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Gene drug constructs for the treatment of mucopolysaccharidosis type 3a

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

Active Publication Date: 2021-04-27
北京瑞希罕见病基因治疗技术研究所
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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 [55] , low immunogenicity

Method used

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  • Gene drug constructs for the treatment of mucopolysaccharidosis type 3a
  • Gene drug constructs for the treatment of mucopolysaccharidosis type 3a
  • Gene drug constructs for the treatment of mucopolysaccharidosis type 3a

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Construction of plasmid vector

[0051] In order to construct the pRDAV-CA-SGSH plasmid required for packaging recombinant AAV virus, we first used the pRDAV-CMV ( figure 1 ) as the basis, the CMV promoter in the pRDAV vector was replaced with the self-designed CA promoter (SEQ ID No.1) to obtain the pRDAV-CA vector. Next, clone the artificially synthesized human SGSH (SEQ ID No.2) sequence into the pRDAV-CA vector Kpn I and EcoR Between the I restriction sites, pRDAV-CA-SGSH and the vector were obtained.

[0052] (1) Construction of pRDAV-CA vector

[0053] The human cytomegalovirus early gene enhancer sequence and the chicken β-actin promoter sequence were spliced ​​to obtain the CA promoter sequence, and the sequence information is shown in SEQ ID No.1. Add at both ends of the CA promoter sequence xho I and Kpn I restriction site. After adding restriction sites, the sequence was synthesized by GenScript Biotechnology Co., Ltd., and the synthetic...

Embodiment 2

[0062] Example 2 In vitro expression verification of pRDAV-CA-SGSH vector

[0063] (1) Expression determination of SGSH protein

[0064] The well-grown Huh-7 cells were evenly spread in 9 wells of a six-well cell culture plate, and when the cell density in each well reached 80%, they were transfected with pRDAV-CA-SGSH and 3 wells for each pscAAV-CMR-SGSH (refer to the instructions for the detailed process). After 48 h of transfection, the cells were collected after digestion, and the total protein of the cells was extracted by repeated freezing and thawing followed by centrifugation. The total protein concentrations of transfected pRDAV-CA-SGSH, pscAAV-CMR-SGSH and blank cells were measured using Pierce BCA Protein Aaasy Kit (ThermoFisher, USA), and the detailed process refers to the kit instructions.

[0065] After extracting the total protein of Huh-7 cells, Western Blotting was used to detect the expression of SGSH protein in the cells. For the method, see "Molecular Clo...

Embodiment 3

[0070] Example 3 Preparation and assay of rAAV-SGSH

[0071] (1) Packaging of different serotype recombinant AAV viruses

[0072] Referring to literature [64], the three-plasmid packaging system was used to package and purify the recombinant AAV virus. Briefly, the AAV vector plasmid (pRDAV-CA-SGSH or pscAAV-CMR-SGSH), the helper plasmid (pHelper) and the AAV Rep and Cap protein expression plasmids (pAAV-R2C5, pAAV-R2C9 or pAAV-R2C10) were prepared according to 1: After mixing at a molar ratio of 1:1, HEK293 cells were transfected by the calcium phosphate method. After 48 hours of 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 to obtain rAAV5-CA-SGSH, rAAV9-CA-SGSH, rAAVrh10-CA-SGSH, rscAAV5-CMR-SGSH, rscAAV9-CMR-SGSH, rscAAVrh10-CMR-SGSH.

[0073] (2) Packaging of rAAVDJ-SGSH

[0074] rAAVDJ-CA-SGSH and rAAVDJ-CMR-SGSH were pack...

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Abstract

The invention of this patent provides a novel gene therapy drug construct that can be used for type 3A mucopolysaccharidosis, which can effectively express recombinant human heparan-N-sulfatase (SGSH), and is used to prepare genes mediated by adeno-associated virus vectors Therapeutic pharmaceutical products.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to two recombinant adeno-associated virus vectors carrying SGSH gene expression cassettes and their application in treating type IIIA mucopolysaccharidosis. Background technique [0002] Mucopolysaccharidosis (MPS) is a kind of acidic mucopolysaccharide (also known as glycosaminoglycan, GAG) cannot be degraded or degraded incompletely due to the lack or activity reduction of related acid hydrolase in lysosomes, resulting in Severely disabled and fatal monogenic genetic metabolic diseases caused by the accumulation of GAG and its intermediate metabolites in the body [1] . GAG is a kind of proteoglycan. The main types of its metabolites and their distribution in the body are as follows: (1) dermatan sulfate (DS), which is mainly distributed in skin, ligaments, arteries and heart valve tissues; (2) ) Chondroitin sulfate (CS) (including 4-CS, 6-CS), mainly present in bone, cartilage, corn...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/55C12N15/864A61K48/00A61K38/46A61P3/00
Inventor 马文豪武志杰董哲岳
Owner 北京瑞希罕见病基因治疗技术研究所
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