Leverage of nuclease-deficient CasX for preventing pathological angiogenesis

Haote Han; Yanhui Yang; Yunjuan Jiao; Hui Qi; Zhuo Han; Luping Wang; Lijun Dong; Jingkui Tian; Bart Vanhaesebroeck; Xiaopeng Li; Junwen Liu; Gaoen Ma; Hetian Lei

doi:10.1016/j.omtn.2023.08.001

Leverage of nuclease-deficient CasX for preventing pathological angiogenesis

Mol Ther Nucleic Acids. 2023 Aug 6:33:738-748. doi: 10.1016/j.omtn.2023.08.001. eCollection 2023 Sep 12.

Authors

Haote Han¹, Yanhui Yang², Yunjuan Jiao^{3

4}, Hui Qi⁵, Zhuo Han¹, Luping Wang¹, Lijun Dong⁵, Jingkui Tian¹, Bart Vanhaesebroeck⁶, Xiaopeng Li⁴, Junwen Liu³, Gaoen Ma⁴, Hetian Lei^{4

5}

Affiliations

¹ Institute of Basic Medicine and Cancer, Chinese Academy of Sciences, Cancer Hospital of the University of Chinese Academy of Sciences, Zhejiang Cancer Hospital, Hangzhou 310000, People's Republic of China.
² Ningxia Key Laboratory of Prevention and Control of Common Infectious Diseases, the School of Basic Medical Sciences, Ningxia Medical University, Yinchuan 750004, People's Republic of China.
³ Department of Histology and Embryology, Xiangya School of Medicine, Central South University, Changsha 410013, People's Republic of China.
⁴ The Third Affiliated Hospital of Xinxiang Medical University, Xinxiang 453003, China.
⁵ Shenzhen Eye Hospital, Jinan University, Shenzhen Eye Institute, Shenzhen 518000, People's Republic of China.
⁶ Cancer Institute, University College London, London NW1 2BU, UK.

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.

Keywords: CRISPR/dCasX-KRAB; MT: RNA/DNA editing; PI3Kδ; Pik3cd; angiogenesis; oxygen-induced retinopathy; rAAV1.