Regulation of Outflow Facility by Gene Transfer

描述(由申请人提供)：该项目的中心，长期目标是确定相关基因/机制对流出设施的贡献，从而开发使用基因转移来调节眼压(IOP)的最佳方案。我们的一般假设是，以特定的cDNA/siRNAs靶向外流通路的细胞将允许调节相关基因的表达(过度表达、沉默和/或诱导靶向启动子)，从而比目前可用的降眼压药物更特异、更持久地控制高眼压。在过去的周期中，我们已经在灌流的人类器官培养中确定了潜在的降低眼压的基因，并确定了针对人类小梁网络(TM)的真核启动子。最重要的是，我们已经确定了TM中缺乏非免疫原腺相关病毒(AAV)转导的机制，并用新一代自互补AAV载体(ScAAV)绕过了它们。具体地说，基于这些发现，我们现在假设，将基因转移到小梁网络需要一个内置的机制，允许控制基因药物的开启和关闭；任何潜在的治疗性基因和机制都需要组织靶向和非免疫原性递送系统；由于在正常动物身上测试TM基因药物的难度，我们需要一个活的动物模型，它将显示出高眼压，而不会破坏抗性组织的物理和功能。为了验证这些新的假设，我们建议研究诱导表达和siRNA沉默对人TM灌流培养的细胞外基质(ECM)重塑和流出便利性的影响。在已鉴定启动子的控制下，将选定的转基因转移到非免疫原性单链AAV载体中，并观察其在活体大鼠和猴体内的降眼压能力。最后，我们建议开始使用基因转移技术来建立功能性TM大鼠高眼压模型。与公共卫生相关：青光眼是导致不可逆转失明的第二大原因，也是非裔美国人最常见的原因。目前，青光眼还没有治愈的方法。我们项目的目标是通过使用基因转移/基因治疗来寻找青光眼的替代长期治疗方法。在短期内，我们试图研究三个强有力的候选基因的潜力，并开发出尽可能好的载体来将它们携带到小梁组织中。为了避免不必要的次要影响，我们正在设计策略，允许有针对性的、可诱导的转基因传递。最后，为了在进行翻译研究之前对这些载体进行适当的分析，我们开始使用相反的策略来创建一个高压动物模型，即向引流组织输送有害的、堵塞的基因。

DESCRIPTION (provided by applicant): The central, long term objective of this project is to determine the contribution of relevant genes/mechanisms to outflow facility and, consequently, to develop the best possible regimen to regulate intraocular pressure (IOP) by the use of gene transfer. Our general hypothesis is that targeting cells of the outflow pathway with specific cDNAs/siRNAs will allow the modulation of relevant genes' expression (overexpressing, silencing, and/or inducing targeted promoters), and thus control elevated IOP in a more specific and prolonged manner that the currently available IOP-reducing pharmacological drugs. During the past cycle, we have identified potential IOP-lowering genes in perfused human organ cultures, and identified eukaryotic promoters targeting the human trabecular meshwork (TM). Most important, we have determined mechanisms for the lack of transduction of the non-immunogenic adeno-associated viruses (AAV) in the TM and bypass them with a new generation self-complementary AAV vector (scAAV). Specifically, and based on these findings, we now hypothesize that gene transfer to the trabecular meshwork would require a built-in mechanism that would allow the controlled turning on and off of the gene drug; that any potentially therapeutic gene and mechanism would need tissue-targeted and non-immunogenic delivery systems; and that, because of the difficulty of assaying TM gene drugs on normotense animals, we would need a living animal model that will exhibit elevated IOP without the physical and functional disruption of the resistance tissue. To test these new hypotheses, here we propose to study the effect of inducible expression and siRNA silencing on extracellular matrix (ECM) remodeling and outflow facility of the human TM perfused cultures. To transfer the selected transgenes to non immunogenic scAAV vectors under the control of the identified promoters, and to investigate their IOP-lowering ability in living rats and monkeys. And finally, we propose to begin using gene transfer technology to develop a functional TM rat model of elevated IOP. PUBLIC HEALTH RELEVANCE: Glaucoma is the second leading cause of irreversible blindness and the most common cause among African- Americans. Currently, there is no cure for glaucoma. The goal of our project is to search for alternative, long- term treatments of glaucoma by the use of gene transfer/gene therapy. In the short term we seek to investigate the potential of three strong candidate genes and to develop the best vector possible to carry them into the trabecular meshwork tissue. To avoid unwanted secondary effects, we are designing strategies which would allow a targeted, inducible delivery of the transgenes. Finally, to properly assay these vectors before translational research can occur, we are beginning to create an elevated pressure animal model using the inverse strategy of delivering a detrimental, clogging gene to the draining tissue.