2022 Fiscal Year Research-status Report
虚血性心筋を標的とするナノボディ修飾細胞外小胞を使用した心筋梗塞治療法の開発
| Project/Area Number |
22K20643
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| Research Institution | Shonan Kamakura General Hospital, Medical Corporation Tokushukai (Center for Clinical and |
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Principal Investigator |
ヤン ジュンジー 医療法人徳洲会湘南鎌倉総合病院(臨床研究センター), 湘南先端医学研究所 再生医療開発研究部, 主任研究員 (20962137)
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| Project Period (FY) |
2022-08-31 – 2024-03-31
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| Keywords | nanobody phage library / biopanning / hypoxia / cell engineering / ischemic heart targeted / mesenchymal stem cells / exosomes |
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| Outline of Annual Research Achievements |
We have generated a novel modular synthetic nanobody phage display library to select for targets including CD8, PD-1 and HER2. We also identified differential proteins of normoxic and hypoxic cardiomyocytes by liquid chromatography mass spectrometry. Among these differential proteins, CD29 and CD166 were identified to have most significant difference and also be involved in cell survival in hypoxic conditions. CD29 and CD166 will be chosen for further 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
CD29 and CD166 are biotinylated and immobilized on Maxisorb plate. Targeting binding, followed by phage binding, washing, elution and amplification are being performed to enrich phage library on CD29 or CD166. Next-generation sequencing will be performed to select most enriched phages.
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| Strategy for Future Research Activity |
Engineer mesenchymal stem cell-derived exosomes with targeting nanobodies and evaluate the therapeutic effects of modified exosomes for the treatment of myocardial infarction. Targeting nanobody will be inserted into an enriched protein, lysosome-associated membrane protein-2b (LAMP2b), on exosome surface by genetic modification. Through forced overexpression of targeting nanobody-LAMP2b in mesenchymal stem cells (MSCs), exosomes secreted by the cells will have targeting nanobody-LAMP2b on their surface. Targeting nanobody-LAMP2b-exosomes will be investigated in vivo by live imaging and multimodal imaging and evaluate the mechanisms and effectiveness for treating MI in mouse models through assessments of cell survival, neovascularization, infarct size, inflammation, and cardiac function.
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| Causes of Carryover |
In the previous fiscal year, the collaborator conducted generation and validation of nanobody phage display library, and identification of differential proteins of normoxic and hypoxic cardiomyocytes by liquid chromatography mass spectrometry. In this fiscal year, we will engineer mesenchymal stem cell-derived exosomes with targeting nanobodies and evaluate the therapeutic effects of modified exosomes for the treatment of myocardial infarction in a mouse model.
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