دورية أكاديمية
Evaluation of trabecular meshwork-specific promoters in vitro and in vivo using scAAV2 vectors expressing C3 transferase
| العنوان: | Evaluation of trabecular meshwork-specific promoters in vitro and in vivo using scAAV2 vectors expressing C3 transferase |
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| المؤلفون: | 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 |
| المصدر: | International Journal of Ophthalmology, Vol 16, Iss 8, Pp 1196-1209 (2023) |
| بيانات النشر: | Press of International Journal of Ophthalmology (IJO PRESS), 2023. |
| سنة النشر: | 2023 |
| المجموعة: | LCC:Ophthalmology |
| مصطلحات موضوعية: | self-complementary aav2, chitinase 3-like 1, matrix gla protein, trabecular meshwork, c3 transferase, Ophthalmology, RE1-994 |
| الوصف: | 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 vector-mediated C3 expression induced morphologic changes, disruption of actin cytoskeleton and reduction of fibronectin expression in TM cells by inhibiting the Rho GTPases/Rho-associated 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 low-injected dose, the IOP was significantly decreased in the scAAV2-Ch3L1-C3-injected eyes but not in scAAV2-MGP-C3-injected and scAAV2-eMGP-C3-injected eyes. At high-injected 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 efficient transduction both in the TM and corneal endothelium. In anterior segment tissues of scAAV2-eMGP-C3-injected eyes, no obvious morphological changes were found except for the TM. Inflammation was absent. CONCLUSION: In scAAV2-transduced TM cells, the promoter-driven efficiency of Ch3L1 is close to that of cytomegalovirus, but obviously higher than that of MGP. In the anterior chamber of rat eye, the transgene expression pattern of scAAV2 vector is presumably affected by MGP promoter, but not by Ch3L1 promoter. These findings would provide a useful reference for improvement of specificity and safety in glaucoma gene therapy using scAAV2 vector. |
| نوع الوثيقة: | article |
| وصف الملف: | electronic resource |
| اللغة: | English |
| تدمد: | 2222-3959 2227-4898 |
| Relation: | http://ies.ijo.cn/en_publish/2023/8/20230803.pdf; https://doaj.org/toc/2222-3959; https://doaj.org/toc/2227-4898 |
| DOI: | 10.18240/ijo.2023.08.03 |
| URL الوصول: | https://doaj.org/article/8772af439d924f33b47576524ad993af |
| رقم الأكسشن: | edsdoj.8772af439d924f33b47576524ad993af |
| قاعدة البيانات: | Directory of Open Access Journals |
Full Text Finder | تدمد: | 22223959 22274898 |
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| DOI: | 10.18240/ijo.2023.08.03 |