| 研究実績の概要 |
Rhabdomyosarcoma (RMS) is the most common pediatric sarcoma in the world arising from skeletal muscle progenitors. Malfunction of the Duchenne muscular dystrophy-related gene, dystrophin was implicated in the growth and metastasis of tumor in RMS.
In this project, the applicants attempted to study dystrophin intron retention in RMS to identify targets for RNA modulation by Antisense Oligonucleotides (AO). Hence, reduce RMS formation and metastasis.
In the previous year, we observed the retention of intron 40 in many RMS.
This year, the applicants successfully identified a candidate long exonic splicing enhancer (LESE) among others, in the retained 3’ end of intron 40, by the web based algorithms, ESE Finder. With this LESE as the target, the applicants designed and screened several AOs and finally succeeded to identify a specific antisense oligonucleotides (AO) to enhance the splicing of the retained 3’ end of intron 40. They subsequently confirmed the specificity and efficiency of this LESE at the transcription level. The results indicated that the splicing of intron 40 retained transcript could be eliminated using this LESE.
Next year, the applicants are going to perform more AO transfection experiments to check for the optimization of AO concentration to eliminate the factor of cell toxicity, as well as a time course experiment to identify the best time that produces the highest percentage of splicing of intron 40 retained transcript. In addition, they will also perform protein analysis of dystrophin expression and animal-based experiments.
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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
The applicants succeeded to identify potential long exon splicing enhancers. In addition, the applicants designed and screened several antisense oligonucleotides (AOs) and finally succeeded to identify a specific (AO) to enhance the splicing of the retained 3’ end of intron 40. They subsequently confirmed the specificity and efficiency of this LESE on the splicing of intron 40 retained sequence at the transcription level. These results were obtained as expected from the proposal. Hence, the work is progressing rather smoothly.
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| 次年度使用額が生じた理由 |
Since the effect of the antisense on intron retention removal was very good, we thought of performing a cell-based experiment as well as animal experiments to test the cell proliferation of the rhabdomyosarcoma tumor.
Due to this new additional experiment, we had to make a new plan, apply to the ethical committee as well as search for potential collaborators for the in vitro experiments. In addition, sufficient time is required for the experiments. Hence, the carrying over of the budget for the next fiscal year.
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