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AAV vector carrying truncated dystrophin gene expression cassette, preparation method and application

A dystrophin, expression cassette technology, applied in the directions of botanical equipment and methods, biochemical equipment and methods, applications, etc., can solve the problems of muscle cell membrane rupture and tear, ion gradient change, increased membrane instability, etc.

Active Publication Date: 2022-02-01
北京京谱迪基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After 48 weeks of treatment, subjects taking ataluren 40 mg / (kg d) orally (walking distance decreased by only 13 m) decreased more slowly compared with placebo (declined by 44 m), and no serious adverse events were reported during the course
The second ataluren phase III clinical trial completed in 2017 failed to achieve the expected stable 6-min walking distance, and most DMD patients had no significant improvement in symptoms (McDonald CM, et al. Lancet.2017; 390(10101): 1489- 1498.)
[0029] 2.1.4 Targeting relevant signaling pathways / molecules Dystrophin loss causes disruption and tearing of muscle cell membranes, leading to increased membrane instability over time, altered critical ion gradients and disruption of second messenger pathways
Borrowing the principle that miRNA inhibits gene expression (Kim VN. Nat Rev Mol Cell Biol.2005;6(5):376-385.), miR-122 molecules in liver cells can inhibit the expression of mini-dystrophin protein, significantly reducing the expression of mini-dystrophin -Expression of dystrophin protein in liver

Method used

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  • AAV vector carrying truncated dystrophin gene expression cassette, preparation method and application
  • AAV vector carrying truncated dystrophin gene expression cassette, preparation method and application
  • AAV vector carrying truncated dystrophin gene expression cassette, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0113] Example 1 Plasmid vector construction

[0114] In order to construct pAAV2-SCMV-Gluc, pAAV2-mMCK-Gluc, pAAV2-HSA-Gluc, pAAV2-MSP-Gluc, pAAV2-SCMV-mini-dystrophin, pAAV2-mMCK-mini-dystrophin, pAAV2 required for obtaining recombinant AAV virus -HSA-mini-dystrophin, pAAV2-MSP-mini-dystrophin, pAAV2-mMCK-mini-dystrophin-122T, pAAV2-HSA-mini-dystrophin-122T, pAAV2-MSP-mini-dystrophin-122T plasmids, we first use the company Saved pAAV2neo ( figure 1 ) as the basis, the secretory luciferase gene coding sequence (Gaussialuciferase, Gluc) was inserted into the pAAV2neo vector to obtain pAAV2-CMV-Gluc ( figure 2 ). Then use the SCMV promoter, mMCK promoter, HSA promoter and MSP promoter sequence to replace the CMV promoter sequence in the pAAV2-CMV-Gluc vector to obtain pAAV2-SCMV-Gluc ( image 3 ), pAAV2-mMCK-Gluc ( Figure 4 ), pAAV2-HSA-Gluc ( Figure 5 ), pAAV2-MSP-Gluc ( Image 6 ).

[0115] Next, pAAV2-SCMV-Gluc ( image 3 ), pAAV2-mMCK-Gluc ( Figure 4 ), pAAV2-...

Embodiment 2

[0136] Example 2 Preparation and assay of recombinant AAV virus

[0137] References (Xiao X, et al . J Virol. 1998;72(3):2224-2232.), the three-plasmid packaging system was used to package the recombinant AAV virus, and the cesium chloride density gradient centrifugation method was used to separate, purify and package the AAV virus. Briefly, AAV vector plasmids (pAAV2-SCMV-Gluc, pAAV2-mMCK-Gluc, pAAV2-HSA-Gluc, pAAV2-MSP-Gluc, pAAV2-SCMV-mini-dystrophin, pAAV2-mMCK-mini-dystrophin, pAAV2-HSA -mini-dystrophin, pAAV2-MSP-mini-dystrophin, pAAV2-mMCK-mini-dystrophin-122T, pAAV2-HSA-mini-dystrophin-122T, pAAV2-MSP-mini-dystrophin-122T), helper plasmid (pHelper) and AAV Rep and Cap protein expression plasmids (pAAV-DJ, pAAV-R2C8, pAAV-R2C9 or pAAV-R2Crh74) were mixed according to the molar ratio of 1:1:1, and transfected into HEK293 cells by calcium phosphate method for 48 hours. Finally, the cells and culture supernatant were harvested, and the recombinant AAV virus was isolated ...

Embodiment 3

[0150] Example 3 In vitro promoter screening experiment

[0151] Using the three-plasmid packaging system, pAAV2-SCMV-Gluc, pAAV2-mMCK-Gluc, pAAV2-HSA-Gluc and pAAV2-MSP-Gluc were packaged to obtain rAAVDJ-SCMV-Gluc, rAAVDJ-mMCK-Gluc, rAAVDJ-HSA-Gluc and 4 kinds of viruses such as rAAVDJ-MSP-Gluc, see Example 2 for details. The expression of Gluc is regulated by different promoters of the 4 kinds of viruses respectively. The function of the promoter was verified by three kinds of cells including mouse muscle-derived myoblast C2C12 cells, human embryonic kidney cells HEK293 cells and liver cancer cells Huh7 cells.

[0152] The cultured C2C12 cells, HEK293 cells and Huh7 cells were divided into 4×10 4 The density of cells / well was inoculated into 96-well cell culture plates respectively. On the second day after inoculation, the four viruses were divided into 1 × 10 4 The infectious dose of vg / cell infected C2C12 cells, HEK293 cells or Huh7 cells. Three replicate wells were m...

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Abstract

The invention provides a series of recombinant adeno-associated viruses carrying truncated dystrophin gene expression cassettes. In vivo experiments show that the recombinant adeno-associated virus vector can be efficiently introduced into the body, continuously and stably express truncated dystrophin, reduce creatine kinase in the blood of animal models, and restore its muscle function. The results suggest that the recombinant adeno-associated virus vector may be developed into a new drug for treating Duchenne muscular dystrophy.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a gene drug for Duchenne muscular dystrophy carrying an artificially designed truncated dystrophin gene expression frame carried by a recombinant adeno-associated virus vector. Background technique [0002] Duchenne muscular dystrophy (DMD) is an X-chromosome-linked recessive hereditary myopathy caused by a pathogenic variant of the dystrophin gene. DMD). The DMD gene is one of the largest human genes, located at Xp21, encoding dystrophin protein, that is, dystrophin protein. The gene spans 2.5Mb in the genome sequence, accounting for 0.1% of the total genome sequence length and 1.5% of the total length of the X chromosome. 99% of the gene sequence is composed of introns, and the coding region includes 79 exons Its mRNA sequence is 14kb long, and it is mainly expressed in skeletal muscle and cardiac muscle, and a small amount is expressed in brain tissue. About 60% to 65% of DMD / B...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/864A61K48/00A61P21/00
CPCC12N15/86A61K48/0008A61P21/00C12N2750/14143
Inventor 田文洪董小岩吴小兵马思思
Owner 北京京谱迪基因科技有限公司