AAV介导EPO基因靶向表达在AMD发病和治疗中的作用

Erythropoietin (EPO) shows a good neuroprotective effect in models of retinal degeneration in vitro and in vivo. Oxidative stress induced retinal pigment epithelium (RPE) dysfunction is hypothesized to be fundamental in the pathogenesis of age-related macular degeneration (AMD). Our previous experiments found that exogenous EPO could protect RPE cells from oxidative damage through regulating the activity of superoxide dismutase (SOD) and other antioxidant enzymes, reducing intracellular reactive oxygen species and the anti-inflammatory effect. We speculated that EPO may serve as a drug for the prevention and treatment of AMD. Adeno-associated virus (AAV) has a proven record of safety and efficacy in animals and patients with retinal degeneration. Our previous studies have shown that tyrosine mutations in the capsid AAV significantly improved infection efficiency in the retina. We plan two sets of experiments: (1) We will establish a conditional gene knockout AMD mice model by inducing the deletion of SOD2 in RPE with the Cre/loxP system. And we will detect expression of EPO and its receptor (EPOR) in the retina, choroid in development of the AMD model. (2) We will construct AAV expression vectors, which carry the shRNA to inhibit EPO-EPOR signal pathway or, conversely, carry a non-erythrogenic variant of the EPO gene to enhance EPO signal. An AAV serotype1 vector will be subretinally injection to infect the RPE layer, and tyrosine to phenylalanine mutants of AAV2 will be injected in the vitreous to infect all retinal layers. We will monitor the time course of retinal degeneration in real time in living mice using electrophysiology (ERG), high resolution structural analysis (SD-OCT), and after sacrifice of the animals we will examine morphological changes to the RPE, Bruch's membrane and the neural retina and measure accumulation of lipofuscin in the RPE after the AMD model infected by AAV vectors. We hypothesize that increased EPO-EPO signaling will have a protective effect while knocking down EPOR will increase the extent of RPE and photoreceptor damage.

促红细胞生成素（EPO）在多种视网膜变性疾病体外和体内模型中显示良好的神经保护作用，但其在AMD发病中的作用尚未见报道。氧化应激诱导RPE功能障碍是公认的AMD始发因素和重要机制。我们前期发现EPO通过调节SOD等抗氧化酶活性，降低细胞内活性氧，抗炎等途径防护RPE氧化损伤,推测其是防治AMD的可能药物。本项目拟:(1)Cre/loxp技术靶向敲除小鼠RPE内SOD2基因,建立条件性基因敲除AMD模型，并检测EPO及受体EPOR在其发生中的变化。(2)其次,构建分别靶向感染RPE层和全视网膜层的新型腺相关病毒（AAV）载体,携带野生型或无红细胞增生作用的变异型EPO基因过表达质粒,以增强EPO信号；或反之,携带shRNA抑制EPOR表达；从正反两方面评价分别靶向RPE和全视网膜内改变EPO信号对AMD发病的影响。并评估眼内长期表达无红细胞增生作用的变异型EPO治疗AMD的有效性和安全性。

促红细胞生成素(EPO)在多种视网膜变性疾病体外和体内模型中显示良好的神经保护作用，其促新生血管作用也有报道，但其在 AMD 发病中的作用尚未见报道。氧化应激诱导 RPE 功能障碍是公认的 AMD 始发因素和重要机制。我们前期发现 EPO 通过调节 SOD 等抗氧化酶活性，降低细胞内活性氧，抗炎等途径防护 RPE 氧化损伤,推测其是防治 AMD 的可能药物。本项目以Cre/loxp 技术靶向敲除小鼠 RPE 内 SOD2 基因,建立了条件性基因敲除 AMD 模型，并检测其发病过程中的变化。另外,我们构建了分别靶向感染 RPE 层的新型腺相关病毒(AAV) 载体,携带无红细胞增生作用的变异型EPO基因过表达质粒,以增强EPO信号;或反之,携带shRNA或microRNA抑制EPOR表达;从正反两方面评价靶向RPE改变EPO信号对干性和湿性两型AMD发病的影响。我们发现，EPO有可能作为治疗干性AMD的新靶点，而下调EPO基因的表达则可能是治疗湿性AMD的新方向。

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