Exosome-mediated cardioprotection and regeneration

描述（由申请人提供）：在心肌梗死（MI）后患者中，球囊衍生细胞（CDC）可有效减少瘢痕大小和再生存活心肌，并在MI动物模型中发挥急性心脏保护作用。CDC的主要益处似乎是通过间接机制介导的。在这里，我们测试的首要假设，CDC的间接影响是由外来体介导的。外泌体由广泛的细胞类型分泌，是30-100 nm的脂质双层囊泡，其富含RNA，包括microRNA（miR）。我们的初步数据表明，在小鼠急性MI模型中，由人CDC分泌的外泌体再现了CDC的治疗益处，并且对于CDC的治疗益处是不可或缺的。将CDC-外来体注射到受损的心脏中模拟CDC移植的结构和功能益处;相反，CDC对外来体分泌的抑制消除了移植的CDC的治疗益处。并非所有的外来体都是有益的：注射来自真皮成纤维细胞的外泌体，对照细胞不改善MI后结果，没有治疗益处。CDC-外泌体减少急性心肌细胞死亡和炎性细胞因子释放，同时减轻MI后心脏的LV重塑和纤维化。来自微小RNA阵列的初步数据揭示了在CDC-外泌体中高度上调的几种“签名miR”。相比之下，质谱表明CDC-外泌体的蛋白质组成是常规的并且与成纤维细胞-外泌体的蛋白质组成相当。因此，我们假设：i）CDC-外来体含有独特的miR补充物，其共同介导CDC的许多或全部治疗效果; ii）CDC-外来体及其组成miR有利地调节MI后心脏中的细胞凋亡、炎症和纤维化; iii）CDC-外来体改善MI后的功能恢复并增加组织活力; iv）CDC-外来体代表心脏修复的可行的“无细胞”治疗候选物。在这里，我们建议对CDC-外泌体的全部RNA内容物进行测序，以确定CDC-外泌体的治疗益处的分子基础，并优化体内递送策略。我们的最终目标是开发CDC-外泌体作为治疗MI和HF的生物产品。由于外泌体与可以主动向靶标迁移的活细胞不同，我们还试图测试新的递送方法，以将CDC-外泌体选择性地靶向损伤的肌细胞。该提案既以假设为驱动，又以产品为导向。我们寻求详细回答机制和转化问题，依靠最先进的科学方法和完善的临床前疾病模型。CDC（自体和同种异体）已经在人体试验中，但细胞作为治疗剂有其局限性。因此，我们的建议，专注于CDC外泌体（作为CDC的无细胞衍生物），具有潜在的重要翻译价值。这些结果也将为急性心肌梗死后细胞介导的心脏保护和心脏再生的基本机制提供新的见解。

DESCRIPTION (provided by applicant): Cardiosphere-derived cells (CDCs) are effective in reducing scar size and regenerating viable myocardium in patients after myocardial infarction (MI), and exert acute cardioprotective effects in animal models of MI. The major benefits of CDCs appear to be mediated by indirect mechanisms. Here we test the overarching hypothesis that the indirect effects of CDCs are mediated by exosomes. Secreted by a wide range of cell types, exosomes are 30-100 nm lipid bilayer vesicles that are enriched in RNAs including microRNAs (miRs). Our preliminary data indicate that exosomes secreted by human CDCs reproduce the therapeutic benefits of CDCs, and are indispensable for the therapeutic benefits of CDCs, in a mouse acute MI model. Injection of CDC-exosomes into the injured heart mimics the structural and functional benefits of CDC transplantation; conversely, inhibition of exosome secretion by CDCs abrogates the therapeutic benefits of transplanted CDCs. Not all exosomes are salutary: Injection of exosomes from dermal fibroblasts, control cells which do not improve post-MI outcomes, had no therapeutic benefit. CDC-exosomes decreased acute cardiomyocyte death and inflammatory cytokine release, while attenuating LV remodeling and fibrosis in the post-MI heart. Preliminary data from microRNA arrays reveal several "signature miRs" that are highly up-regulated in CDC- exosomes. In contrast, mass spectrometry indicates that the protein composition of CDC-exosomes is conventional and comparable to that of fibroblast-exosomes. Thus, we hypothesize that: i) CDC-exosomes contain a unique complement of miRs that, collectively, mediate many, or all, of the therapeutic effects of CDCs; ii) CDC-exosomes and their constituent miRs favorably modulate apoptosis, inflammation and fibrosis in the post-MI heart; iii) CDC-exosomes improve functional recovery and increase tissue viability post-MI; iv) CDC-exosomes represent a viable "cell free" therapeutic candidate for cardiac repair. Here we propose to sequence the full RNA content of CDC-exosomes, to determine the molecular basis underlying the therapeutic benefit of CDC-exosomes, and to optimize delivery strategies in vivo. Our ultimate goal is to develop CDC-exosomes as a biologic product to treat MI and HF. Because exosomes are unlike live cells that can migrate actively towards a target, we also seek to test novel delivery methods to target CDC-exosomes selectively to injured myocytes. The proposal is both hypothesis-driven and product-oriented. We seek to answer both mechanistic and translational questions in detail, relying upon state-of-the-art scientific methods and well-established preclinical disease models. CDCs (both autologous and allogeneic) are already in human trials, but cells have their limitations as therapeutic agents. Thus, our proposal, focusing on CDC-exosomes (as cell-free derivatives of CDCs), is of potentially significant translational value. The results will also open up novel insights into the fundamental mechanisms of cell-mediated cardioprotection and cardiac regeneration after acute MI.