Regulation of Outflow Facility by Gene Transfer

描述（由申请人提供）：本项目的中心、长期目标是确定相关基因/机制对流出设施的贡献，从而开发出通过基因转移调节眼压（IOP）的最佳方案。我们的一般假设是，用特定的cdna / sirna靶向流出通道的细胞，将允许调节相关基因的表达（过表达、沉默和/或诱导靶向启动子），从而以比目前可用的降低眼压的药理学药物更特异性和更持久的方式控制升高的眼压。在过去的周期中，我们已经在灌注的人体器官培养中发现了潜在的降低iopo的基因，并鉴定了针对人类小梁网（TM）的真核启动子。最重要的是，我们已经确定了非免疫原性腺相关病毒（AAV）在TM中缺乏转导的机制，并用新一代自互补的AAV载体（scAAV）绕过它们。具体地说，基于这些发现，我们现在假设基因转移到小梁网需要一种内置的机制来控制基因药物的开启和关闭；任何潜在的治疗基因和机制都需要组织靶向和非免疫原性的传递系统；而且，由于在正常运动的动物身上检测TM基因药物的困难，我们需要一种活的动物模型，它将表现出更高的IOP，而不会对抵抗组织造成物理和功能上的破坏。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灌注培养细胞外基质（ECM）重塑和流出设施的影响。在鉴定的启动子控制下，将选择的转基因转移到非免疫原性的scAAV载体上，并研究其在活体大鼠和猴子体内的降io能力。最后，我们建议开始使用基因转移技术来建立一个功能性的大鼠IOP升高模型。公共卫生相关性：青光眼是不可逆失明的第二大原因，也是非裔美国人中最常见的原因。目前，还没有治愈青光眼的方法。我们项目的目标是通过基因转移/基因治疗寻找青光眼的替代长期治疗方法。在短期内，我们寻求调查三个强大的候选基因的潜力，并开发最好的载体可能携带它们进入小梁网组织。为了避免不必要的继发性影响，我们正在设计一种策略，允许有针对性的、可诱导的转基因传递。最后，为了在转化研究开始之前对这些载体进行适当的检测，我们开始创建一个高压动物模型，使用相反的策略，将有害的堵塞基因传递到引流组织中。

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.