HTRA1 as a Therapeutic Target in the Treatment of Wet AMD

 描述（由申请人提供）：视网膜相关性黄斑变性（AMD）是美国老年人视力损害的最常见原因。这项研究旨在从分子水平上寻找其原因。在AMD中，像许多其他与年龄有关的疾病一样，遗传风险因素和环境攻击都是关键因素。先前已确定10号染色体中的显著遗传变化与AMD易感性密切相关。这种遗传变异位于一种蛋白质的启动子区域，称为高温要求因子A1（HTRA 1）。这种疾病基因型导致HTRA 1表达增加。我们还使用HTRA 1敲除（htra 1-/-）小鼠进行了功能研究，发现HTRA 1的缺失导致视网膜血管发育减少，沿着血管内皮生长因子（VEGF）表达的显著下调。相反，我们的实验表明，在RPE中HTRA 1的表达增加导致AMD样病理和VEGF表达升高。高VEGF刺激眼睛的视网膜色素上皮（RPE）层和布鲁赫膜中的异常血管生长，这可导致湿性AMD。我们最近的一项研究发表在《生物化学杂志》上，揭示了HTRA 1对VEGF的上调与组织生长因子β（TGF-β）家族中称为生长分化因子6（GDF 6）的成员的下调呈负相关。该提案的长期目标是阐明HTRA 1导致AMD风险增加的分子机制，并开发AMD的潜在治疗方法。我们的假设是，氧化应激可以诱导高水平的HTRA 1的表达，这有助于通过调节信号转导，血管发育，血管生成和细胞外基质的AMD的风险。在此假设的指导下，我们计划进行以下方面的研究：1）我们将研究遗传变异如何影响RPE细胞对血管生成因子和HTRA 1表达的氧化应激反应。2)我们将确定HTRA 1和氧化应激是否影响视网膜和脉络膜血管和病理。我们还将尝试确定在小鼠中去除HTRA 1是否对htra 1-/-小鼠和对照小鼠中氧化应激增强的新血管形成具有保护作用，这将用于检查视网膜和脉络膜中的形态学和基因表达。3)我们将利用我们克隆的抗HTRA 1的单克隆抗体，通过组织特异性递送到RPE细胞中来研究它们的治疗潜力，旨在抑制异常的血管生长。在这种情况下，所选择的抗体将被转化为单链肽，其将被进一步“进化”以最大化其对HTRA 1的亲和力和抑制蛋白酶活性的能力，同时使用新型蛋白质支架进化技术最小化其细胞毒性。基因治疗策略将用于将此类抗体递送到RPE细胞中并测试其抑制脉络膜新生血管形成（CNV）的能力，旨在开发治疗湿性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.