Exon splicing enhancer related to Duchenne muscular dystrophy, sgRNA, gene editing tool and application

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

Active Publication Date: 2020-12-11
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 enhancer related to Duchenne muscular dystrophy, sgRNA, gene editing tool and application
  • Exon splicing enhancer related to Duchenne muscular dystrophy, sgRNA, gene editing tool and application
  • Exon splicing enhancer related to Duchenne muscular dystrophy, sgRNA, gene editing tool and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 AAV virus carrying gene editing tool

[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 thereto) was double-digested based on the restriction sites of XhoI and NotI. Simultaneously design the amino acid sequence of AID and Cas9 fusion protein in the gene editing tool, the amino acid sequence and nucleic acid sequence are respectively shown in SEQ ID NO.1 and SEQ ID NO.2. After codon optimization, double-stranded DNA fragments were directly synthesized, and elements such as the Syn100 promoter and tailing signal were connected to the AAV backbone vector to obtain an AAV vector plasmid expressing the AID-Cas9 mutant fusion protein. Its sequence is SEQ ID NO.3 shown. In addition, using primer synthesis and PCR methods...

Embodiment 2

[0049] Embodiment 2 Utilizes AAV carrying gene editing tools to treat DMD model mice in vivo

[0050] In this example, Dmd-E4, a novel DMD mouse disease model with abnormal cardiac function, was selected. This model can be purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., but is not limited thereto. Dmd-E4 showed myocardial 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 simulates the cardiac pathological process of DMD patients well. For this model, we applied cytosine deaminase and Cas9 to design gene editing tools, targeting exons carrying pathogenic mutations, and inducing mutations near their 5' splicing sites, causing them to skip reads. On the basis of not affecting the open reading frame of the protein, the expression of Dystrophin protein is retained to the maximum extent, and its biological function is restored.

[0051] Specifically, the method of E...

Embodiment 3

[0065] Example 3 The gene editing of human induced pluripotent stem cell iPSC DMD model 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 exon 50 Exon50 of the Dystrophin-encoding gene DMD, making the Dystrophin protein encoding The sequence produces a frameshift mutation, thereby constructing a mutation type that simulates a DMD patient, and becomes a good DMD disease model cell. For this cell, we designed the sequence of AID and Cas9 fusion protein and the corresponding sgRNA, which is used to target a series of potential regulatory exon splicing elements of exon 51 Exon51 of the DMD gene, which was used in this example The sgRNA is sgRNA-12 as shown in SEQ ID No.19 and sgRNA-...

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Abstract

The invention discloses an exon splicing enhancer related to Duchenne muscular dystrophy, sgRNA, a gene editing tool and an application. The related exon splicing enhancer, the sgRNA and the gene editing tool can be applied to preparation of drugs for treating Duchenne muscular dystrophy. According to the gene editing tool designed on the basis of mutants of cytosine deaminase AID and Cas9 mutants, an adeno-associated virus AAV serves as a carrier, and site-specific modification can be conducted on a mammal genome. By optimizing the coding nucleic acid sequence and the element composition structure of the editing tool, fixed-point targeted transformation of mammalian genetic material DNA can be efficiently realized; and through performing targeted genetic manipulation on the nucleic acid sequence carrying disease mutation, the pathogenic mutation cannot be retained in a mature protein amino acid sequence or cannot exert the function, so that the purpose of treating various genetic mutation hereditary rare diseases is achieved, and the advantages of high efficiency, safety and stability are achieved.

Description

technical field [0001] The invention belongs to the field of gene therapy, and relates to an exon splicing enhancer, sgRNA, and gene editing tools related to Duchenne muscular dystrophy, which can be used in mammals (experimental animal models and human patients) to cause disease in mammals (experimental animal models and human patients) for gene mutation genetic diseases Transformation therapy for mutations. In particular, it involves the gene editing treatment 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 that account for 0.065% to 0.1% of the total population in a certain area. The pathogenesis of such diseases is often difficult to find, and there is a lack of targeted treatment drugs, which bring great harm to the patient's health and a great burden on their families and society. Due to the extremely high popu...

Claims

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Application Information

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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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