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

产品说明：干性老年性黄斑变性（AMD）是成年人群视力丧失的主要原因，但这种疾病病理学的分子机制仍不清楚。目前还没有针对AMD的疾病修饰疗法，但对疾病过程的更好理解无疑将为这种高度流行和破坏性的疾病带来新的治疗选择。目前的拨款申请旨在创建一个基因靶向的人类多能干细胞（hiPSC）库，以更好地阐明AMD的病理生理机制。在这项工作中产生的初始文库将利用来自全基因组关联研究（GWAS）的AMD的已知基因连锁。为该文库产生的克隆细胞系将是同基因的，仅在先前在GWAS数据库中鉴定的特定单核苷酸多态性（SNP）方面不同。将细胞分化为视网膜色素上皮（RPE）将使得对AMD的炎症反应和玻璃疣产生特征的分子发病机制的前所未有的研究成为可能。为了有效地产生基因靶向的hiPSC的文库，将需要开发用于鉴定和分选hiPSC的克隆集落的先进技术，所述克隆集落已经经历了与所需核苷酸序列的同源重组。为此，一个多学科的研究小组已经组装，以开发仪器，使快速识别和有效的分离和收集基因靶向细胞 产生hiPSC文库所必需的。该系统将集成最先进的细胞操作和并行聚合酶链反应（PCR）的性能的微型技术。该平台的设计、开发、集成和验证将按照三个目标完成。首先，微制造的细胞阵列将适用于使用自动化算法识别单个hiPSC集落，然后对显微镜下的集落进行采样和分离。其次，微孔板将用于通过PCR对基因靶向hiPSC样品进行平行分析。然后将微制造的采样阵列和微孔PCR装置集成，以仅采样、鉴定和收集由适当的基因靶向细胞组成的那些集落。为了对系统性能进行基准测试，该平台将与传统技术平行用于基因靶向hiPSC细胞系的生产，并将对这两种方法进行比较。一旦验证，该平台将用于创建在补体系统中含有特定SNP的单个纯合细胞系的文库，其中眼睛中蛋白质产物的表达与AMD风险的增加或降低有关。使用该系统，细胞将在所有其他基因座上是等基因的。然后将这些细胞系分化为RPE细胞，用于未来AMD病理生理学的研究。这项技术将使大量特异性基因靶向多能细胞系的有效创造成为可能，这些细胞系将在眼科研究中具有广泛的应用，包括理解基础细胞生物学以及药物筛选。

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.