A High Content Screening Approach for the Retinal Degenerative Diseases

视网膜退行性疾病的高内涵筛查方法

• 批准号: 9187475
• 负责人: Cynthia Berlinicke
• 金额: $ 40.5万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-12-01 至 2019-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9187475
• 关键词: Affect; Age related macular degeneration; Alpha Cell; Biological; Biological Assay; Cell Count; Cell Culture System; Cell Culture Techniques; Cell Death; Cell Differentiation process; Cells; Cluster Analysis; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Coupled; Cultured Cells; Defect; Development; Disease; Dose; Endoplasmic Reticulum; Epithelial; Evaluation; Future; Gene Expression; Genes; Human; Immunohistochemistry; In Vitro; Inherited; Knock-in Mouse; Lead; Molecular Probes; Molecular Profiling; Morphology; Mus; Mutate; Mutation; Pathogenesis; Pathway interactions; Phagocytosis; Pharmaceutical Chemistry; Phenotype; Photoreceptors; Property; Protein Tyrosine Kinase; Proto-Oncogenes; Rare Diseases; Rattus; Receptor Cell; Replacement Therapy; Retinal; Retinal Degeneration; Retinal Ganglion Cells; Retinal Pigments; Retinitis Pigmentosa; Rhodopsin; Stem cells; Stress; Structure of retinal pigment epithelium; Surgeon; System; Time; Translating; Tyrosine-Kinase Oncogenes; XBP1 gene; activating transcription factor; base; cell preparation; college; design; drug development; endoplasmic reticulum stress; established cell line; experimental study; human embryonic stem cell; human stem cells; in vivo; mutant; neovascular; neuroprotection; novel; optical spectra; photoreceptor degeneration; public health relevance; response; screening; small molecule; therapeutic development; treatment strategy

DESCRIPTION (provided by applicant): The retinal degenerative (RD) diseases are a heterogeneous group of conditions that range from the Mendellian inherited orphan diseases such as retinitis pigmentosa (RP) to the genetically complex disease age-related macular degeneration (AMD). With the exception of the neovascular ("wet") form of AMD, these diseases remain essentially untreatable. As one approach to both increase our understanding of these diseases and develop novel treatment strategies, we propose to perform phenotypic screens for small molecules that could serve as molecular probes and/or lead molecules for drug development. In our first aim we propose to conduct high content screens (HCS) for photoreceptor (PR) differentiation-promoting and PR neuroprotective compounds using mouse primary retinal cultures, which have significant advantages over the use of established cell lines. As proof of principle for this approach, we have successfully used this strategy in the past to identify compounds that are neuroprotective for retinal ganglion cells both in vitro and in vivo. I aim 2, we propose a variety of secondary assays to characterize, classify and prioritize the active molecules identified in our primary screens. Another PR biological pathway that is sensitive to perturbation is the clearance of PR outer segments (OS). More than a dozen different mutations in the gene for MER tyrosine kinase (MERTK), which encodes the retinal pigment epithelial (RPE) cell receptor necessary for PR OS phagocytosis, have been shown to cause RP. In aim 3, using an assay based upon human stem cell derived MERTK -mutant RPE cells, we will screen for compounds that can increase MERTK-independent RPE phagocytosis.

描述（由申请人提供）：视网膜退行性（RD）疾病是一组异质疾病，范围从门德尔遗传的孤儿疾病（例如色素性视网膜炎）到遗传复杂的疾病年龄相关的黄斑变性（AMD）。除了AMD的新血管（“湿”）形式之外，这些疾病基本上仍然无法治疗。当一种方法是提高我们对这些疾病的理解并制定新的治疗策略时，我们建议对可以用作分子探针和/或铅分子进行药物开发的小分子进行表型筛选。在我们的第一个目的中，我们建议使用小鼠初级视网膜培养物进行光感受器（PR）分化促进和PR神经保护化合物的高含量筛选（HCS），这些化合物在使用已建立的细胞系方面具有显着优势。 作为这种方法的原理证明，我们过去成功地使用了该策略来识别体外和体内视网膜神经节细胞神经保护的化合物。我瞄准2，我们提出了各种辅助测定法，以表征，对我们的主要筛选中确定的活性分子进行表征。对扰动敏感的另一种PR生物学途径是PR外段（OS）的清除。编码视网膜色素上皮（RPE）细胞受体的Mer酪氨酸激酶（MERTK）的基因中有十多个不同的突变，这是PR OS吞噬作用所必需的，这会引起RP。在AIM 3中，使用基于人类干细胞衍生的MERTK突变RPE细胞的测定，我们将筛选可以增加与MERTK独立的RPE吞噬作用的化合物。