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mRNA-liposome complex and application thereof

A technology of liposome complexes and liposomes, which can be used in liposome delivery, DNA/RNA vaccination, complete cell/virus/DNA/RNA components, etc., and can solve the problem of high toxicity

Active Publication Date: 2020-12-22
ZHEJIANG ZHIDA PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Other lipid-based polymers, such as Lipofectamine (invitrogen) or Mirus-Trans IT-mRNA, can effectively transfect mRNA under cultured cell conditions, but these transfection reagents are highly toxic[23]

Method used

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  • mRNA-liposome complex and application thereof
  • mRNA-liposome complex and application thereof
  • mRNA-liposome complex and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0109] Example 1 Preparation of transfection reagent

[0110] In the following examples, the liposome (transfection reagent) InstantFECT comes from PGR-Solutions, Inc.

[0111] InstantFECT contains 1:2w / w lipopolyamine described in formula II and DOPE.

[0112] InstantFECT is a liposome transfection reagent with a unique formula, which can show good transfection efficiency in a relatively wide range of nucleic acid to liposome ratio. At recommended doses, it has very low cytotoxicity and is resistant to serum components during transfection. When using, add the recommended amount (200uL) of the reconstitution solution into the glass bottle, hydrate for one minute, and then vortex for one minute to form a translucent liposome suspension, which is ready to use.

Embodiment 2

[0113] Example 2 mRNA synthesis

[0114] The 5' and 3' UTR regions of TMV are inserted into the normal pUC57 plasmid, and the poly A tail is inserted at the 3' end to form a DNA backbone. Use NEB's T7Hiscribe mRNA synthesis kit to perform in vitro transcription of plasmid DNA encoded by EGFP (green fluorescent protein), ADSa (Staphylococcus aureus adenosine synthase A) and OVA (ovalbumin) (see the kit instructions for specific steps) . A mixture of DNA backbone (containing gene, TMV 5'UTR, TMV 3'UTR, and poly A tail), NTPS (ATP, CTP, GTP, UTP, and pseudouridine), T7 polymerase, and buffer was incubated at 37 °C Incubate for 2-3 hours and purify the mRNA product by LiCl precipitation. The modified in vitro transcribed mRNA was then capped using the NEBCenovia viral capping enzyme cap1 system. After purification by Licl precipitation, the 5'cap-modified mRNA products were stored at -20°C.

[0115] We chose the portion encoding amino acid 241-amino acid 339 in the ovalbumin g...

Embodiment 3

[0120] The preparation of embodiment 3 mRNA liposome complexes

[0121] The method of mRNA lipid nanocomplex was as follows: 5 μg mRNA was diluted with 0.9% NaCl, mixed with 4 μl of InstantFECT and used within 15 minutes.

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Abstract

The invention belongs to the field of bioengineering and medicine, and particularly relates to an mRNA-liposome complex for introducing mRNA with modification into cells as well as application, a preparation method and the like of the mRNA-liposome complex. The mRNA-liposome complex contains a transfection reagent and at least one mRNA with modification. Inconspicuously, the mRNA-liposome complexcan efficiently mediate in vivo transfection of mRNA to a plurality of organs in a topical delivery manner. According to the mRNA-liposome complex, intracellular delivery of modified mRNA, resulting in gene expression, can be used to study biological functions of specific genes, can also correct / treat diseases caused by gene deletion or low gene expression. In addition, the mRNA-liposome complex can be used as a nucleic acid vaccine to prevent infectious diseases and treat cancer. In addition, administration of the mRNA-liposome complex is very simple and convenient, and is safe and effective.

Description

technical field [0001] The invention belongs to the fields of bioengineering and medicine, and relates to an mRNA-liposome complex containing modified mRNA and liposomes and its application and preparation method. Background technique [0002] At present, recombinant protein biopharmaceuticals are becoming more and more important in the application of disease treatment and prevention. It is a simple, effective and more economical method to introduce protein-coding DNA or RNA into cells through a suitable carrier to guide the synthesis of cellular proteins. Viral vectors and non-viral vectors have been developed to deliver DNA or RNA exogenous genes to cells. Viral vectors have a certain risk of mutation from replication-deficient to wild-type, and may even lead to mutagenesis of cells. The preparation of viral vectors is complex, tedious, and highly immunogenic [1]. Non-viral vectors represented by cationic liposomes and polymers deliver biologically active macromolecules...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00A61K39/02A61K39/12A61K9/127A61K47/18A61K47/24A61P31/04A61P31/10A61P31/12A61P35/00A61P37/00
CPCA61K39/0011A61K39/0002A61K39/0005A61K39/0008A61K39/02A61K39/12A61P35/00A61P31/04A61P31/12A61P31/10A61P37/00A61K9/0019A61K9/127A61K47/18A61K47/24A61K2039/53A61K2039/6025A61K2039/54Y02A50/30
Inventor 斯姆瑞蒂·阿莉亚高翔黄建东
Owner ZHEJIANG ZHIDA PHARM CO LTD
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