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Invest Ophthalmol Vis Sci. 2020 Feb 7;61(2):11. doi: 10.1167/iovs.61.2.11.
To study the potential effect of a gene therapy, designed to rescue the expression of dystrophin Dp71 in the retinas of Dp71-null mice, on retinal physiology.
We recorded electroretinograms (ERGs) in Dp71-null and wild-type littermate mice. In dark-adapted eyes, responses to flashes of several strengths were measured. In addition, flash responses on a 25-candela/square meters background were measured. On- and Off-mediated responses to sawtooth stimuli and responses to photopic sine-wave modulation (3-30 Hz) were also recorded. After establishing the ERG phenotype, the ShH10-GFP adeno-associated virus (AAV), which has been previously shown to target specifically Müller glial cells (MGCs), was delivered intravitreously with or without (sham therapy) the Dp71 coding sequence under control of a CBA promoter. ERG recordings were repeated three months after treatment. Real-time quantitative PCR and Western blotting analyses were performed in order to quantify Dp71 expression in the retinas.
Dp71-null mice displayed reduced b-waves in dark- and light-adapted flash ERGs and smaller response amplitudes to photopic rapid-on sawtooth modulation and to sine-wave stimuli. Three months after intravitreal injections of the ShH10-GFP-2A-Dp71 AAV vector, ERG responses were completely recovered in treated eyes of Dp71-null mice. The functional rescue was associated with an overexpression of Dp71 in treated retinas.
The present results show successful functional recovery accompanying the reexpression of Dp71. In addition, this experimental model sheds light on MGCs influencing ERG components, since previous reports showed that aquaporin 4 and Kir4.1 channels were mislocated in MGCs of Dp71-null mice, while their distribution could be normalized following intravitreal delivery of the same ShH10-GFP-2A-Dp71 vector.
研究一种基因治疗的潜在效果，该治疗旨在挽救 Dp71 缺失小鼠视网膜中 Dp71 Dp71 的表达，以研究其对视网膜生理学的影响。
我们记录了 Dp71 缺失和野生型同窝小鼠的视网膜电图 (ERG)。在暗适应的眼睛中，测量了几个强度的闪光反应。此外，还测量了 25 坎德拉/平方米背景下的闪光反应。还记录了锯齿波刺激的 On- 和 Off-介导反应以及光调制正弦波 (3-30 Hz) 的反应。在建立 ERG 表型后，用 ShH10-GFP 腺相关病毒 (AAV) 进行玻璃体内给药，该病毒先前已被证明可特异性靶向 Müller 胶质细胞 (MGC)，并在 CBA 启动子控制下携带 Dp71 编码序列或不携带 (假治疗)。治疗后三个月重复 ERG 记录。进行实时定量 PCR 和 Western blot 分析以定量视网膜中 Dp71 的表达。
Dp71 缺失小鼠在暗适应和明适应闪光 ERG 中显示出 b 波减少，并且对光调制快速锯齿调制和正弦波刺激的反应幅度较小。在 ShH10-GFP-2A-Dp71 AAV 载体玻璃体内注射三个月后，Dp71 缺失小鼠治疗眼的 ERG 反应完全恢复。功能恢复与 Dp71 在治疗视网膜中的过度表达有关。
本研究结果显示成功的功能恢复伴随着 Dp71 的重新表达。此外，该实验模型揭示了 MGC 对 ERG 成分的影响，因为先前的报道表明 Dp71 缺失小鼠的 MGC 中存在水通道蛋白 4 和 Kir4.1 通道定位错误，而在用相同的 ShH10-GFP-2A-Dp71 载体进行玻璃体内给药后，其分布可以恢复正常。
