ATGC Inc. — Department of Health and Human Services STTR Phase I: NIGMS

Description

Abstract Gene correction therapy is one of the most important application directions in regenerative medicine Emerging technologies such as CRISPR clustered regularly interspaced short palindromic repeats Cas CRISPR associated protein Zinc Finger Nuclease ZFN and Transcription Activator Like Effector Nuclease TALEN have enabled efficient and precise gene editing in a wide spectrum of species and hold promises for eventually achieving gene correction therapy in therapeutic settings However several major challenges remain to be addressed including low knock in efficiency off targeting effect and lack of an efficient delivery system in vivo The present proposal focuses on the challenge of low knock in efficiency Recently we reported that RS a homology directed repair HDR enhancer improves the efficiency of Cas or TALEN mediated knock in in rabbit embryos Microinjecting human RAD hRAD mRNA to the embryos mimicked the beneficial effects of RS treatment In the present project we propose experiments to further improve nuclease mediated HR rates In Aim we will first develop a RAD augmentation method to improve Cas mediated HR On RAD Threonine T and Serine S are the two best known sites that are phosphorylated activated in DNA repair processes So we hypothesize that the replacement of T and S with their phosphomimetics T E and S D and the use of such mutant RAD mRNAs will lead to consecutively active RAD which leads to enhanced Cas mediated HR rate BRCA is a key player in HR It is recruited to processed double strand breaks DSBs and facilitates the assembly of RAD In Aim we will develop a TALE and BRCA exon fusion protein TALE BE to help recruiting RAD at the DSB to further improve the HR rate In Aim we will validate these HR improving methods in rabbit embryos The proposal aims to address a bottleneck problem in regenerative medicine i e low knock in efficiency Its success will have significant impacts on the entire field as a majority of stem cell based therapy will require targeted gene modifications Project Narrative Emerging technologies such as CRISPR clustered regularly interspaced short palindromic repeats associated protein Cas have enabled high efficient gene knockout KO in human cells and model animals However the knock in efficiency remains to be further improved We propose novel methods to improve the Cas mediated knock in efficiency