Extracellular Vesicles Mediate Cross-Species Transgene Expression

背景：细胞外小泡本质上是所有生物体中几乎每种细胞类型都会释放的细胞碎片，是一个日益增长的研究领域。这是因为观察到，一定比例的电动汽车将RNA等遗传物质作为货物运输。越来越多的文献描述了它们通过将RNA(信使RNA，长非编码RNA)从供体细胞转移到受体细胞而在细胞间通信中所起的作用。然而，目前还没有确凿的证据表明这种基因重新编程发生在受体细胞中，因为表达的RNA可能被混淆为在受体细胞中表达相同的RNA。很难区分什么是由于RNA货物表达的，什么是由于细胞的正常过程而表达的。为了解决这一问题，我们在体外用鼠/人EVS和鼠/人受体细胞进行了跨物种过继移植实验。我们的初步数据显示，在小鼠到人类的实验中，小鼠电动汽车携带的mRNA可以在人类受体细胞中翻译。大多数的mRNA是全长的，并保留了完整的编码序列。此外，转录本的长度并不重要，因为同时产生了小蛋白和大蛋白。我们后来确定这些蛋白质中的大多数具有核功能，这一点通过小鼠特定蛋白质的免疫组织化学得到证实。这是第一个证据表明，EVS可以介导mRNA货物运送到受体细胞，从而导致翻译。假设：我们的假设是EVS携带特定的mRNA亚群，目的是在受体细胞中以高翻译效率进行翻译。方法：目的1)我们将继续进行体外实验，对小鼠和人细胞系及其释放的EV(小鼠胚胎成纤维细胞/黑色素瘤和人内皮细胞/前列腺上皮细胞)进行蛋白质组学和RNA测序。只要存在鼠与人的配对，就会评估EVS+受体细胞的不同物种组合。我们将通过免疫组织化学方法证实EV转基因在受体体内的表达(例如，人类细胞中的小鼠蛋白)。目的2)我们将重点研究EV衍生的长非编码RNA(LncRNA)及其对受体细胞表观遗传学的影响。基于EV处理引起的表观遗传格局的变化，我们将分析可能负责的lncRNA转录本，并进行过度表达实验，以概括因lncRNA而获得的功能(S)。目的3)我们建立了一种独特的体内模型系统，以确定EV特异性转基因表达的有效性。蛋白质组学和RNA测序将在治疗或模拟注射后在各种样本(细胞系异种移植，EVS)中进行。这些实验将告诉我们，EV是否是从一个位置“送来”的，以及是否可以在生物的远程位置对另一种类型的细胞进行基因重新编程。

Background: Extracellular vesicles (EVs) are essentially cell fragments released by almost every cell type in all organisms and are a growing area of research. This is due to the observation that a proportion of EVs carry genetic material such as RNA as cargo. A growing body of literature describes their role in cell-to-cell communication by transferring RNA (messenger RNA, long noncoding RNA) from donor cells to recipient cells. However, there has been no conclusive evidence that such genetic reprogramming occurs in recipient cells because the RNA being expressed could be confused for the same expression of RNA in the recipient cells. It is difficult to distinguish what is expressed due to the RNA cargo versus what is expressed due to the cell's normal processes. To address this, we have performed cross-species adoptive transfer experiments with mouse/human EVs and mouse/human recipient cells in vitro. Our preliminary data has revealed that in mouse to human experiments, mouse EVs carry mRNA that can be translated in human recipient cells. The majority of mRNA was full length and retained the entire coding sequence. Moreover, the length of the transcript did not matter, as both small and large protein sizes were generated. We later determined that most of these proteins had a nuclear function and this was confirmed by immunohistochemistry for mouse specific proteins. This is the first proof that EVs can mediate mRNA cargo delivery to recipient cells to result in translation. Hypothesis: Our hypothesis is that EVs carry specific subsets of mRNA destined for translation in recipient cells with high translation efficiency. Methods: Objective 1) We will continue in vitro experiments in which proteomics and RNA sequencing is performed on mouse and human cell lines and the EVs that they release (mouse embryonic fibroblasts/melanoma and human endothelial cells/prostatic epithelial cells). Different species combinations of EVs+recipient cells will be evaluated as long as there is a mouse to human pairing. We will confirm expression of EV-transgenes via immunohistochemistry of cross-species protein in the recipient (eg., mouse protein in human cells). Objective 2) We will focus on EV-derived long noncoding RNA (lncRNA) and its impact on the recipient cell's epigenetic profile. Based on the alterations in the epigenetic landscape due to EV treatments, we will then analyze the lncRNA transcripts potentially responsible and perform overexpression experiments to recapitulate gain of function due to the lncRNA(s). Objective 3) We have developed a unique in vivo model system to determine the efficacy of EV-specific transgene expression. Proteomics and RNA sequencing will be performed in various samples (cell line xenograft, EVs) after treatment or mock injection. These experiments will tell us if EVs are being "sent" from one site and can genetically reprogram another cell type at a distant site of the organism.