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.

Rationale: Exosomes are emerging as a promising drug delivery carrier. However, rapid uptake of exosomes by the mononuclear phagocyte system (MPS) remains an obstacle for drug delivery into other targeted organs, including the heart. We hypothesized that prior blocking of uptake of exosomes by the MPS would improve their delivery to the targeted organs.Methods: Exosomes were isolated from the cell culture medium. Fluorescence-labeled exosomes were tracked in vitro and in vivo by fluorescence imaging. The expression of clathrin heavy chain (Cltc), cavolin1, Pak1 and Rhoa, known genes for endocytosis, were profiled in various cell lines and organs by qPCR. The knockdown efficiency of siRNA against Cltc was analyzed by Western blotting. Exosomecontrol and exosomeblocking were constructed by encapsulating isolated exosomes with siControl or siClathrin via electroporation, while exosometherapeutic was constructed by encapsulating isolated exosomes with miR-21a. Doxorubicin-induced cardiotoxicity model was used to verify the therapeutic efficiency of the exosome-based miR-21a delivery by echocardiography.Results: Exosomes were preferentially accumulated in the liver and spleen, mainly due to the presence of abundant macrophages. Besides the well-known phagocytic effect, efficient endocytosis also contributes to the uptake of exosomes by macrophages. Cltc was found to be highly expressed in the macrophages compared with other endocytosis-associated genes. Accordingly, knockdown of Cltc significantly decreased the uptake of exosomes by macrophages in vitro and in vivo. Moreover, prior injection of exosomeblocking strikingly improved the delivery efficiency of exosomes to organs other than spleen and liver. Consistently, compared with the direct injection of exosometherapeutic, prior injection of exosomeblocking produced a much better therapeutic effect on cardiac function in the doxorubicin-induced cardiotoxicity mouse model.Conclusions: Prior blocking of endocytosis of exosomes by macrophages with exosomeblocking successfully and efficiently improves the distribution of following exosometherapeutic in targeted organs, like the heart. The established two-step exosome delivery strategy (blocking the uptake of exosomes first followed by delivery of therapeutic exosomes) would be a promising method for gene therapy.

BackgroundThe cultivated peanut is an important oil and cash crop grown worldwide. To meet the growing demand for peanut production each year, genetic studies and enhanced selection efficiency are essential, including linkage mapping, genome-wide association study, bulked-segregant analysis and marker-assisted selection. Specific locus amplified fragment sequencing (SLAF-seq) is a powerful tool for high density genetic map (HDGM) construction and quantitative trait loci (QTLs) mapping. In this study, a HDGM was constructed using SLAF-seq leading to identification of QTL for seed weight and size in peanut.ResultsA recombinant inbred line (RIL) population was advanced from a cross between a cultivar ‘Huayu36’ and a germplasm line ‘6–13’ with contrasting seed weight, size and shape. Based on the cultivated peanut genome, a HDGM was constructed with 3866 loci consisting of SLAF-seq and simple sequence repeat (SSR) markers distributed on 20 linkage groups (LGs) covering a total map distance of 1266.87 cM. Phenotypic data of four seed related traits were obtained in four environments, which mostly displayed normal distribution with varied levels of correlation. A total of 27 QTLs for 100 seed weight (100SW), seed length (SL), seed width (SW) and length to width ratio (L/W) were identified on 8 chromosomes, with LOD values of 3.16–31.55 and explaining phenotypic variance (PVE) from 0.74 to 83.23%. Two stable QTL regions were identified on chromosomes 2 and 16, and gene content within these regions provided valuable information for further functional analysis of yield component traits.ConclusionsThis study represents a new HDGM based on the cultivated peanut genome using SLAF-seq and SSRs. QTL mapping of four seed related traits revealed two stable QTL regions on chromosomes 2 and 16, which not only facilitate fine mapping and cloning these genes, but also provide opportunity for molecular breeding of new peanut cultivars with improved seed weight and size.

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Copeptin levels are a novel and complementary biomarker to predict functional outcome and mortality 1 year after acute ischemic stroke.

Therapeutically intervening the function of RNA in vivo remains a big challenge. We here developed an exosome-based strategy to deliver engineered RNA-binding protein for the purpose of recruiting specific RNA to the lysosomes for degradation. As a proof-of-principle study, RNA-binding protein HuR was fused to the C-terminus of Lamp2b, a membrane protein localized in both exosome and lysosome. The fusion protein was able to be incorporated into the exosomes. Moreover, exosomes engineered with Lamp2b-HuR successfully decreased the abundance of RNA targets possibly via lysosome-mediated degradation, especially when the exosomes were acidified. The system was specifically effective in macrophages, which are lysosome enriched and resistant to routine transfection mediated RNAi strategy. In the CCl4-induced liver injury mouse model, we found that delivery of acidified exosomes engineered with Lamp2b-HuR significantly reduced liver fibrosis, together with decreased miR-155 and other inflammatory genes. In summary, the established exosome-based RNA-binding protein delivery strategy, namely “exosome-mediated lysosomal clearance”, takes the advantage of exosome in targeted delivery and holds great promise in regulating a set of genes in vivo.

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