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A method for loading mRNA in extracellular vesicles in vitro

A cell and vesicle technology, applied in the field of biological products, can solve the problems of complex preparation process, high experimental cost, long cycle, etc., and achieve the effects of low operating technical level requirements, wide application range and short experimental cycle.

Active Publication Date: 2022-07-26
天津外泌体科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the traditional solution for loading extracellular vesicles with mRNA in vivo has certain limitations, which are reflected in three aspects: (1) mRNA must be expressed in vivo in cells, and for some modern biological industry application scenarios, such as the preparation of nucleic acids for mRNA vaccines, among them There are necessary artificial chemical modifications to enhance biological activity, reduce toxicity, and improve in vivo stability, but artificial chemical modification components cannot be produced in vivo, such as using pseudouridine as a raw material for mRNA synthesis, in order to reduce the natural immune response of cells (2) Cells derived from extracellular vesicles must be easily gene-edited and can be cultured on a large scale, which limits the source of extracellular vesicles. For example, some primary cells are difficult to gene-edit, such as milk-derived extracellular vesicles and urine. The engineering transformation of liquid-derived extracellular vesicles is difficult to obtain through in vivo transformation of cells; (3) loading mRNA in vivo requires the design and synthesis of gene expression vectors, and the transformation of cells at the genetic level requires high levels of experimental operation skills and experimental costs. High and long period, it cannot be used for rapid screening at the experimental level to judge the loading efficiency of target mRNA in extracellular vesicles, because the preparation process is complicated and it is difficult to scale up the production in industry

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  • A method for loading mRNA in extracellular vesicles in vitro
  • A method for loading mRNA in extracellular vesicles in vitro
  • A method for loading mRNA in extracellular vesicles in vitro

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Preparation method of extracellular vesicles with low toxicity cationic liposome Dlin-MC3-DMA loaded with mRNA efficiently in vitro

[0047] Select low-toxic cationic lipid Dlin-MC3-DMA (4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester) (Aiwei Tuo (Shanghai) Pharmaceutical Technology Co., Ltd.), dissolved in Trichloromethane at a final concentration of 1 mM. After drying with nitrogen, reconstitute in PBS and vortex for 10 minutes. Extrude 20 times using a manual extruder with a membrane pore size of 100 nm, such as image 3 is the electron microscope morphological diagram of the Dlin-MC3-DMA liposome, and the charged property of the liposome is determined to be weakly negative, such as Figure 4 Shown are the charged properties of the cationic liposome Dlin-MC3-DMA at different pH. Because as a loading reagent, liposomes must carry a positive charge to adsorb negatively charged nucleic acids, so by adjusting the pH of the buffer, in the case of...

Embodiment 2

[0057] Example 2: In vitro mRNA-loaded extracellular vesicle preparation using two commercial loading reagents

[0058] Two commercial liposome-based cell loading reagents are DOTAP Liposome Loading Reagent (Roche TM ) and Lipofectamine TM 3000 loading reagent (Thermo Scientific TM ), the original intention of these two reagents is to load cells, these two transfection reagents are liposomes, and this example tests their effect on loading extracellular vesicles.

[0059] The loading method is the same as in Example 1, that is, take DOTAP liposome loading reagent or Lipofectamine TM 3000 loading reagent 3 uL, add 300 ng mRNA, incubate at room temperature for 15 minutes, add 400 uL milk-derived extracellular vesicles (the number of particles is about 1E11 / mL), incubate at 37 °C for 2 hours, load mRNA into in milk-derived extracellular vesicles. After quantification by nuclease treatment and RT-qPCR, calculation by DOTAP liposome loading reagent or Lipofectamine TM The en...

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Abstract

The invention discloses a method for loading mRNA in extracellular vesicles in vitro. The method carries weakly negatively charged or neutrally charged liposomes under neutral conditions, carries out mRNA loading under weakly acidic conditions, and co-incubates mRNAs with cationic liposomes. The extracellular vesicles were loaded, and the loaded extracellular vesicles were restored to neutrality by ultrafiltration and medium exchange; RNA in the sample was extracted, and the mRNA copy number was quantitatively detected by RT-qPCR, and the mRNA copy number before and after loading was detected, and the ratio was mRNA Encapsulation efficiency in extracellular vesicles. Compared with the traditional method, the method provided by the present invention is simple to operate, requires low technical level of operation, and has a short experimental period; has less restrictions on the mRNA to be reproduced, and accordingly has a wider application range; Electroneutral lipids, mRNA loading is carried out under weakly acidic conditions, and the loaded extracellular vesicles are restored to a neutral environment by changing the medium, which can avoid the problem of cytotoxicity caused by the positive charge of cationic liposomes. For the preparation of biological products.

Description

technical field [0001] The invention belongs to the technical field of biological products, in particular to a method for loading mRNA in extracellular vesicles in vitro. Background technique [0002] Extracellular vesicles (Extracellular vesicles) are small vesicles with a saucer-shaped structure between 30 and 150 nm in diameter, containing RNA, protein, microRNA, DNA fragments and other components. All eukaryotic cells and some prokaryotic cells can secrete, mainly distributed in blood, saliva, urine, amniotic fluid and breast milk and other body fluids. It is formed by the invagination of the cytoplasmic membrane to form the early endosome, which wraps the material to form multivesicular bodies, and then the multivesicular bodies are released after fusion with the plasma membrane. Although extracellular vesicles were discovered as early as 1983, it was generally believed that it was a waste product of cellular metabolism, and its main role was to take on the role of thr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K9/50A61K47/46A61K31/7105C12N15/88
CPCA61K9/5068A61K31/7105C12N15/88
Inventor 葛啸虎陆路张权柴天聪田应洲王达尹建新王淼韩春乐
Owner 天津外泌体科技有限公司