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Ldlr negative packaging cell line for the production of vsv-g pseudotyped retroviral vector particles or virus particles thereof

A technology for packaging cell lines and retroviruses, applied in the field of producing VSV-G pseudotyped retrovirus vector particles or vector-like particles, which can solve problems such as exhaustion

Pending Publication Date: 2021-04-27
ミルテニイビオテックベーファーウントコーカーゲー
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These systems do not address the issue of cytotoxicity, but avoid the need for transient transfection and limit the burden of VSV-G toxicity to only at harvest
However, additional purification may be required for therapeutic applications to deplete material from viral harvests

Method used

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  • Ldlr negative packaging cell line for the production of vsv-g pseudotyped retroviral vector particles or virus particles thereof
  • Ldlr negative packaging cell line for the production of vsv-g pseudotyped retroviral vector particles or virus particles thereof
  • Ldlr negative packaging cell line for the production of vsv-g pseudotyped retroviral vector particles or virus particles thereof

Examples

Experimental program
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Effect test

Embodiment approach

[0095] In one embodiment of the present invention, the LDLR gene of the HEK 293T packaging cell line has been knocked out by methods well known in the art, thereby generating an LDLR-negative HEK 293T packaging cell line (HEK 293T K.O.). The HEK293T K.O. packaging cell line is made with a psi-negative lentiviral expression vector encoding the gag / pol gene, a psi-negative lentiviral expression vector encoding the rev gene, a psi-positive lentiviral expression vector encoding a transgene, and a psi-negative lentiviral expression encoding VSV-G Vectors are co-transfected, and the transgene may encode, for example, a chimeric antigen receptor. The HEK 293T K.O. packaging cell line produces high rates of VSV-G pseudotyped lentiviral vector particles carrying the genetic information of a transgene (eg, chimeric antigen receptor). These particles can be used to transduce stem cells or immune cells, such as T cells or NK cells, designed to express a transgene (eg, a chimeric antigen r...

Embodiment 1

[0142] Example 1: Production of VSV-G pseudotyped lentiviral vector

[0143] VSV-G pseudotyped lentiviral vectors were generated by transient transfection of HEK 293T cells. On the day of transfection, HEK 293T cells were seeded at 1.0E06 cells / mL in 25 mL of serum-free cell culture medium in 125 mL of Shakerflask (185 rpm) with plasmids encoding VSV-G, gag / pol, rev and encoding GFP (Complete plasmid system) or psi-positive transfer vector plasmid of therapeutically relevant CD20-CAR for transfection. 24 h after transfection, 10 mM sodium butyrate (Sigma-Aldrich, catalog number: 3034 10-5004) was added. 48 h after transfection, the lentiviral supernatant was harvested by centrifugation at 200 x g to remove cell debris, aliquoted and stored at -80°C for subsequent use.

[0144]In order to transfect cell lines stably expressing VSV-G, HEK 293T cells were seeded in DMEM / 10% FCS / 0.8 μg / mL puromycin (Biowest, catalog number 12362; Biochrom, Cat. No. S0415; Gibco, Cat. No. A111...

Embodiment 2

[0145] Example 2: Titration of VSV-G pseudotyped lentiviral vectors on HT1080

[0146] For titration of lentiviral vectors, HT1080 cells cultured in DMEM / 10% FCS were seeded at 1.1E05 cells / well in 24-well plates one day before transduction. On the day of transduction, determine the cell number, wash the cells with DMEM, and use [8 μg / mL] (Sigma Aldrich, catalog number: H9268-5G) serially diluted particles encoding GFP or CD20-CAR in DMEM were transduced. 72 h after transduction, the transduction efficiency was determined by determining the ratio of GFP or CD20-CAR positive cells by flow cytometry. The ratio of GFP positive cells, the dilution factor, and the volume of lentiviral vector applied were used to calculate the titer of the lentiviral vector (ie, transducing units per volume (TU / ml)).

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Abstract

The present invention provides the use of a packaging cell line for the production of VSV-G pseudotyped retroviral vector particles or virus like particles thereof, wherein said packaging cell line is negative for Low-Density Lipoprotein Receptor (LDLR), optionally said packaging cell line stably expresses VSV-G. A method for producing said VSV-G pseudotyped retroviral vector particles or virus like particles thereof is disclosed as well as said particles obtained by said method.

Description

technical field [0001] The present invention relates to the field of packaging cell lines for the production of retroviral vector particles or virus-like particles thereof, in particular packaging cell lines which do not express low density lipoprotein (LDLR) on their surface, for the production of VSV-G Pseudotyped retroviral vector particles or vector-like particles (VLPs) thereof. Background technique [0002] In the field of T cell and stem cell gene therapy, retroviral vectors such as lentiviral vectors (LV) are considered to be efficient tools for delivering therapeutic nucleic acid molecules to target cells and inducing long-term expression. Typically, retroviral vectors contain a heterologous envelope protein from a foreign viral species within the retroviral membrane. The process of exchanging viral vector envelope proteins is called "pseudotyping". The most commonly used envelope protein for pseudotyping is the G protein of Vesicular Stomatitis Virus (VSV-G). It...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/02C12N15/867
CPCC12N15/86C12N2740/16043C12N2740/16052C12N2810/6081C12N7/00C12N2740/15023C12N2740/15043C12N2740/15052
Inventor T·斯卡赛尔J-C·德特曼M·迈耶I·约翰斯顿
Owner ミルテニイビオテックベーファーウントコーカーゲー
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