2023 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

Research Project

Project/Area Number	22K20643
Research Institution	Shonan Kamakura General Hospital, Medical Corporation Tokushukai (Center for Clinical and
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)
Project Period (FY)	2022-08-31 – 2025-03-31
Keywords	exosomes / nanobody phage library / ischemic heart disease / targeted delivery / phage screening
Outline of Annual Research Achievements	Cardiomyocytes were treated with 1% O2, 5% CO2, 94% N2 for 48 hours. Membrane proteins of normoxic and hypoxic cardiomyocytes were isolated by Mem-PER Plus Membrane Protein Extraction Kit and then visualized on stain-free gel. Differential bands on stain-free gel were cut out and proceeded with Mass Spectrometry analysis. Totally 449 proteins were identified, and differential proteins were sorted out by fold change (>1.5) and P value (<0.05). Combined with protein functions under hypoxia, CD29 and CD166 were selected as candidate proteins for the further experiments. CD29 and CD166 were first biotinylated and then biotinylated CD29 and CD166 were immobilized on Maxisorb plate. After 2 rounds of target binding, phage binding, washing, elution and amplification, enriched phages were collected and processed for Next Generation Sequencing.
Current Status of Research Progress	Current Status of Research Progress 2: Research has progressed on the whole more than it was originally planned. Reason We are working on the bioinformatic analysis of Next Generation Sequencing data.
Strategy for Future Research Activity	Next Generation Sequencing will be analyzed by bioinformatics. Peptide sequences with high affinity and binding capacity to CD29 or CD166 will be selected and testified by in vitro and in vivo experiments. Targeting peptide sequence will be inserted into an enriched protein, lysosome-associated membrane protein-2b (LAMP2b), on EV surface by genetic modification. I will investigate the biodistribution of targeting peptide-LAMP2b-EVs 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.
Causes of Carryover	Next Generation Sequencing will be analyzed by bioinformatics. Peptide sequences with high affinity and binding capacity to CD29 or CD166 will be selected and testified by in vitro and in vivo experiments. Research fund will be used for the bioinformatic analysis and in vitro and in vivo validation.