Zhu Xinlei, Qi Benxiang, Ren Zhongmei, Cong Lin, Pan Xiaojing, Zhou Qingjun, Zhang Bi Ning, Xie Lixin
Qingdao Medical College, Qingdao University, Qingdao, China.
Eye Institute of Shandong First Medical University, Qingdao Eye Hospital of Shandong First Medical University, Qingdao, China.
Invest Ophthalmol Vis Sci. 2025 Apr 1;66(4):48. doi: 10.1167/iovs.66.4.48.
The purpose of this study was to evaluate the neuroprotective effects of delivering nerve growth factor (NGF) to retinal ganglion cells (RGCs) through adeno-associated virus serotype 2 (AAV2) carrying a neuronal-specific human synapsin (hSyn) promoter.
AAV2-hSyn-NGF was injected intravitreally in three glaucoma models: optic nerve crush (ONC), microbead-induced ocular hypertension (MB), and genetic glaucoma model (DBA). Quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA) determined the optimal injection concentration of AAV vector. Flow cytometry monitored immune responses. Transduction efficiency was quantified using green fluorescent protein (GFP) co-localization with RGC-specific marker RNA-binding protein with multiple splicing (RBPMS). The RGCs' density, retinal nerve fiber density, ganglion cell complex thickness, and positive scotopic threshold response (pSTR) were measured to assess structural and functional outcomes of the RGCs. Non-parametric Mann-Whitney U tests or Kruskal-Wallis tests were utilized to ascertain the statistical significance (P < 0.05).
The optimal concentration of AAV vector for intravitreal injection was determined to be 1 × 1010 vector particles (VPs) per eye. The use of the hSyn promoter significantly enhanced targeting specificity to RGCs, resulting in a transduction efficiency of 46.64% ± 2.18%. Administration of AAV2-hSyn-NGF effectively preserved the RGCs' density, nerve fiber layer integrity, and the thickness of ganglion cell complex, while maintaining the RGCs' function across three glaucoma models. Furthermore, this gene delivery system did not elicit detectable immune responses or structural damage to the retina.
The AAV2-hSyn-NGF gene therapy offers a safe and effective neuroprotective strategy for RGCs across multiple glaucoma models, making it a promising candidate for future clinical trials in patients with glaucoma.
本研究旨在评估通过携带神经元特异性人突触素（hSyn）启动子的2型腺相关病毒（AAV2）将神经生长因子（NGF）递送至视网膜神经节细胞（RGCs）的神经保护作用。
将AAV2-hSyn-NGF玻璃体内注射到三种青光眼模型中：视神经挤压（ONC）、微珠诱导的高眼压（MB）和遗传性青光眼模型（DBA）。定量聚合酶链反应（qPCR）和酶联免疫吸附测定（ELISA）确定AAV载体的最佳注射浓度。流式细胞术监测免疫反应。使用绿色荧光蛋白（GFP）与RGC特异性标志物多剪接RNA结合蛋白（RBPMS）的共定位来量化转导效率。测量RGCs的密度、视网膜神经纤维密度、神经节细胞复合体厚度和阳性暗视阈值反应（pSTR），以评估RGCs的结构和功能结果。采用非参数曼-惠特尼U检验或克鲁斯卡尔-沃利斯检验确定统计学意义（P < 0.05）。
确定玻璃体内注射AAV载体的最佳浓度为每只眼1×10¹⁰载体颗粒（VPs）。hSyn启动子的使用显著提高了对RGCs的靶向特异性，转导效率为46.64%±2.18%。在三种青光眼模型中，给予AAV2-hSyn-NGF有效地保留了RGCs的密度、神经纤维层完整性和神经节细胞复合体的厚度，同时维持了RGCs的功能。此外，这种基因递送系统未引发可检测到的免疫反应或对视网膜的结构损伤。
AAV2-hSyn-NGF基因疗法为多种青光眼模型中的RGCs提供了一种安全有效的神经保护策略，使其成为未来青光眼患者临床试验的有希望的候选方案。
