| 研究実績の概要 |
In order to achieve the research theme of the Cas9 delivery for genomic correction, Duchenne muscular dystrophy (DMD) disease, which is caused by mutation in the gene encoding dystrophin, was selected as a target disease. First, single-strand guide RNA (sgRNA) was designed for the cleavage of target mutation region after complexation with Cas9 protein. In a cell-free experiment, the significant cleavage of dystrophin gene was observed using the designed sgRNA. Second, six types of cationic polymers were synthesized for the construction of polyion complexes (PICs) with Cas9-coding mRNA, as well as sgRNA. The successful PIC formation was confirmed by the size measurement. Finally, several PIC formulations elicited the significant mRNA expression in cultured C2C12 (Mus musculus muscle) cells.
|
| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
1) The sgRNA was successfully designed for degradation of target dystrophin in genome, as evidenced by gel electrophoresis, where two cleavage sites of intron (22 and 23 intron) were observed after treatment with Cas9.
2) Thee types of N-substituted polyasparamide (PAsp) homopolymers, two types of poly(ethylene glycol) (PEG)-PAsp block copolymers, and PEG-polylysine block copolymer were successfully synthesized and applied for polyion complex (PIC) formation with RNA species. Among them, PAsp homopolymers and PEG-PAsp could form PICs with a diameter of ~100 nm with narrow size distributions.
3) The significant transfection efficiencies of the PICs prepared from PAsp homopolymers were observed for cultured C2C12 cells.
|
| 今後の研究の推進方策 |
For more efficient Cas9/sgRNA delivery, RNA-loaded PICs (or cationic polymers) are modified with hydrophobic moieties or lipids to enhance the complex stability and/or cellular uptake efficiency. The obtained PICs are evaluated for simultaneous loading of Cas9 mRNA and sgRNA. Then, they are tested for in vitro transfection against C2C12 cells to determine the genome editing activity.
|
