Extracellular Vesicles Mediate Cross-Species Transgene Expression

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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.