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

CRISPR-Cas13 nucleases are programmable RNA-targeting effectors that can silence gene expression in a transient manner. Recent iterations of Cas13 nucleases are compact for adeno-associated virus (AAV) delivery to achieve strong and persistent expression of various organs in a safe manner. Here, we report significant transcriptomic signatures of Cas13bt3 expression in retinal cells and show all-in-one AAV gene therapy with Cas13bt3 can effectively silence <i>VEGFA</i> mRNA in human retinal organoids and humanized <i>VEGF</i> transgenic mouse (trVEGF029, Kimba) models. Specifically, human embryonic stem cells (hESC)-derived retinal pigment epithelium cells show high expression of Cas13bt3 from virus delivery corresponding to a significant reduction of <i>VEGFA</i> mRNA. We further show that intravitreal delivery of Cas13bt3 by AAV2.7m8 can efficiently transduce mouse retinal cells for specific knockdown of human <i>VEGFA</i> in the Kimba mouse. Our results reveal important considerations for assessing Cas13 activity, and establish the Cas13bt3 RNA editing system as a potential anti-VEGF agent that can achieve significant control of VEGFA for the treatment of retinal neovascularization.