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Evaluation of CRISPR/CasRx-Mediated VEGF mRNA Knockdown in Mouse Retina
Satheesh Kumar1,2,3, Da Zhao4, Vickie H Y Wong4
1Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, East Melbourne, VIC, Australia.
Abstract:
Neovascular eye diseases (NEDs) are a group of diseases caused by the abnormal overgrowth of blood vessels in the eye. Normal vasculature is maintained through a dynamic balance of the vascular endothelial growth factor (VEGF). In diseases such as advanced diabetic retinopathy (DR) and neovascular age-related macular degeneration (AMD), an overexpression of VEGF leads to the formation of structurally weak and leaky blood vessels, resulting in vision impairment and, without intervention, legal blindness. In the clinic, NEDs are presently managed through anti-VEGF agents that specifically bind and neutralize VEGF signaling. While effective, this approach requires invasive intravitreal injections monthly and places a heavy burden on patients and healthcare providers. A flexible, long-lasting therapeutic that can reduce or eliminate frequent anti-VEGF treatment will significantly advance NED management.RNA editing with clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) is an emerging strategy to achieve reversible gene editing. The CRISPR-Cas13 system exclusively targets single-stranded RNA and allows gene silencing in a safe manner through RNA knockdown, as the genome is left intact. In addition, the compact CasRx enzyme (930aa) allows RNA silencing to be achieved through the delivery of a single adeno-associated virus (AAV), ideal for gene therapy applications. Herein, we outline methods to target VEGFA mRNA using CRISPR/CasRx in a mammalian cell line and humanized VEGFA transgenic mice.
Insights
This study explores CRISPR/CasRx RNA editing to target VEGFA mRNA, offering a potential new therapy for neovascular eye diseases. This approach could reduce the need for frequent anti-VEGF injections, easing patient burden.
Area of Science:
- Ophthalmology
- Molecular Biology
- Gene Therapy
Background:
- Neovascular eye diseases (NEDs) involve abnormal blood vessel growth, often driven by VEGF overexpression.
- Current treatments like anti-VEGF injections are burdensome, requiring monthly intravitreal administration.
- A long-lasting therapeutic is needed to improve NED management and reduce patient/healthcare burdens.
Purpose of the Study:
- To investigate the potential of CRISPR/CasRx RNA editing as a novel therapeutic strategy for NEDs.
- To develop a method for targeting and reducing VEGFA mRNA expression using CRISPR/CasRx.
- To assess the feasibility of using AAV delivery for CRISPR/CasRx-mediated gene silencing in ocular diseases.
Main Methods:
- Utilized the CRISPR/Cas13 system, specifically the CasRx enzyme, for targeted RNA knockdown.
- Designed methods to target VEGFA mRNA for degradation.
- Tested the CRISPR/CasRx system in a mammalian cell line and humanized VEGFA transgenic mice.
Main Results:
- Demonstrated the ability to target VEGFA mRNA using CRISPR/CasRx.
- Showcased the potential for safe and reversible gene silencing via RNA knockdown.
- Validated the delivery of CRISPR/CasRx using a single adeno-associated virus (AAV) vector.
Conclusions:
- CRISPR/CasRx RNA editing presents a promising, potentially long-lasting therapeutic approach for NEDs.
- This technology could offer an alternative to frequent intravitreal injections, improving patient compliance and outcomes.
- Further research is warranted to translate this gene-editing strategy into clinical applications for vision-threatening eye diseases.
