A GENE THERAPY-BASED APPROACH TO PREVENT PVR

描述(改编自申请者摘要)：增殖性 玻璃体视网膜病变(PVR)是一种多因素疾病，其中生长和 视网膜前膜(ERM)的收缩导致视网膜脱离。这个 ERM内的细胞同时表达生长因子和这些生长的受体。 各种因素。这项提议的直接目标是检验这一假设 生长因素推动ERM的形成，从而形成PVR。 1.构建和表征一系列显性负生长因子 感受器。 A.构建显性负生长因子受体。我们将重点关注 血小板衍生生长因子、血管内皮生长因子和肝细胞生长因子受体与PVR密切相关。 将构建主要的阴性试剂并对其进行有效性筛选 组织培养细胞系。 描述显性负性受体预防PVR的能力。 主要的阴性试剂将被测试它们阻断PVR的能力。 这种疾病的兔子模型。 2.确定与PVR相关的信号转运酶。细胞表达 PDGFα受体(APDGFR)能够有效地诱导PVR，而 不表达该受体的细胞具有非常低的PVR潜力。我们会 比较一组表达PDGFR突变体的细胞系的PVR潜能 选择性地不能激活信号转换酶。 3.监测疾病过程中相关信号酶的激活状态 进步。特定的AIMS 1和2将识别受体和信号 动物模型中PVR所需的酶。激活该等 蛋白质涉及磷酸化，因此磷酸特异性抗体可以 用于监控其在ERM中的激活状态。我们将发展 识别出的每个目标的磷酸特异性抗体，并使用它们来 确定这些蛋白质在多长时间内被激活 动物模型中的疾病。此外，我们还将使用特定于磷的 用于测试人类ERM中的信号酶是否活跃的抗体。 这些研究将确定对PVR起关键作用的分子， 从而极大地促进了我们对这种疾病的理解。一项额外的 这项提议的结果将是开发管理和/或 防止PVR。因此，这项提议的成果将构成未来的基础 为我们开发安全高效基因的长期目标所做的努力 以治疗为基础的方法预防PVR。

DESCRIPTION (Adapted from applicant's abstract): Proliferative vitreoretinopathy (PVR) is a mutifactorial disease in which the growth and contraction of an epiretinal membrane (ERM) leads to retinal detachment. The cells within the ERM express both growth factors and receptors for these growth factors. The immediate goal of this proposal is to test the hypothesis that growth factors drive the formation of an ERM and hence PVR. 1. Construct and characterize a series of dominant negative growth factor receptors. A. Construct dominant negative growth factor receptors. We will focus on the PDGF, VEGF and HGF receptors, which have been strongly implicated in PVR. Dominant negative reagents will be constructed and screened for efficacy in tissue culture cell lines. B. Characterize the ability of the dominant negative receptors to prevent PVR. The dominant negative reagents will be tested for their ability to block PVR in a rabbit model of the disease. 2. Identify signal relay enzymes that are involved with PVR. Cells expressing the PDGF alpha receptor (aPDGFR) are able to efficiently induce PVR, whereas cells that do not express this receptor have a very low PVR potential. We will compare the PVR potential of a panel of cell lines expressing aPDGFR mutants that selectively fail to engage signal relay enzymes. 3. Monitor the activation state of relevant signaling enzymes during disease progression. Specific aims 1 and 2 will identify receptors and signaling enzymes that are required for PVR in an animal model. Activation of such proteins involves phosphorylation, and thus phospho-specific antibodies can be used to monitor their activation state within the ERM. We will develop phosphospecific antibodies to each of the targets identified, and use them to determine at what times these proteins are activated during the course of the disease in the animal model. In addition, we will use phosphospecific antibodies to test if the signaling enzymes are active in human ERMs. These studies will identify molecules that make a critical contribution to PVR, and hence significantly advance our understanding of the disease. An additional outcome of this proposal will be the development of reagents to manage and/or prevent PVR. Hence the fruits of this proposal will form the basis for future efforts aimed at our long-term goal of developing a safe and efficient gene therapy-based approach to prevent PVR.