2017 Fiscal Year Research-status Report
Dystrophin intron retention analysis to identify new targets for Antisense Oligonucleotide mediated RNA modulation in Rhabdomyosarcoma
| Project/Area Number |
16K07216
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| Research Institution | Kobe University |
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Principal Investigator |
ニバ タベ・エマ・エコ 神戸大学, 医学研究科, 助教 (00727810)
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| Co-Investigator(Kenkyū-buntansha) |
松尾 雅文 神戸学院大学, 総合リハビリテーション学部, 教授 (10157266)
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| Project Period (FY) |
2016-04-01 – 2019-03-31
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| Keywords | Rhabdomyosarcoma / Dystrophin / Intron retention |
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| Outline of Annual Research Achievements |
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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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
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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| Strategy for Future Research Activity |
Based on the findings, protein analysis of dystrophin expression will be performed. Finally, the cell proliferation in the pressence of AO as a factor of growth and metastasis of RMS will be investigated.
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| Causes of Carryover |
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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[Journal Article] Intron-retained transcripts of the spinal muscular atrophy genes, SMN1 and SMN22018
Author(s)
Harahap NIF, Niba ETE, Ar Rochmah M, Wijaya YOS, Saito T, Saito K, Awano H, Morioka I, Iijima K, Lai PS, Motsuo M, Nishio H, Shinohara M
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Journal Title
Brain & Development
Volume: 40 (8)
Pages: 670-677
DOI
Peer Reviewed / Open Access / Int'l Joint Research
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[Journal Article] New, improved version of the mCOP-PCR screening system for detection of spinal muscular atrophy Gene (SMN1) deletion.2017
Author(s)
Shinohara M, Ar Rochmah M, Nakanishi K, Harahap NIF, Niba ETE, Saito T, Saito K, Takeuchi A, Bouike Y, Nishio H
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Journal Title
Kobe J Med Sci
Volume: 63(2)
Pages: E37-E40
Open Access
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[Journal Article] Genetic screening of spinal muscular atrophy using a real-time modified COP-PCR technique with dried blood-spot DNA2017
Author(s)
Ar Rochmah M, Harahap NIF, Niba ETE, Nakanishi K, Awano H, Morioka I, Iijima K, Saito T, Saito K, Lai PS, Takeshima Y, Takeuchi A, Bouike Y, Okamoto M, Nishio H, Shinohara M
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Journal Title
Brain Dev.
Volume: 39(9)
Pages: 774-782
DOI
Open Access / Int'l Joint Research
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[Journal Article] SMA diagnosis: Detection of SMN1 deletion with real-time mCOP-PCR system using fresh blood DNA2017
Author(s)
Niba ETE, Ar Rochmah M, Harahap NIF, Awano H, Morioka I, Iijima K, Saito T, Saito K, Takeuchi A, Lai PS, Bouike Y, Nishio H, Shinohara M.
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Journal Title
Kobe J Med Sci
Volume: 63(3)
Pages: E80-E83
Open Access
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[Journal Article] Gender effects on the clinical phenotype in Japanese patients with spinal muscular atrophy.2017
Author(s)
Ar Rochmah M, Shima A, Harahap NIF, Niba ETE, Morisada N, Yanagisawa S, Saito T, Kaneko K, Saito K, Morioka I, Iijima K, Lai PS, Bouike Y, Nishio H, Shinohara M
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Journal Title
Kobe J Med Sci
Volume: 63(2)
Pages: E41-E44.
Open Access
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