A pipeline for rapid functional determination and drug discovery of UDP genes

UDP 基因快速功能测定和药物发现的管道

基本信息

批准号：
8680859
负责人：
WENBIAO CHEN
金额：
$ 23.55万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-15 至 2016-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8680859
关键词：
Adopted Advertising Affect Alleles Animal Disease Models Animals Attention Behavior Behavioral Biological Assay Boxing Cells Chemicals Chromosomes Clinical Cloning Clustered Regularly Interspaced Short Palindromic Repeats Collection Communities DNA Defect Development Disease Disease Pathway Dose Drug Targeting Embryonic Development Engineering Enhancers Exons Explosion Fertilization Gene Expression Profile Gene Targeting Generations Genes Genetic Genome engineering Goals Guide RNA Heart Hereditary Disease In Situ Hybridization Information Networks Injection of therapeutic agent International Knock-out Knowledge Laboratories Libraries Life Light Messenger RNA Metabolic Methods Midbrain structure Modeling Mutagenesis Mutation Neuroanatomy Oligonucleotides Orthologous Gene Pathogenesis Pathway interactions Patients Pattern Pharmacologic Substance Phenotype Physiological Physiology Procedures Production Proteins Reagent Regulation Research Research Personnel Resources Staging System Techniques Technology Testing Therapeutic Time United States National Institutes of Health Zebrafish cell motility cost design disease phenotype drug discovery gene function genome sequencing hindbrain improved loss of function mutation mutant nervous system disorder novel programs public health relevance research study response screening small molecule small molecule libraries tool zebrafish genome

项目摘要

DESCRIPTION (provided by applicant): The rapid explosion in genetic knowledge coming from high-throughput genome sequencing of patient DNA is generating unprecedented numbers of novel alleles correlated with both rare and common diseases. Many of these newly discovered alleles are in genes whose function is not known. In order to build models of these genetic diseases, geneticists generate knockout mutations in the gene and study the resulting phenotype. Phenotypic analysis can provide confirmation that a particular gene can contribute to the disease state and provide a tool to better understand the disease. However, a better understanding of gene function requires identification of the genetic pathways that the disease gene is required for. To identify these genetic pathways many approaches can be taken, but unbiased screening approaches have the advantage that they are model independent and can be applied to many different disease-associated genes in an analytical pipeline. Classically, genetic suppressor/enhancer screens have been used to identify pathways and genetic interactions, however this approach is time consuming and laborious. More recently, chemical interaction screens have been developed in which well-characterized, pharmacologically-active small molecule libraries are screened against the mutants. This approach is amenable to high throughput pipelines. Interactors are those small molecules that ameliorate or specifically exacerbate the phenotype. Since the protein targets of these libraries are well characterized, the pathway can be identified from these screening techniques. These results can then be confirmed through genetic interaction with the drug target genes. Our laboratories have developed new genome engineering technologies that allow for the rapid generation of mutations in the zebrafish genome. We will utilize this crispr-directed mutagenesis system to generate mutations in each of the zebrafish orthologs of the genes identified by the NIH Undiagnosed Disease Program. To begin to characterize these largely uncharacterized genes, the expression pattern of the genes will be analyzed during the first 5 days of life by in situ hybridization. The generated mutants will be analyzed for anatomical/developmental defects as well as functional differences in behavior and physiology. Many of the UDP-identified alleles are associated with neurological disease and particular attention for those genes will be placed on midbrain and hindbrain neuroanatomy, optokinetic response, motility, autonomic regulation of the heart, and ss cell mass changes as appropriate for the disease phenotype. The phenotypes most amenable to small molecule library screening will then be tested for interaction against a small molecule library of pharmacologically active and well characterized compounds. This compound screen will serve two purposes: First, the screen will identify compounds that interact with the allele which will assist in gene function model building. Second, the compound screen may identify pharmaceuticals that may be of direct clinical use to the patient.

描述（由申请人提供）：来自患者DNA的高通量基因组测序的遗传知识的快速爆炸正在产生前所未有的新等位基因与稀有疾病和常见疾病相关。这些新发现的等位基因中有许多是在尚不清楚的基因中。为了建立这些遗传疾病的模型，遗传学家在基因中产生敲除突变并研究所得的表型。表型分析可以确认特定基因可以有助于疾病状态，并提供一种更好地理解疾病的工具。但是，对基因功能的更好理解需要鉴定需要疾病基因的遗传途径。为了识别这些遗传途径，可以采用许多方法，但是公正的筛选方法的优势是它们独立于模型，并且可以应用于分析管道中许多不同疾病相关的基因。从经典上讲，遗传抑制剂/增强子筛选已被用来识别途径和遗传相互作用，但是这种方法是耗时且艰辛的。最近，已经开发了化学相互作用筛选，其中将良好的，药理活性的小分子文库筛选了突变体。这种方法适合高通量管道。相互作用者是那些改善或特别加剧表型的小分子。由于这些文库的蛋白质靶标有很好的特征，因此可以从这些筛选技术中识别出该途径。然后可以通过与药物靶基因的遗传相互作用来确认这些结果。我们的实验室开发了新的基因组工程技术，可以在斑马鱼基因组中快速产生突变。我们将利用这种CRISPR指导的诱变系统在NIH未诊断疾病计划鉴定的基因的斑马鱼直系同源物中产生突变。为了开始表征这些很大程度上未表征的基因，将通过原位杂交在生命的前5天分析基因的表达模式。将分析产生的突变体的解剖/发育缺陷以及行为和生理学的功能差异。许多被UDP识别的等位基因与神经疾病有关，这些基因的特别关注将放在中脑和后脑神经解术，光动力学反应，运动性，心脏的自主性调节以及SS细胞质量变化适合于疾病表型。然后将测试最适合小分子库筛选的表型与药理学活性和表征良好的化合物的小分子库进行测试。该复合屏幕将有两个目的：首先，屏幕将识别与等位基因相互作用的化合物，这些化合物将有助于基因功能模型构建。其次，复合屏幕可以识别可能对患者直接临床使用的药物。