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Genetic vector material and preparation method and application thereof

A technology of gene carrier and hydroxyl position, which is applied in the field of gene carrier materials and its preparation, can solve the problems of low operating efficiency of safe synthetic carriers, application restrictions of non-viral vectors, doubts about the safety of viral vectors, etc., and achieve good liver targeting , High gene transfection efficiency, the effect of high gene transfection efficiency

Inactive Publication Date: 2013-04-03
ZHEJIANG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, with the deepening of research, in clinical application and research, the safety of viral vectors has become more and more doubted, and the problems of immunogenicity and tissue accumulation in the application of viral vectors, as well as serious problems in clinical trials Side effects made researchers turn their attention to another transfection system - non-viral vector transfection system
[0005] The non-viral transfection system is favored by researchers because of its low toxicity and immunogenicity, and easy synthesis and acquisition. However, the low transfection efficiency is an insurmountable bottleneck for non-viral vectors, which makes non-viral vectors widely used in gene therapy. The application of the
[0006] Therefore, in gene therapy, the safety of the vector and the low operating efficiency of the synthetic vector are the main limitations at present.

Method used

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  • Genetic vector material and preparation method and application thereof
  • Genetic vector material and preparation method and application thereof
  • Genetic vector material and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1 prepares gene carrier material

[0047](1) Preparation of gene carrier materials

[0048] 1) Dissolve 25mg of mannan (average molecular weight 100kDa) in 13mL of anhydrous dimethyl sulfoxide, stir at 60°C until completely dissolved; add 213mg of carbonyldiimidazole for acylation for 30min to obtain acylated mannan solution ;

[0049] 2) Dissolve 798 mg of spermine in 15 mL of anhydrous dimethyl sulfoxide, slowly add acylated mannan solution, and stir at room temperature for 18 hours;

[0050] 3) Collect the product, dialyze it with a dialysis bag with a molecular weight of 8000-12000WM for 48 hours, lyophilize it into white color and store it at 4°C.

[0051] (2) Elemental analysis

[0052] The obtained gene carrier material was taken for elemental analysis test, and the results are shown in Table 1. The grafting rate of spermine can be calculated to be 12.07%.

[0053] Table 1 Elemental analysis table of gene carrier material

[0054]

[0055] (3)...

Embodiment 2

[0060] Example 2 Liver Targeted Accumulation Experiment

[0061] (1) Preparation of gene transfection system

[0062] Using the gene carrier material obtained in Example 1 to prepare a gene transfection system, the specific steps are as follows:

[0063] 1) The gene carrier material was prepared into a solution with a concentration of 4 mg / mL with ultrapure water, and then diluted with phosphate buffer (pH 7.4) to a concentration of 0.236 mg / mL, 0.586 mg / mL, and 1.172 mg / mL;

[0064] 2) Prepare isothiocyanate-labeled DNA with phosphate buffer (pH 7.4) to a solution with a concentration of 0.2 mg / mL;

[0065] 3) Mix each diluted solution in step 1) with the DNA solution in step 2) in equal volumes (the ratios of nitrogen and phosphorus between the gene carrier material and the gene are 2:1, 5:1, and 10:1, respectively), and incubate at room temperature 15min, the gene transfection system was prepared;

[0066] 4) Dilute the obtained gene transfection system 5 times with phos...

Embodiment 3

[0069] Example 3 Gene transfection efficiency and toxicity evaluation experiment

[0070] (1) Preparation of gene transfection system

[0071] Using the gene carrier material obtained in Example 1 to prepare a gene transfection system, the specific steps are as follows:

[0072] 1) The gene carrier material was prepared into a solution with a concentration of 4mg / mL with ultrapure water, and then diluted with phosphate buffer (pH 7.4) to a concentration of 0.059mg / mL, 0.293mg / mL, 0.411mg / mL, and 0.587mg / mL, respectively. mg / mL, 1.174mg / mL, 1.761mg / mL solution;

[0073] 2) Prepare the plasmid pGl3-Control encoding luciferase with phosphate buffer (pH 7.4) to make a solution with a concentration of 0.1 mg / mL;

[0074] 3) Mix each diluted solution in step 1) with the plasmid solution in step 2) in equal volumes (the ratio of nitrogen and phosphorus between the gene carrier material and the gene is 1:1, 3:1, 5:1, 7:1, 10:1, 20:1, 30:1), incubate at room temperature for 15 minut...

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Abstract

The invention discloses a genetic vector material, and a preparation method and application thereof. The genetic vector material is a polymer formed by polysaccharide 6-hydroxyl grafted polyamine. The method comprise the steps of (1) acylating polysaccharide with an acylation reagent to prepare an acylation intermediate, and (2) allowing the acylation intermediate to react with polyamine, so as to obtain the genetic vector material. The invention further discloses the application of the genetic vector material in a gene preparation transfection system. The genetic vector material can be subjected to specific recognition by mannose receptor on a cell surface, and has good liver targeting, low toxicity, higher gene transfection efficiency, and good research and application prospects in in-vivo / vitro transfection of the receptor.

Description

technical field [0001] The invention belongs to the technical field of gene therapy, and in particular relates to a gene carrier material and its preparation method and application. Background technique [0002] Gene therapy is a biomedical technology that introduces exogenous normal genes into target cells to correct or compensate diseases caused by gene defects and abnormalities, so as to achieve therapeutic purposes. The way to correct it can be to repair the defective gene in situ, or to transfer a functional normal gene into a certain part of the cell genome to replace the defective gene to play a role. [0003] Since the first attempt of gene therapy in 1990, gene therapy has been researched and applied in tumors, immunodeficiency diseases, AIDS and other difficult diseases. Gene therapy mostly relies on a fixed gene transfection system to transfect the required target gene into target cells to express the desired product. Gene transfection systems mainly include vir...

Claims

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Application Information

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IPC IPC(8): C12N15/87C08B37/08C08B37/02C08B37/00
Inventor 高建青阮桂鑫苗佩宏胡忠杰
Owner ZHEJIANG UNIV
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