Promoters for Glaucoma Gene Therapy

青光眼是一种进行性视神经病变，通常伴有眼压升高，是世界范围内致盲的主要原因。发病率随着年龄的增长而增加，在某些种族群体中发病率很高。虽然有治疗方法，但并不理想。青光眼也发生在没有眼压升高的情况下，目前的治疗方法对这些患者无效。鉴于这些问题，需要改进治疗策略。青光眼是基因治疗方法的主要候选者。青光眼的病理涉及整个眼睛的结构，包括小梁网（TM）、睫状体上皮（CE）、睫状肌（CM）和视网膜神经节细胞（RGC），为基因传递提供了几个候选靶点。许多基因治疗青光眼的方法是可行的，但不知道具体的疾病相关基因。例如，可以靶向TM或CM来增加流出量，可以靶向睫状体上皮细胞来减少液体产生，可以应用神经保护策略来防止细胞死亡。青光眼基因治疗的进展严重阻碍了缺乏适当的启动子来表达转基因。目前还没有组织特异性启动子，也没有任何启动子在相关细胞中表达相当长的时间。我们设计了一个基于单纯疱疹病毒（HSV）扩增子载体的新系统，使我们能够在体内条件下分离潜在的基因治疗启动子。总的目标是分离出适合眼部基因治疗的启动子。特异性目的1：我们将测试一个或多个适合用于TM基因治疗的启动子可以使用我们的HSV扩增子系统分离的假设。我们将使用染色质免疫沉淀法从原代TM细胞中分离表达DNA，将选择的DNA克隆到我们的无启动子GFP扩增子载体中，并在用启动子文库扩增子转导后从大鼠前房细胞中选择表达GFP的细胞。特异性目标2：我们将通过结合TM细胞的转录因子分析和生物信息学分析Aim 1中分离的文库中的启动子以及其他人鉴定的基因启动子（EST和微阵列数据库）来验证小梁网络中存在一组特异性转录调节蛋白的假设。

DESCRIPTION (provided by applicant): Glaucoma, a progressive optic neuropathy usually involving elevated intraocular pressure, is a leading cause of blindness worldwide. The incidence increases with age and is high among certain ethnic groups. Although treatments are available, they are not ideal. Glaucoma also occurs in the absence of increased intraocular pressure and current treatments are not effective for these patients. Given these problems, improved strategies for therapy are needed. Glaucoma is a prime candidate for gene therapy approaches. The pathology of glaucoma involves structures throughout the eye including the trabecular meshwork (TM), ciliary body epithelium (CE), ciliary muscle (CM), and retinal ganglion cells (RGC) providing several candidate targets for gene delivery. Numerous gene therapy approaches for glaucoma are feasible without knowing the specific disease related gene. For example, the TM or CM could be targeted to increase outflow, ciliary body epithelial cells could be targeted to reduce fluid production, and neuroprotective strategies could be applied to prevent cell death. Progress in glaucoma gene therapy is severely hampered by a lack of appropriate promoters to express the transgenes. Tissue specific promoters are not yet available nor are any promoters known to express for considerable lengths of time in relevant cells. We have designed a novel system based on Herpes simplex virus (HSV) amplicon vectors that will allow us to isolate potential gene therapy promoters under in vivo conditions. The overall goal is to isolate promoters that are suitable for ocular gene therapy. The specific aims are: Specific Aim 1: We will test the hypothesis that one or more promoters suitable for use in gene therapy in the TM can be isolated using our HSV amplicon system. We will do this by using chromatin immunoprecipitation to isolate expressing DNA from primary TM cells, cloning the selected DNA into our promoterless GFP amplicon vector, and selecting GFP expressing cells from rat anterior chamber cells following transduction with the promoter library amplicons. Specific Aim 2: We will test the hypothesis that a specific set of transcriptional regulatory proteins exist in the trabecular meshwork by combining transcription factor profiling of TM cells with bioinformatic analysis of promoters from the library isolated in Aim 1 and the promoters of genes identified by others (EST and microarray databases).