A GENE THERAPY-BASED APPROACH TO PREVENT PVR

描述（改编自申请人摘要）：说明性 玻璃体视网膜病变（PVR）是一种多因素疾病， 视网膜前膜（ERM）的收缩导致视网膜脱离。的 ERM内的细胞表达生长因子和这些生长因子的受体 因素这项提议的直接目标是检验以下假设： 生长因子驱动ERM和PVR的形成。 1.构建并表征一系列显性负增长因子 受体。 A.构建显性负性生长因子受体。重点抓好 PDGF、VEGF和HGF受体与PVR密切相关。 将构建显性阴性试剂，并筛选其在以下方面的有效性： 组织培养细胞系 B。描述显性负性受体预防PVR的能力。 将检测显性阴性试剂阻断PVR的能力， 一只兔子的疾病模型。 2.识别参与PVR的信号传递酶。细胞表达 PDGF α受体（aPDGFR）能够有效诱导PVR，而 不表达该受体的细胞具有非常低的PVR潜能。我们将 比较一组表达aPDGFR突变体的细胞系的PVR潜力 选择性地不参与信号传递酶。 3.监测疾病期间相关信号酶的激活状态 进展具体目标1和2将确定受体和信号传导 在动物模型中PVR所需的酶。激活此类 蛋白质涉及磷酸化，因此磷酸化特异性抗体可以被 用于监控其在ERM中的激活状态。我们将开发 磷酸特异性抗体的每一个目标确定，并使用它们来 确定这些蛋白质是在什么时候被激活的过程中， 动物模型中的疾病。此外，我们将使用磷酸特异性 抗体来测试信号酶是否在人ERMs中有活性。 这些研究将确定对PVR起关键作用的分子， 从而极大地推进了我们对疾病的理解。额外 该提案的结果将是开发试剂，以管理和/或 预防PVR。因此，这项建议的成果将成为未来的基础。 我们的长期目标是开发一种安全有效的基因， 以治疗为基础的方法来预防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.