Method for constructing CAR-T cell by silencing human Tim-3 gene through shRNA and application of CAR-T cell

A technology of tim-3 and tim3-siRNA-1, applied in the field of CAR-T cells, can solve problems such as suboptimal efficacy, inability to exert anti-tumor immune response, and dysfunction of CAR-T cells

Pending Publication Date: 2022-05-13
SUZHOUEVERHEALTH BIOMEDICAL CO LTD
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Problems solved by technology

CAR-T cells have made impressive achievements in hematological malignancies, but the efficacy in solid tumors is still not ideal
One of the main reasons is that the immunosuppressive microenvironment of solid tumors and the immune e

Method used

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  • Method for constructing CAR-T cell by silencing human Tim-3 gene through shRNA and application of CAR-T cell
  • Method for constructing CAR-T cell by silencing human Tim-3 gene through shRNA and application of CAR-T cell
  • Method for constructing CAR-T cell by silencing human Tim-3 gene through shRNA and application of CAR-T cell

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Embodiment Construction

[0043] The following examples are to further illustrate some preferred embodiments of the present invention, but not all of them. Other embodiments based on the present invention made by professionals in the field without creative work all belong to the protection scope of the present invention. The present invention will be further described below in conjunction with the accompanying drawings.

[0044] 1. Design and synthesis of siRNA sequence for Tim-3 gene silencing

[0045] According to BLOCK-iT TMRNAi Designer (https: / / rnaidesigner.thermofisher.com / rnaiexpress / design.do) designed siRNA to Tim-3 coding sequence (Table 1). The target sequence was subjected to BLAST (NCBI) non-homology query, showing no other homologous sequences. Send the designed sequence to Suzhou Jinweizhi Biotechnology Co., Ltd. for siRNA synthesis (Table 2)

[0046] Table 1 Tim3-siRNA sequence

[0047]

[0048] 2. Screen the siRNA with the highest efficiency for Tim-3 gene silencing

[0049] 1...

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Abstract

The invention provides a method for constructing a targeting ROR1 CAR-T cell by using shRNA (short hairpin ribonucleic acid) to silence a human Tim-3 gene and application of the targeting ROR1 CAR-T cell. The HKP55 CAR-T cell prepared by the method can be applied to cancer treatment. According to the invention, three different targeting Tim-3 genes with small hairpin RNA (shRNA) sequence specificity are designed, and the shRNA-3 sequence with the best silencing effect is obtained through screening. Therefore, the shRNA-3 sequence and the CAR sequence of the targeted ROR1 are co-transfected into the T cell, and the ROR1 CAR-T (HKP55 CAR-T) cell with the silent Tim-3 gene is prepared. The Tim-3 expression level of HKP55 CAR-T cells is greatly reduced, and compared with ROR1 CAR-T (HKP69 CAR-T) cells, the in-vitro killing of the ROR1 CAR-T cells on target cells with high expression of Galectin-9 and cell proliferation dependent on the target cells are remarkably enhanced through silencing of the Tim-3 gene. Therefore, the HKP55 CAR-T cell can overcome a complex immunosuppressive tumor microenvironment, continuously proliferate and exert an anti-tumor effect function.

Description

technical field [0001] The present invention relates to the fields of immunology and molecular biology, in particular to CAR-T cells. Background technique [0002] At present, chimeric antigen receptor (CAR) modified T cell therapy is a new therapy targeting immune cells developed in recent years. CAR-T cells have made remarkable achievements in hematological malignancies, but the efficacy in solid tumors is still not satisfactory. One of the main reasons is that the immunosuppressive microenvironment of solid tumors and the immune escape mechanism of tumor cells lead to the dysfunction of CAR-T cells and the inability to exert an anti-tumor immune response. However, the research and application of general-purpose CAR-T in China is still in its infancy. stage. [0003] T cell immunoglobulin and mucin-domain containing-3 (T cell immunoglobulin and mucin-domain containing-3, Tim-3) is a transmembrane protein encoded by Havcr2. It was first discovered in CD4+ and CD8+ effect...

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Application Information

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IPC IPC(8): C12N5/10C12N15/113C12N15/62C12N15/867A61K39/00A61P35/00
CPCC07K14/70503C07K14/7051C07K16/2803C12N15/1138C12N5/0636C12N15/86A61K39/001111A61P35/00C07K2319/33C12N2510/00C12N2310/531C12N2310/14C12N2740/15043A61K2039/5158
Inventor 吴炯夏玉龙师春青金昕
Owner SUZHOUEVERHEALTH BIOMEDICAL CO LTD
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