Universal-type CAR-T cell based on base editing and preparation method and application thereof

A general-purpose, cell-based technology, applied in the field of preparation of general-purpose CAR-T cells, can solve problems such as complex preparation process, off-target effects, and immune system damage, and achieve simplified preparation procedures, improved transfection efficiency, and simple preparation process Effect

Pending Publication Date: 2019-05-03
SUZHOU MAXIMUM BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, CAR-T cells also have some disadvantages in the treatment of tumors: first, it causes side effects such as cytokine storm; second, the current adoptive cell therapy is still based on autologous cell reinfusion therapy, and a certain amount of peripheral blood needs to be drawn from the patient, and then in vitro T lymphocytes are modified and expanded in vitro, and the modified autologous T cells proliferate to a certain number before being infused back into the patient. However, most patients, especially tumor patients, have undergone radiotherapy and chemotherapy before undergoing CAR-T cell therapy. etc., the immune system has been destroyed, and the number, activity and proliferation of its own T cells have been attenuated. These obstacles limit the use of autologous peripheral blood mononuclear cells and affect the application of CAR-T cell therapy and clinical tumor treatment. And development
The preparation process of this method is complicated, and there is a serious off-target effect in gene editing mediated by CRISPR-Cas9, making it a certain safety hazard in clinical application.

Method used

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  • Universal-type CAR-T cell based on base editing and preparation method and application thereof
  • Universal-type CAR-T cell based on base editing and preparation method and application thereof
  • Universal-type CAR-T cell based on base editing and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Construction of recombinant expression vectors.

[0057] The single base editing system used in the present invention is BE-Plus: a stop codon is introduced through CT mutation to achieve gene knockout. BE-Plus two-plasmid system (scFv-APOBEC-UGI, GCN4-D10A), 10 GCN4s are connected to the N-terminal of D10A, and one scFv is connected to the N-terminal of APOBEC. In this way, GCN4-D10A can recruit 10 scFv-APOBECs, increasing the probability of mutation. The work window is expanded from 4-8 to 4-16, making the efficiency higher.

[0058] Use SV201-pGL3-U6-gRNA-Puromycin mut Bsa1 ACCG in the present invention (see figure 1 ) as a backbone carrier, linearized by restriction endonuclease Bsa1 (NEB), and ligated with artificially synthesized sgRNA under the action of T4 ligase to form a recombinant vector.

[0059] Synthesis of TRAC-sgRNA and B2M-sgRNA: Select sgRNA as reverse sgRNA (CCA is the target codon, GGG is the PAM sequence). TRAC-sgRNA synthesized unidi...

Embodiment 2

[0060] Example 2 sgRNA functional verification.

[0061] 1) Cell culture and transfection.

[0062]A. Use the electroporation kit to transfect HEK293T cells to verify the targeting efficiency of sgRNA. 2 µg pST1374-scFv-APOBEC-UGI-GB1 (see figure 2 ), 2.0 µg of pST1374-N-NLS-GCN4-D10A (see image 3 ), 2µgpGL3-U6-TRAC sgRNA BE plasmid was mixed with the electroporation solution, and the cells were co-transfected. After 6-8 hours, the medium was changed, and Blasticidin (invitrigen, ant-bl-1) and Puromycin (invitrigen, ant-pr-1) and Puromycin (invitrigen, ant-pr- 1) Drug screening, collect cells after 48 hours.

[0063] B. Use the electroporation kit to transfect HEK293T cells to verify the targeting efficiency of sgRNA, add 2 µg pST1374-scFv-APOBEC-UGI-GB1 (see figure 2 ), 2.0 µg of pST1374-N-NLS-GCN4-D10A (see image 3 ), 2µgpGL3-U6-B2M sgRNA BE plasmid was mixed with the electroporation solution, and the cells were co-transfected. After 6-8 hours, the medium was changed...

Embodiment 3

[0067] Example 3 Preparation of PBMC cells and sorting of CD3 positive T cells.

[0068] 1) Use a blood collection bag to collect 80-100 mL of peripheral blood from healthy people (shake while collecting to fully mix the peripheral blood with the anticoagulant), and isolate PBMCs in a sterile cell preparation workshop. Transfer the peripheral blood to a 50ml centrifuge tube, centrifuge at 800g at room temperature for 15 minutes, absorb the upper layer of yellow autologous plasma into a new 50ml centrifuge tube, inactivate at 56°C for 30 minutes, and store it at -20°C for later use; add to the lower cell layer Equal volume of PBS, mix up and down;

[0069] 2) Draw 15ml of lymphocyte separation medium (Ficoll-Paque Plus, GE) at the bottom of a new 50ml centrifuge tube, and slowly add 30ml of diluted blood along the tube wall to the upper layer of the lymphocyte separation medium, put the centrifuge tube in a centrifuge, 450g, slowly Rise and descend slowly for 25 minutes, no br...

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Abstract

The present invention belongs to the field of immunotherapy and discloses a universal-type CAR-T cell based on base editing and a preparation method thereof, and an application of the universal-type CAR-T cell in preparing drugs for treating tumor cells. A BE-Plus system is used to knock out TRAC gene and B2M gene on allogeneic T cells and optimizes a technical scheme for preparing the universal-type CAR-T cell, the obtained CAR-T cell does not produce host rejection reaction, and allogeneic infusion-back of the universal-type CAR-T cell can be realized.

Description

technical field [0001] The invention belongs to the field of immune cell preparation and immunotherapy, and more specifically relates to a preparation method and application of a universal CAR-T cell that uses a base editing system to knock out both genes of TRAC and B2M. Background technique [0002] Tumor has always been a major disease that plagues the world. Traditional treatment methods include surgery, radiotherapy, chemotherapy, and targeted therapy, all of which have their own limitations. Tumor immunotherapy is a new treatment after surgery, radiotherapy, and chemotherapy, especially with the rise of immune checkpoint monoclonal antibody therapy and CAR-T cell immunotherapy, and the efficacy of immunotherapy has gradually been affirmed. In 2013, "Science" magazine selected tumor immunotherapy as the top ten scientific breakthroughs of the year. In January 2016, based on the development of immunotherapy and targeted therapy, the United States proposed a "moon landin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85C12N15/90A61K35/17A61P35/00
Inventor 蒋海娟尚小云刘慧莹徐凡丽马少文赵丹
Owner SUZHOU MAXIMUM BIO TECH CO LTD
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