CRISPR Cas9 protein delivery via virus-like particles for in vivo treatment of Duchenne muscular dystrophy
| 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
|
| Project Status |
Completed (Fiscal Year 2018)
|
| Budget Amount *help |
¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000)
Fiscal Year 2018: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
Fiscal Year 2017: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
|
|---|
| Keywords | CRISPR / DMD / Genome editing / Delivery / Virus-like particles / CRISPR Cas9 / ゲノム / 遺伝子 / ウィルス / 再生医療 / 細胞 |
|---|
| 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.
|
| 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.
|
Report
(3 results)
Research Products
(4 results)
