| 研究実績の概要 |
In order to achieve genomic correction by Cas9 activity, we continued to check double strand break (DSB) efficacy of dystrophin gene, which is the main target gene in Duchenne muscular dystrophy. At first, Cas9 protein and single-stranded guide RNA (sgRNA) formed a Cas9/sgRNA complex and then this protein complex was loaded into a pre-formed micelle, which was confirmed by SDS gel electrophoresis. The Cas9/sgRNA-loaded micelle was transfected into C2C12 murine myoblast cells and then the Cas9 activity was checked by surveyor’s assay. However, we could not obtain DSB effects of the micelle. As an alternative way, we assumed that delivery vehicles (or cationic polymers) were not enough to be efficient to generate significant gene editing effect. We synthesized novel amphiphilic polymers for Cas9 messenger RNA (mRNA) transfection.
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| 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) We concluded that a more versatile Cas9 activity assay was necessary for the delivery carrier screening. To this end, we obtained a luciferase-based Cas9 activity assay system from our collaborator. This assay system consists of three types of plasmid DNAs (pDNA), i) sgRNA-expressing pDNA, stop codon-included luciferase-expressing pDNA, and luciferase-donor DNA-expressing pDNA. When Cas9 eliminates the stop codon in the stop codon-included luciferase-expressing pDNA and inserts the luciferase-donor DNA, luciferase will be expressed and its activity can be quantified by a luciferase assay.
2) For efficient Cas9 mRNA transfection into C2C12 cells, we synthesized novel amphiphilic polymers. Poly(beta-benzyl L-aspartate) (PBLA) was aminolyzed with diethylenetriamine and primary aliphatic amines. Indeed, pentylamine, heptylamine, octylamine, nonylamine, and decylamine were used as primary aliphatic amine candidates. Various substitution degrees of the primary aliphatic amines were tested to optimize hydrophobicity of amphiphilic polymers.
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| 今後の研究の推進方策 |
1) Cas9/sgRNA-loaded micelles will be transfected into C2C12 cells and their Cas9 activity will be checked by the luciferase-based Cas9 activity assay. Efficacy of Cas9 protein will be checked for various delivery carriers in comparison with commercially available transfection reagent as a control.
2) We continue to synthesize new amphiphilic polymers. Their transfection efficacy will be checked by Gaussian luciferase assay and the luciferase-based Cas9 activity assay. Also, we will investigate the mechanism of efficient polymers (or delivery carriers) in terms of cellular uptake efficiency, nanoparticle stability, and endosomal escapability.
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