Investigating the transfer of miRNAs by exosomes during inflammation

摘要 哺乳动物microRNA是几种人类疾病的关键调节因子。因此，治疗 在患病组织中操纵miRNAs已经成为对抗人类免疫缺陷病毒的有希望的方法。 在某些情况下取得了成功。然而，有效地将miRNA或其抑制剂递送到许多人， 细胞和组织类型仍然是一个主要的挑战，这强调了改进方法的必要性 将小RNA传递到相关细胞类型。最近的报道表明，miRNA可以被释放， 来自一种叫做外泌体的小脂质囊泡中的细胞。反过来，外泌体可以将miRNA递送到受体细胞， 并且该系统被认为构成了细胞间通信的新形式。这个过程包括一个 内源性miRNA传递机制在很大程度上是未知的，但可以提供有价值的见解 如何改善治疗性miRNA的递送以及如何调节炎症反应。我们 初步数据表明小鼠骨髓来源的树突状细胞（BMDC）产生外泌体 含有特定的miRNAs，包括炎症调节因子miR-155和mIR-146 a， 递送至受体免疫细胞并随后在体外和体内介导靶敲低。这 导致体外和体内对内毒素的反应改变，这提供了实验证据， 外泌体转移的miRNA提供了一种新的炎症调节层。此外，我们还 能够成功地将特定的miRNA模拟物加载到外泌体中，并证明它们可以被递送到 受体细胞以功能相关的方式。根据这些发现，我们假设外泌体 miRNAs在生理相关的炎症反应中发挥着新的调节作用， 随着对这一过程的进一步理解，外泌体最终可以被选择来递送特定的miRNA。 鸡尾酒治疗相关的方式。我们将进行以下具体目标，以测试这些 预测。首先，我们将定义外泌体miRNA递送到免疫细胞的过程。接下来我们就 使用辐射嵌合体确定炎症期间外泌体miRNA的功能相关性， 具有受损的外泌体产生的Rab 27 a/B DKO小鼠。最后，我们将设计定制的外泌体， 它们对炎症性疾病的影响。总之，这个项目将揭示外泌体如何转移miRNAs 调节，确定该系统在生理相关炎症过程中的作用，并开始 理解这个新过程的转化潜力。

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.