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Circular RNA (Ribonucleic Acid) molecule for tissue specific expression and application of circular RNA molecule

A molecular and cyclic technology, applied in the field of prevention or treatment of diseases, can solve problems such as tissue and organ damage, achieve broad application prospects, improve targeted expression and therapeutic effects

Pending Publication Date: 2022-06-07
SUZHOU CUREMED BIOMEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, circular RNA molecules have shown important application value in the treatment of clinical diseases. However, limited by the way of administration, circular mRNAs also show high abundance expression in non-pathological parts after delivery in vivo (for example, Liver usually has a high abundance tendency), causing certain damage to normal tissues and organs of the body

Method used

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  • Circular RNA (Ribonucleic Acid) molecule for tissue specific expression and application of circular RNA molecule
  • Circular RNA (Ribonucleic Acid) molecule for tissue specific expression and application of circular RNA molecule
  • Circular RNA (Ribonucleic Acid) molecule for tissue specific expression and application of circular RNA molecule

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0281] Example 1: Preparation of circular mRNA containing miR-122 recognition region and its expression in A549 cells

[0282] (1) Plasmid construction

[0283] To construct target Luciferase genes with different sequences, this step is entrusted to Suzhou Jinweizhi Biotechnology Co., Ltd. for gene synthesis and cloning. The resulting gene fragment was ligated into the pUC57 vector. The following plasmids were obtained:

[0284] pUC57-EV29-LUC,

[0285] pUC57-EV29-LUC-3'UTR,

[0286] pUC57-EV29-LUC-3'UTR+1×miR-122.

[0287] (2) Preparation of linear plasmid template

[0288] 1) Plasmid extraction

[0289] ①Activate the externally synthesized puncture bacteria under the condition of 37℃ / 220rpm / 3~4h.

[0290] ② Take the activated bacterial liquid to expand the culture, culture conditions: 37℃ / 220rpm / overnight.

[0291] ③Plasmid extraction (Tiangen Endotoxin-Free Small Medium Extraction Kit), and the OD value was determined.

[0292] 2) Plasmid digestion

[0293] Adopt ...

Embodiment 2

[0346] Example 2: Expression of circular mRNA containing multiple miR-122 recognition regions in A549 cells

[0347] On the basis of the above Example 1, more miR-122 recognition regions are added on the basis of circular mRNA EV29-LUC-3'UTR+1×miR-122, for example, 3×miR-122 is added. Plasmid DNA linearization, in vitro transcription of circular precursor mRNA, purification of circular precursor mRNA, mRNA cyclization reaction, purification of circular mRNA, cell culture and Luciferase detection are the same as those in 1.1 of Example 1.

[0348] Cell transfection steps:

[0349] A549 cells were plated at 1 × 10 before transfection 5 Cells / well were seeded in 24-well plates at 37°C, 5% CO 2 Cultivated in an incubator. After the cells reach 70-90% confluence, use Lipofectamine Messenger Max (Invitrogen) transfection reagent to transfect A549 cells with 500ng / well of mRNA. The specific operations are as follows:

[0350] ① Dilute Messenger MAX TM Reagent

[0351] Table 8 ...

Embodiment 3

[0360] Example 3: The effect of circular mRNA containing different tissue-specific miRNA recognition regions on the expression of Luciferase in specific tissue cells

[0361] On the basis of the above Example 1, ev29-Luc circular RNAs containing different micoRNA sequences were constructed, and statistics were calculated by their expression in different cell lines. The linearization of plasmid DNA, the in vitro transcription of circular precursor mRNA, the purification of circular precursor mRNA, the circling reaction of mRNA, the purification of circular mRNA, and the methods of cell transfection and Luciferase detection are all the same as those in Example 1. 1.1 is the same.

[0362] 1) Recovery and subculture of MRC-5 and IMR-90 human lung fibroblasts

[0363] Take out the MRC-5 and IMR-90 human lung fibroblast cell lines from the liquid nitrogen storage tank, quickly put the cryopreservation tube into a 37°C water bath, and shake quickly until the cryopreservation soluti...

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Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a tissue-specific expression circular RNA molecule, a cyclized precursor RNA molecule, a recombinant nucleic acid molecule, a recombinant expression vector, a recombinant host cell, a composition, a pharmaceutical preparation and application of the tissue-specific expression circular RNA molecule, the cyclized precursor RNA molecule, the recombinant nucleic acid molecule, the recombinant expression vector, the recombinant host cell, the composition and the pharmaceutical preparation in preparation of drugs for preventing or treating diseases. And a method of preventing or treating a disease. According to the circular RNA molecule provided by the invention, the coding region is operably connected with the expression regulation element, so that specific high expression of a target polypeptide in a target cell or a target tissue and low expression of the target polypeptide in a non-target cell or a non-target tissue are realized, and the circular RNA molecule has high tissue expression specificity; a safe and effective treatment strategy is provided for clinical targeted treatment of diseases such as tumors.

Description

technical field [0001] The present disclosure belongs to the technical field of biomedicine, and in particular, the present disclosure relates to a tissue-specifically expressed circular RNA molecule, a circularized precursor RNA molecule, a recombinant nucleic acid molecule, a recombinant expression vector, a recombinant host cell, a composition, and a drug The preparation and its application in the preparation of medicines for preventing or treating diseases, and a method for preventing or treating diseases. Background technique [0002] With the emergence of synthetic mRNA as an emerging tool for in vivo protein expression, mRNA therapy brings more possibilities for the diagnosis and treatment of tumors, genetically deficient diseases, and epidemic prevention. Since mRNA can be synthesized in vitro, the synthesis of mRNA is more efficient, fast, open and low-cost compared to other current biotechnology treatments. However, mRNA still faces many technical difficulties in ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/13C12N15/26C12N15/85C12N5/10A61K38/17A61K48/00A61P35/00
CPCC12N15/113C07K14/55C07K16/2818C12N15/85A61K48/005A61K38/1709A61K48/0075A61P35/00C12N2310/532C12N2840/203C12N2840/007C12N2840/002C12N2800/107A61K48/0058A61K31/7105C12N2800/50
Inventor 左炽健仇宗浩杨嘉丽朱佳凤李娜孙振华
Owner SUZHOU CUREMED BIOMEDICAL TECH CO LTD
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