Engineered immune cell with antiviral activity and construction method and application thereof

An antiviral activity, immune cell technology, applied in blood/immune system cells, antiviral agents, genetically modified cells, etc., can solve problems such as difficult antiviral therapy

Pending Publication Date: 2020-10-30
英基生物医药(香港)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although NK cells have the potential as a therapeutic agent against infectious diseases or cancer, most NK cells in normal human body exist in a dormant state, and NK cells are difficult to be cultured in large quantities in vitro and are difficult to apply to antiviral therapy

Method used

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  • Engineered immune cell with antiviral activity and construction method and application thereof
  • Engineered immune cell with antiviral activity and construction method and application thereof
  • Engineered immune cell with antiviral activity and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] The present embodiment provides a construction of engineered immune cells with antiviral activity, said cells are denoted as ITNK cells, and its construction method comprises the following steps:

[0068] (1) Construction of gene knockout vectors: In this embodiment, the gRNAs of SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:36 and SEQ ID NO:37 were used to construct BCL11B gene knockout plasmids respectively and It is mixed, and the partial structure of the obtained plasmid PX458-gBCL11B is as follows figure 1 shown;

[0069] Transfect PX458-gBCL11B in the 293T cell line, and pick a single clone, and detect the base deletion and dislocation of the BCL11B knockout target by gene sequencing (such as figure 2 ), the results showed that the target site was successfully knocked out.

[0070] (2) Sorting and activation of T cells

[0071] In this embodiment, T cells are separated from the peripheral blood and umbilical cord blood of human mature T cells: (i) respectively centr...

Embodiment 2

[0079] A kind of SARS-CoV-2 pseudovirus containing green fluorescent protein gene is provided in the present embodiment, and the specific packing method is:

[0080] (1) Cultivate 293T cells in a 10cm culture dish, and when the density of 293T cells in the culture dish reaches 80%, replace the culture medium, and after cultivating for 2 hours, start step (2); the culture medium described in this implementation all contains 10 %FBS and 1% double antibody (100× penicillin-streptomycin mixed solution) in DMEM high glucose medium.

[0081](2) Take 500 μL of opti-DMEM into a 15 mL centrifuge tube, and add 7.2 μL of PEI (linear polyethyleneimine) with a concentration of 10 μg / μL, mix slightly, and let stand for 5 minutes;

[0082] (3) Put 500 μL of opti-DMEM into a 15 mL centrifuge tube, take 9 μg of plasmid expressing green fluorescent protein, 15 μg of SARS-CoV-2S protein helper plasmid, and 12 μg of psPAX, add them to the centrifuge tube, and mix well;

[0083] (4) After step (2...

Embodiment 3

[0090] In this example, the SARS-CoV-2 pseudovirus expressing green fluorescent protein prepared in Example 2 was used to infect the ACE2-tCD19 cell line.

[0091] (1) Digest the BGC823 cells overexpressing ACE2-tCD19 in the culture dish with 0.25% trypsin for 5 min, add culture medium to stop the digestion and collect the cell suspension, put it in a 15 mL centrifuge tube and centrifuge at 300 g for 5 min, discard the supernatant and use 3 mL of fresh medium was resuspended; the medium described in this example was 1640 medium containing 5% FBS and 1% double antibody.

[0092] (2) Count the cells and divide 1×10 5 A BGC823 cell suspension was seeded in a 24-well plate, and 1 mL of medium was added to each well;

[0093] (3) The SARS-CoV-2 pseudovirus with the green fluorescent protein gene purified in Example 2 was added to the BGC823 cells overexpressing ACE2-tCD19, 50 μL per well;

[0094] (4) After 12 hours of culture, 1 mL of fresh medium was replaced;

[0095] (5) Dis...

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Abstract

The invention provides an engineered immune cell with antiviral activity and a construction method and application thereof. The engineered immune cell is obtained by reprogramming through a gene editing method and is an ITNK cell for simultaneously expressing an NK cell marker and a T cell marker, has good antiviral activity, can secrete IFN gamma, and realizes inhibition and killing effects on viruses including coronaviruses; meanwhile, the engineered immune cells can be massively propagated in vitro, and the problem that the NK cells are difficult to amplify in vitro is solved.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an engineered immune cell with antiviral activity and its construction method and application. Background technique [0002] Cell reprogramming refers to the process in which differentiated cells are reversed under specific conditions and then returned to a pluripotent state, or form an embryonic stem cell line, or further develop into a new individual. In the field of immunotherapy of diseases, there have been reports on the conversion of immune cell types through cell reprogramming. For example, the Gladstone Institutes have reported that pro-inflammatory effector T cells are reprogrammed into anti-inflammatory regulatory T cells through a specific reprogramming method; this reprogramming is important for the treatment of autoimmune diseases Significantly, specifically, in autoimmune diseases, over-stimulated effector T cells can cause damage to the bod...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/90C12N15/85C12Q1/02A61K35/17A61P31/14A61P31/22
CPCC12N5/0636C12N5/0646C12N15/907C12N15/85C07K14/4703G01N33/505A61K35/17A61P31/14A61P31/22C12N2510/00C12N2800/107
Inventor 不公告发明人
Owner 英基生物医药(香港)有限公司
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