A method of preparing a lentivirus vector

A technology of lentiviral vectors and vector plasmids, applied in the biological field, can solve the problems of small host range of lentiviral products, and achieve the effects of reducing transduced cell death, good gene expression or RNAi interference, and stable expression

Inactive Publication Date: 2017-08-15
SHANGHAI GENECHEM
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  • Abstract
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  • Claims
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Problems solved by technology

Therefore, the host range that can be transduced by the final prepared lentiviral product is also small, and it is not suitable for animal experiments / in vivo injections and other clinical-level experiments

Method used

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  • A method of preparing a lentivirus vector
  • A method of preparing a lentivirus vector
  • A method of preparing a lentivirus vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] The preparation of cGMP grade lentiviral vector particles comprises the following steps:

[0048] (1) Culture of HEK293T cells:

[0049] The production of lentiviral vector particles is in the 10-layer CellFactory (6320cm 2 , NUNC). HEK293T cells were divided into 2 × 10 per well 5 / cm 2 The density was inoculated in DMEM medium containing 10% FBS, and placed at 37°C, 5% CO 2 The culture was carried out in an environment with saturated humidity, and the transfection was carried out 3 days after inoculation.

[0050] (2) Transfection of HEK293T cells:

[0051] Before transfection, prepare 580 ml of DMEM medium containing 2% FBS, and add chloroquine to a final concentration of 20-30 mmol / L. Prepare calcium phosphate transfection system: Add 5865 μg DNA plasmid (pHelper 1.0: pHelper2.0: pLenti-EGFP=15:12:24) to 5ml Opti-MEM in sequence, then add 5750 μl of 2.5M CaCl 2 , and finally 60ml of 2x HBS was added dropwise on a stirring shaker. After the transfection syste...

Embodiment 2

[0058]The lentiviral vector particles prepared in Example 1 were tested for activity titer and physical titer, nucleic acid residue detection, serum protein residue detection, LDH detection, RCL detection, sterility detection and mycoplasma detection.

[0059] (1) Activity titer detection:

[0060] HEK293T cells were divided into 2 × 10 per well 3 Cells were seeded in 96-well plates and cultured in DMEM medium containing 10% FBS. After 24 hours of cell plating, the lentiviral vector particle products I, II, III, and IV obtained in different purification process steps were respectively transduced into cells according to the optimal MOI value. For the four groups of samples I, II, III, and IV, five consecutive 10-fold serial dilutions were performed with DMEM medium containing 10% FBS (MOI=10). And add one well of untransduced empty cells as a control for each group of samples. After 24 hours of transduction, 100 μl of DMEM medium containing 10% FBS was supplemented to each w...

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Abstract

The invention relates to the field of biotechnology, particularly to a method of preparing a lentivirus vector. The method includes (1) transfecting a plasmid comprising a lentivirus vector genomic sequence into a culture cell; (2) culturing the culture cell obtained in the step (1), and collecting a cell culture supernatant fluid that is a lentivirus vector culture liquid; and (3) subjecting the lentivirus vector culture liquid obtained in the step (2) to tangential flow filtration, and purifying a filtrate obtained by the tangential flow filtration to obtain a lentivirus vector product. The lentivirus vector product has characteristics of high quality and high stability, meets cGMP-level (clinical-level) related standards (with the standards being from the Chinese Pharmacopoeia and the American FDA), and can be used for I- and II-stage clinical experiments, academic research at home and abroad, clinical research of medical mechanisms, large-scale production, and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a preparation method of a lentiviral vector. Background technique [0002] Lentivirus vectors (Lentivirus vectors, LVs) are gene therapy vectors developed on the basis of HIV-1 (human immunodeficiency virus type I). Lentiviral vectors can infect not only cells in the active phase of mitosis, but also cells that divide slowly and are in the terminal phase of division, including hematopoietic stem cells, neural stem cells, neurons at the end of differentiation, liver parenchymal cells, etc. In in vitro cell culture experiments and in vivo transplantation experiments, the target gene introduced by the lentiviral vector can be expressed stably for a long time. In addition, lentiviral vectors do not express any HIV-1 proteins and have low immunogenicity. The modified lentiviral vector can accommodate about 10kb of foreign genes, so most of the cDNA can be cloned into the lentiviral vect...

Claims

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Application Information

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IPC IPC(8): C12N15/867
CPCC12N15/86C12N2740/15043C12N2800/107
Inventor 姜军李敏陈鑫潘付晶吴涛
Owner SHANGHAI GENECHEM
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