HTRA1 and Age-Related Macular Degeneration

HTRA1 和年龄相关性黄斑变性

• 批准号: 8763871
• 负责人: KANG ZHANG
• 金额: --
• 依托单位: VA SAN DIEGO HEALTHCARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-10-01 至 2017-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8763871
• 关键词: Affinity; Age related macular degeneration; Antibodies; Atrophic; Binding; Blood Vessels; Bruch' s basal membrane structure; Cells; Choroidal Neovascularization; Chromosomes; Complement Factor H; Complex; Data; Development; Diagnostic; Disease; Down-Regulation; ERG gene; Early Intervention; Elderly; Environmental Risk Factor; Evolution; Extracellular Matrix; Family; Funding; Gene Expression; Generations; Genes; Genetic; Genetic Predisposition to Disease; Genetic Risk; Genotype; Haplotypes; High temperature of physical object; Histology; Human; Immunoglobulin Variable Region; Immunohistochemistry; In Vitro; Knock-out; Knockout Mice; Lasers; Lead; Link; Modeling; Molecular; Molecular Profiling; Monoclonal Antibodies; Mus; Ophthalmoscopy; Pathogenesis; Pathology; Pathway interactions; Play; Predisposition; Proteins; Public Health; Regulation; Retina; Retinal; Risk; Role; Scanning; Signal Pathway; Signal Transduction; Single Nucleotide Polymorphism; Social Impacts; Specificity; Structure of retinal pigment epithelium; TGFBR1 gene; Technology; Testing; Therapeutic; Tissues; Transforming Growth Factor beta; Transforming Growth Factor beta Receptors; Transgenic Mice; Transgenic Organisms; United States; Up-Regulation; VEGFA gene; Vascular Endothelial Growth Factors; Visual impairment; Western Blotting; angiogenesis; gene therapy; genetic association; genetic variant; growth differentiation factor 6; insight; loss of function; member; novel; photoreceptor degeneration; public health relevance; receptor; research study; risk variant

DESCRIPTION (provided by applicant): Age-related macular degeneration (AMD) is the most common cause of visual impairment of the elderly in the United States. We have previously identified a genetic variant in the chromosome region of HTRA1/ARMS2 was associated with major susceptibility to AMD. This disease genotype results in increased expression of high temperature requirement factor A1 (HTRA1). In our previous funding period, we systematically scanned all single nucleotide polymorphisms (SNPs) related to AMD and investigated their regulation of expression of HTRA1 and ARMS2. Our functional study reveals loss of HTRA1 leads to decreased retinal vascular development and significant down-regulation of vascular endothelial growth factor (VEGF) gene expression in HTRA1 knockout (htra1-/-) mice. Conversely, we show Increased expression of HTRA1 in RPE leads to Bruch's membrane pathology and elevated VEGF expression. We further revealed that the up-regulation of VEGF by HTRA1 is inversely correlated to the down-regulation of a member of the TGF-b family, the growth differentiation factor 6 (GDF6). The long term objectives of this proposal are to characterize the normal function of HTRA1, elucidate molecular mechanism by which HTRA1 contributes increased risk of AMD, and development potential therapies. Our central hypothesis is that HTRA1 contributes to AMD risk by regulation of vascular development, angiogenesis, TGF-b signaling, and extracellular matrix modeling. Guided by this hypothesis, we propose to conduct the following specific aims. Specific Aim 1: Generation of monoclonal antibodies to HTRA1 and investigating their therapeutic potential by tissue specific delivery into the RPE cells. Increased HTRA1 expression induced PCV and retinal pigment epithelium atrophy and photoreceptor degeneration. We will generate monoclonal antibodies specifically bind to HTRA1. The selected antibody strains with the high affinity to HTRA1 will be converted into a single chain format (scFv), which will be further "evolved" to maximize binding specificity and affinity using a state-of-the-art novel protein in vitro evolution technology. We will use a gene therapy strategy to deliver such optimized HTRA1 antibodies into RPE and test its effect on inhibition in HTRA1 transgenic mice and a laser-CNV model. Specific Aim 2: To determine the role of TGF-b signal pathway in retinal development and pathology. We show that GDF6 significantly associated with AMD and demonstrated that the GDF6 AMD risk allele is associated with decreased expression of the GDF6 and increased expression of HTRA1. We also showed that TGF-b receptor 1 (TGF-b R1) is associated with AMD. We will generate conditional knockout mice which delete GDF6 and TGF-b R1 in either retina or RPE to examine the role of TGF-b signal pathway in retinal development and pathogenic changes, including ophthalmoscopy, histology, ERG, and expression profile, immunohistochemistry and choroidal neovascularization (CNV) formation. PUBLIC HEALTH RELEVANCE: AMD represents a major public health burden with economical and social impacts. Identification and functional studies of genes that have substantial impact on the risk of AMD may define key molecular pathways involved in its pathogenesis. The experiments we propose will yield novel mechanistic insight relevant to the pathogenesis of both early and late AMD, which may lead to therapies directed at the underlying cause and pre-symptomatic diagnostics to allow for earlier intervention with those therapies.

描述（由申请人提供）： 年龄相关性黄斑变性（AMD）是美国老年人视力障碍的最常见原因。我们之前已经发现 HTRA1/ARMS2 染色体区域的遗传变异与 AMD 的主要易感性相关。这种疾病基因型导致高温需求因子 A1 (HTRA1) 表达增加。在我们之前的资助期间，我们系统地扫描了与AMD相关的所有单核苷酸多态性（SNP），并研究了它们对HTRA1和ARMS2表达的调控。我们的功能研究表明，在 HTRA1 敲除 (htra1-/-) 小鼠中，HTRA1 缺失会导致视网膜血管发育减少，血管内皮生长因子 (VEGF) 基因表达显着下调。相反，我们发现 RPE 中 HTRA1 表达增加会导致 Bruch 膜病理学和 VEGF 表达升高。我们进一步发现，HTRA1 对 VEGF 的上调与 TGF-b 家族成员生长分化因子 6 (GDF6) 的下调呈负相关。该提案的长期目标是表征 HTRA1 的正常功能，阐明 HTRA1 增加 AMD 风险的分子机制，并开发潜在的治疗方法。我们的中心假设是 HTRA1 通过调节血管发育、血管生成、TGF-b 信号传导和细胞外基质建模而导致 AMD 风险。在这一假设的指导下，我们建议实现以下具体目标。 具体目标 1：生成 HTRA1 单克隆抗体，并通过组织特异性递送至 RPE 细胞来研究其治疗潜力。 HTRA1 表达增加诱导 PCV 和视网膜色素上皮萎缩和光感受器变性。我们将产生特异性结合 HTRA1 的单克隆抗体。选定的与HTRA1具有高亲和力的抗体株将被转化为单链形式（scFv），并利用最先进的新型蛋白质体外进化技术进一步“进化”以最大限度地提高结合特异性和亲和力。我们将使用基因治疗策略将这种优化的 HTRA1 抗体传递到 RPE 中，并在 HTRA1 转基因小鼠和激光 CNV 模型中测试其抑制效果。 具体目标 2：确定 TGF-b 信号通路在视网膜发育和病理学中的作用。我们发现 GDF6 与 AMD 显着相关，并证明 GDF6 AMD 风险等位基因与 GDF6 表达减少和 HTRA1 表达增加相关。我们还发现 TGF-b 受体 1 (TGF-b R1) 与 AMD 相关。我们将产生条件性基因敲除小鼠，在视网膜或RPE中删除GDF6和TGF-b R1，以检查TGF-b信号通路在视网膜发育和致病性变化中的作用，包括检眼镜检查、组织学、ERG和表达谱、免疫组织化学和脉络膜新生血管（CNV）形成。 公共卫生相关性： AMD 是一个重大的公共卫生负担，具有经济和社会影响。对 AMD 风险有重大影响的基因的鉴定和功能研究可能会定义参与其发病机制的关键分子途径。我们提出的实验将产生与早期和晚期 AMD 发病机制相关的新机制见解，这可能导致针对根本原因的治疗和症状前诊断，以便更早地干预这些治疗。