2023 Fiscal Year Annual Research Report
Development of transcription therapy for skin cell rejuvenation using design-on-demand artificial transcription factors
| Project/Area Number |
23H02607
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| Allocation Type | Single-year Grants |
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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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| 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 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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