Evaluation of trabecular meshwork-specific promoters in vitro and in vivo using scAAV2 vectors expressing C3 transferase  

作　　者：Jun-Kai Tan Ying Xiao Guo Liu Long-Xiang Huang Wen-Hao Ma Yan Xia Xi-Zhen Wang Xian-Jun Zhu Su-Ping Cai Xiao-Bing Wu Yun Wang Xu-Yang Liu 

机构地区：[1]Xiamen Eye Center,Xiamen University,Xiamen 361004,Fujian Province,China [2]Department of Pathology,Hospital of Chengdu University of Traditional Chinese Medicine,Chengdu 610032,Sichuan Province,China [3]Sichuan Provincial Key Laboratory for Human Disease Gene Study,Sichuan Provincial People’s Hospital,University of Electronic Science and Technology of China,Chengdu 610072,Sichuan Province,China [4]The First Affiliated Hospital of Fujian Medical University,Fuzhou 350004,Fujian Province,China [5]Beijing FivePlus Molecular Medicine Institute Co.,Ltd.,Beijing 102600,China [6]Shenzhen Key Laboratory of Ophthalmology,Shenzhen Eye Hospital,School of Optometry,Jinan University,Shenzhen 518040,Guangdong Province,China [7]Department of Ophthalmology,Shenzhen People’s Hospital,the 2nd Clinical Medical College,Jinan University,Shenzhen 518020,Guangdong Province,China

出　　处：《International Journal of Ophthalmology(English edition)》2023年第8期1196-1209,共14页国际眼科杂志（英文版）

基　　金：Supported by the National Natural Science Foundation of China(No.81900829,No.82070963);the Xiamen Medical and Health Guiding Project Fund Project(No.3502Z20214ZD1214);the Guangdong Basic and Applied Basic Research Foundation(No.2019A1515011234);the Science and Technology Innovation Committee of Shenzhen(No.JCYJ20210324125614039)。

摘　　要：AIM:To evaluate the potential of two trabecular meshwork(TM)-specific promoters,Chitinase 3-like 1(Ch3L1)and matrix gla protein(MGP),for improving specificity and safety in glaucoma gene therapy based on self-complementary AAV2(scAAV2)vector technologies.METHODS:An scAAV2 vector with C3 transferase(C3)as the reporter gene(scAAV2-C3)was selected.The scAAV2-C3 vectors were driven by Ch3L1(scAAV2-Ch3L1-C3),MGP(scAAV2-MGP-C3),enhanced MGP(scAAV2-eMGP-C3)and cytomegalovirus(scAAV2-CMV-C3),respectively.The cultured primary human TM cells were treated with each vector at different multiplicities of infections.Changes in cell morphology were observed by phase contrast microscopy.Actin stress fibers and Rho GTPases/Rho-associated protein kinase pathway-related molecules were assessed by immunofluorescence staining,real-time quantitative polymerase chain reaction and Western blot.Each vector was injected intracamerally into the one eye of each rat at low and high doses respectively.In vivo green fluorescence was visualized by a Micron III Retinal Imaging Microscope.Intraocular pressure(IOP)was monitored using a rebound tonometer.Ocular responses were evaluated by slit-lamp microscopy.Ocular histopathology analysis was examined by hematoxylin and eosin staining.RESULTS:In TM cell culture studies,the vectormediated C3 expression induced morphologic changes,disruption of actin cytoskeleton and reduction of fibronectin expression in TM cells by inhibiting the Rho GTPases/Rhoassociated protein kinase signaling pathway.At the same dose,these changes were significant in TM cells treated with scAAV2-CMV-C3 or scAAV2-Ch3L1-C3,but not in cells treated with scAAV2-eMGP-C3 or scAAV2-MGP-C3.At lowinjected dose,the IOP was significantly decreased in the scAAV2-Ch3L1-C3-injected eyes but not in scAAV2-MGPC3-injected and scAAV2-eMGP-C3-injected eyes.At highinjected dose,significant IOP reduction was observed in the scAAV2-eMGP-C3-injected eyes but not in scAAV2-MGP-C3-injected eyes.Similar to scAAV2-CMV-C3,scAAV2-Ch3L1-C3 vector showed ef

关 键 词：self-complementary AAV2 chitinase 3-like 1 matrix gla protein trabecular meshwork C3 transferase 

分 类 号：Q78[生物学—分子生物学]