Biology subjects

Wong, R. C. B.

Publications and source records attributed to Wong, R. C. B..

5 recordsLinked to original sources

bioRxiv2026

Characterising retinal function with optomotor visual performance in P23H rodent models of retinitis pigmentosa

Rhodopsin (RHO) P23H is one of the most common mutations causing autosomal dominant retinitis pigmentosa (adRP), yet the relationship between retinal electrophysiology, structure and visually guided behaviour in rodent models remains unclear. We characterised changes in heterozygous P23H (Sakami line) mice and P23H line 3 (P23H-3) rats using full-field electroretinography (ERG), optomotor response (OMR) assays and, in rats, optical coherence tomography (OCT). ERG assessed rod- and cone-mediated responses relative to wild-type controls, whereas OMR under scotopic and photopic conditions quantified contrast sensitivity and visual acuity. In P23H mice, scotopic ERG responses were significantly reduced from postnatal day 16 and declined further from 4 months. Scotopic OMR contrast sensitivity remained largely preserved until 2 months, and photopic acuity was comparable to wild-type up to 6 months. In 13-week-old P23H-3 rats, ERG amplitudes were significantly reduced, and OCT revealed retinal thinning. OMR showed a decline in contrast sensitivity at 7 and 15 weeks, whereas photopic acuity was maintained. Thus, in both models, electrophysiological and structural abnormalities precede detectable OMR deficits, with implications for the selection of outcome measures in preclinical studies. Summary StatementThis study compares electrical and behavioural measures of vision in rodent models of inherited blindness, revealing that retinal dysfunction appears well before measurable vision loss.

animal behavior and cognition↗

bioRxiv2026

Optimization of AAV tools to target M&uumlller glial cells for retinal gene therapy

Reprogramming of Muller glial (MG) cells into retinal neurons has the potential to treat vision loss by regenerating the retina. Development of efficient gene delivery systems to target the MG cells is critical. Adeno-associated virus (AAV) serotypes and promoter specificity are important factors that influence AAV transduction profile in the retina. However, studies that optimize these parameters to specifically target MG cells are limited, in particular in rats which are commonly used for eye research. Here we tested 4 AAV serotypes and 14 promoters to optimize gene delivery to human MG cells in vitro and/or rat MG cells in vivo. We showed that the combinatorial use of MG-specific serotypes and promoters achieved high specificity for MG cell targeting, with ShH10Y serotype and the GFAP (gfaABC1D) promoter as the best performing tool to target rat MG cells in vivo. We developed new AAV vectors using known and novel MG-specific promoters and engineered short promoter variants to improve the cargo capacity of AAV delivery. Our results highlighted a number of promoters that can target MG cells in vitro or in vivo. This study further expands the AAV toolbox to target MG cells, which has important implications for retinal gene therapy development.

bioengineering↗

bioRxiv2024

Transcriptomic analysis of TGFβ-mediated fibrosis in primary human Tenon's fibroblasts

Glaucoma filtration surgery (GFS) is performed to slow down disease progression in glaucoma, a leading cause of irreversible blindness worldwide. Following surgery, pathological wound healing may lead to conjunctival fibrosis and filtering failure. Myofibroblasts are the key cells responsible for postoperative conjunctival scarring. This study aims to further understand the molecular mechanisms of conjunctival fibrosis following GFS. We utilised RNA-sequencing (RNA-seq) to delineate the TGF{beta}1 induced changes in the transcriptome of human Tenons fibroblasts (HTFs). RNA sequencing was performed on HTFs after 5 days of TGF{beta}1 treatment. Following quality control, 3,362 differentially expressed genes were identified, of which 1,532 were upregulated and 1,820 were downregulated. We identified signaling pathways associated with the pathogenesis of conjunctival fibrosis. The DEGs (differentially expressed genes) were enriched in pathways including myofibroblast differentiation, TGF{beta}-signaling, collagen and extracellular matrix organization, epithelial to mesenchymal transition, and cell cycle regulation. The results of this study identified the transition from HTF to myofibroblast is characterised by the upregulation of key genes including LDLRAD4, CDKN2B, FZD8, MYOZ1, and the downregulation of SOD3, LTBP4 and RCAN2. This unprecedented insight into the transcriptional landscape of HTFs and myofibroblast differentiation is essential to understand the pathophysiology of conjunctival scarring and develop new therapeutic agents.

bioinformatics↗

bioRxiv2024

Spatial transcriptomic profiling of human retinoblastoma

Retinoblastoma (RB) represents one of the most prevalent intraocular cancers in children. Understanding the tumor heterogeneity in RB is important to design better targeted therapies. Here we used spatial transcriptomic to profile human retina and RB tumor to comprehensively dissect the spatial cell-cell communication networks. We found high intratumoral heterogeneity in RB, consisting of 10 transcriptionally distinct subpopulations with varying levels of proliferation capacity. Our results uncovered a complex architecture of the tumor microenvironment that predominantly consisted of cone precursors, as well as glial cells and cancer-associated fibroblasts. We delineated the cell trajectory underlying malignant progression of RB, and identified key signaling pathways driving genetic regulation across RB progression. We also explored the signaling pathways mediating cell-cell communications in RB subpopulations, and mapped the spatial networks of RB subpopulations and region neighbors. Altogether, we constructed the first spatial gene atlas for RB, which allowed us to characterize the transcriptomic landscape in spatially-resolved RB subpopulations, providing novel insights into the complex spatial communications involved in RB progression.

systems biology↗

bioRxiv2018

A simple cloning-free method to efficiently induce gene expression using CRISPR/Cas9

Gain-of-function studies often require the tedious cloning of transgene cDNA into vectors for overexpression beyond the physiological expression levels. The rapid development of CRISPR/Cas technology presents promising opportunities to address these issues. Here we report a simple, cloning-free method to induce gene expression at endogenous locus using CRISPR/Cas9 activators. Our strategy utilises synthesized sgRNA expression cassettes to direct a nuclease-null Cas9 complex fused with transcriptional activators (VP64, p65 and Rta) for site-specific induction of endogenous genes. This strategy allows rapid initiation of gain-of-function studies in the same day. Using this cloning-free approach, we tested two CRISPR activation systems, dSpCas9VPR and dSaCas9VPR, for induction of multiple genes in human and rat cells. Our results showed that both CRISPR activators allow efficient induction of six different neural development genes (CRX, RORB, RAX, OTX2, ASCL1 and NEUROD1) in human cells, whereas the rat cells exhibit a more variable and less efficient levels of gene induction, as observed in three different genes (Ascl1, Neurod1, Nrl). Altogether, this study provides a simple method to efficiently activate endogenous gene expression using CRISPR/Cas9 activators, which can be applies as a rapid workflow to initiate gain-of-function studies for a range of molecular and cell biology disciplines.

molecular biology↗