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Circular RNA molecule and application thereof in targeted degradation of target protein

A target protein and molecular technology, applied in the direction of DNA/RNA fragments, medical preparations containing active ingredients, peptide/protein components, etc., can solve problems such as difficult to achieve transmembrane delivery, difficult to achieve target protein degradation in vivo, and achieve good results Effects of tumor cell membrane permeability, release of ubiquitin ligase dependence, and ease of delivery

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

Circular RNA molecules have good membrane permeability, are easy to deliver into cells, and have high-efficiency in vivo protein targeting degradation activity; effectively solve the existing protein targeting degradation chimeric molecules that are difficult to achieve transmembrane delivery, and difficult to The problem of achieving the degradation of the target protein in vivo

Method used

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  • Circular RNA molecule and application thereof in targeted degradation of target protein
  • Circular RNA molecule and application thereof in targeted degradation of target protein
  • Circular RNA molecule and application thereof in targeted degradation of target protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0253] Example 1: Validation of the degradation effect of the fusion protein encoded by circular RNA targeting EGFP protein degradation in 293T cells

[0254] 1.1 Experimental methods and steps:

[0255] (1) Construction of plasmids such as EV29-H2B-EGFP

[0256] To construct the target gene that expresses H2B-EGFP and targets H2B-EGFP protein degradation, this step entrusts Suzhou Jinweizhi Biotechnology Co., Ltd. to carry out gene synthesis and cloning. The DNA vector used here for the construction of circular RNA contains T7 promoter, 5' homology arm, 3' intron, second exon E2, 5' spacer, IRES element, H2B-EGFP and targeting Sequence coding region for EGFP protein degradation, downstream spacer region, 5' intron, first exon E1, 3' homology arm, and restriction enzyme cleavage site XbaI for plasmid linearization. The resulting gene fragment for linear mRNA TRNP-EGFP-DASP was ligated into the pUC57 vector.

[0257] Table 1

[0258] protein sequence name RNA se...

Embodiment 2

[0316] Example 2: Validation of the degradation effect of a fusion protein encoded by circular RNA targeting PCNA protein degradation in A549 cells

[0317] 2.1 Experimental methods and steps

[0318] (1) Construction of plasmids such as EV29-CON1-SP-WT

[0319] On the basis of the above Example 1, the same circular RNA element as in the above Example 1 was used to construct the fusion protein gene sequence EV29-CON1-SP-WT encoding targeted PCNA protein degradation, and the fusion protein gene sequence with loss of function pUC57-EV29-CON1-SP-mu and pUC57-EV29-CON1-mu-SP, the information is shown below. The DNA synthesis was entrusted to Suzhou Jinweizhi Biotechnology Co., Ltd.

[0320] Table 8

[0321] protein sequence name RNA sequence name Plasmid name RNASEQ.ID CON1-SP-WT EV29-CON1-SP-WT pUC57-EV29-CON1-SP-WT SEQ.ID No: 4 CON1-SP-mu EV29-CON1-SP-mu pUC57-EV29-CON1-SP-mu SEQ.ID No: 5 CON1-mu-SP EV29-CON1-mu-SP pUC57-EV29-CON...

Embodiment 3

[0348] Example 3: In vivo degradation effect verification of fusion protein encoded by circular mRNA targeting EGFP protein degradation

[0349] 3.1 Experimental method

[0350] 1) The plasmid DNA linearization, the in vitro transcription of the circularized pre-mRNA, the purification of the circularized pre-mRNA, the circularization reaction of the mRNA, the purification of the circular mRNA, and the cell culture are all the same as those in 1.1 of Example 1, except that the cell type is changed. For the tumor cell line A549.

[0351] 2) Cell collection: The cells were digested with 0.25% trypsin-EDTA (Gibco), centrifuged at 1000 rpm for 5 min, the supernatant was discarded, the cells were resuspended in phosphate buffered saline supplemented with Matrigel (Corning), and the cells were counted.

[0352] 3) A549 tumor model: female CD-1Nude nude mice (6-7 weeks old) were subcutaneously inoculated with 3×10 6 For each A549 cell, tumor size was measured with a vernier caliper ...

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Abstract

The invention belongs to the field of biological medicine, and particularly relates to a circular RNA molecule, a cyclized precursor RNA molecule, a recombinant nucleic acid molecule, a recombinant expression vector, a recombinant host cell, a composition, application of the composition in targeted degradation of target protein and a method for preventing or treating diseases. The circular RNA molecule has good membrane permeability, is easy to be delivered into cells, and has efficient in-vivo protein targeted degradation activity. According to the circular RNA molecule disclosed by the invention, tumor growth inhibition is successfully realized, the in-vivo protein degradation activity of the bio-PROTACs is proved for the first time, and a positive and effective treatment scheme is provided for treatment of diseases such as tumors.

Description

technical field [0001] The present disclosure belongs to the field of biomedicine, and in particular, the present disclosure relates to a circular RNA molecule, a circularized precursor RNA molecule, a recombinant nucleic acid molecule, a recombinant expression vector, a recombinant host cell, a composition and the targeting thereof in a target protein degradation, and a method for preventing or treating disease. Background technique [0002] Proteins in cells are constantly in the process of metabolic renewal of synthesis, modification and degradation. Maintaining normal protein metabolism is essential for the normal function of life. Degradation or intervention of specific proteins is an important way to study protein function, and it is also an important measure to treat related diseases caused by abnormal protein expression. At present, the degradation or intervention of pathogenic proteins has become one of the most effective disease treatment strategies. [0003] Tra...

Claims

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Application Information

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IPC IPC(8): C12N15/62A61K38/53A61K48/00A61P35/00
CPCC12N9/93C12Y603/02019A61K38/53A61K48/005A61K48/0075A61P35/00C07K2319/00A61K31/7105
Inventor 左炽健杨嘉丽朱佳凤杜雅冉赵阳孙振华
Owner SUZHOU CUREMED BIOMEDICAL TECH CO LTD
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