Extracellular Vesicles Mediate Cross-Species Transgene Expression

细胞外囊泡介导跨物种转基因表达

• 批准号: RGPIN-2020-05887
• 负责人: Leong, Hon
• 金额: $ 2.62万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=760723
• 关键词: Extracellular; Vesicles; Mediate; Cross; Species

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.

背景资料：细胞外囊泡（EV）基本上是所有生物体中几乎每种细胞类型释放的细胞碎片，并且是一个不断增长的研究领域。这是由于观察到一部分EV携带遗传物质，如RNA作为货物。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讯中的作用。然而，没有确凿的证据表明这种遗传重编程发生在受体细胞中，因为表达的RNA可能与受体细胞中相同的RNA表达混淆。很难区分什么是由于RNA货物表达与什么是由于细胞的正常过程表达。为了解决这个问题，我们已经在体外用小鼠/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是全长的，并保留了整个编码序列。此外，转录本的长度并不重要，因为产生了小和大的蛋白质大小。我们后来确定这些蛋白质中的大多数具有核功能，并且通过小鼠特异性蛋白质的免疫组织化学证实了这一点。这是EV可以介导mRNA货物递送到受体细胞以导致翻译的第一个证据。假设：我们的假设是，EV携带特定的mRNA亚群，注定在受体细胞中以高翻译效率翻译。研究方法：目的1）我们将继续进行体外实验，其中对小鼠和人类细胞系及其释放的EV（小鼠胚胎成纤维细胞/黑色素瘤和人类内皮细胞/前列腺上皮细胞）进行蛋白质组学和RNA测序。只要存在小鼠与人配对，就将评价EV+受体细胞的不同种属组合。我们将通过受体中跨物种蛋白的免疫组织化学来确认EV转基因的表达（例如，人细胞中的小鼠蛋白）。目的2）研究EV来源的长链非编码RNA（lncRNA）及其对受体细胞表观遗传学的影响。基于EV治疗引起的表观遗传景观的改变，我们将分析可能负责的lncRNA转录本，并进行过表达实验以重现由于lncRNA而获得的功能。目的3）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功效。将在处理或模拟注射后的各种样品（细胞系异种移植物，EV）中进行蛋白质组学和RNA测序。这些实验将告诉我们，电动汽车是否是从一个地点“发送”的，并且可以在生物体的远处对另一种细胞类型进行基因重编程。