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

描述：干型老年性黄斑变性（AMD）是导致成人视力丧失的主要原因，但这种疾病的分子机制尚不清楚。目前还没有针对AMD的疾病改善疗法，但更好地了解疾病过程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建立基因靶向人类多能干细胞（hiPSC）文库，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全基因组关联研究（GWAS）中已知的AMD基因联系。为该文库生产的克隆细胞系将是等基因的，仅在GWAS数据库中先前鉴定的特定单核苷酸多态性（snp）上存在差异。细胞分化为视网膜色素上皮（RPE）将使前所未有的研究AMD炎症反应的分子发病机制和drusen产生特征成为可能。为了有效地产生基因靶向的hipsc文库，需要发展先进的技术来鉴定和分类hipsc克隆菌落，这些克隆菌落经过了与所需核苷酸序列的同源重组。为此，已经组建了一个多学科的研究小组来开发能够快速识别和有效分离和收集基因靶向细胞的仪器

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.