2020 Fiscal Year Annual Research Report
心筋症関連ミトコンドリア・核内遺伝子の協奏的制御を可能にする人工転写因子の開発
| Project/Area Number |
19H03349
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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, 理学研究科, 教授 (50183843)
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| Project Period (FY) |
2019-04-01 – 2022-03-31
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| Keywords | ピロールイミダゾールポリアミド / エピジェネティクス / 人工転写因子 / ナノテクノロジー / 心筋症 |
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| Outline of Annual Research Achievements |
This fiscal year, we have productively developed different versions of SMART-TFs capable of switching ON and OFF the therapeutically important and cell fate regulating nuclear and mitochondrial genes. We have developed a biomimetic epigenetic code termed En-PGC-1 to induce targeted epigenetic activation of peroxisome proliferator-activated receptor-gamma coactivator 1 alpha/beta, a regulator of mitochondrial biogenesis and AMP-activated protein kinase pathway. En-PGC-1 enhanced mitochondrial activation, energy metabolism, proliferation of CD8+ T cells in vitro, and enhanced oxidative phosphorylation thereby synergizing PD-1 blockade immunotherapy in a mouse model. We developed and published an epigenetic code capable of activating cardiac-related genes in mouse embryonic stem cells and differentiate them into spontaneous beating cardiomyocytes. Microarray and chromatin immunoprecipitation sequencing substantiated the targeted epigenetic modulation as the reason behind the mechanism of the pre-version of these nuclear ON SMART-TFs. Regarding the OFF SMART-TFs, we have also published that incorporating tri-arginine moiety can enhance the nuclear accumulation of our molecular codes and trigger the efficient repression of genes in live cells. We have also advanced the use of our biomolecular codes as probes and have successfully visualized telomere length and dynamics in live cells. We also explored the natural products to neutralize reactive oxygen species and have published a preliminary analysis of differentially expressed genes in mouse Leydig cells.
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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
Encouraged with the strong foundation laid by integrating diverse disciplines including machine learning in FY2019, this fiscal year we accelerated our research and promptly achieved the milestones mentioned in the application to perform biological evaluation of SMART-TF in regulating the AMP-activated protein kinase (AMPK) pathway. Furthermore, we have successfully accomplished a milestone of the work package originally planned for FY2021 regarding cardiomyogenesis in advance and is exploring new applications in immunotherapy. In particular, while constructing mitochondrial SMART-TF, we made a serendipitous discovery that the incorporation of tri-arginine moiety can remarkably enhance the nuclear accumulation of our molecular codes. Considering the correlation between telomere length and mitochondrial DNA copy number, we have also explored a new direction and constructed a near infra-red fluorogenic probe capable of visualizing telomere length and dynamics in live cells. These two important major leaps aided us to effectively program our SMART-TF components as probes and/or gene regulators on-demand and broaden their applications in the uncharted domains. We have also attained the results by using nanoparticle version of our SMART-TFs with potential application in neurodegenerative diseases and have used machine learning programs to advance our biomolecular codes. While exchange visits were not possible due to the pandemic, the online meetings and webinars facilitated us to expand our network and further foster international collaborations.
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| Strategy for Future Research Activity |
The project's current status offers us an ideal platform to advance our chemical biology approach to create potential therapeutics and diagnostics. We plan to functionalize our molecular codes and validate their biological efficacy in different cell lines and disease models to realize this aim. Reactive oxygen species (ROS) is a common phenomenon occurring in several diseases, and so we can extend our synthetic strategy to regulate them and the therapeutically important genes associated with the intractable diseases. We plan to develop a chemical probe-based nanopore DNA sequencing protocol to evaluate 8-Oxoguanine, one of the most common DNA lesions resulting from ROS. We will also supplement the nanomaterials lignin in the cell culture setup to have an external control over ROS and demonstrate the cooperative control in wide range of cell lines including cardiomyopathy cell lines. To further promote our biomimetic epigenetic codes targeting the right place at the genome, we plan to administer them at the right time by deciphering the circadian gene expression of the target genes using the machine learning algorithms. Because mitochondria and bacteria have common features like circular DNA, we are also fine-tuning our SMART-TFs to trigger promoter-specific transcription suppression on demand. We plan to make good use of our international collaborators and accelerate our ongoing research topics. We will summarize the research results and publish them in high impact research journals, which is expected to be frequently cited
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