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Serum-free suspension system for lentiviral production

A production system and lentivirus technology, which are applied in the field of serum-free suspension systems for lentivirus production, and can solve problems such as increasing costs

Pending Publication Date: 2019-06-14
LIFE TECH CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Culturing adherent cells is more difficult for the operator and requires more effort to generate large quantities of lentiviral vector, increasing costs

Method used

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  • Serum-free suspension system for lentiviral production
  • Serum-free suspension system for lentiviral production
  • Serum-free suspension system for lentiviral production

Examples

Experimental program
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Effect test

example 1

[0176] Example 1. Cell Culture

[0177] Suspend cells in LV on an orbital shaker at 30% to 40% of the shake flask size volume in polycarbonate, disposable sterile Vent-up shake flasks (125 mL to 1 L) at 125 rpm in a cell culture incubator (37 °C, 8% CO2, 70% to 80% humidity). Cells were isolated every 3 to 4 days at a density of 4 to 5 x 10 6 / mL.

example 2

[0178] Example 2. Lentivirus production experiment

[0179] Based on development needs, lentivirus (LV) production experiments were performed in two formats: 96-well deep-well plates and 125 mL shake flasks, with 1 mL and 30 mL culture volumes, respectively. The cell density was 4 x 10 6 / mL. Lentiviral vectors (LVV) were packaged by co-transfecting lentiviral expression (transfer) plasmid-pLenti6.3 / V5-GW / EmGFP and lentiviral packaging plasmid-ViraPower lentiviral packaging mixture with a total DNA of 3 μg / mL. For both transfection reagent and DNA diluent, Opti- I is the DNA / reagent complex medium at 75uL / mL; the total complex volume is 150ul / mL. The complexation time is 10 minutes to 20 minutes. ExpifectamineTM 293 is a transfection reagent developed by LV culture supplement and LV enhancer, the dosage is 5ul / mL to 8ul / mL. 5% LV Culture Supplement is included in all assays under development. Prior to the development described here, 5 mM caffeine was used as an LV enha...

example 3

[0180] Example 3: Lentivirus titer measurement

[0181] Lentivirus titers were measured by infecting Ht1080 cells. Four hours before infection, 7000 cells per well were seeded in 96-well plates. Upon infection, cells were attached to the culture vessel at approximately 30% confluency. Containing 8ug / mL in the culture medium for 10 1 to 10 5 Serial dilutions of LVV. Pass 10 4 and 10 5 Virus dilutions infect cells. After infecting the cells, the infected Ht1080 plates were spun at 2000 rpm for 30 minutes at room temperature. 18 hours after infection, use without Replace the medium with fresh medium. Cells were incubated for an additional 72 hours. And GFP positive cells were measured by Attune flow cytometry. Titers were calculated based on the percentage of GFP cells between 1% and 20% of wells.

[0182] On the day of transfection, prepare 30 mL cultures of 3 different cell densities in 125 mL shake flasks: 2.5 x 10 6 / mL, 4×10 6 / mL and 5×10 6 / mL. Each dens...

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PUM

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Abstract

A lentiviral vector production system comprises (a) a lentiviral culture supplement to control cell growth, (b) a transfection reagent comprising DHDMS, DOPE, and cholesterol to increase transfectionefficiency, (c) a lentiviral production enhancer comprising sodium propionate, sodium butyrate, and caffeine to boost lentiviral production, wherein the lentiviral vector production system is serum-free. A method of lentiviral vector production comprises using the lentiviral production system. Another method for lentiviral vector production comprises (a) culturing eukaryotic cells in a serum-freemedium, (b) providing a lentiviral culture supplement to control cell growth, (c) transfecting the cells with a lentiviral vector using a transfection reagent comprising DHDMS, DOPE, and cholesterol to increase transfection efficiency, and (d) providing a lentiviral production using a lentiviral production enhancer comprising sodium propionate, sodium butyrate capable of boosting lentiviral production.

Description

technical field [0001] The novel lentiviral vector production system employs large-scale serum-free suspensions. Background technique [0002] Recently, lentiviral vectors have been at the center of attention for use as gene transfer vehicles in gene therapy. A current next-generation therapy, CAR-T cell therapy uses lentiviral vectors as effective gene transfer tools to express engineered chimeric antigen receptors (CARs) on the surface of T cells to recognize and kill cancer cells. Therefore, preclinical and clinical researchers have requested lentivirus production on a larger scale, at high titers, and in animal serum-free media. Lentiviral production contributes to the high cost of developing CAR-T cell therapies. [0003] Current lentiviral production systems primarily use adherent cells, which require fetal bovine serum to support cell growth in flasks or cell factories. This system is suitable for small-scale but not large-scale research purposes requiring large in...

Claims

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Application Information

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IPC IPC(8): C12N7/02C12N15/86
CPCC12N2740/16043C12N2740/16051C12N15/86C12N7/00C12N2500/90C12N2740/15052C12N2740/15043
Inventor 于昕X·德莫尔拉特杜杰
Owner LIFE TECH CORP
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