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 of the 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 human iPS 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 well as 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

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 first attempts to identify small molecules through phenotypic screening approach for improving HR efficiency in human iPS cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Small Business Innovation Research Grants (SBIR) - Phase I (R43)
Project #
1R43GM103049-01A1
Application #
8454894
Study Section
Special Emphasis Panel (ZRG1-IMST-N (11))
Program Officer
Maas, Stefan
Project Start
2013-04-01
Project End
2014-09-30
Budget Start
2013-04-01
Budget End
2014-09-30
Support Year
1
Fiscal Year
2013
Total Cost
$224,700
Indirect Cost
Name
Cellomics Technology
Department
Type
DUNS #
020067527
City
Rockville
State
MD
Country
United States
Zip Code
20850