HTRA1 as a Therapeutic Target in the Treatment of Wet AMD

 描述(申请人提供)：老年性黄斑变性(AMD)是美国老年人视力受损的最常见原因。本研究旨在从分子水平上找出其原因。在AMD中，与许多其他与年龄相关的疾病一样，遗传风险因素和环境攻击都是关键因素。10号染色体上的一个显著的遗传变化已经被发现与AMD的易感性密切相关。这种基因变异位于一种蛋白质的启动子区域，称为高温需求因子A1(HTRA1)。这种疾病的基因型导致HTRA1的表达增加。我们还使用HTRA1基因敲除(HTRA1-/-)小鼠进行了功能研究，发现HTRA1基因的缺失导致视网膜血管发育减少，同时血管内皮生长因子(VEGF)的表达显著下调。相反，我们的实验表明，HTRA1在RPE中的表达增加会导致AMD样病理和VEGF表达的增加。高水平的血管内皮生长因子刺激眼部视网膜色素上皮(RPE)层和Bruchs膜的异常血管生长，从而导致湿性AMD。我们最近在《生物化学杂志》上发表的一项研究显示，hTRA1上调血管内皮生长因子与下调组织生长因子-β(转化生长因子-家族)中的一种被称为生长分化因子6(GDF6)的成员呈负相关。这项建议的长期目标是阐明HTRA1导致AMD风险增加的分子机制，并开发潜在的AMD治疗方法。我们的假设是，氧化应激可以诱导HTRA1的高水平表达，这通过调节信号转导、血管发育、血管生成和细胞外基质而导致AMD的风险。在这一假说的指导下，我们拟在以下几个方面进行研究：1)我们将研究基因变异如何影响RPE细胞对血管生成因子和hTRA1表达的氧化应激反应。2)我们将确定HTRA1和氧化应激是否影响视网膜和脉络膜血管和病理。我们还将尝试确定移除小鼠的HTRA1是否对HTRA1-/-小鼠和对照组小鼠的氧化应激增强的新生血管具有保护作用，这将用于检测视网膜和脉络膜的形态和基因表达。3)我们将利用克隆的抗hTRA1的单抗，通过组织特异性的导入RPE细胞，研究其治疗RPE细胞的潜能，旨在抑制异常的血管生长。在这种情况下，选定的抗体将被转化为单链多肽，该单链多肽将通过一种新的蛋白质支架进化技术进行进一步的“进化”，以最大限度地提高其与HTRA1的亲和力和抑制蛋白酶活性的能力，同时将其细胞毒性降至最低。基因治疗策略将被用来将这种抗体导入RPE细胞，并测试它们抑制脉络膜新生血管(CNV)的能力，旨在开发一种治疗湿性AMD的新方法。

 DESCRIPTION (provided by applicant): Age-related macular degeneration (AMD) is the most common cause of visual impairment among the elderly in the United States. This research aims to find its cause at the molecular level. In AMD, like many other age- related diseases, both genetic risk factors and environmental assaults are key contributors. A prominent genetic change in chromosome 10 has been previously identified to be strongly associated with susceptibility to AMD. This genetic variant is located in the promoter region of a protein, called the high temperature requirement factor A1 (HTRA1). This disease genotype results in an increased expression of HTRA1. We also performed functional studies using the HTRA1 knockout (htra1-/-) mice, and found that the loss of HTRA1 leads to decreased retinal vascular development along with a significant down-regulation of vascular endothelial growth factor (VEGF) expression. Conversely, our experiments show that increased expression of HTRA1 in RPE leads to an AMD-like pathology and elevated VEGF expression. High VEGF stimulates abnormal vascular growth in the retinal pigment epithelium (RPE) layer and Bruch's membrane of the eye, which can result in wet AMD. One of our more recent studies, published in the Journal of Biological Chemistry revealed that the up- regulation of VEGF by HTRA1 is inversely correlated to the down-regulation of a member of the tissue growth factor-beta (TGF- family called Growth Differentiation Factor 6 (GDF6). The long term objectives of this proposal are to elucidate the molecular mechanism by which HTRA1 contributes to an increased risk of AMD, and to develop potential therapies for AMD. Our hypothesis is that oxidative stress can induce a higher level expression of HTRA1, which contributes to the risk of AMD by regulating signal transduction, vascular development, angiogenesis, and extracellular matrix. Guided by this hypothesis, we propose to conduct studies in following areas: 1) We will investigate how genetic variants can influence the RPE cells to respond to oxidative stress for the expression of angiogenic factors and HTRA1. 2) We will determine whether HTRA1 and oxidative stress impact retinal and choroidal vasculature and pathology. We will also try to determine whether removal of HTRA1 in mouse is protective to oxidative stress enhanced neovascularization in htra1-/- mice and control mice, which will be used to examine morphology and gene expression in the retina and choroid. 3) We will utilize our cloned monoclonal antibodies against HTRA1 to investigate their therapeutic potential by tissue-specific delivery into the RPE cells, aiming to inhibit the abnormal vascular growth. In this scenario, the selected antibody will be converted into a single chain peptide, which will be further "evolved" to maximize its affinity to HTRA1 and ability to inhibit the protease activity while minimizing its cytotoxicity using a novel protein scaffold evolution technology. A gene therapy strategy will be used to deliver such antibodies into the RPE cells and test their ability to inhibit choroidal neovascularization (CNV) aiming to develop a novel approach to treat wet AMD.