APPLIED STEMCELL, INC. — Department of Health and Human Services STTR Phase I: 200

Description

DESCRIPTION provided by applicant Gene correction therapy is one of the most important application directions in regenerative medicine Scientists are exploring various approaches to introduce targeted mutations corrections to pluripotent stem cells PSCs to establish disease models and or to develop therapeutic agents Emerging technologies such as Zinc Finger Nucleases ZFNs Transcription Activator Like Effector Nucleases TALENs and CRISPR clustered regularly interspaced short palindromic repeats associated protein Cas have enabled high efficient gene knockout KO in pluripotent stem cells in humans and model animal species Indeed these meganucleases especially Cas are becoming the mainstream choice for gene targeting in stem cells and in transgenic animal production However the knock in efficiency remains to be further improved even in the presence of these meganucleases We hypothesize that inhibiting non homologous end joining NHEJ and or enhancing the homology directed repair HDR via small molecules will improve the meganuclease mediated knock in efficiency in pluripotent stem cells In this project we will test the feasibility by screening smll molecules using a reporter cell line to identify candidate compounds followed by validation of the efficacy of these compounds in human iPSCs In Aim we will first establish a reporter cell line that will express luciferase upon successful Ca mediated homologous recombination HR We will then screen small molecule compounds on this reporter cell line After identifying candidate compounds in Aim we will validate their efficacy on human induced PSCs iPSCs in Aim We will work to determine the Cas mediated knock in efficiency in gene correction on multiple human iPSC lines using candidate compounds identified in Aim This proposal aims to address a bottleneck problem in regenerative medicine i e low knock in efficiency in PSCs Its success will have significant impacts on the entire field as a majority o stem cell based therapy will require targeted gene modifications Notably it may even enable multiplex gene corrections in human PSCs PUBLIC HEALTH RELEVANCE Emerging technologies such as CRISPR clustered regularly interspaced short palindromic repeats associated protein Cas have enabled high efficient gene knockout KO in pluripotent stem cells in humans and model animal species However the knock in efficiency remains to be further improved even in the presence of these meganucleases We propose to screen small molecule library on a reporter cell line to identify candidate compound that can dramatically improve the efficiency of Cas mediated homologous recombination