Investigating the transfer of miRNAs by exosomes during inflammation

摘要 哺乳动物的microRNAs是几种人类疾病的关键调节因子。因此，具有治疗作用 在疾病组织中操纵miRNAs已成为对抗人类 疾病，并在一些环境中取得了成功。然而，将miRNAs或其抑制剂有效地传递给许多人 细胞和组织类型仍然是一个重大挑战，这突出了改进方法的必要性 将小RNA传递给相关类型的细胞。最近的报告表明，miRNAs可以被释放 来自被称为外体的小脂泡中的细胞。反过来，外切体可以将miRNAs运送到受体细胞， 这一系统被认为构成了一种新的细胞间通信形式。此过程包括一个 内源miRNA递送机制在很大程度上尚未确定，但可以提供有价值的见解 研究如何改进治疗性miRNA传递以及如何调节炎症反应。我们的 初步研究表明，小鼠骨髓来源的树突状细胞(BMDCs)可产生外切体 含有特定的miRNA，包括炎症调节剂miR-155和miR-146a，可以是 递送到受体免疫细胞，随后在体外和体内介导靶向击倒。这 结果在体外和体内对内毒素的反应都发生了变化，这提供了实验证据 外切体转移的miRNAs提供了一层新的调节炎症的层。此外，我们也一直在 能够成功地将特定的miRNA模拟物装载到外体中，并证明它们可以被输送到 以功能相关的方式接收细胞。作为这些发现的结果，我们假设外体 MiRNAs在生理上相关的炎症反应中发挥着新的调节作用，并且通过 如果对这一过程有更好的理解，外体最终可以被增选来传递特定的miRNA 鸡尾酒在治疗方面的作用。我们将执行以下具体目标来测试这些 预测。首先，我们将定义外体miRNA递送到免疫细胞的过程。接下来，我们将 用放射嵌合体和放射免疫技术确定外体miRNAs在炎症过程中的功能相关性 Rab27a/b DKO小鼠外切小体产生受损。最后，我们将设计定制的外显子并测试 它们对炎症性疾病的影响。综上所述，该项目将阐明外体如何转移miRNAs 是调节的，确定这个系统在生理相关炎症中的作用，并开始 理解这一新过程的翻译潜力。

Abstract Mammalian microRNAs are critical regulators of several human diseases. As a result, therapeutic manipulation of miRNAs in diseased tissues has emerged as a promising approach to combating human disorders, and has had success in some settings. Yet, effective delivery of miRNAs or their inhibitors to many cell and tissue types continues to be a major challenge, and this underscores the need for improved methods of small RNA delivery to relevant cell types. Recent reports have demonstrated that miRNAs can be released from cells in small lipid vesicles called exosomes. In turn, the exosomes can deliver miRNAs to recipient cells, and this system is thought to constitute a novel form of intercellular communication. This process includes an endogenous miRNA delivery mechanism that is largely uncharacterized, yet could provide valuable insights into how therapeutic miRNA delivery can be improved and how inflammatory responses are regulated. Our preliminary data demonstrate that mouse bone marrow derived dendritic cells (BMDCs) produce exosomes that contain specific miRNAs, including the inflammatory regulators miR-155 and mIR-146a, that can be delivered to recipient immune cells and subsequently mediate target knockdown both in vitro and in vivo. This results in an altered response to endotoxin both in vitro and in vivo, which provides experimental evidence that exosome-transferred miRNAs provide a novel layer of regulating inflammation. Further, we have also been able to successfully load specific miRNA mimics into exosomes and demonstrate that they can be delivered to recipient cells in a functionally relevant manner. As a result of these findings, we hypothesize that exosomal miRNAs play novel regulatory roles during physiologically relevant inflammatory responses, and that through an improved understanding of this process, exosomes can ultimately be coopted to deliver specific miRNA cocktails in a therapeutically relevant manner. We will carry out the following specific aims to test these predictions. First, we will define the process of exosomal miRNA delivery to immune cells. Next, we will determine the functional relevance of exosomal miRNAs during inflammation using radiation chimeras and Rab27a/b DKO mice with impaired exosome production. Finally, we will engineer custom exosomes and test their impact on inflammatory disease. Together, this project will shed light on how exosome transfer of miRNAs is regulated, determine the role of this system during physiologically relevant inflammation, and begin to understand the translational potential of this novel process.