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Engineered exosome with on-demand anti-inflammatory function and loaded with inflammatory response type mRNA as well as construction method and application of engineered exosome
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An exosome, engineered technology, applied in the field of genetic engineering, to achieve the effect of avoiding translation, ideal tissue specificity, and strong tissue specificity
Active Publication Date: 2022-03-01
FOURTH MILITARY MEDICAL UNIVERSITY
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However, most of these therapeutic agents are administered systemically and often require freq
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Embodiment 1
[0045] 1. Entrusted Nanjing Jinsi Biotechnology Co., Ltd. Synthesis SEQ ID NO.4 (PME1-IRES-IL-10-miR-122Recognition Site-Linker-mir-122Recognition Site-Linker-mir-122Recognition site- BSTBI);
[0046] 2. Insert the sequence shown in SEQ ID NO.4 into the PWPI vector by PME1 and BSTBi double-cutting and connection, and the sequence shown in SEQ ID NO is inserted into the PWPI vector;
[0047] 3, using liposome transfection, HEK293T cells are transfected;
[0048] 4, transfected cells, placed in 37 ° C with 5% CO 2 The cell incubator, after 4- ~ 6h, exchanged to serum-free fully medium, and collected cell supernatants after 48 hours;
[0049] 5, filter the cell epitherent with 0.22 μm filter, remove cell fragments;
[0050] 6. Cell supernatant 500 g Centrifuge 10 min removed cells, 10000 g from 20 min to remove residual cell debris, 100,000 g of ultracenter centrifugation 2 h, and the resulting precipitate is an outer body.
[0051] Table 1 Primer sequence required by QPCR
[0052] ...
Embodiment 2
[0061] Exploring the loading, delivery, targeting and intervention efficiency in cells and in vivo; injecting the extracted exotic body into an atherosclerosis model mouse (ApoE) - / - Mouse) body, EXO IRES-Il-10 Treatment of mouse aorta in the aortic pulse, the level of inflammation decreased, and atherosclerosis was obviously alleviated. In addition, liver and kidney function and octor H & E stain show, EXO IRES-Il-10 Treatment without obvious toxicity reactions.
[0062] 1. Cellular Verification EXO IRES-Il-10 Deliver miR-155 response type IRES-IL-10 mRNA to receptor cells
[0063] In vitro verification, IRES-IL-10 can be delivered to the receptor cells in vitro. First, the exosomes labeled with DII are co-cultured with macrophage lines, and the laser confocal results show that macrophages can be swallowed. . Cell dynamic testing proves that there is no obvious cytotoxicity in the foreign body (see figure 2 . After the total culture of the prolonged body and the transfection of M...
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Abstract
The invention discloses an engineered exosome based on an on-demand anti-inflammatory function and loaded with inflammation response type mRNA as well as a construction method and application of the engineered exosome, and relates to the technical field of gene engineering. The structure of the fusion gene comprises IRES, IL-10 and miR-122 pairing sequences, a recombinant vector and a recombinant cell are constructed based on the fusion gene, the recombinant cell is used for producing the engineered exosome, the engineered exosome has ideal tissue specificity and a good treatment effect, the preparation method of the exosome is simple, the cost is low, and the application prospect is wide. The method has the advantages of high tissue specificity, low off-target rate and the like, can be used as a vector for gene therapy, and is of great significance.
Description
Technical field [0001] The present invention belongs to the field of genetic engineering technology, and in particular, an engineering episode based on an anti-inflammatory function, loading inflammatory response type mRNA, and its construction methods and its construction methods. Background technique [0002] Atherosclerosis (AS) is the main cause of cardiovascular disease (CVD), is a chronic inflammatory disease, characterized in that the aorta and medium artery inner plaque accumulation are accumulated. At present, the clinical treatment of atherosclerosis mainly includes lifestyle intervention, lipid-lipid-lipid-lipid-proof treatment and anti-thrombus treatment. More and more studies have shown that anti-inflammatory treatment has opened a new window for the treatment of atherosclerosis. For example, the efficacy and safety of interleukinum-1β inhibitors CANAKINUMAB, lipase A2 inhibitors and P38MAPK inhibitors Losmapimod were studied in clinical trials. However, most of thes...
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