Department of Ophthalmology, Medical University of South Carolina, Charleston, SC, USA.
Department of Neuroscience, Medical University of South Carolina, Charleston, SC, USA.
Transl Vis Sci Technol. 2023 Jul 3;12(7):17. doi: 10.1167/tvst.12.7.17.
Risk for developing age-related macular degeneration (AMD) is linked to an overactive complement system. In the mouse model of laser-induced choroidal neovascularization (CNV), elevated levels of complement effector molecules, including complement C3, have been identified, and the alternative pathway (AP) is required for pathology. The main soluble AP regular is complement factor H (fH). We have previously shown that AP inhibition via subretinal AAV-mediated delivery of CR2-fH using a constitutive promoter is efficacious in reducing CNV. Here we ask whether the C3 promoter (pC3) effectively drives CR2-fH bioavailability for gene therapy.
Truncated pC3 was used to generate plasmids pC3-mCherry/CR2-fH followed by production of corresponding AAV5 vectors. pC3 activation was determined in transiently transfected ARPE-19 cells stimulated with H2O2 or normal human serum (+/- antioxidant or humanized CR2-fH, respectively). CNV was analyzed in C57BL/6J mice treated subretinally with AAV5-pC3-mCherry/CR2-fH using imaging (optical coherence tomography [OCT] and fundus imaging), functional (electroretinography [ERG]), and molecular (protein expression) readouts.
Modulation of pC3 in vitro is complement and oxidative stress dependent, as shown by mCherry fluorescence. AAV5-pC3-CR2-fH were identified as safe and effective using OCT and ERG. CR2-fH expression significantly reduced CNV compared to mCherry and was correlated with reduced levels of C3dg/C3d in the retinal pigment epithelium/choroid fraction.
We conclude that complement-dependent regulation of AP inhibition ameliorates AMD pathology as effectively as using a constitutive promoter.
The goal of anticomplement therapy is to restore homeostatic levels of complement activation, which might be more easily achievable using a self-regulating system.
与年龄相关性黄斑变性（AMD）发展相关的风险与补体系统过度活跃有关。在激光诱导脉络膜新生血管（CNV）的小鼠模型中，已鉴定出补体效应分子（包括补体 C3）水平升高，并且病理学需要替代途径（AP）。AP 的主要可溶性调节剂是补体因子 H（fH）。我们之前已经表明，通过使用组成型启动子通过视网膜下 AAV 介导的 CR2-fH 传递来抑制 AP 可有效减少 CNV。在这里，我们想知道 C3 启动子（pC3）是否有效地驱动 CR2-fH 的基因治疗的生物利用度。
使用截断的 pC3 生成质粒 pC3-mCherry/CR2-fH，然后生产相应的 AAV5 载体。通过用 H2O2 或正常人血清（分别用抗氧化剂或人源化 CR2-fH 刺激）瞬时转染 ARPE-19 细胞来确定 pC3 的激活。使用成像（光学相干断层扫描[OCT]和眼底成像）、功能（视网膜电图[ERG]）和分子（蛋白表达）读数分析用 AAV5-pC3-mCherry/CR2-fH 经视网膜下处理的 C57BL/6J 小鼠的 CNV。
体外 pC3 的调节依赖于补体和氧化应激，如 mCherry 荧光所示。使用 OCT 和 ERG 鉴定 AAV5-pC3-CR2-fH 是安全有效的。与 mCherry 相比，CR2-fH 的表达显着降低了 CNV，并且与视网膜色素上皮/脉络膜部分的 C3dg/C3d 水平降低相关。
我们得出结论，AP 抑制的补体依赖性调节可有效改善 AMD 病理，与使用组成型启动子一样有效。
抗补体治疗的目标是恢复补体激活的动态平衡，这可能更容易通过自我调节系统实现。
