A novel CRISPR-Cas13bt3 system has demonstrated effective silencing of the VEGFA gene in retinal cells, offering a potential therapeutic avenue for retinal diseases. The research, published in the Proceedings of the National Academy of Sciences, details the successful reduction of VEGFA mRNA and protein expression using adeno-associated virus (AAV)-delivered Cas13bt3 and sgRNA plasmids in both human retinal organoids and VEGF-transgenic mouse models.
The study, spearheaded by Satheesh Kumar and Guei-Sheung Liu from the Centre for Eye Research Australia and the University of Melbourne, addresses the critical need for targeted therapies in retinal diseases characterized by VEGFA overexpression.
Targeted VEGFA Knockdown
The CRISPR-Cas13bt3 system was designed to selectively target and degrade VEGFA mRNA, a key driver of angiogenesis and vascular permeability in retinal disorders. The researchers employed AAV vectors to deliver the Cas13bt3 and sgRNA plasmids, ensuring efficient transduction of retinal cells. Results showed a significant reduction in both VEGFA mRNA and protein levels, indicating successful gene silencing.
Specificity and Off-Target Effects
To assess the specificity of the Cas13bt3 system, single-cell RNA sequencing and transcriptomic analysis were performed. The data revealed that the system, when combined with a targeting sgRNA, maintained high specificity, primarily reducing VEGFA in retinal pigment epithelium (RPE) cells. Importantly, the study found limited unintended gene expression impacts across other cell types, suggesting a favorable safety profile.
Multiplexed sgRNA Delivery
The researchers also explored the potential of multiplexed sgRNA delivery to enhance VEGFA knockdown efficiency. Single AAV vectors were used to deliver multiple sgRNAs targeting VEGFA. However, the study found that multiplexed sgRNA delivery did not result in a significant increase in VEGFA knockdown compared to single sgRNA delivery, suggesting that a single, well-designed sgRNA is sufficient for effective gene silencing in this context.
Clinical Implications
These findings highlight the potential of the CRISPR-Cas13bt3 system as a targeted gene therapy approach for retinal diseases. By selectively silencing VEGFA, this system could offer a novel strategy for managing conditions characterized by abnormal angiogenesis and vascular leakage in the retina. Further research is needed to evaluate the long-term efficacy and safety of this approach in preclinical and clinical studies.
