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A gene medicine for the treatment of severe hypertriglyceridemia

A technology of gene medicine and gene expression, applied in drug combination, gene therapy, pharmaceutical formula, 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

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

Method used

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  • A gene medicine for the treatment of severe hypertriglyceridemia
  • A gene medicine for the treatment of severe hypertriglyceridemia
  • A gene medicine for the treatment of severe hypertriglyceridemia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1 Plasmid vector construction

[0073] In order to construct the pscAAV8-LP15-ApoC2 plasmid required for packaging recombinant AAV virus, we first use the pscAAV8-LP15-EGFP vector kept by the company [the ITR sequence on one side of the vector is trs (terminal resolution site) in the ITR of AAV2 deleted and the mutant sequence of D sequence, named ΔITR, as shown in SEQ ID No.7] as the basis, select the liver-specific high-efficiency expression promoter to regulate the expression of ApoC2 gene, optimize the sequence of ApoC2 coding region, and introduce in the optimized sequence Short introns improve gene transcription efficiency. Introducing four completely complementary target sequences of miR-142-3p in the 3'UTR region, reduced the expression of ApoC2 gene in antigen-presenting cells, and weakened the immune response caused by the overexpression of ApoC2. A short liver-specific enhancer was inserted at the 3' end of the expression frame to enhance the transcr...

Embodiment 2

[0078] Example 2 Preparation and assay of recombinant AAV virus

[0079] Three different packaging systems were used to package scAAV8-LP15-ApoC2 recombinant virus respectively.

[0080] Packaging system 1 Three-plasmid co-transfection HEK293 packaging system

[0081] 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 plasmid (pscAAV8-LP15-ApoC2), helper plasmid (pHelper) and AAV Rep and Cap protein expression plasmid (pAAV-R2C8) were mixed at a molar ratio of 1:1:1, followed by the calcium phosphate method HEK293 cells were transfected, and 48 hours after transfection, the cells and culture supernatant were harvested, and the recombinant AAV virus was isolated and purified by cesium chloride density gradient centrifugation. Packaged and purified ...

Embodiment 3

[0091] Example 3 ApoC2 - / - Establishment of the hamster model

[0092] The ApoC2 knockout hamster model was provided by Mr. Liu Guoqing from Peking University Health Science Center. Patent name: Kit and method for constructing ApoC2 gene knockout hamsters. Patent application number: 201811179674.7.

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Abstract

The invention provides a recombinant adeno-associated virus-mediated gene medicine for treating severe hypertriglyceridemia and its complication acute pancreatitis. The recombinant adeno-associated virus vector carries the ApoC2 gene expression cassette containing the human miR‑142‑3p target sequence. In vivo experiments show that the recombinant adeno-associated virus vector can be efficiently introduced into the body, express apolipoprotein C2 continuously and stably, significantly reduce the content of triglyceride in blood and restore it to normal, and effectively prolong the survival period of hamsters. The results suggest that the recombinant adeno-associated virus vector gene medicine can be used to treat severe hypertriglyceridemia and its complication acute pancreatitis.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant adeno-associated virus vector carrying the ApoC2 gene expression cassette of the human miR-142-3p target sequence, which is used for the treatment of severe hypertriglyceridemia and its complication acute pancreatitis gene medicine. Background technique [0002] Hypertriglyceridemia (HTG) is a heterogeneous triglyceride protein synthesis or degradation disorder. Deposits gradually form small plaques, namely atherosclerosis. According to the etiology, hypertriglyceridemia can be divided into primary and secondary. Primary hypertriglyceridemia includes familial hypertriglyceridemia, familial mixed dyslipidemia, familial abnormal β - Lipoproteinemia, etc.; the etiology of secondary hypertriglyceridemia mainly includes metabolic diseases, certain disease states, hormones and drugs, etc. [0003] Hypertriglyceridemia is the second leading cause of acute pancreatitis (AP) ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00A61K38/17A61P3/06A61P1/18A61P37/06
CPCA61K48/005A61K38/1709A61P3/06A61P1/18A61P37/06
Inventor 田文洪董小岩马思思
Owner BEIJING GENECRADLE PHARM CO LTD
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