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Exosome‐mediated small RNA delivery for gene therapy

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Small RNAs, including small interfering RNAs (siRNA) and microRNAs (miRNA), are emerging as promising therapeutic drugs against a wide array of diseases. The key obstacle for the successful clinical application of small RNAs is to develop a safe delivery system directed at the target tissues only. Current small RNA transfer techniques use viruses or synthetic agents as delivery vehicles. The replacement of these delivery vehicles with a low toxicity and high target‐specific approach is essential for making small RNA therapy feasible. Because exosomes have the intrinsic ability to traverse biological barriers and to naturally transport functional small RNAs between cells, they represent a novel and exciting delivery vehicle for the field of small RNA therapy. As therapeutic delivery agents, exosomes will potentially be better tolerated by the immune system because they are natural nanocarriers derived from endogenous cells. Furthermore, exosomes derived from genetically engineered cells can deliver small RNAs to target tissues and cells. Thus, exosome‐based delivery of small RNAs may provide an untapped, effective delivery strategy to overcome impediments such as inefficiency, nonspecificity, and immunogenic reactions. In this review, we briefly describe how exosomal small RNAs function in recipient cells. Furthermore, we provide an update and overview of new findings that reveal the potential applications of exosome‐based small RNA delivery as therapeutics in clinical settings. WIREs RNA 2016, 7:758–771. doi: 10.1002/wrna.1363 This article is categorized under: RNA Export and Localization > RNA Localization Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA in Disease and Development > RNA in Disease
Strategy to endow exosomes with the potential for targeted small RNA delivery. The efficacy of exosome‐based small RNA delivery can be improved by incorporating selected targeting peptides on the outer membrane surface of exosomes. Because targeting peptides exhibit superior binding and entry specificity, the modified exosomes are destined to engage specific receptors on target cells. Then, exosomes are taken up by target cells and release their small RNA cargo into the cytoplasm where the small RNA can block mRNA translation into protein.
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Schematic diagram of small RNA transfer by exosomes. Cytosolic small RNAs (siRNAs and miRNAs) are incorporated into the intraluminal vesicles of MVBs during the invagination of the endosomal membrane. When the MVB fuses with the plasma membrane, exosomal small RNA is released into the circulation simultaneously with the release of exosomes. Subsequently, exosomes reach their destinations, a process usually determined by the binding of specific ligands on their surfaces, and then they enter target cells through multiple pathways: phagocytosis, membrane fusion, caveolin‐mediated endocytosis, clathrin‐mediated endocytosis, lipid raft‐mediated endocytosis, macropinocytosis, etc. All pathways result in the delivery of the exosomal small RNA to the cytosol of the target cell, where it may associate with and silence corresponding mRNA.
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Biogenesis of exosomes. The plasma membrane buds inward, forming a membrane‐bound vacuole termed the early endosome. The early endosome then undergoes several changes as it matures to a late endosome. Subsequently, the endosomal membrane buds inward and pinches off to form membrane‐bound vesicles inside the endosome, which is now called a multivesicular body (MVB). This process is mediated by ESCRT complex and other molecules. The MVB may travel to the lysosome and degrade its contents, and this process is believed to be important for ‘cell trash removal.’ Alternatively, the presence of high concentrations of the ceramide lipid family helps the MVB contents escape lysosomal digestion and selectively package protein, small RNAs, and lipids as cargo. This process is regulated by several molecules (e.g., nsMase2 and hnRNPA2B1) but the detailed mechanisms are still largely unknown. The MVBs then travel along the cytoskeleton and fuse with the plasma membrane under the regulation of molecular motor and small GTPase Rab protein. Finally, MVBs bud and release its contents, which are called exosomes once they exist outside the cell.
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Biogenesis of siRNA and miRNA. For siRNA processing, double‐stranded RNA (dsRNA) is processed into siRNA between 19 and 26 base pairs in length by the RNase III enzyme Dicer. After the siRNA unwinds, one of the strands is degraded, and the other strand is incorporated into RISC and guides the complex to recognize messenger RNA (mRNA) sequences with perfect or nearly perfect complementarity, resulting in cleavage and degradation of the target, thereby interrupting protein synthesis of the target gene. For miRNA processing, the miRNA is synthesized by RNA polymerase II as long primary miRNA (pri‐miRNA) transcripts. Pri‐miRNA is then cleaved by Drosha into ~70 nucleotide hairpin known as miRNA precursor (pre‐miRNA) in the nucleus. Pre‐miRNA is then transported from the nucleus into the cytoplasm by exportin‐5. Once in the cytoplasm, pre‐miRNA is cut by Dicer into a 22‐nucleotide duplex. Next, one strand is incorporated into RISC while the other strand is degraded. Mature miRNA binds to target mRNAs by partial base‐pairing and silences gene expression either by promoting mRNA degradation or by attenuating protein translation.
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RNA in Disease and Development > RNA in Disease
Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs
RNA Export and Localization > RNA Localization

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