Biology subjects

Luu, C. D.

Publications and source records attributed to Luu, C. D..

3 recordsLinked to original sources

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↗

bioRxiv2025

Engineering single-AAV CRISPR-Cas13 RNA base editors for treatment of inherited retinal diseases

Site-directed RNA editing, especially RNA base editing, allows for specific manipulation of RNA sequences, making it a useful approach for the correction of pathogenic mutations. Correction of RNA transcripts allows therapeutic gene editing in a safe and reversible manner and avoids permanent alterations in the genome. RNA-targeting CRISPR-Cas nucleases (e.g., CRISPR-Cas13) enable delivery within a single adeno-associated virus (AAV) vector for RNA base editing, making the approach clinically feasible. Here, we used the inactive CRISPR-Cas13bt3 (also known as Cas13X.1) fused to the ADAR2 deaminase domain (ADAR2DD) for targeted correction of inherited retinal disease (IRD) mutations. First, we show in vitro that dCas13bt3-ADAR2DD can efficiently correct a pathogenic nonsense mutation (c.130C>T [p.R44X]) found in the mouse Rpe65 gene and recover protein expression in retinal pigment epithelium cells (RPEs). Across clinically reported RPE65 mutations, we observed editing efficiencies ranging from 0% to 60%. In the Rpe65-deficient mouse model of retinal degeneration (rd12), we observed that RNA base editing can recover Rpe65 expression in RPEs and rescue retinal function with no observable adverse effects. We further employed our RNA base editor against the large USH2A gene to assess the promise of RNA base editing for addressing untreatable IRDs caused by genes too large for AAV gene delivery. Against the human USH2A in vitro, we observed up to 60% on-target efficiency. We further found that gRNA mismatches, domain-inlaid ADAR2DD design and nucleocytoplasmic shuttling of the RNA base editor optimised on-target and bystander editing for a highly precise base editor. Against the mouse Ush2a in vitro, we similarly observed up to 60% on-target editing in mammalian cells, while in the Ush2aW3947X mice, we observed [~]12% on-target editing, with no impact on retinal structure or function, or transcriptome-wide editing. Overall, our findings demonstrate dCas13bt3-ADAR2DD as a potent tool for gene therapy against IRDs, addressing a significant unmet clinical need in ophthalmology.

bioengineering↗

bioRxiv2020

Comparison of CRISPR/Cas endonucleases for in vivo retinal gene editing.

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 sgRNA targeting YFP and a single vector system with SaCas9/YFP sgRNA were generated and validated in YFP-expressing HEK293A cell by flow cytometry and 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 vitro and in vivo.

bioengineering↗