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）： 与年龄相关的黄斑变性（AMD）是美国老年人视力障碍的最常见原因。我们先前已经确定了HTRA1/ARM2染色体区域中的遗传变异与AMD的主要敏感性有关。该疾病基因型导致高温需求因子A1（HTRA1）的表达增加。在以前的资金期间，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的表达调节。我们的功能研究表明，HTRA1的丧失导致视网膜血管发育降低和HTRA1敲除（HTRA1-/ - ）小鼠中血管内皮生长因子（VEGF）基因表达的显着下调。相反，我们显示了HTRA1在RPE中的表达增加导致BRUCH的膜病理学和VEGF表达升高。我们进一步揭示了HTRA1对VEGF的上调与TGF-B家族成员的下调成反比，即生长分化因子6（GDF6）。该提案的长期目标是表征HTRA1的正常功能，阐明了HTRA1促进AMD风险增加和发育潜在疗法的分子机制。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，我们建议进行以下特定目标。 特定目的1：对HTRA1的单克隆抗体产生，并通过组织特异性递送到RPE细胞中研究其治疗潜力。 HTRA1表达增加诱导的PCV和视网膜色素上皮萎缩和光感受器变性。我们将生成特异性结合HTRA1的单克隆抗体。对HTRA1具有高亲和力的选定抗体菌株将转换为单链格式（SCFV），该格式将进一步“进化”，以使用最先进的新型蛋白质在体外进化技术中最大化结合特异性和亲和力。我们将使用一种基因治疗策略将这种优化的HTRA1抗体传递到RPE中，并测试其对HTRA1转基因小鼠和激光CNV模型的抑制作用。 特定目的2：确定TGF-B信号途径在视网膜发育和病理学中的作用。我们表明GDF6与AMD显着相关，并证明GDF6 AMD风险等位基因与GDF6表达降低和HTRA1表达增加有关。我们还表明，TGF-B受体1（TGF-B R1）与AMD相关。我们将生成有条件的敲除小鼠，这些小鼠在视网膜或RPE中删除GDF6和TGF-B R1，以检查TGF-B信号途径在视网膜发育和致病性变化中的作用，包括眼镜检查，组织学，ERG和表达谱，和表达曲线和表达型，Immunoyoloid组织和脉络膜化学化学和脉络膜固定性Nocasculinal（CNV）。 公共卫生相关性： AMD代表了具有经济和社会影响的重大公共卫生负担。对AMD风险具有重大影响的基因的鉴定和功能研究可能定义与其发病机理有关的关键分子途径。我们提出的实验将产生与早期和晚期AMD发病机理有关的新型机械洞察力，这可能导致针对基本原因和症状前诊断的疗法，以便对这些疗法进行更早的干预。