CRISPR Cas9 protein delivery via virus-like particles for in vivo treatment of Duchenne muscular dystrophy

2018 Fiscal Year Final Research Report

• Project/Area Number: 17K15048
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: System genome science
• Research Institution: Kyoto University
• Principal Investigator: Gee Peter David 京都大学, iPS細胞研究所, 特定研究員 (00754227)
• Project Period (FY): 2017-04-01 – 2019-03-31
• Keywords: CRISPR / DMD / Genome editing / Delivery

Outline of Final Research Achievements

We developed a novel protein delivery system based on virus-like particles that we term NanoMEDIC (nanomembrane-derived extracellular vesicles for the delivery of macromolecular cargo) which can be used to deliver proteins of interest in a variety of cell types including human iPS cells, monocytes, T cells, cancer cell lines, and also in vivo. In particular, genome editing technology such as CRISPR-Cas9 could be delivered into a variety of cells and achieved high editing efficiency. The system utilizes the FKBP12 and FRB dimerization domains fused with HIV-Gag and SpCas9, respectively. We optimized the FRB-SpCas9 fusion protein by testing the incorporation of SpCas9 when FRB was fused either to the N-, C- or N- and C- terminal ends of the protein. Furthermore, mutation of the FRB domain abrogated the dimerization activity.

Free Research Field

Gene Therapy

Academic Significance and Societal Importance of the Research Achievements

Transient delivery of CRISPR-Cas9 RNP will allow for safer use of genome editing technology in vivo and may be applicable future clinical work. We also developed a serum-free culture system using flow electroporation to potentially apply the production of our NanoMEDIC system for industrial use.