Regulation of Outflow Facility by Gene Transfer

描述(申请人提供)：特定细胞的生理学为 由其基因的协调表达决定的。我们的将军，正在进行 假说是操控细胞内相关蛋白质的浓度 小梁网(TM)的细胞将允许我们调制房水 体内的流出设施。最重要的是，引入了核酸 编码这些蛋白质的序列(正义或反义定向)进入 TM细胞将有助于阐明它们对细胞功能的基本贡献。 具体地说，我们假设一个共同的机制路径，通过它 细胞骨架和应激相关蛋白(TIGR/MYOC)影响流出机制 可能是通过NF-kappaB信号通路对分泌的影响而介导的。 类似地，我们假设在我们的TM图谱文库中有丰富的基因产物 但在其Unigene集群中具有较少数量的条目(对于所有其他 组织)，可能表明了其他重要的影响机制 眼压(IOP)。我们还假设，通过结合使用 人眼前节器官灌流的重组腺病毒载体 我们将优先识别针对不同基因的启动子序列 完整的人TM组织的细胞类型。最后，我们假设 棕色挪威大鼠是研究TM基因转移的理想动物模型 活体动物和食蟹猴将有助于更好地阐明 在青光眼中进行这样的基因转移。 为了验证这些假设，我们建议转导人类TM(HTM)细胞和 携带野生型、突变型和反义Ad的灌流器官培养 TIGR/MYOC基因的表达及其在应激中的作用 保护、地塞米松(DEX)干扰、细胞分泌和调节 核因子-kappaB。从我们的TM基因谱中选择1-2个基因并研究它们的 对流出设施的影响。利用融合启动子构建重组腺病毒载体 (α-晶体蛋白、TIGR/MYOC、MGP和HC GP-39)与lacZ基因连接并注射 它们被注入到灌流的人体器官培养中。最后，我们建议将 不同的病毒载体(AdS和腺相关病毒(AAV))在Brown 并研究我们现有的TM基因ADS对眼压的影响。我们会 使用大鼠模型预先选择用于食蟹猴的载体。 即使这些机制和/或选定的基因中的一些最终不会 对流出的调节有主要的意义，他们的调查 在TM中的功能及其转移的优化将潜在地 提供了关于基因表达参与的重要新见解 房水生理学。

DESCRIPTION (provided by applicant): The physiology of a given cell is determined by the coordinate expression of its genes. Our general, ongoing hypothesis is that manipulating the concentration of relevant proteins in the cells of the trabecular meshwork (TM) will allow us to modulate aqueous humor outflow facility in vivo. Most important, that introducing the nucleic acid sequences (sense or antisense orientation) coding for those proteins into the TM cells will help to elucidate their basic contribution to cellular function. Specifically, we hypothesize that a common pathway of the mechanisms by which cytoskeletal and stress related proteins (TIGR/MYOC) influence outflow facility might be mediated through the NF-kappaB signaling pathway effect on secretion. Similarly, we hypothesize that gene products abundant in our TM profile library but with a lower number of entries in their UniGene cluster (for all other tissues), might be indicative of additional important mechanisms influencing intraocular pressure (IOP). We also hypothesize that by combining the use of recombinant adenoviral (Ad) vectors with perfused human anterior segment organ cultures we will identify promoter sequences preferentially targeting distinct cells types of the intact human TM tissue. Finally, we hypothesize that the Brown Norway rats represent an ideal animal model to study TM gene transfer in vivo and that Cynomolgus monkeys will help to better elucidate the relevance of such gene transfer in glaucoma. To test these hypotheses we propose to transduce human TM (HTM) cells and perfused organ cultures with Ad carrying wild type, mutant and antisense forms of TIGR/MYOC cDNA and study the effects of their over-expression in stress protection, dexamethasone (DEX) interference, cellular secretion and modulation of NF-kappaB. To select 1-2 genes from our TM gene profile and study their impact on outflow facility. To construct Ad vectors with fused promoters (alphaB-crystallin, TIGR/MYOC, MGP and HC gp-39) to the LacZ gene and inject them into perfused human organ cultures. Finally, we propose to characterize different viral vectors (Ads and Adeno-associated viruses (AAV)) in Brown Norway rats and study the effect of our existing TM genes Ads in IOP. We will use the rat model to pre-select the vectors for use in cynomolgus monkeys. Even if some of these mechanisms and/or selected genes would turn out not to have primary relevance on the regulation of outflow, the investigation of their function in the TM and the optimization of their transfer will potentially provide important new insights concerning the involvement of gene expression in aqueous humor physiology.