-derived cardiomyocytes display electrophysiological abnormalities and calcium handling deficiency
    Crombie, DE ; Curl, CL ; Raaijmakers, AJA ; Sivakumaran, P ; Kulkarni, T ; Wong, RCB ; Minami, I ; Evans-Galea, MV ; Lim, SY ; Delbridge, L ; Corben, LA ; Dottori, M ; Nakatsuji, N ; Trounce, IA ; Hewitt, AW ; Delatycki, MB ; Pera, MF ; Pebay, A (IMPACT JOURNALS LLC, 2017-05)
    We sought to identify the impacts of Friedreich's ataxia (FRDA) on cardiomyocytes. FRDA is an autosomal recessive degenerative condition with neuronal and non-neuronal manifestations, the latter including progressive cardiomyopathy of the left ventricle, the leading cause of death in FRDA. Little is known about the cellular pathogenesis of FRDA in cardiomyocytes. Induced pluripotent stem cells (iPSCs) were derived from three FRDA individuals with characterized GAA repeats. The cells were differentiated into cardiomyocytes to assess phenotypes. FRDA iPSC- cardiomyocytes retained low levels of FRATAXIN (FXN) mRNA and protein. Electrophysiology revealed an increased variation of FRDA- cardiomyocyte beating rates which was prevented by addition of nifedipine, suggestive of a calcium handling deficiency. Finally, calcium imaging was performed and we identified small amplitude, diastolic and systolic calcium transients confirming a deficiency in calcium handling. We defined a robust FRDA cardiac-specific electrophysiological profile in patient-derived iPSCs which could be used for high throughput compound screening. This cell-specific signature will contribute to the identification and screening of novel treatments for this life-threatening disease.