Department of Ophthalmology, University of Pennsylvania, Philadelphia, PA, USA.
F.M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA.
Transl Vis Sci Technol. 2024 Aug 1;13(8):37. doi: 10.1167/tvst.13.8.37.
Adeno-associated virus (AAV) demonstrates promise in delivering therapeutic genes to retinal ganglion cells (RGCs). Delivery of neuroprotective genes is constrained by packaging size and/or cell selectivity. This study compares the ability of the RGC-selective gamma-synuclein (SNCG) promoter and the smaller RGC-selective neurofilament heavy chain (NEFH) promoter, as well as portions of the RGC-selective atonal bHLH transcription factor 7 (ATOH7) enhancer, to drive gene expression in RGCs.
AAV2 constructs with green fluorescent protein (GFP) or human sirtuin 1 (hSIRT1) driven by cytomegalovirus (CMV) enhancer and NEFH promoter (AAV2-eCMV-NEFH) or distal active sequences of the ATOH7 enhancer (DiATOH7) with the SNCG promoter (AAV2-DiATOH7-SNCG) were intravitreally injected into C57BL/6J mice. RGCs were immunolabeled with Brn3a antibodies and counted. AAV constructs with the utmost transduction efficiency were used to test the therapeutic efficacy of the hSIRT1 gene in 12-week-old C57BL/6J mice subjected to microbead (MB)-induced intraocular pressure (IOP) elevation. Visual function was measured using optokinetic responses (OKRs).
The eGFP transduction efficiency of AAV2-eCMV-NEFH was similar to that of AAV2-eCMV-SNCG and AAV2-DiATOH7-SNCG. When combined with the SNCG promoter, a larger ATOH7 enhancer was less efficient than the shorter DiATOH7 enhancer. Similarly, the hSIRT1 efficiency of AAV2-eCMV-NEFH was similar to that of AAV2-eCMV-SNCG. The latter two vectors were equally efficient in increasing RGC survival and improving visual function in the mouse model of MB-induced IOP elevation.
SNCG and NEFH promoters represent two equally efficient and comparable RGC selective promoter sequences; however, the NEFH promoter offers a smaller packaging size.
Smaller enhancer-promoter combinations can be used to deliver larger genes in human cells and for treatment in optic neuropathies including glaucoma.
腺相关病毒（AAV）在向视网膜神经节细胞（RGC）传递治疗基因方面显示出潜力。神经保护基因的传递受到包装大小和/或细胞选择性的限制。本研究比较了 RGC 选择性 γ-突触核蛋白（SNCG）启动子和较小的 RGC 选择性神经丝重链（NEFH）启动子的能力，以及 RGC 选择性的 ATOH7 增强子的较小部分，以驱动 RGC 中的基因表达。
用巨细胞病毒（CMV）增强子和 NEFH 启动子（AAV2-eCMV-NEFH）或 ATOH7 增强子的远端活性序列（DiATOH7）驱动绿色荧光蛋白（GFP）或人 SIRT1（hSIRT1）的 AAV2 构建体通过玻璃体内注射到 C57BL/6J 小鼠中。用 Brn3a 抗体对 RGC 进行免疫标记并计数。使用最大转导效率的 AAV 构建体测试 hSIRT1 基因在 12 周龄 C57BL/6J 小鼠中对微珠（MB）诱导的眼内压（IOP）升高的治疗效果。使用视动反应（OKR）测量视觉功能。
AAV2-eCMV-NEFH 的 eGFP 转导效率与 AAV2-eCMV-SNCG 和 AAV2-DiATOH7-SNCG 相似。当与 SNCG 启动子结合时，较大的 ATOH7 增强子不如较短的 DiATOH7 增强子有效。同样，AAV2-eCMV-NEFH 的 hSIRT1 效率与 AAV2-eCMV-SNCG 相似。在后两种载体中，在 MB 诱导的 IOP 升高的小鼠模型中，增加 RGC 存活和改善视觉功能的效率相等。
SNCG 和 NEFH 启动子代表两种同样有效且可比的 RGC 选择性启动子序列；然而，NEFH 启动子提供了更小的包装尺寸。
杨可欣
