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Exon splicing enhancers, sgRNAs, gene editing tools and applications associated with Duchenne muscular dystrophy

A Duchenne muscular nutrition, gene editing technology, applied in genetic engineering, DNA/RNA fragments, applications, etc., can solve rare problems

Active Publication Date: 2022-06-28
WESTLAKE UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Not only diseases such as DMD, but currently the application of gene editing tools for the treatment of rare genetic diseases is extremely rare in the world

Method used

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  • Exon splicing enhancers, sgRNAs, gene editing tools and applications associated with Duchenne muscular dystrophy
  • Exon splicing enhancers, sgRNAs, gene editing tools and applications associated with Duchenne muscular dystrophy
  • Exon splicing enhancers, sgRNAs, gene editing tools and applications associated with Duchenne muscular dystrophy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 AAV virus carrying gene editing tools

[0046] Gene editing tools designed according to the present invention such as figure 1 As shown, taking AID as an example, we cloned the corresponding sequence into the AAV plasmid, including the following steps:

[0047] First, the pAAV2 backbone vector (purchased from addgene, but not limited to) was double-digested based on the restriction sites of XhoI and NotI. At the same time, the amino acid sequences of the AID and Cas9 fusion proteins in the gene editing tool were designed, and the amino acid sequences and nucleic acid sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. After codon optimization, the double-stranded DNA fragment was directly synthesized, and the elements such as Syn100 promoter and tailing signal were connected to the AAV backbone vector to obtain the AAV vector plasmid expressing the AID-Cas9 mutant fusion protein, the sequence of which was SEQ ID NO.3 shown. In addition, using ...

Embodiment 2

[0049] Example 2 In vivo treatment of DMD model mice with AAV carrying gene editing tools

[0050] In this example, a novel DMD mouse disease model Dmd-E4 with abnormal cardiac function was selected, which can be purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., but is not limited thereto. Dmd-E4 showed cardiac hypertrophy, fibrosis and other phenotypes in the heart at 6-8 weeks, and showed severe degeneration of cardiac function at about 8 months. This process closely mimics the cardiac pathology of DMD patients. In response to this model, we used cytosine deaminase and Cas9 to design gene editing tools to target the exons carrying pathogenic mutations, induce mutations near their 5' splice sites, and make them skip reads. On the basis of not affecting the open reading frame of the protein, the expression of Dystrophin protein can be retained to the maximum extent and its biological function can be restored.

[0051] Specifically, the method of Example 1 was use...

Embodiment 3

[0065] Example 3 Gene editing of the DMD model of human induced pluripotent stem cell iPSC successfully restored the expression of Dystrophin protein

[0066] At the same time, the present invention has successfully implemented the gene editing therapy of human cells. First, we constructed induced pluripotent stem cells (iPSCs) from normal human peripheral blood mononuclear cells, and then used the CRISPR-cas9 method to specifically delete the exon 50 of the Dystrophin-encoding gene DMD, Exon50, so that the dystrophin protein encoding The sequence produced a frameshift mutation, thereby constructing a mutation type that mimics a DMD patient, making it a good DMD disease model cell. For this cell, we designed the sequences of AID and Cas9 fusion proteins and corresponding sgRNAs to target a series of elements potentially regulating exon splicing in Exon51 of exon 51 of the DMD gene. sgRNA is sgRNA-12 shown in SEQ ID No. 19 and sgRNA-13 shown in SEQ ID No. 20, sgRNA-12 mainly t...

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Abstract

The invention discloses an exon splicing enhancer, sgRNA, gene editing tool and application related to Duchenne muscular dystrophy. Related exon splicing enhancers, sgRNA and gene editing tools can be used in the preparation of drugs for treating Duchenne muscular dystrophy. Among them, the gene editing tool designed based on the mutant of cytosine deaminase AID and the mutant of Cas9, using the adeno-associated virus AAV as the carrier, can carry out targeted modification of the genome of mammals. By optimizing the coding nucleic acid sequence and component structure of the editing tool, the present invention can efficiently realize the fixed-point targeted transformation of mammalian genetic material DNA, and carry out targeted genetic manipulation on the nucleic acid sequence carrying the disease mutation, so that the pathogenic mutation cannot be retained Into the mature protein amino acid sequence or make the pathogenic mutation unable to perform its function, so as to achieve the purpose of treating a variety of genetic rare diseases such as gene mutations, which has the advantages of high efficiency, safety and stability.

Description

technical field [0001] The invention belongs to the field of gene therapy, and relates to a Duchenne muscular dystrophy-related exon splicing enhancer, sgRNA, and a gene editing tool in mammals (experimental animal models and human patients) targeting gene mutation type hereditary diseases. Mutational transformation therapy. In particular, it relates to the gene editing therapy of Duchenne muscular dystrophy DMD in mouse models and human patients. Background technique [0002] According to the definition of the World Health Organization (WHO), rare diseases are uncommon diseases in a certain area, with the number of patients accounting for 0.065% to 0.1% of the total population. The pathogenesis of these diseases is often difficult to find, and there is a lack of targeted treatment drugs, which brings great harm to the health of patients and brings a great burden to their families and society. Due to the extremely high population base in my country, the absolute number of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/864A61K48/00A61K31/7088A61P21/00A61P9/00
CPCC12N15/113C07K14/4708C12N15/86A61K31/7088A61P21/00A61P9/00C12N2310/20C12N2750/14143C12N2800/107A61K48/005C12N9/22C07K2319/00C12N9/78C12Y305/04005C12N15/90
Inventor 常兴李甲邱晗
Owner WESTLAKE UNIV
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