Promoters for Glaucoma Gene Therapy

描述(申请人提供)：青光眼，一种进行性视神经病变，通常涉及眼压升高，是世界范围内导致失明的主要原因。发病率随着年龄的增长而增加，在某些民族中发病率较高。虽然治疗方法是可用的，但它们并不理想。在眼压不升高的情况下也会发生青光眼，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。青光眼是基因治疗的首选方法。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)，为基因输送提供了几个候选靶点。许多青光眼的基因治疗方法在不了解特定疾病相关基因的情况下是可行的。例如，TM或CM可以针对增加流出，睫状体上皮细胞可以针对减少液体产生，神经保护策略可以应用来防止细胞死亡。由于缺乏合适的启动子来表达转基因，青光眼基因治疗的进展受到严重阻碍。目前还没有组织特异性启动子，也没有任何已知的启动子在相关细胞中表达相当长的时间。我们设计了一种基于单纯疱疹病毒(HSV)扩增载体的新型系统，使我们能够在体内条件下分离潜在的基因治疗启动子。总体目标是分离出适合眼部基因治疗的启动子。具体目标是：特定目标1：我们将利用我们的HSV扩增系统来检验这样的假设，即可以在TM中分离出一个或多个适合用于基因治疗的启动子。我们将通过染色质免疫沉淀从原代TM细胞中分离出表达GFP的DNA，将所选择的DNA克隆到我们的无启动子GFP扩增载体中，并通过启动子文库扩增从大鼠前房细胞中选择表达GFP的细胞。特定目标2：我们将通过结合TM细胞的转录因子图谱与从Aim 1中分离的文库中分离的启动子和其他基因的启动子的生物信息学分析(EST和微阵列数据库)来验证这一假设，即在小梁网络中存在一组特定的转录调控蛋白。

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).