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AAV vector carrying atp7b gene expression frame and variants and its application

A technology of gene expression and viral vector, applied in the direction of vector, application, nucleic acid vector, etc., can solve the problem of low immunogenicity

Active Publication Date: 2021-12-28
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 viral packaging in the wild-type virus, and does not contain the protein-coding genes in the wild-type virus genome (Salgenik M, et al. Microbiols Spectr . 2015; 3(4).), low immunogenicity

Method used

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  • AAV vector carrying atp7b gene expression frame and variants and its application
  • AAV vector carrying atp7b gene expression frame and variants and its application
  • AAV vector carrying atp7b gene expression frame and variants and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1 Plasmid vector construction

[0064] In order to package the AAV virus used in the invention process of this patent, a series of AAV plasmid vectors need to be constructed. These AAV plasmid vectors can be divided into three categories, the first category is the plasmid vector pAAV2-LP15-EGFP required for the packaging of the control virus; the second category is the AAV plasmid vector pAAV2-LP15-ATP7B of the ATP7B gene expression cassette; AAV plasmid vector pAAV2-LP15-ΔC4ATP7B with short ATP7B gene expression cassette. All AAV plasmid vector constructions are based on the pAAV2neo vector constructed and preserved by our company. The structure of pAAV2neo is as follows: figure 1 shown.

[0065] (1) Construction of plasmid vector pAAV2-LP15-EGFP required for control virus packaging

[0066] First, add an XhoI restriction site "5'-CTCGAG-3'" to the 5' end of the liver-specific promoter LP15 designed and preserved by our company (see SEQ ID NO.1) and add a K...

Embodiment 2

[0074] Example 2 Preparation and assay of recombinant AAV virus

[0075] Referring to literature (Xiao X, et al. J Virol. 1998;72(3):2224-2232.), AAV virus was packaged using a three-plasmid packaging system and cesium chloride density gradient centrifugation, purification and packaging to obtain recombinant AAV virus. Briefly, the AAV vector plasmid (referring to the plasmid whose name contains "pAAV2" in the present invention) (pAAV2-LP15-EGFP, pAAV2-LP15-ATP7B or pAAV2-LP15-ΔC4ATP7B), helper plasmid (pHelper) and AAV Rep and The Cap protein expression plasmid (referring to the plasmid whose name contains "pAAV-R" in the present invention) was mixed according to the molar ratio of 1:1:1, and then transfected into HEK293 cells by the calcium phosphate method. After 48 hours of transfection, the cells were harvested and The culture supernatant was separated and purified by cesium chloride density gradient centrifugation. Packaged and purified to obtain 6 kinds of recombinant ...

Embodiment 3

[0081] Example 3 Different doses of rAAV8-LP15-ATP7B virus injection model animal experiment results

[0082] Taking the rAAV8-LP15-EGFP virus as a control, the recombinant AAV8 virus carrying the ATP7B expression cassette was injected into hepatolenticular via tail vein at high, medium and low levels (1E+10vg / monkey, 3E+10vg / bird and 9E+10vg / bird). Nuclear degeneration model mice, detection of transaminase in serum, copper ion content in urine and copper ion content in liver tissue, ATP7B mRNA expression level in liver, to verify the effectiveness of rAAV8-LP15-ATP7B and the effect of different doses on effectiveness influences. At the same time, wild-type C57BL / 6 mice were set as controls to observe the changes of transaminase levels in normal mice.

[0083] Hepatolenticular degeneration mice atp7b - / - Prepared by mating rats prepared from hepatolenticular degeneration model. Breeding mice were purchased from Jackson Laboratory, USA, and were heterozygous for B6; 129S1-At...

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Abstract

The invention provides a recombinant AAV vector containing an ATP7B gene expression frame or a variant thereof. The AAV vector is intravenously injected into the hepatolenticular degeneration model mice, and it is highly expressed in the liver to produce ATP7B or its variant protein, effectively reducing the copper ion content in the liver and urine of the model mice, and significantly reducing the model small The level of alanine aminotransferase in mouse blood relieves the adverse symptoms caused by the accumulation of copper ions in model mice, and shows the potential for treating hepatolenticular degeneration.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an AAV vector containing an ATP7B gene expression frame and a variant, which is applied to the development of gene therapy drugs for hepatolenticular degeneration. Background technique [0002] Hepatolenticular degeneration, also known as Wilson's disease, is an autosomal single-gene recessive genetic disease characterized by copper metabolism disorder. Basal ganglia damage, kidney damage and corneal pigment ring, etc. (Kodama H, et al. Curr Drug Metab. 2012;13(3): 237-250.). Hepatolenticular degeneration is caused by mutations in the ATP7B gene, which encodes copper-transporting P-type ATPase (ATP7B). ATP7B is a membrane protein expressed in multiple organs, and its main function is to promote the excretion of copper with blood and bile. When the ATP7B gene is mutated, the localization of ATP7B in the cell changes, and its ability to transport copper decreases, resulting in decrea...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/864A61K48/00A61K38/46A61P1/16
CPCC12N15/86A61K48/0025A61K48/005A61K38/46C12Y306/03004A61P1/16C12N2750/14143C12N2800/107C12N2800/60C12N2800/22C12N2830/50
Inventor 田文洪董小岩吴小兵马思思
Owner BEIJING GENECRADLE PHARM CO LTD