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Method for enhancing lentivirus vector production

A technology of lentiviral vectors and lentiviruses, applied in the direction of viruses/bacteriophages, biochemical equipment and methods, viruses, etc., to achieve the effect of improving the production of lentiviral vectors, enhancing the production of lentiviral vectors, and producing them safely

Pending Publication Date: 2021-07-23
NAT UNIV CORP TOKYO MEDICAL & DENTAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in order to prepare high-titer lentiviral vectors, further improvements are still needed

Method used

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  • Method for enhancing lentivirus vector production
  • Method for enhancing lentivirus vector production
  • Method for enhancing lentivirus vector production

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0109] [Example 1] Enhancement of Lentiviral Vector Production by HTLV-1 Tax

[0110] Materials and methods

[0111] cell

[0112] Cell line HEK293T (Invitrogen Corporation, Carlsbad, CA) was cultured in Dulbeco's modified Eagle's medium (Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS), 100 U / ml penicillin and 100 μg / ml streptomycin. MT-4 cells were maintained in complete RPMI 1640 medium (Nacalai Tesque) supplemented with 10% FBS, 100 U / ml penicillin and 100 μg / ml streptomycin.

[0113] plasmid

[0114] pCSII-CMV-MCS-IRES2-Bsd, pCAG-HIVgp, and pCMV-VSV-G-RSV-Rev were provided by Riken BRC through the Ministry of Education, Culture, Sports, Science and Technology's National Bioresource Project. To generate pCSII-CMV-luc-IRES2-Bsd, pCSII-CMV-MCS-IRES2-Bsd and pCMV-luc were digested with XhoI and NotI. Next, the XhoI-NotI fragment holding the firefly luciferase gene derived from pCMV-luc was inserted into the XhoI-NotI site of pCSII-CMV-MCS-IRES2-Bsd. The res...

Embodiment 2

[0137] [Example 2] Enhanced production of lentiviral vectors when HTLV-1Tax, Tat or NF-κB RelA are expressed individually or simultaneously

[0138] Next, using the third-generation lentiviral vector system pCAG-HIVgp, pCMV-VSV-G-RSV-Rev, pCSII-CMV-luc-IRES2-Bsd, using the luciferase activity in MT4 cells as an indicator, the following combinations The effect of increasing the production of lentiviral vectors caused by co-expression was studied (Fig. 7).

[0139] Figure 7-1 In means that Tax is co-expressed alone ( Figure 7-1 A), Tat alone co-expression ( Figure 7-1 B) Co-expression with RelA alone ( Figure 7-1 C) The effect on the yield of the third generation lentiviral vector.

[0140] Figure 7-2 In means co-expression in Tax, Tat and RelA ( Figure 7-2 A: Tax and Tat; Figure 7-2 B: Tax and RelA; Figure 7-2 C: Effects of Tat and RelA) on the yield of the third-generation lentiviral vector.

[0141] Figure 7-2 D shows the effect of the co-expression of Tax,...

Embodiment 3

[0143] [Example 3] The production of lentiviral vector is enhanced when Tat or NF-κB RelA is expressed alone or simultaneously

[0144] (1) In a collagen-coated 24-well plate, human embryonic kidney (Human Embryonic Kidney) (HEK) 293T cells were mixed with 0.5 mL of DMEM containing 10% bovine fetal serum at 3.0×10 5 cells / well for seeding and cell adhesion.

[0145](2) After 2 hours, mix the following plasmid with Opti-MEM (Gibco) 900 μL, add 1 μg / μL polyethyleneimine (Polyethyleneimine, PEI) 30 μL and incubate at room temperature for 30 minutes, and then drop into the cells .

[0146] (i) 0.25 μg pCAG-HIVgp

[0147] (ii) 0.125 μg pCMV-VSV-G-RSV-Rev

[0148] (iii) 0.375 μg pCSII-CMV-luc-IRES2-Bsd

[0149] (iv) 0.05 μg pSV2Tat or pSV2

[0150] (v) 0.025 µg of pRC / CMVRelA or pRC / CMV (manufactured by Invitrogen)

[0151] The structure of the pSV plasmid is as follows Figure 8 shown.

[0152] (3) After 24 hours, replace the culture medium.

[0153] (4) After 48 hours, re...

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Abstract

The purpose of the present invention is to provide a method and a reagent for enhancing lentivirus vector production in a 293T cell. The invention relates to a method for producing a lentivirus vector, which enhances the amount of lentivirus vector production by expressing HTLV-1 Tax, HIV-1 Tat or NF-kB RelA in a 293T cell when cotransfecting the 293T cell with a packaging mix that includes multiple plasmids each comprising a gene that encodes a protein essential for formation of a lentivirus particle, and a plasmid comprising a gene that transcribes an RNA included in the lentivirus particle and a target transgene to be expressed.

Description

technical field [0001] The invention relates to a high-efficiency production method of a lentiviral vector. Background technique [0002] Lentiviral vectors derived from human immunodeficiency virus type 1 (HIV-1) are important tools for introducing foreign genes into dividing and non-dividing cells, both in vitro and in vivo. The safety and convenience of lentiviral vectors have been improved by various methods (Non-Patent Document 1). Vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral vector is produced in HEK293T cells under the regulation of the earliest promoter of human cytomegalovirus (CMV), which can mediate the very Efficient transduction (Non-Patent Document 2). Lentiviral vectors targeting hematopoietic stem cells and T cells have been very successful in recent clinical applications (Non-Patent Document 3). In in vivo experiments and transduction of non-dividing cells, high-titer lentiviral vectors are required in many cases, so they must be...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N5/10
CPCC12N15/86C12N2740/16043C12N2740/16051C12N2740/16322C12N2740/14022C07K14/005C12N7/00C12N2740/15043C12N2740/15052
Inventor 山冈昇司铃木尚人芳田刚
Owner NAT UNIV CORP TOKYO MEDICAL & DENTAL UNIV
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