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AAV1 virus medicated PCK1 gene expression vector with skeletal muscle specificity and application thereof

A gene and virus technology, applied in the biological field, can solve problems such as unclear metabolism

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

In addition to being mainly present in the liver, PCK1 is also widely present in mammalian tissues, including the small intestine, colon, mammary gland, adrenal gland, lung and muscle, but the metabolic role in these tissues is not clear

Method used

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  • AAV1 virus medicated PCK1 gene expression vector with skeletal muscle specificity and application thereof
  • AAV1 virus medicated PCK1 gene expression vector with skeletal muscle specificity and application thereof
  • AAV1 virus medicated PCK1 gene expression vector with skeletal muscle specificity and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Construction of AAV1-HSA-PCK1 recombinant expression vector

[0059] According to literature reports, the human α-skeletal muscle actin promoter sequence was obtained (Mol Cell Biol. 1987; 7(11):4089-4099. J Biol Chem. 2007; 282(45): 32844-32855.), and the The promoter sequence was mutated to eliminate the XhoI and SalI restriction sites in the sequence. Then the MVM intron sequence was added behind the promoter to obtain the complete promoter sequence required to express the PCK1 gene. This sequence is named HSA.

[0060] According to the GenBank database, the cDNA sequence of the PCK1 gene was obtained. According to the principle of codon degeneracy, the obtained PCK1 gene coding sequence was mutated to eliminate the restriction sites of EcoRI and BglII. Four fully complementary target sequences of miR-122 (highly expressed in the liver) were then introduced after the stop codon of the PCK1 gene. The sequence was named PCK1-4×miR-122T.

[0061] The two s...

Embodiment 2A

[0063] Packaging and assay of embodiment 2AAV1-HSA and AAV1-HSA-PCK1 recombinant virus

[0064] Referring to literature (Xiao X, et al. Production of High-Titer Recombinant Adeno-Associated Virus Vectors in the Absence of Helper Adenovirus. J Virol. 1998; 72(3):2224-2232.), the application of three plasmid packaging system packaging and Purification of recombinant AAV virus. Briefly, the AAV vector plasmid (pAAV2neo-HSA or pAAV2neo-HSA-PCK1-4×miR-122T), the helper plasmid (pHelper) and the AAV1 Rep and Cap protein expression plasmid pAAV-R2C1 were used in a molar ratio of 1:1:1 After mixing, 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. Two recombinant viruses, AAV1-HSA (packaged from pAAV2neo-HSA plasmid) and AAV1-HSA-PCK1 (packaged from pAAV2neo-HAS-PCK1-4×miR-122T plasm...

Embodiment 3

[0070] Example 3 Establishment of hyperlipidemia mouse animal model

[0071] Twelve mice of C57BL / 6J strain, half male and half male, weighing 18-22g, were raised under the conditions of relative humidity 60±10% and temperature 22±1ºC. After adaptively feeding the mice with ordinary feed for 3 days, they switched to high-fat feed (33.5% corn meal, 5.0% fish meal, 2.0% cholesterol, 4.0% milk powder, 10.0% palm oil, 16.7% soybean and other nutrients), The hyperlipidemia model was successfully established by continuous feeding for 16 weeks. After that, the mice were randomly divided into control group and experimental group, with half male and half male in each group. The dose of multi-point injection into the muscle of the control group and the experimental group was 1×10 11 vg / AAV1-HSA and AAV1-HSA-PCK1 virus, continue to feed for one month, remove the eyeball to take blood (12 hours before blood collection, fasting without water), centrifuge at 3000rpm for 10min to take seru...

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Abstract

The invention provides a vector for expressing and transferring gene with muscle tissue specific expression of phosphoenolpyruvatecarboxykinase (PEPCK) for reducing hyperlipidemia, delaying aging, andimproving reproductive capacity. The vector contains a reconstructed promoter of human alpha-skeletal actin gene, an intron of minute virus of mice (MVM), reconstructed cDNA of human phosphoenolpyruvatecarboxykinase 1 (PCK1), and four human miR-122 target sequences which are connected in series connection. The recombinant vector is mediated by an adeno-associated virus vector, and comprises a type 1 adeno-associated virus and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a skeletal muscle-specific PCK1 gene expression vector mediated by an AAV1 virus vector and its application, including a recombinant expression unit and a gene therapy method of the vector. Background technique [0002] Phosphoenolpyruvatecarboxykinase (PEPCK) is a rate-limiting enzyme that catalyzes the reaction of gluconeogenesis, that is, the conversion of oxaloacetate and GTP into phosphoenolpyruvate, GDP and CO 2 Reaction. The tricarboxylic acid cycle is carried out in the mitochondria, and PEPCK has two isozymes, namely PEPCK-M (mitochondrial form) in the mitochondria and PEPCK-C (cytosolic form) in the cytoplasm. The PEPCK-C gene is called PCK1 in humans and plays an important role in gluconeogenesis. At present, PCK1 has become the first clear marker of liver gluconeogenesis, and the transcription level of this gene in the liver is an important indicator for clinical evalua...

Claims

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Application Information

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IPC IPC(8): C12N15/864A61K48/00A61P3/06A61P39/06A61P15/08
CPCA61K48/005C12N9/1205C12N15/86C12N2750/14143C12Y207/0104
Inventor 田文洪董小岩吴小兵马思思
Owner BEIJING GENECRADLE PHARM CO LTD
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