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Preparation formula of gene therapy medicament taking recombinant adeno-associated virus (rAAV) as vector

A technology of gene therapy and preparation, applied in the field of molecular medicine and disease prevention and molecular pharmacology

Inactive Publication Date: 2014-04-02
HUAQIAO UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although inspiration can be obtained from the formulation of vaccines and recombinant protein products, gene medicines based on viral vectors are fundamentally different from vaccines and recombinant protein medicines. Existing research results also show that the formulation and stability of viral vector gene medicines are A new challenge for formulation researchers

Method used

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  • Preparation formula of gene therapy medicament taking recombinant adeno-associated virus (rAAV) as vector
  • Preparation formula of gene therapy medicament taking recombinant adeno-associated virus (rAAV) as vector
  • Preparation formula of gene therapy medicament taking recombinant adeno-associated virus (rAAV) as vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Preparation of stock solution of rAAV gene medicine

[0021] To facilitate the determination of the transduction titer (biological activity) of the rAAV gene drug, the transgene of the drug in this example is the firefly luciferase (Fluc) gene (purchased from Promega).

[0022] The preparation method of the rAAV gene therapy vector used in this example is: preparation by multi-plasmid co-transfection method without the participation of helper virus, that is, the calcium phosphate method is used to combine the vector plasmid with the helper plasmid (such as H22, H23, H24, H25, H26, H27, H28, H29, pXX6, etc.) (purchased from the University of Pennsylvania) were co-transfected into 293 cells (purchased from the American Type Culture Collection (ATCC)), and the recombinant virus was harvested after 60-72 hours, and chlorinated cesium gradient centrifugation and purification for later use. AAV helper plasmids (H22, H23, H24, H25, H26, H27, H28, H29) and adenovirus helper pl...

Embodiment 2

[0028] rAAV gene drug transduction titer (biological activity) determination

[0029] ①Cultivate 293 cells in a 6-well cell culture plate with a cell density of 10 5 / hole.

[0030] ② Take the rAAV gene drug and add it to the cell culture plate in step ① after appropriate dilution. The amount of rAAV gene drug added to each well is 10 6 Genome titer (vg).

[0031] ③The transduction titer of rAAV gene drug rAAV / Fluc was measured using the luciferase reporter gene detection kit produced by Beyontian Factory, and calculated based on the expression level (pg) of Fluc in each well.

Embodiment 3

[0033] The stock solution of the rAAV gene drug prepared in Example 1 was taken, and the solvent was replaced by ultrafiltration to obtain the preparations in Table 1 respectively. PBS buffer (pH 7.4) commonly used in the laboratory was used as the control solvent. Table 1 is the preparation table of rAAV gene drug stability research preparation, wherein the preparation of PBS buffer solution: Na 2 HPO 4 12H 2 O 3.868 mg, NaH 2 PO 4 2H 2 O 0.395mg, NaCl 6.8mg, dissolved in 1ml of water for injection. Of course, the amount of adding excipient components in the prescription can be any value in the following range and can realize the purpose of the present invention: add human serum albumin 0.5-5mg, sodium citrate 10-50mg, citrate in every 1ml water for injection Citric acid 0.1-1.0mg, polyether F-68 0.001-0.1mg.

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Abstract

The invention discloses a preparation formula of a gene therapy medicament taking a recombinant adeno-associated virus (rAAV) as a vector. A preparation in the formula comprises a rAAV gene therapy medicament as well as excipient components such as a protective human serum albumin for preventing container adsorption, a buffer pair of citric acid and sodium citrate for maintaining the pH value of a solution in a neutral range and a surfactant polyether F-68 for preventing polymerization among rAAV gene vectors. The preparation does not cause the rapid descending of the biological activity under a room temperature condition, and does not require cryopreservation.

Description

technical field [0001] The invention relates to a preparation prescription of a gene therapy drug with a recombinant adeno-associated virus as a carrier, and belongs to the fields of molecular pharmacology, molecular medicine and disease prevention and treatment. Background technique [0002] Gene therapy provides a new solution for the treatment of diseases caused by gene defects. The recent success of gene therapy for several major diseases marks a major breakthrough in gene therapy, and was rated as one of the top ten scientific advances in 2009 by Science magazine[ Alberts B. Science, 2009, 326(5960): p1589]. Successful cases include the clinical research on the successful cure of Leber's congenital amaurosis with recombinant adeno-associated virus (rAAV) gene medicine. The formulation research of viral vector-based gene medicine is a relatively new field, and so far few systematic studies have been done to solve the problem of formulation optimization and stability of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00A61K47/42A61K47/34A61K47/12A61K9/19A61K9/08A61P27/02
Inventor 刁勇许瑞安
Owner HUAQIAO UNIVERSITY
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