| Project/Area Number |
23K27298
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| Project/Area Number (Other) |
23H02607 (2023)
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Multi-year Fund (2024)
Single-year Grants (2023) |
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| Section | 一般 |
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| Review Section |
Basic Section 47010:Pharmaceutical chemistry and drug development sciences-related
Basic Section 47050:Environmental and natural pharmaceutical resources-related
Sections That Are Subject to Joint Review: Basic Section47010:Pharmaceutical chemistry and drug development sciences-related , Basic Section47050:Environmental and natural pharmaceutical resources-related
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| Research Institution | Kyoto University |
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Principal Investigator |
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| Co-Investigator(Kenkyū-buntansha) |
杉山 弘 京都大学, 高等研究院, 研究員 (50183843)
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| Project Period (FY) |
2023-04-01 – 2026-03-31
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| Project Status |
Granted (Fiscal Year 2024)
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| Budget Amount *help |
¥18,460,000 (Direct Cost: ¥14,200,000、Indirect Cost: ¥4,260,000)
Fiscal Year 2025: ¥6,110,000 (Direct Cost: ¥4,700,000、Indirect Cost: ¥1,410,000)
Fiscal Year 2024: ¥5,980,000 (Direct Cost: ¥4,600,000、Indirect Cost: ¥1,380,000)
Fiscal Year 2023: ¥6,370,000 (Direct Cost: ¥4,900,000、Indirect Cost: ¥1,470,000)
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| Keywords | ミトコンドリア / 転写療法 / 活性酸素種 / 皮膚細胞の若返り / エピジェネティックコード |
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| Outline of Research at the Start |
細胞レベルでは加齢による皮膚の老化に伴って、ミトコンドリアのレドックス恒常性が低下し、遺伝子転写機構の変化・エピゲノムの乱れ・活性酸素種 (ROS) の過剰な産生が行われる。既存の生物活性化合物により、核やミトコンドリアによるROSを調節する複雑な遺伝子転写機構の分子成分を個別に標的とすることは可能である。しかし、複数の標的をオンデマンドで協調的に調節するプログラム可能な合成ツールは存在していない。本研究では、皮膚細胞の若返りに関与する複雑な核およびミトコンドリア遺伝子転写機構の協調的な調節を可能にするナノ粒子ベースの人工転写因子 (PRO-TF) を構築し、評価することを目標とする。
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| Outline of Annual Research Achievements |
During this fiscal year, we conducted proof-of-concept studies to verify the primary purpose of our research, which is to create a transcription therapy approach using programmable transcription factors (PRO-TFs). We screened and characterized various nanoparticles and identified biomass-derived carbon dots as the optimal platform to construct the PRO-TFs. Furthermore, we have successfully synthesized and verified a designer pyrrole imidazole polyamides (PIP) and an epigenetic modulator capable of causing promoter-specific transcription suppression and activation of genes described in the proposal, including human interleukin-6 (IL-6) on-demand. We have verified the biocompatibility of the PRO-TF components and performed confocal microscopy studies to optimize their maximum localization using differentially aged human dermal fibroblasts, artificially inflamed chondrocytes and in vivo medaka fish model. Furthermore, we have extended our transcription-targeting tools to sense messenger RNA in live cells. We have summarized part of these results in original research articles and review papers and presented the results at various conferences, including the World Immune Regulation meeting.
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| Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
Over the course of this year, we have successfully achieved all the proposed milestones in sequence and conducted screening studies to identify the bioactive components require to create programmable transcription factors (PRO-TFs), which is expected to simultaneously regulate mitochondrial and nuclear genes associated with cellular rejuvenation. We have successful isolated biomass-derived quantum dots and demonstrated their ability to selectively target the mitochondria and differentially accumulate in the healthy and senescence-induced human dermal fibroblasts. Furthermore, we have developed a slightly different version of PRO-TFs by assembling oligonucleotide strands onto silica nanoparticles and verified their efficacy to simultaneously detect and quench interleukin-6 messenger RNA. Furthermore, we optimized the analytical and sequencing methodology required to assess PRO-TF`s functionality. Through the JSPS exchange program, Dr. Petta Dalila from the switzerland visited us for three months and introduced self-renewable scaffold model in microfluidic platform. In exchange, Dr. Namasivayam visited and gave an invited lecture at the World Immune Regulation meeting to extend networking. Also, our team member Mahima Kumar visited University of Zurich for two weeks to accelerate the collaboration and cover more new research areas this year.
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| Strategy for Future Research Activity |
Encouraged by the results obtained in the last fiscal year, we verify the bioefficacy of the PRO-TFs to achieve stepwise control of mitochondrial (MITO-PRO-TF) and nuclear (Nu-PRO-TF) genes in 97-year-old human dermal fibroblasts and in H2O2-induced senescent skin cells. After optimization studies, the quantification will be performed by measuring changes in transcript levels using PCR. We also plan to construct a cytoplasmic hybrid (cybrid) model containing mtDNA mutations to assess the effect on reversing mtDNA heteroplasmy.
We plan to perform transcriptome analysis using microarray and obtain indicators for improving Nu- and Mito-PRO-TF through pathway analysis. Next-generation sequencing analysis (Bind-n-Seq and Chem-Seq) will be performed using biotin-labeled PRO-TF to identify signal motifs that control signal pathways and the expression of target genes. We will use telomere targeting PIP to decode telomere length in young and old human dermal fibroblasts.
The effect of PRO-TF on mitochondrial function will be evaluated by measuring the parameters such as mitochondrial mass (fluorescent MitoTracker dye), NAD/NADH ratio (colorimetric kit), and mitochondrial DNA (quantitative PCR).
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