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Preparation method and application of engineered exosome for drug targeted delivery

An exosome, engineered technology, applied in biochemical equipment and methods, chemical instruments and methods, drug combinations, etc., can solve problems such as cancer cell apoptosis

Pending Publication Date: 2021-09-28
路宝特(南京)环保科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In the prior art, targeted drug delivery (for example, targeting cancer) is the focus and difficulty of research and development, and the technology of directly targeting cancer cells through engineered exosomes to induce apoptosis of cancer cells has not been reported yet.

Method used

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  • Preparation method and application of engineered exosome for drug targeted delivery
  • Preparation method and application of engineered exosome for drug targeted delivery

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] (1) Construction of target gene expression vector; acquisition or construction of exosome cytoplasmic transport auxiliary plasmid (pGmL4), exosome secretion-promoting plasmid (pGmL6), exosome targeting plasmid (pGmL7), target gene mRNA packaging Plasmid (pGmL5); the target gene is a toxic protein gene (pGmL1 or pGmL2) (perforin or granzyme); in the target gene expression vector, the target gene 5'-UTR is inserted into the encrypted sequence;

[0037] (2) HEK293F cells were co-transfected and cultured with a mixed plasmid consisting of the target gene expression vector, the exosome cytoplasmic transport auxiliary plasmid, the exosome secretion-promoting plasmid, the exosome targeting plasmid, and the target gene mRNA packaging plasmid;

[0038] (3) Isolation of engineered exosomes that facilitate targeted drug delivery:

[0039] The culture medium of HEK293F cells after the above transfection was collected and filtered to obtain exosomes in the culture medium.

[0040] ...

Embodiment 2

[0051] Similar to Example 1, the only difference is that this example does not require a targeting plasmid, and the expression of the target gene (perforin or granzyme) only depends on the characteristic miRNA that recognizes the encrypted sequence in the target cancer cell. Obtain exosome product 3 (containing encrypted sequence-target gene perforin mRNA, Cx43S368A protein, CD63-L7Ae protein and STEAP3 / SDC4 / NadB protein) and exosome product 4 (containing encrypted sequence-target gene granzyme mRNA, Cx43S368A protein, CD63-L7Ae protein and STEAP3 / SDC4 / NadB protein).

Embodiment 3

[0053] (1) Construction of target gene expression vector; obtain or construct exosome cytoplasmic transport auxiliary plasmid, exosome secretion-promoting plasmid, target gene miRNA packaging plasmid, and exosome targeting plasmid; the target gene is miR -Y (if the effector Y is miR-9-3p, it can promote the apoptosis of glioma cells; if Y is miR-1469, it can induce the apoptosis of laryngeal cancer cells); in the target gene expression vector, the target gene 5 '-UTR is inserted into the encrypted sequence;

[0054] (2) HEK293F cells were co-transfected and cultured with a mixed plasmid consisting of the target gene expression vector, the exosome cytoplasmic transport auxiliary plasmid, the exosome secretion-promoting plasmid, the target gene miRNA packaging plasmid, and the exosome targeting plasmid;

[0055] (3) Isolation of engineered exosomes that facilitate targeted drug delivery:

[0056] The culture medium of HEK293F cells after the above transfection was collected and...

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Abstract

The invention relates to a preparation method and application of an engineered exosome for drug targeted delivery. Messenger RNA (mRNA) carrying miRNA or encoded toxic protein (such as perforin or granzyme) capable of inducing specific cell apoptosis is loaded into the exosome. The secreted exosome loaded with specific nucleic acid cargo is collected and purified. A molecular switch encryption device using target cell characteristic miRNA as a triggering signal is preset in 5'-UTR of a target gene, so that the miRNA or mRNA does not have the function in host cells. When the engineered exosome is used as a carrier for conveying a carrier-effector (i.e., the miRNA or toxic protein mRNA capable of inducing target cell apoptosis) to the target cells, the specific miRNAs in cancer cells are used as a triggering switch for starting the release of the effector (miRNA or toxic protein); the apoptosis program is started, so that the target cancer cells are killed.

Description

technical field [0001] The invention relates to a preparation method and application of engineered exosomes for drug targeted delivery. Background technique [0002] Exosomes are microscopic vesicles of 40-100 nanometers secreted by various cells. Exosomes, or insoluble microvesicle structures, are an important pathway for intercellular signal communication discovered in recent years, and are important by-products of soluble cytokines. secreted form. The characteristics of Exosome mainly include: (1) the diameter is 40-100nm; (2) has the cytoplasm and lipid membrane components of the source cell; (3) the density is 1.13-1.19g / ml; (4) contains the source cell Specific proteins and exosome-related proteins such as CD9, CD81, Alix, etc. Exosome components mainly include: microRNA, mRNA, mtDNA, protein, surface markers, etc. Proteomic analysis found that the protein composition of exosome is relatively complex, and the composition of exosome protein from different cell sources...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10A61K47/46A61K35/28A61P35/00
CPCC12N15/1135C07K14/47C12N5/0686C12N5/0662A61K47/46A61K35/28A61P35/00C12N2310/141C12N2510/00
Inventor 安文林贾守义
Owner 路宝特(南京)环保科技有限公司
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