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Transfection method

A technology of bubbles and immune cells, applied in the field of transfection, to achieve the effect of improving the introduction efficiency, realizing cost production and low-cost production

Pending Publication Date: 2022-04-08
TAKEDA PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there have been no reports on the selective introduction of nucleic acids (e.g., mRNA, DNA) encoding CARs or exogenous TCRs into immune cells (such as T cells, etc.) by using the sonoporation effect

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0162] Reference Example 1: Preparation of ultrafine sparkling water

[0163] Polysorbate 80 (2g) (0.1% polysorbate 80) was dissolved in water for injection (2L) or diluted Mclivaine buffer (pH 3.0, 2L), and the following settings were used using ultrafine granules manufactured by IDEC. bubble generator (nanoGALF TM FZ1N-02) to prepare ultrafine bubble aqueous solution. Positively charged ultrafine bubbles can be generated when using an acidic buffer (diluted Mclivaine buffer: pH 3.0), and negatively charged ultrafine bubbles can be generated when using water for injection.

[0164] Gas used for preparation: Air

[0165] Sparkling water flow: about 4.0L / min

[0166] Dissolution pressure: 300KPa±5%(s.d.)

[0167] The prepared ultrafine bubble aqueous solution was subjected to high pressure steam sterilization for 30 min at 121°C-124°C using an autoclave appropriately. After sterilization, the ultrafine bubble average diameter, ultrafine bubble density, and d90 / d10 ratio w...

Embodiment 2

[0176] Example 2 Introduction of proteins into T cells

[0177] Jurkat E6.1 (3 x 10) in RPMI 1640 medium with 10% FBS 4 cells, 0.5 mL) were seeded in 48 wells. After removing the medium, 0.5 mL of ultrafine bubble / RPMI medium containing 5 μg of IgG-FITC was added (4 mL of ultrafine bubble / RPMI medium was added to 40 μL of IgG-FITC). The ultrafine bubble / RPMI medium prepared by adding 1 L of the ultrafine bubble aqueous solution prepared in Reference Example 1 to the RPMI medium powder and adding 2 g / L NaHCO was used as the transfection medium 3 and 10% FBS.

[0178] After addition of IgG-FITC-containing medium, a sonicator (NEPAGENE) was used at a frequency of 1 MHz and 0.5 W / cm 2 The output intensity was subjected to ultrasonic irradiation for 10 s. The cells were cultured for 2 h, then the transfection medium was removed, and the cells were cultured in RPMI medium (culture medium) for 48 h. The fluorescence intensity of IgG-FITC was measured with a spectrofluorometer. ...

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Abstract

In order to provide a novel method for safely and efficiently introducing nucleic acids, proteins or other target substances into T cells or other immune cells, the present invention provides a system for delivering target substances to immune cells comprising a combination of an ultrasound generating device and an aqueous solution or water of ultra-fine bubbles, the aqueous ultrafine bubble solution or ultrafine bubble water includes ultrafine bubbles having an average diameter of 200 nm or less and not containing phospholipids. The present invention provides, inter alia, a method for improving the delivery ability of nucleic acids or proteins to immune cells using ultrasound and the above-mentioned ultrafine bubble water, etc.

Description

[Technical Field] [0001] The present invention relates to a system for delivering a target substance to immune cells, the system comprising ultrafine bubble water containing ultrafine bubbles or a combination of an aqueous solution of ultrafine bubbles and an ultrasonic generator; a system for delivering a target substance by using the A method of increasing the delivery of nucleic acid or protein to immune cells by ultrafine sparkling water or aqueous solution and ultrasound; a formulation containing a combination of nucleic acid or protein and said ultrafine sparkling water or aqueous solution for use in combination with ultrasound irradiation delivery of the nucleic acid or protein to immune cells; and a method for delivering the nucleic acid or protein to the cells by contacting immune cells with the formulation and sonicating the cells; and the like. (Background technique) [0002] Ultrasound has been primarily used as an ultrasound imaging modality in the medical field...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K9/10A61K48/00A61K38/17A61K41/00A61P35/00C12N15/87
CPCA61P37/00C12N15/87A61K9/0009A61K9/08A61K9/5138A61K47/26C07K14/7051C12N15/64
Inventor 村田直之S·丘阿诺伊柳井薫雄
Owner TAKEDA PHARMA CO LTD
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