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Recombinant adeno-associated virus vector, gene composition and application for treating atherosclerosis

A technology of atherosclerosis and viral vectors, applied in the direction of viruses/bacteriophages, using vectors to introduce foreign genetic material, viruses, etc.

Active Publication Date: 2022-07-05
XIEHE HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI & TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the application of this measure still cannot prevent the occurrence of severe cardiovascular diseases to a large extent, especially for patients with atherosclerosis in the middle and late stages, so it is imperative to find a new direction for the treatment of atherosclerosis

Method used

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  • Recombinant adeno-associated virus vector, gene composition and application for treating atherosclerosis
  • Recombinant adeno-associated virus vector, gene composition and application for treating atherosclerosis
  • Recombinant adeno-associated virus vector, gene composition and application for treating atherosclerosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The preparation and packaging process of the adeno-associated virus vector of AAV8-TBG-LOX-1 in Example 1 are as follows:

[0038] (1) Obtaining the target gene: It was found from the Gene database in NCBI that the LOX-1 gene was mainly expressed in human umbilical vein endothelial cells. The cells were used to extract RNA (Trizol extraction), and after reverse transcription (reverse transcription kit) Use synthesized primers with protected bases and restriction sites (see the end of the text for the design method of primers with restriction sites) for PCR (use high-fidelity enzymes for PCR to avoid mismatches). PCR conditions were adjusted and optimized as needed. The PCR product was then subjected to agarose electrophoresis (1%), and the gel pieces corresponding to the target gene fragments were excised, and the gel was recovered (using a gel recovery kit), and the DNA concentration was measured after the gel recovery. At this point, we have obtained a gene fragment ...

Embodiment 2

[0053]Example 2: The adeno-associated virus vector of AAV8-TBG-LOX-1 with a virus titer of 2×1013vg(virus genomes) / ml prepared in Example 1 was diluted with sterile PBS buffer to 2×1011vg / ml Only / 100μl, take 10ul virus stock solution + 90ul sterile PBS, prepare it in the ultra-clean bench, mix it with a pipette and mix it well, and store it in an ice box for low temperature storage. The titer had an effect; then, 100ul of virus dilution was drawn with an insulin needle and injected into the mice through the tail vein, and the transfection effect was waited for. Under normal circumstances, the virus can be stored in a -80 refrigerator. After injection, the virus is stably expressed about 2 weeks after injection, and the duration of stable expression can be at least 2 months.

[0054] The following is a combination of different experiments to study the expression of AAV8-TBG-LOX-1 adeno-associated virus vector in the liver, its transfection effect and its therapeutic effect on a...

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Abstract

The invention provides a recombinant adeno-associated virus vector, gene composition and application of the gene composition for treating atherosclerosis. The recombinant adeno-associated virus vector is obtained by inserting lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) into the adeno-associated virus vector, and combining it with the TBG promoter to form a gene composition for the preparation of treatment Gene therapy injections for atherosclerosis. The sequence of the LOX-1 gene is shown in SEQ ID NO: 1 in the sequence listing. The present invention utilizes the adeno-associated virus vector to efficiently introduce the drug effect element into the body through intravenous injection, realizes the ectopic high-efficiency expression of the therapeutic effect protein LOX-1 of the drug effect element expression product, and uses the TBG promoter to transfer the drug effect element that is not originally expressed in the liver through the TBG promoter. The LOX-1 receptor is successfully expressed in hepatocytes. With the help of the liver's powerful lipid metabolism and metabolic pathways, it can phagocytose and clear the overloaded OX-LDL in the circulation of patients with atherosclerosis, thereby inhibiting the progression of atherosclerosis.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant adeno-associated virus vector, gene composition and application for treating atherosclerosis, in particular to an AAV virus vector system that mediates the specific ectopic expression of LOX-1 in the liver , the LOX-1 receptor that is not originally expressed in the liver is successfully expressed in hepatocytes. With the help of the liver's powerful lipid metabolism and metabolic pathways, the overloaded OX-LDL in the circulation of patients with atherosclerosis is phagocytosed and cleared, thereby inhibiting the Atherosclerosis progression. Background technique [0002] Atherosclerosis is a systemic pathological change caused by abnormal deposition of cholesterol-rich lipoproteins on the blood vessel wall. Continued to rise by 21.1%. In more than 100 years of atherosclerosis research and practice, modern clinical practice has been able to use statin drugs that inhib...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/864C12N15/12A61K48/00A61K38/17A61P9/10
CPCC12N15/86C07K14/705A61K48/0008A61K48/0058A61K38/177A61P9/10C12N2750/14143C12N2800/107C12N2830/008
Inventor 王志文郭小朋魏宇淼曾状林孙谛曹炳鑫张青杜高辉陈娟陈磊任衍乔张卫华景熙瑞李嘉熙
Owner XIEHE HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI & TECH UNIV
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