Generation of a Gene-Targeted Human iPS Cell Library for Macular Degeneration

生成针对黄斑变性的基因靶向人类 iPS 细胞库

• 批准号: 8748459
• 负责人: Nancy L. Allbritton
• 金额: $ 61.4万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8748459
• 关键词: Address; Adult; Advanced Development; Affect; Age; Age related macular degeneration; Algorithms; Animal Model; Applications Grants; Atrophic; Automobile Driving; Base Sequence; Benchmarking; Biochemical; Biological Models; Biopsy; Blindness; Cell Differentiation process; Cell Line; Cell model; Cells; Cellular biology; Characteristics; Chemical Synapse; Choroid; Chronic; Cloning; Collection; Complement; Complement Activation; Complement Factor H; Complex; Data; Databases; Defect; Deposition; Development; Devices; Disease; Disease model; Drusen; Electrical Synapse; Environment; Epithelial; Eye; Eye diseases; Functional disorder; Future; Gene Targeting; Generations; Genes; Genetic; Genetic Engineering; Genetic Polymorphism; Genome; Glycolipids; Grant; Human; In Vitro; Individual; Inflammatory Response; Lead; Libraries; Link; Liver; Location; Macular degeneration; Methods; Microscopic; Modeling; Molecular; Molecular Target; Mus; Mutation; Partner in relationship; Pathogenesis; Pathology; Pathway interactions; Patients; Performance; Pharmacologic Substance; Photoreceptors; Pluripotent Stem Cells; Polymerase Chain Reaction; Population; Preclinical Drug Evaluation; Prevalence; Process; Production; Proteins; Reagent; Regenerative Medicine; Research; Research Personnel; Retinal; Retinal Pigments; Risk; Role; Sampling; Single Nucleotide Polymorphism; Site; Sorting - Cell Movement; Source; Stem cells; Structure; Structure of retinal pigment epithelium; System; Techniques; Technology; Therapeutic; Therapeutic Human Experimentation; Time; Transformed Cell Line; Transplantation; Validation; Vision; Visual; Work; aging population; base; complement system; design; disease-causing mutation; effective therapy; gene repair; genetic association; genome wide association study; high risk; high throughput technology; homologous recombination; human stem cells; improved; induced pluripotent stem cell; insight; instrumentation; multidisciplinary; new technology; novel; novel therapeutics; protein expression; public health relevance; regenerative; response; retinal progenitor cell; screening; tool

DESCRIPTION: The dry form of age-related macular degeneration (AMD) is the leading cause of visual loss in the adult population, but the molecular mechanisms underlying the pathology of this disease remain unclear. There are no disease-modifying therapies for AMD, and yet a better understanding of the disease process will undoubtedly lead to new therapeutic options for this highly prevalent and devastating condition. The current grant application aims to create a library of gene-targeted human pluripotent stem cells (hiPSC) to better elucidate the pathophysiologic mechanisms of AMD. The initial library to be produced in this work will take advantage of known gene linkages for AMD from genome-wide association studies (GWAS). The clonal cell lines produced for this library will be isogenic differing only in specific single-nucleotide polymorphisms (SNPs) previously identified in the GWAS database. Differentiation of the cells into retinal pigmented epithelial (RPE) will make possible unprecedented studies into the molecular pathogenesis of the inflammatory response and drusen production characteristic of AMD. To efficiently produce libraries of gene- targeted hiPSCs will require the development of advanced techniques for identifying and sorting clonal colonies of hiPSCs which have undergone homologous recombination with the desired nucleotide sequence. To this end, a multidisciplinary team of investigators has been assembled to develop the instrumentation that will enable the rapid identification and efficient separation and collection of gene-targeted cells needed to produce hiPSC libraries. The system will integrate state-of-the-art microtechnologies for cell manipulation and the performance of parallel polymerase chain reactions (PCR). Design, development, integration and validation of the platform will be accomplished in three aims. First, microfabricated cell arrays will be adapted for identification of individual hiPSC colonies using automated algorithms followed by sampling and separation of the microscopic colonies. Second, a microwell plate will be utilized for parallel analyses of gene- targeted hiPSC samples by PCR. The microfabricated sampling array and microwell PCR device will then be integrated to sample, identify, and collect only those colonies composed of properly gene-targeted cells. To benchmark system performance, the platform will be utilized in the production of gene-targeted hiPSC cell lines in parallel with conventional technology, and the two approaches will be compared. Once validated, the platform will be employed to create a library of individual homozygous cell lines containing specific SNPs in the complement system for which expression of the protein products in the eye have been linked to increased or decreased risk of AMD. Using this system the cells will be isogenic at all other loci. These cell lines will then be differentiated into RPE cells for future study of AMD pathophysiology. This technology will make possible the efficient creation of large numbers of specific gene-targeted pluripotent cell lines that will have widespread application in eye research including understanding basic cell biology as well as pharmaceutical screens.

描述：与年龄相关的黄斑变性（AMD）的干燥形式是成年人口视觉丧失的主要原因，但是该疾病病理学的分子机制尚不清楚。没有针对AMD的疾病改良疗法，但是对疾病过程的更好理解无疑会为这种高度普遍且毁灭性的疾病带来新的治疗选择。当前的赠款应用旨在创建一个靶向基因的人多能干细胞（HIPSC）的库，以更好地阐明AMD的病理生理机制。这项工作中要生产的最初文库将利用来自基因组关联研究（GWAS）的已知基因链接。为该库产生的克隆细胞系仅在GWAS数据库中先前鉴定出的特定单核苷酸多态性（SNP）中，仅在异基因源性上有所不同。细胞将细胞分化为视网膜色素上皮（RPE），将使对AMD的炎症反应和DRUSEN生产特征的分子发病机理进行前所未有的研究。为了有效地生产基因靶向hipsc的文库，将需要开发高级技术，以识别和分类HIPSC的克隆菌落，这些HIPSC具有与所需的核苷酸序列同源重组的HIPSC。为此，已经组装了一个多学科研究人员团队，以开发仪器，以使基因靶向细胞的快速识别和有效的分离和收集 需要生产HIPSC库。该系统将集成最新的微技术，以进行细胞操作和平行聚合酶链反应（PCR）的性能。平台的设计，开发，集成和验证将以三个目标来完成。首先，将使用自动化算法进行微生物细胞阵列，以鉴定单个HIPSC菌落，然后对微观菌落进行采样和分离。其次，通过PCR对基因靶向HIPSC样品进行平行分析，将使用Microwell板。然后，将集成微生物的采样阵列和Microwell PCR设备，以采样，识别和收集由适当的基因靶向细胞组成的菌落。为了进行基准测试系统性能，该平台将用于与常规技术并行的基因靶向HIPSC细胞系的生产中，并将两种方法进行比较。经过验证后，该平台将被用来创建一个单个纯合细胞系的库，该细胞系中包含特定SNP的补体系统中，蛋白质产物在眼中的表达与AMD风险的增加或降低有关。使用此系统，细胞在所有其他基因座都将是同源性的。然后，这些细胞系将被分化为RPE细胞，以进行未来的AMD病理生理学研究。这项技术将使有效地创建大量特定基因的多能细胞系，这些细胞系将在眼睛研究中广泛应用，包括了解基本细胞生物学以及药物筛选。