Rho GTPase siRNA as a Therapeutic Strategy in Regulating Outflow Resistance

 描述(由申请人提供)： 原发性开角型青光眼(POAG)是世界范围内导致失明的主要原因，在美国有200多万45岁或以上的人受到影响。VHA内青光眼的个人、社会和医疗负担仍然是一个非常重要的问题。尽管POAG具有压倒性的发病率和社会经济影响，但退伍军人POAG的治疗目前仅限于降低升高的眼压(IOP)的非特异性干预。对于许多青光眼退伍军人来说，眼压的药物治疗在临床上仍然是屈光不正的。这些退伍军人通常需要更广泛的干预，包括侵入性手术操作。针对高眼压原因制定有针对性的治疗策略对于青光眼退伍军人的管理至关重要。在健康的眼睛中，眼压通过平衡的房水产生和流出(AH)来维持。小梁网(TM)对AH流出的阻力增加被认为是退伍军人POAG患者眼压异常升高的主要原因。发生这种情况的机制仍不明确，但很可能涉及TM细胞收缩能力增强和细胞外基质沉积改变的组合。独立研究表明，促进Rho GTPase介导的细胞外基质沉积和TM细胞肌动蛋白应激纤维组织的细胞因子转化生长因子2在POAG患者的AH中显著升高。内皮素-1(ET-1)是一种有效的血管收缩因子，在POAG患者的AH中也有升高。然而，转化生长因子-β2、ET-1和Rho GTPase信号与POAG之间的关系尚不清楚。来自我们实验室的同行评审和发表的研究表明，在人类TM细胞中，转化生长因子2和内皮素-1的产生之间存在显著的关联。我们在本申请中的初步发现首次进一步表明，通过选择性地靶向和干扰Rho GTPase信号，我们可以成功地阻止人TM细胞中转化生长因子-β2和内皮素-1表达的有害增加。这些发现有力地表明，Rho GTPase信号的异常升高了眼压，部分原因是促进了退伍军人POAG患者眼前节中转化生长因子-β2和内皮素-1的病理性增加。迫切需要新的翻译研究来开发治疗和康复策略，为因青光眼而虚弱的退伍军人提供高级护理。SiRNA技术是一个令人兴奋和有前途的新领域，在包括青光眼在内的眼部疾病的治疗/康复方面具有现实的治疗/康复潜力。在这项为期两年的SPIRE研究中，我们将确定siRNA介导的Rho GTP酶基因敲除是否在治疗上降低了实验升高的眼压。假设：选择性靶向单体Rho GTP酶的siRNA治疗将通过干扰转化生长因子-β2和ET-1的表达和信号转导，显著降低病理性升高的眼压。具体目标1将确定给予针对关键单体Rho GTP酶的siRNAs是否能保护通过人类和猪眼前段血流灌注分析量化的流出阻力实验升高的变化。具体目标2将确定针对关键单体Rho GTP酶的siRNAs的注射是否会减少ProET-1、ETA、ETB、转化生长因子-2或转化生长因子βRI在人和猪球中的表达，这些人和猪球的流出阻力通过免疫组织化学和共聚焦显微镜进行了量化。这项研究的直接目标是建立siRNA介导的Rho GTP酶下调作为一种新的策略，通过它来管理与POAG相关的高眼压。这项研究的成功完成将是为管理患有POAG的退伍军人开发这项技术的关键的第一步。

 DESCRIPTION (provided by applicant): Primary open-angle glaucoma (POAG) is a leading cause of blindness worldwide, affecting over 2 million individuals 45 years of age or older in the US. The personal, social, and medical burden of glaucoma within the VHA remains an extraordinarily significant concern. Despite its overwhelming prevalence and socioeconomic impact, the treatment of Veterans with POAG is currently restricted to non-specific interventions that lower elevated intraocular pressure (IOP). For many glaucomatous Veterans, pharmacological management of IOP remains clinically refractive. These Veterans often require more extensive interventions, including invasive surgical manipulation. The development of targeted therapeutic strategies directed at the cause of elevated IOP is critical for the management of our glaucomatous Veterans. In healthy eyes, IOP is maintained through balanced production and outflow of aqueous humor (AH). Increased resistance to AH outflow through the trabecular meshwork (TM) is considered to be a major contributor of aberrant elevation of IOP in Veterans with POAG. The mechanism by which this occurs remains poorly defined, but most likely involves a combination of enhanced TM cell contractility and altered ECM deposition. Independent studies have shown that TGF-ß2, a cytokine that facilitates Rho GTPase-mediated ECM deposition and actin stress fiber organization in TM cells, is markedly elevated in the AH of POAG patients. Endothelin-1 (ET-1) a potent vasoconstrictor, is also increased in the AH from POAG patients. The association between TGF-ß2, ET-1, and Rho GTPase signaling with POAG, however, remains unclear. Peer-reviewed and published studies from our laboratory demonstrate a striking association between TGF-ß2 and ET-1 production within human TM cells. Our preliminary findings included in this application further demonstrate for the first time that by selectively targeting and disrupting Rho GTPase signaling, we can successfully prevent harmful increases in TGF-ß2 and ET-1 expression in human TM cells. These findings strongly suggest that aberrant Rho GTPase signaling elevates IOP, in part, by facilitating pathologic increases in TGF-ß2 and ET-1 in the anterior segment of POAG Veterans. Novel translational studies are critically needed to develop therapeutic and rehabilitative strategies for the advanced care of our Veterans debilitated by glaucoma. siRNA technology is an exciting and promising new field that has realistic therapeutic/rehabilitative potential for the management of ocular disorders, including glaucoma. In this two-year SPiRE study, we will determine whether siRNA-mediated Rho GTPase knockdown therapeutically lowers experimentally-elevated IOP. Hypothesis: Therapeutically administered siRNA selectively targeting monomeric Rho GTPases will markedly lower pathologically elevated intraocular pressure by disrupting TGF-ß2 and ET-1 expression and signaling. Specific Aim 1 will determine whether administration of siRNAs targeting key monomeric Rho GTPases protects against experimentally elevated changes in outflow resistance as quantified by human and porcine anterior segment perfusion analyses. Specific Aim 2 will determine whether administration of siRNAs targeting key monomeric Rho GTPases attenuates expression of proET-1, ETA, ETB, TGF-ß2 or TGFßRI in human and porcine globes with experimentally elevated outflow resistance as quantified by immunohistochemistry and confocal microscopy. The immediate goal of this study is to establish siRNA-mediated Rho GTPase knockdown as a novel strategy by which to manage elevated IOP associated with POAG. Successful completion of this study will serve as a critical first step toward developing this technology for the management of Veterans with POAG.