Extracellular Vesicles Mediate Cross-Species Transgene Expression

细胞外囊泡（EVs）本质上是所有生物体中几乎所有细胞类型释放的细胞片段，是一个不断发展的研究领域。这是由于观察到一部分电动汽车携带遗传物质，如RNA作为货物。越来越多的文献描述了它们在细胞间通信中的作用，通过将RNA（信使RNA，长链非编码RNA）从供体细胞转移到受体细胞。然而，没有确凿的证据表明这种基因重编程发生在受体细胞中，因为被表达的RNA可能与受体细胞中相同的RNA表达相混淆。很难区分哪些是由于RNA货物而表达的，哪些是由于细胞的正常过程而表达的。为了解决这个问题，我们在体外用小鼠/人ev和小鼠/人受体细胞进行了跨物种过继转移实验。我们的初步数据显示，在小鼠到人类的实验中，小鼠ev携带可以在人类受体细胞中翻译的mRNA。大部分mRNA为全长，保留了完整的编码序列。此外，转录物的长度并不重要，因为产生的蛋白质大小不同。我们后来确定，大多数这些蛋白具有核功能，这是由小鼠特异性蛋白的免疫组织化学证实。这是首次证明ev可以介导mRNA货物递送到受体细胞并导致翻译。假设：我们的假设是ev携带特定的mRNA亚群，目的是在受体细胞中翻译，翻译效率高。目的1)我们将继续对小鼠和人类细胞系及其释放的内皮细胞（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测序的体外实验。只要有小鼠与人配对，就可以评估ev +受体细胞的不同物种组合。我们将通过受体体内跨种蛋白的免疫组化来证实ev -转基因的表达(例如：（人类细胞中的小鼠蛋白）。目的2)我们将关注ev衍生的长链非编码RNA （lncRNA）及其对受体细胞表观遗传谱的影响。基于EV处理导致的表观遗传格局的改变，我们将分析可能负责的lncRNA转录本，并进行过表达实验，以概括lncRNA(s)的功能增益。目的3)我们开发了一种独特的体内模型系统来确定ev特异性转基因表达的功效。蛋白质组学和RNA测序将在治疗或模拟注射后的各种样品（细胞系异种移植，ev）中进行。这些实验将告诉我们，电动汽车是否从一个地方“发送”过来，并能在生物体的另一个遥远的地方对另一种细胞类型进行基因重组。

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.