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Method for delivering exogenous molecules into T cells

An exogenous and molecular technology, applied in the field of biomedicine and medical devices, can solve the problems of unclear gene insertion site tumorigenic risk, low delivery efficiency, high cytotoxicity, etc., to improve cell harvest rate and delivery efficiency , the effect of a large number of cell treatments

Active Publication Date: 2021-01-19
BIOMODI BIOTECH SUZHOU CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Genetic modification of primary T cells through lentiviral infection is the most mature method for preparing CAR-T cells, but the preparation cost of viral vectors in this method is high, and there are many uncertain factors, including gene insertion sites Unclear, potential tumorigenic risk, etc.
Electroporation is not suitable for substances other than nucleic acids, such as proteins, and more studies have shown that electroporation can change the gene expression profile of cells, resulting in long-term defects in normal cell function
Microfluidic gene delivery technology has certain limitations in delivery efficiency and yield
In addition, there are also reports in the literature that use gold nanoparticles to deliver small interfering RNA to T cells by photothermal method, but a large number of gold nanoparticles enter the cells, resulting in high cytotoxicity of this method, and its delivery efficiency is also low
[0006] Although the global demand for CAR-T cell therapy is huge, the above-mentioned current situation and bottlenecks in T cell transformation technology have caused the current gene therapy manufacturing speed to be relatively slow, the cost is high, and the manufacturing aspect is not scalable to meet market demand.

Method used

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  • Method for delivering exogenous molecules into T cells
  • Method for delivering exogenous molecules into T cells
  • Method for delivering exogenous molecules into T cells

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0035]

[0036] In the present invention, a photothermal transfer substrate is firstly prepared, and a polydopamine layer is deposited on its surface. The substrate with the polydopamine layer deposited on the surface of the present invention can be used to deliver exogenous molecules into T cells.

[0037] Dopamine (dopamine, DA, chemical structural formula: C 6 h 3 (OH) 2-CH 2 -CH 2 -NH 2 , CAS No.: 62-31-7) As one of the catechol derivatives, it is a neurotransmitter, a chemical substance used to help cells transmit pulses. In recent years, dopamine has been widely used in biomedicine and biomaterials because of its strong adhesion to the surface of substrates. Dopamine is easily oxidized by dissolved oxygen in aqueous solution, and then triggers a self-polymerization cross-linking reaction, which can form a closely adhered polydopamine composite layer on the surface of almost any solid material. Polydopamine is a melanin-like substance. It not only has good adhesi...

Embodiment 1

[0056] Step 1: Preparation of CAR-T cells

[0057] (1) Dissolve 0.08 g of dopamine hydrochloride in 40 mL of deionized water, and adjust the pH value to 8.5 with alkaline solution. Immerse the gold flakes in the above solution and let stand at 37° C. for 24 hours to obtain a substrate deposited with a polydopamine deposition layer.

[0058] (2) Mix T cells and CAR gene (plasmid DNA encoding CAR protein, Lenti-EF1a-CD19(FMC63)-2nd-CAR(4-1BB)-EGFRt, Aikangde Bio) in a certain ratio in serum-free In the cell culture medium, the density of T cells is 200,000 / cm 2 , wherein the final concentration of the CAR gene was 0.006 μg / mL.

[0059] (3) The photothermal substrate is sterilized with 75% ethanol, and the above cell suspension is dropped onto the surface of the photothermal substrate to form a micron-sized liquid thin layer.

[0060] (4) Use a laser light source in the near-infrared band at 1W / cm 2 The cells on the sample were irradiated within the power density range for 3 ...

Embodiment 2

[0074] The CAR gene in Step 1 (2) of Example 1 was replaced with rhodamine-labeled dextran (molecular weight 4.4kDa), and Step 1 (1), (3)-(5) and Step 2 were the same as in Example 1.

[0075] Replace (6) in Step 1 of Example 1 with: after 30 minutes of laser irradiation, stain the cell nucleus with 4',6-diamidino-2-phenylindole, and observe the entry of dextran into the cell with a fluorescence microscope . Cells in blue are stained nuclei, and red is the color emitted by rhodamine-labeled dextran, representing successfully delivered cells. The transfer efficiency was obtained by quantifying the fluorescence micrographs. In addition, the cell viability was detected by CCK-8 48 hours after laser irradiation. For delivery efficiency and cell viability histograms, see figure 1 .

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Abstract

The invention relates to a method of delivering exogenous molecules into T cells. The method comprises the following steps of mixing T cells with a reagent containing exogenous molecules; dropwise adding an obtained cell suspension to the surface of a photo-thermal substrate; and irradiating the cells with a laser light source in a near-infrared band. The method has the advantages of high universality, high delivery efficiency, high transfection efficiency, low cytotoxicity, high modified cell yield and high one-time cell treatment flux. The killing efficiency of CART cells prepared by the method provided by the invention on tumor cells can reach up to 95 percent.

Description

technical field [0001] The invention relates to the fields of biomedicine and medical equipment, in particular to a method for transferring exogenous molecules into T cells. Background technique [0002] Immune cell therapies include cytokine therapy, immune checkpoint blockade approaches, adaptive immunotherapy, and chimeric antigen receptor-modified T cell immunotherapy (CAR-T). [0003] Chimeric antigen receptor (chimeric antigen receptor) is an artificially synthesized T cell receptor, which is structurally composed of an extracellular targeting junction region and a T cell activation signal domain (hinge region, transmembrane region, intracellular signal transduction region, etc. guide area) composition. Chimeric antigen receptor T cells are one of the most promising approaches for tumor cell immunotherapy. By "recoding" the patient's T cells, it can specifically recognize the tumor-associated antigen target, and after the recognition and binding, the signal to activa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/87C12N13/00C12N5/0783
CPCC12N15/87C12N13/00C12N5/0636C12N2533/30C12N2529/10C12N2510/00
Inventor 陈红王蕾郁李胤
Owner BIOMODI BIOTECH SUZHOU CO LTD
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