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Gene therapy medicament for treating hyperuricemia

A gene drug, uric acid technology, 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

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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  • Gene therapy medicament for treating hyperuricemia
  • Gene therapy medicament for treating hyperuricemia
  • Gene therapy medicament for treating hyperuricemia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 Plasmid vector construction

[0063] In order to construct the pscAAV-CAM-AUO and pscAAV-CAM-AUO-142T plasmids required for packaging recombinant AAV viruses, we first replaced the pAAV2neo with the self-designed CAM promoter (SEQ ID No.6) based on the company’s preserved pAAV2neo For the CMV promoter in the pAAV2neo vector, replace one side of the ITR sequence in the pAAV2neo vector with a mutated ITR sequence (named ΔITR) (SEQ ID No.8) that deletes the trs (terminal resolution site) and D sequences in the ITR of AAV2 , to obtain the pscAAV-CAM vector. Next, artificially synthesized CS SP-AUO (SEQ ID No.5) and CS SP-AUO-142T (SEQ ID No.9) (CS SP-AUO plus four tandem miR-142-3p target sequences , between the two are connected by EcoRI restriction site) sequences were cloned into the pscAAV-CAM vector between KpnI and EcoRI and KpnI and BglII restriction sites, respectively, to obtain pscAAV-CAM-AUO and pscAAV-CAM-AUO-142T carrier.

[0064] (1) Construction ...

Embodiment 2

[0070] Example 2 Preparation and assay of recombinant AAV virus

[0071] References (Xiao X, et al . J Virol. 1998; 72(3): 2224-2232.), using a three-plasmid packaging system to package recombinant AAV virus, and using cesium chloride density gradient centrifugation to separate, purify and package AAV virus. Briefly, the AAV vector plasmid (pscAAV-CAM-AUO or pscAAV-CAM-AUO-142T), the helper plasmid (pHelper), and the AAV Rep and Cap protein expression plasmids (pAAV-R2C1, pAAV-RC, 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-AUO, scAAV1-CAM-AUO-142T, scAAV2-CAM-AUO, scAAV2-CAM-AUO-142T, scAAV8-CAM-AUO, scAAV8-CAM-AUO-142T, scAAV9-CAM-AUO and scAAV9-CAM-AUO-142T and other 8 kind...

Embodiment 3

[0077] Example 3 Establishment of mouse hyperuricemia model

[0078] References (Wu X, et al . Proc Natl Acad Sci USA. 1994; 91(2): 742-746.), to establish a mouse model of hyperuricemia. The homologous recombination method is used to knock out the urate oxidase gene in mouse embryonic stem cells to prepare transgenic mice heterozygous for the urate oxidase gene. The preparation of transgenic mice was completed by Beijing Biocytogen Biotechnology Co., Ltd. The urate oxidase gene heterozygous mouse C57BL / 6J prepared by Beijing Biocytogen Biotechnology Co., Ltd. + / - Transferred to Beijing Wujiahe Institute of Molecular Medicine Co., Ltd. Mice heterozygous for the urate oxidase gene appeared normal. Male and female C57BL / 6J heterozygous for the urate oxidase gene + / - Mating to obtain homozygous mice C57BL / 6J lacking urate oxidase gene - / - , as a model of hyperuricemia. Screening to identify male and female C57BL / 6J by measuring mouse phenotype + / - Homozygous mice C57BL / 6J...

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Abstract

The invention provides a recombinant adeno-associated virus mediated medicament for treating hyperuricemia. A recombinant adeno-associated virus vector carries a gene expression cassette of artificialurate oxidase (AUO for short) containing a human miR-142-3p target sequence. An internal experiment shows that the recombinant adeno-associated virus vector can be efficiently introduced into body, in order to continuously and stably express urate oxidase, reduce content of uric acid, and effectively alleviate gout symptom due to excessive uric acid in blood. A result shows that the recombinant adeno-associated virus vector is hopeful to be developed into a novel medicament for treating hyperuricemia.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a hyperuricemia gene therapy drug carrying an artificially designed urate oxidase (AUO) gene expression cassette with a recombinant adeno-associated virus vector. Background technique [0002] With the improvement of economic level and the change of people's lifestyle and dietary structure, the incidence of gout and hyperuricemia is increasing year by year worldwide (Guo Lixin. Drug Evaluation. 2014; 11(1): 21-23. Weaver AL. Cleve Clin J Med. 2008; 75(s5): s9-s12.). Hyperuricemia is caused by the disorder of purine metabolism leading to the increase of uric acid production and / or the decrease of excretion, which makes the concentration of uric acid in the blood higher than the normal range. It is the biochemical basis of gout (Zhang Yumei, et al. Medical Review. 2012; 18( 15): 2441-2444.), the clinical diagnosis standard is under normal purine diet, male blood uric acid content> ...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N15/864A61K48/00A61K31/711A61P19/06
CPCA61K48/005C12N9/0048C12N15/86C12N2750/14143C12Y107/03003
Inventor 田文洪董小岩吴小兵马思思
Owner BEIJING GENECRADLE PHARM CO LTD
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