In Vitro and in Vivo RNA Inhibition by CD9-HuR Functionalized Exosomes Encapsulated with miRNA or CRISPR/dCas9
In Vitro and in Vivo RNA Inhibition by CD9-HuR Functionalized Exosomes Encapsulated with miRNA or CRISPR/dCas9
复制标题
封装有 miRNA 或 CRISPR/dCas9 的 CD9-HuR 功能化外泌体的体外和体内 RNA 抑制
DOI：
10.1021/acs.nanolett.8b02689
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发表时间：
2019-01-01
期刊：
影响因子：
10.8
通讯作者：
Yuan, Lijun
中科院分区：
文献类型：
--
作者：
Li, Zhelong;Zhou, Xueying;Yuan, Lijun
体外和体内递送感兴趣的RNA为基因治疗带来了希望。近年来，外泌体被认为是一种理想的RNA载体，尤其是miRNA和/或siRNA，但其装载效率有限。在这项研究中，我们通过构建融合蛋白来设计外泌体以装载RNA，其中外泌体膜蛋白CD 9与RNA结合蛋白融合，而感兴趣的RNA要么天然携带，要么经过设计以具有结合元件。通过原理验证实验，我们在这里将CD 9与HuR融合，HuR是一种与miR-155以相对高的亲和力相互作用的RNA结合蛋白。在外泌体包装细胞中，当miR-155过量表达时，融合的CD 9-HuR成功地将miR-155富集到外泌体中。此外，包裹在外泌体中的miR-155反过来可以有效地递送到受体细胞中并识别内源性靶标。此外，我们还揭示了当RNA被工程化以具有富含Au的元件时，CD 9-HuR外泌体可以富集功能性miRNA抑制剂或CRISPR/dCas 9。总之，我们在这里建立了一种新的策略，用于增强RNA货物封装到工程化的外泌体中，而外泌体又在受体细胞中发挥作用。
In vitro and in vivo delivery of RNAs of interest holds promise for gene therapy. Recently, exosomes are considered as a kind of rational vehicle for RNA delivery, especially miRNA and/or siRNA, while the loading efficiency is limited. In this study, we engineered the exosomes for RNA loading by constructing a fusion protein in which the exosomal membrane protein CD9 was fused with RNA binding protein, while the RNA of interest either natively harbors or is engineered to have the elements for the binding. By proof-of-principle experiments, we here fused CD9 with HuR, an RNA binding protein interacting with miR-155 with a relatively high affinity. In the exosome packaging cells, the fused CD9-HuR successfully enriched miR-155 into exosomes when miR-155 was excessively expressed. Moreover, miR-155 encapsulated in the exosomes in turn could be efficiently delivered into the recipient cells and recognized the endogenous targets. In addition, we also revealed that the CD9-HuR exosomes could enrich the functional miRNA inhibitor or CRISPR/dCas9 when the RNAs were engineered to have the AU rich elements. Taken together, we here have established a novel strategy for enhanced RNA cargo encapsulation into engineered exosomes, which in turn functions in the recipient cells.