Design Of Exosomes Carrying RNA Using L7Ae Proteins

Exosomes,a kind of extracellular vesicle that is approximately 40-200 nm in diameter,have good biocompatibility,circulation stability,biological barrier permeability,low toxicity,and low immunogenicity.These natural advantages make it an ideal drug delivery system for RNA and small molecules.Electroporation,chemical transfection,and incubation are the met hods commonly used in loading exosomes with RNA in exogenous method.However,there are some problems with these methods,such as poor quality of exosomes,restriction in electroporation condition,and low transfection efficiency.Due to the complex mechanism of exosomes entry for drugs and the uncertainty of the exosomes' loading efficiency,there are few studies in the area of loading exosomes with RNA in endogenous method.Here,we attempted to explore a new method for efficient RNA incorporation into exosomes by utilizing the characteristic of specific binding between L7 Ae and the K-turn structure in the gene.First,recombinant plasmid that expresses CD63-L7 Ae fusion protein in HEK293 T cells was constructed.Then the recombinant plasmid(p CMV-CD63-L7Ae)was transfected HEK293 T cells,and the exosomes secreted by the HEK293 T cells were extracted and identified by Western blot and Nanoparticle tracking analysis.It was found that exosomes were rich in CD63-L7 Ae proteins.What's more,the diameter of exosomes was the same as that of natural exosomes,indicating that the physical properties of exosomes were not affected by the expression of CD63-L7 Ae.Second,sh RNA vector that can silence EGFP protein fused with the K-turn motif was constructed which was demonstrated that it could reduce EGFP expression in HEK293 T cells.Subsequently,the recombinant plasmid(p CMV-L7Ae)was constructed,and HEK293 T cells were cotransfected transiently with p CMV-L7 Ae and sh RNA vector,providing the proof that L7 Ae could bind to K-turn in HEK293 T cells.HEK293 T cells were then cotransfected with the recombinant plasmid(p CMV-CD63-L7Ae)and the sh RNA vector(p RNAT-U6.1 / Neo-Kt-sh EGFP)containing K-turn,followed by extration of exosomes(Kt-CD63L7Ae-Exo)secreted by the HEK293 T cells.Comparing with the control(control-Exo?Kt-p CMV-Exo),the copy number of Kt-sh EGFP in Kt-CD63L7Ae-Exo increased significantly by q RT-PCR assay.The Kt-sh EGFP transfered by exsomes to recipient cells was found to remain the silencing function of EGFP.Finally,using NAMPT m RNA,we tried to verify whether this strategy could be applied to loading m RNA into exosomes.According to some reports,NAMPT is secreted extracellularly through the form of exosomes,and only Ev(Extracellular vesicle)-contained e NAMPT can be internalized into cells.Thus,we chose NAMPT as the target m RNA loaded into exosomes.The m RNA expression plasmid fused with K-turn(p CMV-NAMPT-Kt)in C-terminal was constructed,and HEK293 T cells were transfected with the recombinant plasmid(p CMV-CD63-L7Ae)and p CMV-NAMPT-Kt.The exosomes(NAMPT-CD63L7Ae-Exo)were extracted and identified by q RT-PCR.It was showed that the copy numbers of NAMPT m RNA in NAMPT-CD63L7Ae-Exo was significantly higher than that in control-Exo or NAMPT-p CMV-Exo.The m RNA of NAMPT was transfered into the recipient cell by transfection of NAMPT-CD63-L7Ae-Exo and it was found that m RNA can be expressed in the recipient cell and has the deacetylation function of NAMPT.In summary,we produce an exosome that can wrap in RNA directionally base on the specific binding between L7 Ae and the K-turn structure.This kind of exosome proves to be an effective drug delivery system,which can deliver RNA into the recipient cells.