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Gene drug for X chromosome-linked myotubular myopathy

A gene expression and microtubule technology, applied in gene therapy, drug combination, genetic engineering, etc., can solve the problem of reducing the expression of MTM1 protein

Active Publication Date: 2018-10-02
BEIJING GENECRADLE PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Borrowing the principle that miRNA inhibits gene expression (Kim VN. Nat Rev Mol Cell Biol.2005;6(5):376-385.), miR-122 molecules in liver cells can inhibit the expression of MTM1 protein, significantly reducing the expression of MTM1 protein in Expression in liver

Method used

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  • Gene drug for X chromosome-linked myotubular myopathy
  • Gene drug for X chromosome-linked myotubular myopathy
  • Gene drug for X chromosome-linked myotubular myopathy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Plasmid vector construction

[0045] In order to construct the pAAV2-MCK-EGFP, pAAV2-MCK-hMTM1 and pAAV2-MCK-hMTM1-122T-142T plasmids required for packaging recombinant AAV viruses, we first used the pAAV2neo ( figure 1 ) as the basis, reference (Shield MA, et al. Mol Cell Biol. 1996; 16(9): 5058-5068.) Replace the CMV promoter in the pAAV2neo vector with the MCK promoter (SEQ IDNo.1) to obtain pAAV2 -MCK. Next, the DNA sequence containing the enhanced green fluorescent protein coding sequence (SEQ ID No.2), the artificially optimized and synthesized hMTM1 sequence (SEQ ID No.3) and the hMTM1-122T-142T sequence (SEQ ID NO.4 ) (2 human miR-122 fully complementary target sequences and 2 human miR-142-3p fully complementary target sequences were added sequentially after the stop codon of the hMTM1 sequence) respectively cloned into KpnI and EcoRI and KpnI and KpnI of the pAAV2-MCK vector Between the BglII restriction sites, pAAV2-MCK-EGFP, pAAV2-MCK-hMTM1 and ...

Embodiment 2

[0056] Example 2 Preparation and assay of recombinant AAV virus

[0057] 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 (pAAV2-MCK-EGFP, pAAV2-MCK-hMTM1 or pAAV2-MCK-hMTM1-122T-142T), helper plasmid (pHelper) and AAV Rep and Cap protein expression plasmid (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 6 kinds of recombinants including AAV8-MCK-EGFP, AAV8-MCK-hMTM1, AAV8-MCK-hMTM1-122T-142T, AAV9-MCK-EGFP, AAV9-MCK-hMTM1, AAV9-MCK-hMTM1-122T-142T Virus...

Embodiment 3

[0064] Embodiment 3 Animal experiments

[0065] X-linked myotubular myopathy mouse model preparation breed mice, purchased from The Jackson Laboratory, USA, trade name B6.Cg- Mtm1 tm1Itl / J, conservation number 018153. The sex of this mouse is female, the X chromosome linked mtm1 (myotubularmyopathy gene 1) gene exon 4 mutation heterozygosity, the mutant mtm1 gene cannot express and produce normal MTM1 protein (Pierson CR, et al. Hum Mol Genet. 2012;21 (4):811-25. ). After the breeding mice were crossed with male wild-type C57BL / 6J mice, the newborn male mice were tested for mtm1 gene mutation. For the method, refer to the literature (Buj-BelloA, et al. PNAS. 2002; 99(23):15060-15065.) . Male mice with mtm1 gene mutation were selected for subsequent experiments.

[0066] Thirty 3-week-old mtm1 mutant male mice were randomly divided into 6 groups, 5 mice in each group, and were injected intravenously with AAV8-MCK-EGFP, AAV8-MCK-hMTM1, AAV8-MCK-hMTM1-122T-142T , AAV9-MCK...

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Abstract

The invention provides a recombinant adeno-associated virus-mediated X chromosome-linked myotubular myopathy treatment drug. A recombinant adeno-associated virus vector carries a myotubularin gene expression box containing human miR-142-3p and miR-122 target sequences. In-vivo experiments show that the recombinant adeno-associated virus vector can be efficiently introduced into a body to continuously and stably express myotubularin, prolong the survival time of an animal model and recover the growth and the development of the animal model. Results prompt that the recombinant adeno-associated virus vector is expected to be developed into the novel X chromosome-linked myotubular myopathy treatment drug.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an X-chromosome-linked myotubular myopathy gene drug carrying an artificially designed myotubulin gene (mtm1) expression frame with a recombinant adeno-associated virus vector. Background technique [0002] X-linked myotubular myopathy (XLMTM) is a congenital muscular dystrophy, which is mainly caused by obstacles in the formation of myofibrils into myotubes, resulting in poor formation of myotubes, thus interrupting the development of muscle tissue And stay in the muscle tissue of the fetus. Muscle cells can be found in the patient's muscle slices, but the nuclei are not scattered around the muscle fibers like mature muscle tissue, but are located in the center of the cells. XLMTM disease is caused by mutations in the X-linked myotubularin (mtm1) gene. The mtm1 gene encodes a phosphatase that regulates PIP and PIP in cells 2 It plays an important role in the process of cell signal...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N15/864A61K48/00A61K38/46A61P21/00
CPCA61K38/465A61K48/005A61P21/00C12N9/16C12N15/86C12N2750/14143
Inventor 田文洪马思思董小岩吴小兵
Owner BEIJING GENECRADLE PHARM CO LTD
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