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Gene therapy medicament for hyperlipidemia

A technology of gene therapy and gene expression, applied in gene therapy, drug combination, genetic engineering, etc., can solve the problem of low immunogenicity

Active Publication Date: 2018-06-01
BEIJING GENECRADLE PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

First, the AAV vector only retains the two ITR sequences required for packaging in the wild-type virus, and does not contain the protein-coding genes in the wild-type virus genome (Salgenik M, et al. Microbiol Spectr. 2015; 3(4).), Immunization Low originality

Method used

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  • Gene therapy medicament for hyperlipidemia
  • Gene therapy medicament for hyperlipidemia
  • Gene therapy medicament for hyperlipidemia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Plasmid vector construction

[0055] In order to construct the pscAAV-CAM-OFat-1 and pscAAV-CAM-OFat-1-142T plasmids required for packaging recombinant AAV viruses, we first used the pAAV2neo preserved by the company as the basis and used the self-designed CAM promoter (SEQ IDNo. 1) Replace the CMV promoter in the pAAV2neo vector, and replace one of the pAAV2neo vectors with a mutated ITR sequence (named ΔITR) (SEQ ID No.2) that deletes the trs (terminal resolution site) and D sequences in the AAV2 ITR flanking ITR sequences to obtain the pscAAV-CAM vector. Next, the artificially synthesized OFat-1 (SEQ ID No.3) and OFat-1-142T (SEQ ID No.4) sequences were respectively cloned into the KpnI and EcoRI and KpnI and BglII restriction sites of the pscAAV-CAM vector Between, pscAAV-CAM-OFat-1 and pscAAV-CAM-OFat-1-142T vectors were obtained.

[0056] (1) Construction of pscAAV-CAM vector

[0057] The human cytomegalovirus early gene enhancer sequence, the chicken...

Embodiment 2

[0062] Example 2 Preparation and assay of recombinant AAV virus

[0063] Referring to the literature (Xiao X, et al. J Virol. 1998;72(3):2224-2232.), the three-plasmid packaging system was used to package and purify the recombinant AAV virus. Briefly, the AAV vector plasmid (pscAAV-CAM-OFat-1 or pscAAV-CAM-OFat-1-142T), the helper plasmid (pHelper), and the AAV Rep and Cap protein expression plasmids (pAAV-R2C1, pAAV-R2C8 or pAAV -R2C9) mixed according to the molar ratio of 1:1:1, transfect HEK293 cells by calcium phosphate method, after 48 hours of transfection, harvest the cells and culture supernatant, and use cesium chloride density gradient centrifugation to separate and purify the recombinant AAV virus . Packaged and purified to obtain scAAV1-CAM-OFat-1, scAAV1-CAM-OFat-1-142T, scAAV8-CAM-OFat-1, scAAV8-CAM-OFat-1-142T, scAAV9-CAM-OFat-1 and scAAV9-CAM - 6 kinds of recombinant viruses such as OFat-1-142T.

[0064] Quantitative PCR method was used to measure the genome...

Embodiment 3

[0069] Example 3 Establishment of Rat Hyperlipidemia Model

[0070] References (Zhao Jinming, et al. Chinese Pharmacology and Clinic. 2012;28(1):177-180.), construct a hyperlipidemia model in rats. First prepare a high-fat emulsion, melt 30g of self-made lard in a water bath at 50°C, add 5g of cholesterol, 5g of egg yolk powder, 1g of sodium hyocholate, 0.5g of propylthiouracil, 5g of glucose and 2g of emetic in turn while stirring. Temperature -80, after uniform emulsification, distill the volume to 200mL with distilled water to obtain the high-fat emulsion needed to induce the hyperlipidemia model in rats. Purchased 120 SPF-grade Wistar rats from Beijing Huafukang Biotechnology Co., Ltd., half male and half male, weighing 180-220 g, randomly selected 10 male and female rats as the control group, and the remaining 100 rats as the experimental group. The high-fat emulsion was melted and stirred evenly in a 50°C water bath, and the experimental group was given the high-fat emu...

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Abstract

The invention provides a recombinant adeno-associated virus mediated medicament for treating hyperlipidemia. A recombinant adeno-associated virus vector carries a gene expression cassette of optimizedFat-1 (oFat-1 for short) containing a human miR-142-3p target sequence. An in vivo experiment shows that the recombinant adeno-associated virus vector can be efficiently introduced into bodies as well as continuously and stably express Fat-1 protein, in order to increase content of omega-3 polyunsaturated fatty acid in vivo, reduce contents of total cholesterol, triglycerides and low density lipoprotein in vivo, and improve content of high density lipoprotein. A result shows that the recombinant adeno-associated virus vector is hopeful to be developed into a novel medicament for treating hyperlipidemia.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a gene therapy drug for hyperlipidemia with a recombinant adeno-associated virus vector carrying an oFat-1 gene expression frame. Background technique [0002] Hyperlipidemia is a disease caused by an increase in blood lipid levels caused by lipid metabolism disorders in the body, that is, one or more lipid components in the blood are abnormally increased and thus lead to a series of clinical pathological manifestations. Usually, a serum total cholesterol > 5.72 mmol / L and a triacylglycerol > 2.3 mmol / L can be diagnosed as hyperlipidemia. [0003] The prevalence of dyslipidemia in my country is as high as 18.6%, about 200 million people, which has caused great harm to people's health. This is because hyperlipidemia is a major risk factor for many diseases. First of all, hyperlipidemia is one of the main risk factors for coronary heart disease. The direct damage of hyperlipid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/864A61K48/00A61P3/06
CPCA61K48/005C12N9/001C12N15/86C12N2750/14143
Inventor 田文洪董小岩吴小兵马思思
Owner BEIJING GENECRADLE PHARM CO LTD