High-Throughput Screening to Identify Small Molecules for Improving Homologous Re

DESCRIPTION (provided by applicant): The present project aims to perform high-throughput screening (HTS) to identify small molecules that could improve the homologous recombination (HR) efficiency in human induced pluripotent (iPS) cells. The emergence ofthe iPS cell technology has dramatically impacted the field of regenerative medicine. However, to successfully utilize iPS cells for gene correction therapy or other cell based therapies in human, we first need to overcome the low efficiency of HR in humaniPS cells. We propose to first establish an efficient and reliable selection platform for our HTS work. Considering the difficulty to maintain single cell colonies of human iPS cells, we will first establish the human fibroblasts that are suitable for homology directed recombination (HDR) assay. We will use lentiviral vectors to transduce the fibroblast cells. The lentiviral vector consists of two copies of mutated Green Fluorescent Protein (GFP), neither of which can express functional GFP and one of them contains a reorganization site for restrictive enzyme I-SceI. The fibroblast cells containing the two mutated copies of GFP are designated HDR-fibroblasts . Upon introduction of I-SceI via a separate lentiviral transduction, HR may take place between the two mutated GFP copies, after which functional GFP will be expressed. Therefore, the HR frequency can be determined through measuring the percentage of GFP positive cells. As the ultimate goal of the present project is to improve HR efficiency in human iPS cells, Aim 2 of the Phase I work is to derive HDR-iPS cells from the HDR- fibroblasts. We plan to generate one or more such iPS cell lines that are characterized with typical iPS morphology, pluripotent cell markers and gene expression profiles, as wellas normal ploidy. We will also validate these cells for HDR assay using RS-1, which is known to enhance HR efficiency in mammalian cells. These HDR-iPSCs will be used in Phase II for large scale HTS of small molecules. PUBLIC HEALTH RELEVANCE PUBLIC HEALTH RELEVANCE: The emergence of the iPS cell technology has dramatically impacted the field of regenerative medicine. However, to successfully utilize these cells for cell based therapies in human, we first need to overcome the low efficiency of homologous recombination (HR) in these cells. The present project aims to perform high-throughput screening (HTS) to identify small molecules that could improve the HR efficiency in human iPS cells. To our knowledge, this project represents one of the firstattempts to identify small molecules through phenotypic screening approach for improving HR efficiency in human iPS cells.