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

Abstract

CRISPR-Cas gene editing technology has revolutionised the development of novel genetic therapies. CRISPR-Cas13 nucleases are programmable RNA-targeting effectors that can silence gene expression in a reversible manner. Recent iterations of Cas13 nucleases are compact and adequate for adeno-associated virus (AAV) delivery to achieve strong and persistent transduction of various organs in a safe manner. Here, we report significant transcriptomic signatures of Cas13bt3 expression and demonstrate that all-in-one AAV gene therapy with Cas13bt3 can effectively silenceVEGFAin human retinal organoids and humanised mouse models. Specifically, human embryonic stem cells (hESC)-derived retinal pigment epithelium cells show high expression of Cas13bt3 after virus delivery corresponding to significant reduction ofVEGFAmRNA. We further show that intravitreal delivery of Cas13bt3 can transduce mouse retinal cells efficiently, reaching the photoreceptor cells for specific knockdown of humanVEGFA, without impacting mouseVEGFAin the trVEGF029 (Kimba) mouse. Our results reveal important considerations for assessing Cas13 activity and establish Cas13bt3 as a potential anti-VEGF agent that can achieve long-term control of VEGF for treatment of diabetic retinopathy and wet age-related macular degeneration.