Han, H; Yang, Y; Jiao, Y; Qi, H; Han, Z; Wang, L; Dong, L; ... Lei, H; + view all Han, H; Yang, Y; Jiao, Y; Qi, H; Han, Z; Wang, L; Dong, L; Tian, J; Vanhaesebroeck, B; Li, X; Liu, J; Ma, G; Lei, H; - view fewer (2023) Leverage of nuclease-deficient CasX for preventing pathological angiogenesis. Molecular Therapy - Nucleic Acids , 33 pp. 738-748. 10.1016/j.omtn.2023.08.001.

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Abstract

Gene editing with a CRISPR/Cas system is a novel potential strategy for treating human diseases. Pharmacological inhibition of phosphoinositide 3-kinase (PI3K) δ suppresses retinal angiogenesis in a mouse model of oxygen-induced retinopathy. Here we show that an innovative system of adeno-associated virus (AAV)-mediated CRISPR/nuclease-deficient (d)CasX fused with the Krueppel-associated box (KRAB) domain is leveraged to block (81.2% ± 6.5%) in vitro expression of p110δ, the catalytic subunit of PI3Kδ, encoded by Pik3cd. This CRISPR/dCasX-KRAB (4, 269 bp) system is small enough to be fit into a single AAV vector. We then document that recombinant AAV serotype (rAAV)1 efficiently transduces vascular endothelial cells from pathologic retinal vessels, which show high expression of p110δ; furthermore, we demonstrate that blockade of retinal p110δ expression by intravitreally injected rAAV1-CRISPR/dCasX-KRAB targeting the Pik3cd promoter prevents (32.1% ± 5.3%) retinal p110δ expression as well as pathological retinal angiogenesis in a mouse model of oxygen-induced retinopathy. These data establish a strong foundation for treating pathological angiogenesis by AAV-mediated CRISPR interference with p110δ expression.

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