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/机制对流出功能的贡献，从而开发通过基因转移调节眼内压（IOP）的最佳方案。我们的一般假设是，用特异性cDNA/siRNA靶向流出途径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IOP的药理学药物更特异和更长的方式控制升高的IOP。在过去的周期中，我们已经确定了灌注的人体器官培养物中潜在的IOP降低基因，并确定了靶向人小梁网（TM）的真核启动子。最重要的是，我们已经确定了在TM中缺乏非免疫原性腺相关病毒（AAV）转导的机制，并用新一代自身互补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个内置机制，允许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递送系统;并且，由于在正常血压的动物上测定TM基因药物的难度，我们需要一种能够表现出升高的IOP而不破坏抵抗组织的物理和功能的活体动物模型。为了验证这些新的假设，在这里，我们建议研究诱导表达和siRNA沉默对细胞外基质（ECM）重塑和流出设施的人TM灌注培养物的影响。将筛选出的转基因转移到无免疫原性的scAAV载体中，并在活体大鼠和猴中研究其降低IOP的能力。最后，我们建议开始使用基因转移技术来开发一种功能性的高眼压TM大鼠模型。公共卫生相关性：青光眼是不可逆失明的第二大原因，也是非洲裔美国人最常见的原因。青光眼的治疗方法有哪些？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性长期治疗方法。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。为了避免不必要的二次效应，我们正在设计策略，以实现转基因的靶向、诱导型递送。最后，为了在转化研究之前正确地检测这些载体，我们开始使用将有害的堵塞基因递送到引流组织的相反策略来创建高压动物模型。

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.