GRNA of targeted CTGF gene and application thereof

A targeted and gene-based technology, applied in the field of gene editing, can solve problems such as increased treatment costs, poor specificity of oligomeric RNA, poor targeting of free molecules, etc., to achieve low long-term treatment costs, long-lasting and stable curative effect, and patient compliance good sex effect

Pending Publication Date: 2022-03-29
GUANGZHOU REFORGENE MEDICINE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The role of oligoRNA is at the RNA level. The main problem is similar to that of protein antibody drugs. It needs long-term continuous administration, which brings inconvenience and increases the cost of treatment.
In addition, siRNA has defects such as liver aggregation (hepatotoxicity), easy clearance by the reticuloendothelial system, poor targeting of free molecules, and the need to engage ligands or be packaged by carriers for targeted delivery.
General oligo RNA specificity is not as good as siRNA

Method used

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  • GRNA of targeted CTGF gene and application thereof
  • GRNA of targeted CTGF gene and application thereof
  • GRNA of targeted CTGF gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] The CTGF gene promoter element or exon 2 was edited by the CRISPR gene editing method.

[0075] 1. Vector preparation.

[0076] 1) Determine the gRNA targeting domain (sequence identical to the target sequence):

[0077]According to the sequence near the SMAD binding site in the human CTGF gene promoter region and the exon 2 (Exon2) sequence, a gRNA with a targeting domain length of 17nt-24nt was designed. In addition, a gRNA (Responsive-sgRNAx) targeting the TGFβ response element GTGTCAAGGGGTC sequence adjacent to the SMAD binding site was designed. Some of them are shown in Table 2.

[0078] Table 2. Designed gRNA targeting domains

[0079] gRNA name targeting domain sequence targeting domain sequence SMAD-sgRNA1 SEQ ID NO:1 GUGCCAGCUUUUUCAGA SMAD-sgRNA2 SEQ ID NO:4 AGUGUGCCAGCUUUUUCAGA SMAD-sgRNA3 SEQ ID NO:6 GGAGUGUGCCAGCUUUUUCAGA SMAD-sgRNA4 SEQ ID NO:8 CUGGAGUGUGCCAGCUUUUUCAGA SMAD-sgRNA5 SEQ ID NO:9 CC...

Embodiment 2

[0130] Gene editing in a cell model of lung fibrosis.

[0131] 1. Experimental materials.

[0132] Nucleic acid purification kit with 2×Accurate Taq master Mix, reverse transcription kit, 2X Green Pro Taq HS Premix was purchased from Hunan Aikerui Bioengineering Co., Ltd., OPTI-MEM and Lipofectamine 3000 transfection reagents were purchased from Thermo, and TGF-β1, SpCas9 protein and SaCas9 protein were purchased from Novoprotein. Primers and related RNAs used in PCR and sequencing were synthesized by reagent companies. A549 cells are commercially available.

[0133] 2. Experimental method.

[0134] 2.1 Experimental grouping

[0135] Group as follows:

[0136] Group A549: blank control without any treatment;

[0137] A549 TGF-β1 group: cells were only treated with TGF-β1 without RNP transfection.

[0138] Each group of SMAD-sgRNAx (x is the sequence number): wherein the gRNA targeting domain is the same as in Example 1, and the complete sequence of the gRNA is: targeti...

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Abstract

The invention relates to gRNA of a targeted CTGF gene and application of the gRNA, and belongs to the technical field of gene editing. The gRNA is used for cutting an SMAD binding site area of a CTGF gene promoter in a targeted manner. According to the gRNA disclosed by the invention, overexpression of the human CTGF gene can be reduced through a CRISPR-Cas gene editing system. The gRNA has the advantages of lasting and stable curative effect, small adverse reaction, low treatment frequency, good patient compliance and low long-term treatment cost when being used for preparing medicines for treating fibrosis diseases.

Description

[0001] This application is a divisional application of the Chinese patent application with the application number 202011002055.8, submitted to the China Intellectual Property Office on September 22, 2020, and the application name is "gRNA targeting CTGF gene and its application". technical field [0002] The invention relates to the technical field of gene editing, in particular to a gRNA targeting CTGF gene and its application. Background technique [0003] Idiopathic pulmonary fibrosis (IPF for short), sometimes also called ordinary interstitial pneumonia (UIP), is a chronic, progressive, fibrotic interstitial lung disease. Lung histology is characterized by common interstitial pneumonia, which is the most common type of idiopathic interstitial pneumonia (IIP). According to the course of the disease, it can be divided into acute, subacute and chronic. The disease is mostly sporadic, and according to statistics, the annual prevalence rate in the whole population is about (...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85C12N15/88C12N15/55C12N9/22A61K48/00A61K38/46A61P11/00
CPCC12N15/1136C12N15/85A61K48/0008A61P11/00C12N2310/20C12N2800/107C12N15/88C12N9/22A61K48/005A61K38/465C12N2320/11C12N2320/30C12N15/90Y02A50/30C12N15/11C12N15/907C12N2800/80
Inventor 梁峻彬徐辉欧家裕古博
Owner GUANGZHOU REFORGENE MEDICINE CO LTD
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