Characterisation of RNA editing and gene therapy with a compact CRISPR-Cas13 in the retina

Abstract CRISPR-Cas13 nucleases are programmable RNA-targeting effectors that can silence gene expression in a reversible manner. Recent iterations of Cas13 nucleases are compact for adeno-associated virus (AAV) delivery to achieve strong and persistent expression in various organs in a safe manner. Here, we report significant transcriptomic signatures of Cas13bt3 expression and show all-in-one AAV gene therapy with Cas13bt3 can effectively silence VEGFA in human retinal organoids and humanised mouse models. Specifically, human embryonic stem cells (hESC)-derived retinal pigment epithelium cells show high expression of Cas13bt3 from virus delivery corresponding to significant reduction of VEGFA mRNA. We further show that intravitreal delivery of Cas13bt3 can transduce mouse retinal cells efficiently, reaching the photoreceptors for specific knockdown of human VEGFA in the trVEGF029 (Kimba) mouse. Our results reveal important considerations for assessing Cas13 activity and establishing Cas13bt3 as a potential anti-VEGF agent that can achieve long-term control of VEGFA for treatment of diabetic retinopathy and wet age-related macular degeneration.