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An adeno-associated virus vector carrying MG53 gene that can alleviate the symptoms of heart failure in mice

A viral vector and virus technology, applied in the field of construction and evaluation of therapeutic effects, can solve the problems of weak targeting and low transduction efficiency.

Active Publication Date: 2018-03-30
汉恒生物科技(上海)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

With the deepening of the research and development of AAV vectors, the problems of immunogenicity, poor targeting and low transduction efficiency in gene therapy, etc. can be solved by modifying AAV coat protein, using pharmaceutical preparations, biological materials and other methods. got a certain solution

Method used

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  • An adeno-associated virus vector carrying MG53 gene that can alleviate the symptoms of heart failure in mice
  • An adeno-associated virus vector carrying MG53 gene that can alleviate the symptoms of heart failure in mice
  • An adeno-associated virus vector carrying MG53 gene that can alleviate the symptoms of heart failure in mice

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Construction of 293 cell line stably and highly expressing MG53 gene

[0049] The 293 cell line with stable and high expression of MG53 gene was constructed by adeno-associated virus vector.

[0050] 1 Experimental materials

[0051] Reagent name

Manufacturer

Escherichia coli strain DH5α

Tiangen

restriction endonuclease

Fermentas

T4 ligase

Fermentas

Plasmid DNA Small, Massive Extraction Kit

Kang Wei Century

Gel Recovery Kit

Axygen

[0052] Agarose

Biowest

DNA ladder

GeneRay

[0053] Carrier and target gene information

[0054] 1) The pHBAAV-CMV-IRES-Zsgreen vector map is as follows figure 1 Shown, wherein EcoRI and BamHI are insertion sites, and its sequence is shown in SEQ ID NO.:3.

[0055] The pHBAAV-CMV-IRES-Zsgreen vector has a CMV promoter to promote the expression of the target gene, and contains the Zsgreen gene as a marker.

[0056] 2) The polyn...

Embodiment 2

[0070] Example 2 Adeno-associated virus packaging

[0071] 1. Experimental process

[0072] The adeno-associated virus shuttle plasmid and its auxiliary packaging original vector plasmid were prepared. The three plasmid vectors were extracted with high purity and no endotoxin, and co-transfected into AAV-293 cells. After 8 hours of transfection, they were replaced with complete medium, and after 48 hours of culture, The cell supernatant rich in adeno-associated virus particles was collected, the virus supernatant was filtered with a 0.45um filter (Millpore Company), and the virus supernatant was concentrated by ultracentrifugation. After it is concentrated, a high-titer adeno-associated virus concentrate is obtained.

[0073] 2. Experimental materials

[0074] 2.1 Adeno-associated virus vectors, packaging cells and strains

[0075] The viral packaging system is a three-plasmid system consisting of RC (the plasmid sequence is shown in SEQ ID NO.: 6), pHelper (the plasmid seq...

Embodiment 3

[0097] Example 3 Injection of adeno-associated virus with high expression of MG53 into heart failure model mice improves the symptoms of heart failure in mice

[0098] 1. Experimental materials

[0099] C57BL / 6 mice (the plasmid can be purchased from Shanghai Slack Experimental Animal Co., Ltd.).

[0100] 2. Experimental method

[0101] 2.1 Modeling of heart failure in mice

[0102] The right anterior external incision was taken to open the chest, and the ascending aorta was exposed, and the ascending aorta was constricted with a constriction ring at 1 cm above the aorta, and the heart failure of the mice could be induced after 4 weeks.

[0103] 2.2 Tail vein injection of pHBAAV-CMV-IRES-Zsgreen-MG53 and control adeno-associated virus

[0104] Using a 5-gauge needle syringe, inject 1x10 per mouse 11 The adeno-associated virus of v.g, the control adeno-associated virus is AAV-GFP (adeno-associated virus with green fluorescent protein).

[0105] 2. Experimental results

[...

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Abstract

The present invention discloses an adeno-associated virus vector carrying the MG53 gene that can alleviate the symptoms of heart failure in mice. After in-depth research by the inventor, it was unexpectedly found that the adeno-associated virus carrying the MG53 gene was expressed in mice with heart failure Injection of adeno-associated virus carrying this gene can effectively alleviate the symptoms of heart failure in mice. All cardiac functions and other indexes of the mice were effectively improved, which indicated that the technical scheme of the present invention was applicable to the treatment of heart failure diseases.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to the construction of an adeno-associated virus vector for treating heart failure and the evaluation of the therapeutic effect. Background technique [0002] 1. The life cycle of AAV virus: [0003] Adeno-associated virus (AAV) is a member of the Parvoviridae family. Members of this family are a class of tiny, non-enveloped, icosahedral viruses. The diameter of virus particles is between 20 and 26 nm, and contains a linear single-stranded DNA genome with a size between 4.7 and 6 kb. Parvoviruses have been isolated from insects to humans. AAV virus belongs to the category of dependent viruses (Dependovirus). It was originally a contaminating component found in purified adenovirus fluid, hence the name. [0004] The life cycle of AAV has two distinct intracellular phases. In the absence of helper virus, AAV virus particles enter the cell, and after uncapsiding, the regulat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/01C12N15/12C12N15/864A61K48/00A61P9/04
Inventor 陈意雄蔡晓龙邹杰白易鑫王颖芝韦唯王丽莎朱鹏飞张静怡
Owner 汉恒生物科技(上海)有限公司
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