Han Haote, Yang Yanhui, Jiao Yunjuan, Qi Hui, Han Zhuo, Wang Luping, Dong Lijun, Tian Jingkui, Vanhaesebroeck Bart, Li Xiaopeng, Liu Junwen, Ma Gaoen, Lei Hetian
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.
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.
Mol Ther Nucleic Acids. 2023 Aug 6;33:738-748. doi: 10.1016/j.omtn.2023.08.001. eCollection 2023 Sep 12.
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%) expression of p110δ, the catalytic subunit of PI3Kδ, encoded by . 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 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.
利用CRISPR/Cas系统进行基因编辑是一种治疗人类疾病的新型潜在策略。在氧诱导视网膜病变小鼠模型中，磷酸肌醇3激酶（PI3K）δ的药理学抑制可抑制视网膜血管生成。在此，我们展示了一种创新系统，即腺相关病毒（AAV）介导的与克勒佩尔相关盒（KRAB）结构域融合的CRISPR/核酸酶缺陷型（d）CasX，用于阻断由 编码的PI3Kδ催化亚基p110δ的表达（81.2%±6.5%）。这种CRISPR/dCasX-KRAB（4269 bp）系统足够小，可以装入单个AAV载体。然后我们证明重组腺相关病毒血清型（rAAV）1能有效地转导病理性视网膜血管中的血管内皮细胞，这些细胞显示出p110δ的高表达；此外，我们还证明，在氧诱导视网膜病变小鼠模型中，通过玻璃体内注射靶向 启动子的rAAV1-CRISPR/dCasX-KRAB来阻断视网膜p110δ的表达，可防止（32.1%±5.3%）视网膜p110δ的表达以及病理性视网膜血管生成。这些数据为通过AAV介导的CRISPR干扰p110δ表达来治疗病理性血管生成奠定了坚实基础。
