Preparation method and application of targeting exosome

An exosome, targeting technology, applied in the field of biomedicine, can solve the problem of low exosome modification efficiency, and achieve the problem of low transfection efficiency, difficulty in large-scale production, simple modification means, and good cell targeting. Effect

Inactive Publication Date: 2018-12-25
来复赛尔(厦门)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In order to solve the problem of low exosome modification efficiency mentioned in the a...

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  • Preparation method and application of targeting exosome
  • Preparation method and application of targeting exosome
  • Preparation method and application of targeting exosome

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preparation example Construction

[0048] The present invention provides a method for preparing targeted exosomes. After the PSP1 polypeptide is covalently combined with any targeting peptide or a short peptide with immune function to form a PSP1 targeting peptide, it is then combined with exosomes , that is, to obtain targeted exosomes; wherein, the amino acid sequence of the PSP1 polypeptide is: CLSYYPSYC, or a polypeptide having a sequence of more than 80% homology with PSP1.

[0049] Now, EGFR targeting peptide (GE11), nAchR targeting peptide (RVG29), liver cancer cell targeting peptide (SP94) and PSP1 polypeptide are combined to form PSP1 targeting peptide: PSP1-GE11, PSP1-RVG29-6His and PSP1-SP94 , and then modify the surface of exosomes as Example 1, Example 2 and Example 3; of course, the present invention describes not only the above targeting peptides, but all targeting peptides under the concept of the present invention or have Short peptides that promote immune function, that is, the above examples ...

Embodiment 1

[0058] Example 1: Preparation of PSP1-GE11

[0059] GE11 is a polypeptide with 12 amino acid residues (amino acid sequence: YHWYGYTPQNVI) with EGFR targeting, and is not derived from EGF, and has no EGFR activation activity.

[0060] Using chemical synthesis method, the amino acid sequence is directly synthesized: CLSYYPSYCYHWYGYTPQNVI 21 amino acid residue polypeptide with bidirectional binding ability of phosphatidylserine and EGFR, that is, PSP1-GE11 is obtained.

[0061] Wherein, the chemical synthesis method can adopt any synthetic method in the field of chemistry or biology. The present invention provides the Fmoc solid-phase polypeptide synthesis method as one of the examples, using 2-Cl-Trt (2-chlorotrityl chloride ) resin as a solid phase carrier. Its synthetic route is as follows figure 1 shown; the specific steps are as follows:

[0062] 1. Add a certain amount of 2-Cl-Trt resin (1eq) and an appropriate amount of DIEA (N, N diisopropylethylamine, 5eq) to an appro...

Embodiment 2

[0066] Example 2: Preparation of PSP1-RVG29-6His

[0067] Neurotropic virus-derived peptide (RVG29) (amino acid sequence: YTIWMPENPRGTPCDIFTNSRGKRASNG) is derived from the RVG protein on the capsid of rabies virus, and can be specifically recognized by the nicotinic acetylcholine receptor (nAchR) on the surface of nerve cells.

[0068] By means of biological expression, the prokaryotic expression system of PSP1-RVG29-6His (CLSYYPSYCYTIWMPENPRPGTPCDIFTNSRGKRASNGHHHHHH) with 6 histidine residues was constructed, and the specific steps are as follows:

[0069] Step a, synthesize 147 pairs of DNA sequences each containing BamHI and XhoI restriction endonuclease sites at the 5' end and the 3' end: 5'-ggatccTGCCTGTCCTATTATCCGTCCTATTGCTACACTATTTGGATGCCGGAAAATCCGCGTCCGGGTACCCCATGCGACATCTTCACCAACTCCGTGGTAAACGCGCGTCTAACGGCCACCATCACCACCACCATTAActcgag-3';

[0070]Step b, 147 pairs of DNA sequences of PSP1-RVG29-6His and pGEX-4T-1 plasmid were double digested with BamHI and XhoI restrictio...

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Abstract

The invention provides a preparation and application of a targeting exosome. The preparation method includes: covalently binding a PSP1 polypeptide with optional one targeting peptide or oligopeptidewith an immunity promoting function to form a PSP1 targeting peptide, and binding the PSP1 targeting peptide with an exosome, wherein the amino acid sequence of the PSP1 polypeptide is CLSYYPSYC or apolypeptide with more than 80% homological sequence of PSP1. The preparation method has the advantages that through a large number of tests and experiments, the PSP1 polypeptide is bound with the targeting peptide or the oligopeptide with an immunity promoting function for the first time, and the formed bidirectional-targeting polypeptide is bound with the exosome; the method solves the problem that existing surface modification methods such as plasmid transfection are low in transfection efficiency and increases the surface modification efficiency of the exosome, large-scale preparation of the targeting exosome is achieved, and the method is quite significant to the researches and application of the exosome serving as the transport vector.

Description

technical field [0001] The invention relates to the field of biomedical technology, in particular to a method for preparing targeted exosomes and its application. Background technique [0002] RNA therapy technologies, especially siRNA, antisense oligonucleotides, and CRISPR-CAS9 genome editing-mediated RNAs are emerging programmable therapeutic technologies, characterized by high specificity and high variability. However, most current programmable RNA therapeutic approaches are not suitable for clinical application due to low uptake efficiency and high cytotoxicity. Therefore, safe and effective strategies for delivering RNA drugs to most primary tissues and cancer cells, including leukemia cells and solid tumor cells, remain underdeveloped (Usman et al., 2018). [0003] Exosomes are small extracellular vesicles with a diameter of 30-150 nm and a circular single-layer membrane structure formed by cells through a series of regulatory processes such as "endocytosis-fusion-ef...

Claims

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Application Information

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IPC IPC(8): C12N5/071C07K19/00C12N15/70A61K35/36A61K35/22A61P35/00
CPCA61K35/22A61K35/36A61P35/00C07K7/06C07K7/08C07K2319/21C12N5/0625C12N5/0686C12N15/70
Inventor 孙坪田云鹏李柱
Owner 来复赛尔(厦门)生物科技有限公司
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