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SiRNA for knocking down human IL-15, CD19 CAR expression vector, CAR-T cell and construction method and application of siRNA

A technology of expression vector and construction method, which is applied in the field of CAR-T cells and its application, CD19CAR expression vector, and siRNA knockdown of human IL-15, which can solve the problems of reducing IL-15 level, so as to reduce the amount of secretion and reduce the risk Effect

Active Publication Date: 2018-12-07
苏州一兮生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Therefore, in order to reduce the risk of cytokine syndrome and cerebral edema, the level of IL-15 in patients can be reduced by lymphodepletion preconditioning with mild chemotherapy regimen, while CAR-T cells themselves activate the IL-15 released by proliferation. -15 but no way to control

Method used

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  • SiRNA for knocking down human IL-15, CD19 CAR expression vector, CAR-T cell and construction method and application of siRNA
  • SiRNA for knocking down human IL-15, CD19 CAR expression vector, CAR-T cell and construction method and application of siRNA
  • SiRNA for knocking down human IL-15, CD19 CAR expression vector, CAR-T cell and construction method and application of siRNA

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Design of the sequence of siRNA knocking down human IL-15:

[0044] The mRNAs of different isoforms of IL-15 were obtained from the official website of NCBI, compared to obtain its common sequence, and the siRNA was designed using this sequence as the targeting sequence.

[0045] Using the design website https: / / portals.broadinstitute.org / gpp / public / seq / search, three pairs of sequences with better specificity were obtained, and the sequence a with better specificity was selected for testing:

[0046] a. The sense strand sequence is the nucleotide sequence described in SEQ ID NO.1, and the antisense strand sequence is the nucleotide sequence described in SEQ ID NO.2;

[0047] b. The sense strand sequence is the nucleotide sequence described in SEQ ID NO.3, and the antisense strand sequence is the nucleotide sequence described in SEQ ID NO.4;

[0048] c. The sense strand sequence is the nucleotide sequence described in SEQ ID NO.5, and the antisense strand sequence is th...

Embodiment 2

[0053] Construction of recombinant lentiviral vector for knocking down human IL-15 siRNA

[0054] (1) Construction of the pCDH vector of CD19 CAR:

[0055] The CD19 CAR gene is composed of a second-generation CAR. The entire CD19 CAR is composed of scFv fragments that recognize CD19 molecules, CD8 transmembrane regions, 4-1BB fragments, and CD3ζ fragments;

[0056] scFv is an Anti-CD19 antibody molecular light chain and heavy chain composed of single-chain molecules, the specific sequence is derived from the patent document PCTUS2011064191, the sequence is shown in SEQ ID NO.9; CD8α hinge region / Transmembrane (CD8α hinge / TM), CD8α hinge region and transmembrane region, the CD8α hinge region is a flexible region containing a large amount of proline, and the CD8α transmembrane region mainly anchors the extracellular region and the intracellular region on the cell membrane. The CD8α hinge region and transmembrane region sequence NM_001768 (1301..1507) was found in the Genebank ...

Embodiment 3

[0077] CD19 CAR-T cells with human IL-15 knockdown:

[0078] T cell culture and transfection of T cells with pCDH-anti-CD19 scFV-CD8α hinge / TM-CD137-CD3ζ and pCDH-anti-CD19scFV-CD8α hinge / TM-CD137-CD3ζ-IL15KD lentivirus

[0079] (1) Recovery of T cells: Take out a PBMC cell from liquid nitrogen, quickly put it into a 37°C water bath to dissolve it quickly, and transfer the PBMC cell into the preheated RPMI1640 medium when the cell ice cube is only the size of a soybean , centrifuge at 300g for 5min, remove the supernatant, resuspend PBMC cells in KBM581 serum-free medium, add to 1μg / mL anti-CD3, 2μg / mL anti-CD28 pre-coated culture flask, 37℃5%CO 2 nourish;

[0080] (2) On the fourth day, transfer the T cells into the lentivirus pre-coated with Retronectin, the MOI value is 10, that is, lentivirus: T cells = 10:1, 37°C 5% CO 2 nourish. CD19 CAR-T cells and IL15-knockdown CD19 CAR-T cells can be obtained.

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Abstract

The invention discloses siRNA for knocking down human IL-15, a CD19 CAR expression vector, a CD19 CAR-T cell and application of siRNA based on the problem that IL-15 released by the CD19 CAR-T cell through self-activating proliferation cannot be controlled by a method, relating to the technical field of tumor immunotherapy. The invention discloses siRNA for knocking down human IL-15, a lentiviralexpression vector of siRNA and the CD19 CAR-T cell for knocking down IL-15. The siRNA has the beneficial effects that the knock-down effect of IL-15 in the CD19 CAR-T cell is realized, and the secretion volume of IL-15 is greatly reduced, so that the risks of cell factor release syndrome and encephaledema are reduced.

Description

technical field [0001] The invention relates to the technical field of tumor immunotherapy, in particular to an siRNA for knocking down human IL-15, a CD19CAR expression vector, CAR-T cells and applications thereof. Background technique [0002] At the beginning of 2017, the National Cancer Center released the latest cancer data in China, each of which tells us with objective facts: China is facing a war against cancer. About 10,000 people are diagnosed with cancer every day, and about 7 people are diagnosed with cancer every minute. The number of new cancer cases continues to rise, from 3.58 million to 3.68 million, an increase of 3%. The number of new cancer cases in the world is about 14.09 million, and China's new cancer cases account for 1 / 4 of the world's total. The treatment of cancer in our country is facing huge challenges. At present, traditional therapies mainly include surgery, chemotherapy, radiotherapy and targeted therapy, which have certain effects in the tr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/867C12N15/62C12N5/10A61P35/00
CPCA61P35/00C07K14/5443C07K14/7051C07K14/70517C07K14/70578C07K16/2803C07K2317/622C07K2319/00C07K2319/03C07K2319/33C07K2319/74C12N5/0636C12N15/1136C12N15/86C12N2310/14C12N2510/00C12N2740/15043C12N2800/107
Inventor 徐华栋刘振云韩江萌王维程丰伟
Owner 苏州一兮生物技术有限公司
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