间充质干细胞来源的外泌体作为新型ncRNA靶向载体治疗心梗的策略及机制研究

Myocardial infarction is a major cause of mortality and morbidity,and there is no effective treatment. Recently, the transplantation of exosomes, which function as cellular messengers, has emerged as a potential approach for the treatment of several devastating diseases. We found that exosomes derived from mesenchymal stem cells with β2-microglobulin knockout restore cardiac function in a myocardial infarction model, and the effects are mediated by non-coding RNA (ncRNA). Based on these findings, the applicant proposed an exosome imprinting theory, published in Nature Reviews Cardiology in 2017. In this grant application, we propose the following studies: 1) To eliminate the immunogenic imprint from parent cells using CRISPR technology in order to obtain exosomes enriched in ncRNAs with therapeutic potential; 2) To identify cardiac homing peptides using the phage display technique and express these peptides in exosomes via genetic engineering of stem cells; 3) To prolong exosome retention in infarcted hearts by encapsulating the exosomes in composite polypeptide nanofiber gel; and 4) To delineate the precise mechanisms by which exosome-derived ncRNAs exert their therapeutic effects, using small-interfering RNA-based gene silencing and lentivirus-based gene overexpression. The goal of this study is to provide both an experimental and a theoretical basis for stem cell-derived exosomes as novel ncRNA carriers targeting the heart and for the treatment of myocardial infarction.

心梗是人类致死致残的主要原因，无有效疗法。近期，细胞间信使外泌体移植治疗心梗备受研究者关注。我们发现敲除脐带间充质干细胞的免疫抗原B2M后，将其外泌体注射到心梗动物模型，能更有效地恢复心功能，且是由外泌体内ncRNA介导。基于此，申请人提出了“外泌体母系印记”理论，于2017年发表在Nature Reviews Cardiology杂志。研究内容：1)利用CRISPR技术清除母代细胞免疫排斥等印记，以期得到富含心梗治疗相关ncRNA的外泌体；2）用噬菌体展示技术体内筛选靶向心肌细胞的归巢肽，用基因工程技术改造干细胞，使其外泌体表达归巢肽，确保外泌体的心肌靶向性；3）用复合多肽纳米纤维胶包封外泌体，提高其在心梗区的驻留率；4）用siRNA、慢病毒过表达等多重策略，阐明外泌体中ncRNA治疗心梗的具体机制。本研究旨在为干细胞来源的外泌体作为新型ncRNA靶向载体治疗心梗提供实验数据和理论基础。

心血管疾病是人类致死致残的主要原因，无有效疗法，且相关机制不完善。本项目的主要研究内容：1）用circRNA/microRNA/mRNA测序、蛋白质测序、circRNA和蛋白质的共沉淀、蛋白质之间的免疫共沉淀、核质分离、siRNA抑制剂、慢病毒过表达系统、分子信息学、组织特异性敲除circRNA动物模型等多重策略， 筛选出富含与心血管疾病治疗相关的ncRNA的外泌体。2）利用CRISPR技术清除母代细胞免疫排斥等印记，使用具有心肌保护作用的纳米多肽材料包封间充质干细胞来源的外泌体(UMSC-Exo)，检测外泌体在心脏的驻留率。3）构建新型他莫昔芬诱导的心肌细胞特异性circHIPK3敲除小鼠，提供研究心肌损伤的新模型，并在体内、体外发掘UMSC-Exo中ncRNA在组织修复再生中的新机制。重要结果：1) 成功用 CRISPR/Cas9 技术敲除UMSC细胞中导致免疫排斥的HLA轻链B2M基因，发现即使在IFN-γ诱导条件下，也未观察到T细胞介导的免疫排斥。2）率先利用PGN复合多肽纳米水凝胶包封外泌体，确保UMSC-Exos的稳定性和持续释放，提高其在心梗区的驻留率。3）成功构建了心肌细胞特异性circHIPK3基因敲除小鼠，发现circHIPK3的缺失导致心肌细胞衰老加剧和心脏功能下降。circHIPK3作为支架增强了细胞质中E3泛素连接酶β-TrCP和HuR的结合，导致HuR的泛素化和降解，从而降低了衰老诱导因子p21活性。此外，UMSC-Exo通过递送circHIPK3发挥了心脏保护作用。4）揭示了新的机制：UMSC-Exo通过ncRNA/Rb1/AMPKα2/NLRP3信号通路抑制NLRP3炎症小体活化及焦亡，修复缺血组织损伤。科学意义及应用前景：通过 B2M 敲除调节外泌体的母系印记是防止 UMSCs 免疫排斥的有效策略， UMSC无需 HLA 匹配，有望临床应用。用复合多肽纳米纤维胶包封外泌体，提高其在心梗区的驻留率，为外泌体临床转化亟待解决的瓶颈问题提供了新方法。这些研究结果为心血管组织修复再生提供了新策略。发表SCI论文19篇，已获得3项授权专利。获得中华医学会医学科技奖一等奖、江苏省科学技术奖一等奖和苏州市优秀论文一等奖。