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Improved transfection of eukaryontic cells with linear polynucleotides by electroporation

A polynucleotide and linear technology, applied in the direction of allergic diseases, cells modified by introducing foreign genetic material, microorganism measurement/testing, etc., can solve problems such as RNA electroporation and RNA transfection not mentioned

Inactive Publication Date: 2013-12-04
UNIVERSITY OF ANTWERP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This patent discloses DNA transfection by electroporation (column 15, Example 2) using the following electrical parameter settings (250 μF; 220-290 V; 100 μl volume, BioRad cell and Gene ), this electrical setup is not commonly used for plasmid electroporation, however, the patent does not mention RNA transfection, let alone RNA electroporation

Method used

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  • Improved transfection of eukaryontic cells with linear polynucleotides by electroporation
  • Improved transfection of eukaryontic cells with linear polynucleotides by electroporation
  • Improved transfection of eukaryontic cells with linear polynucleotides by electroporation

Examples

Experimental program
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Effect test

Embodiment 1

[0136] A. Optimizing transfection of IVT mRNA in K562 cells: In preliminary experiments optimizing mRNA electroporation, we used leukemic K562 cells because of their convenient transfection by plasmid electroporation (Baum, C. et al., Biotechniques, 17:1058-1062 (1994)). The mRNA transfection efficiency was assessed with the EGFP reporter gene. Various electroporation parameters were tested, and transfection efficiency was determined by FCM analysis of EGFP expression ( Figure 1A ). A voltage of 300 V combined with a capacitance of 150 μF in a total sample cell volume of 200 μl resulted in the highest EGFP expression of all electrical setups tested (Table 1).

[0137] Table 1: Optimizing mRNA Electroporation Parameters in K562 Cells

[0138] electroporation

[0139]

Voltage (V)

Capacitance (μF)

cell volume

(μl)

efficiency(%)

Viability

(%)

dna

260

1050

500

40

49

300

...

Embodiment 2

[0159] Immature monocyte-derived dendritic cells (prepared with leukapheresis products and treated with IL-1β+IL-6+TNFα+PEG) by electroporation 2 The mixture was matured under clinical GMP conditions) for EGFP RNA-transfection. Monocyte-derived immature dendritic cells (DC) were prepared from leukapheresis products according to published methods (Feuerstein, B. et al., J. Immunol. Methods 245: 15-29 (2000)). Wash immature DC (d6) twice with RPMI, and wash with -Wash once with the washing solution of the kit (EQUIBIO, Maidstone Kent, U.K.). exist - Adjust the DC to 10×10 in the medium 6 / ml final cell concentration. Then 0.2 ml of cell suspension was mixed with 20 μg of in vitro transcribed EGFP RNA in a 1.5 ml reaction tube. Incubate for a maximum of 3 minutes at room temperature before transferring the cell suspension to the 0.4 cm gap electroporation sample cell. Pulses were triggered with a Gene Pulser II (BioRad, Munich, Germany) at a voltage of 300 V and a capacit...

Embodiment 3

[0163] EGFP RNA-transfection-potential titration of monocyte-derived dendritic cells by electroporation

[0164] A： Effect of Voltage on Cell Size and Granularity

[0165] Monocyte-derived immature dendritic cells (DC) were prepared using leukapheresis products according to published methods (Feuerstein, B. et al., J. Immunol. Methods 245: 15-29 (2000)). Wash immature DC(d6) twice with RPMI, and wash with The washing solution of the kit (EQUIBIO, Maidstone Kent, U.K.) was washed once. exist - Adjust the DC to 10×10 in the medium 6 / ml final cell concentration. 0.2 ml of the cell suspension was then mixed with or without 20 μg of in vitro transcribed EGFP RNA in a 1.5 ml reaction tube. Incubate at room temperature for a maximum of 3 minutes, then transfer the cell suspension into a 0.4 cm gap electroporation sample cell. Pulses were triggered with a Gene Pulser II (BioRad, Munich, Germany) at the indicated voltages and a capacitance of 150 [mu]F, resulting in pulse tim...

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Abstract

The present invention provides an improved method of gene delivery in human hematopoietic cells, particularly dendritic cells, by electroporation. The method of the present invention is superior to lipofection and passive pulsing of mRNA, and to electroporation of plasmid cDNA for gene delivery, including tumor antigen loading of dendritic cells.

Description

[0001] The present invention provides an improved method of gene delivery by electroporation in eukaryotic cells, preferably human hematopoietic cells, especially dendritic cells. The method of the present invention is superior to lipofection and passive pulsing of mRNA, and to electroporation of plasmid cDNA for gene delivery, including tumor antigen loading of dendritic cells. Background of the invention [0002] Dendritic cells (DCs) are bone marrow-derived leukocytes that function as specialized antigen capture and presentation cells for initiating primary immune responses in vitro and in vivo (Banchereau, J., Steinman, R.M., Nature, 392:245 -252 (1998)). Because of their central role in cell-mediated immunity in vivo, they are very attractive targets for molecular immunotherapy of acquired diseases such as AIDS and cancer. Recent developments in the generation of DCs ex vivo, and the ability to modulate DC function, provide a rationale for designing DC-based tumor vaccin...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/87C12N5/10A61K48/00A61P37/00C12N15/09A61K38/00A61K39/00A61P35/00A61P37/02C12P21/02C12Q1/02C12Q1/68
CPCA61K2039/5152A61K2039/5154A61K2039/5156A61P35/00A61P37/00A61P37/02A61P43/00C12N15/87
Inventor 杰罗尔德·许勒Z·N·伯内曼V·F·I·范特德鲁P·庞萨特斯I·斯特罗贝尔
Owner UNIVERSITY OF ANTWERP
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