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SiRNA capable of knocking down human PD-1, recombinant expression CAR-T vector as well as construction method and application thereof

An expression vector, PD-1 technology, applied in DNA/RNA fragments, recombinant DNA technology, retro RNA virus, etc., can solve problems such as high price

Active Publication Date: 2017-08-18
SHANGHAI UNICAR THERAPY BIOPHARM TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The price is quite expensive and unaffordable for ordinary families

Method used

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  • SiRNA capable of knocking down human PD-1, recombinant expression CAR-T vector as well as construction method and application thereof
  • SiRNA capable of knocking down human PD-1, recombinant expression CAR-T vector as well as construction method and application thereof
  • SiRNA capable of knocking down human PD-1, recombinant expression CAR-T vector as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1 Construction of recombinant lentiviral vector

[0082] 1. Materials

[0083] 1. Lentiviral backbone plasmid pLenti-3G ​​silencer, lentiviral packaging plasmids pPac-GP, pPac-R and membrane protein plasmid pEnv-G, HEK293T / 17 cells, homologous recombination enzyme, Oligo Annealing Buffer are provided by Shiao (Shanghai) Bio Provided by Medical Technology Co., Ltd.;

[0084] 2. Primers: According to the principles of primer design, the primers required for amplifying DNA fragments and target sites are designed. The primers are synthesized by Shanghai Biological Company, specifically:

[0085] EF1α-F: 5'-ATTCAAAATTTTATCGATGCTCCGGTGCCCGTCAGT-3' (SEQ ID NO.25)

[0086] EF1α-R: 5'-TCACGACACCTGAAATGGAAGA-3' (SEQ ID NO.26)

[0087] CD8leader-F: 5'-GGTGTCGTGAGGATCCGCCACCATGGCCTTACCAGTGACCGC-3' (SEQ ID NO.27)

[0088] CD8leader-R: 5'-GTGTCATCTGGATGTCCGGCCTGGCGGCGTG-3' (SEQ ID NO.28)

[0089] VL-F: 5'-CACGCCGCCAGGCCGGACATCCAGATGACCCAGAGCC-3' (SEQ ID NO.29)

[0090] VL...

Embodiment 2

[0190] Example 2 Concentration and detection of recombinant lentiviral vector

[0191] 1. Purification of recombinant lentiviral vectors by ion exchange chromatography (such as Figure 8 shown);

[0192] (1) Use a Thermo vacuum pump to filter the collected supernatant through a 0.22 μm-0.8 μm PES filter to remove impurities;

[0193] (2) Add 1.5M NaCl 250mM Tris-HCl (pH 6-8) to the supernatant at a ratio of 1:1 to 1:10;

[0194] (3) Place two ion exchange columns in series, and pass through the columns sequentially with 4ml 1M NaOH, 4ml 1M NaCl, 5ml 0.15M NaCl25mM Tris-HCl (pH 6-8);

[0195] (4) The solution obtained in step 2 is loaded on the ion exchange column with a speed of 1-10ml / min by a peristaltic pump;

[0196] (5) After passing all the supernatant through the column, wash it once with 10ml 0.15M NaCl 25mM Tris-HCl (pH 6-8) solution;

[0197] (6) Use 1-5ml 1.5M NaCl 25mM Tris-HCl (pH 6-8) for elution according to the loading amount, and collect the eluate;

[01...

Embodiment 3

[0251] Example 3 Functional detection of recombinant lentiviral vector lvCARbcma-1453~lvCARbcma-1460

[0252] 1. Cell-level expression detection of CAR gene:

[0253] (1) After the recombinant lentiviral vector lvCARbcma-1453~lvCARbcma-1460 and the control virus MOCK infected PBMC cells, the collected cells were detected by RT-PCR for CAR mRNA transcription level to verify the expression of CAR gene. If the CAR mRNA transcription level increased, then It shows that the transcription level expression of CAR gene is successful;

[0254] (2) After the recombinant lentiviral vectors lvCARbcma-1453~lvCARbcma-1460 and the control virus MOCK infected PBMC cells, the cells were collected to detect the expression level of CAR protein by western blot to verify the expression of CAR gene. If the expression level of CAR protein increased, then It shows that the translation level expression of CAR gene is successful;

[0255] (3) Cells were infected with lvCARbcma-1453-lvCARbcma-1460 wit...

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Abstract

The invention discloses siRNA capable of knocking down human PD-1, a recombinant expression CAR-T vector as well as a construction method and an application thereof. The PD-1 knocking-down siRNA expression cassette and an siRNA expression product thereof can be applied to CAR-T therapy of multiple myeloma (MM) for eliminating or relieving an immune escape mechanism of tumors and can be also used for inhibiting the immune escape mechanism in the CAR-T therapy of such tumors as pancreatic cancer, brain glioma, myeloma and the like.

Description

technical field [0001] The invention belongs to the technical field of tumor immunotherapy, and specifically relates to an siRNA for knocking down human programmed death receptor 1 (PD-1), a recombinant expression CAR-T vector (especially an A CAR-T transgene carrier for tumor immune escape mechanism) and its construction method and application. Background technique [0002] The theoretical basis of tumor immunotherapy is that the immune system has the ability to recognize tumor-associated antigens and regulate the body's ability to attack tumor cells (highly specific cytolysis). In the 1950s, Burnet and Thomas put forward the theory of "immune surveillance", thinking that the mutated tumor cells that often appear in the body can be recognized and eliminated by the immune system, which laid the theoretical foundation for tumor immunotherapy [Burnet FM. Immunological aspects of malignant disease. Lancet, 1967; 1:1171-4]. Subsequently, various tumor immunotherapies, includin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/867C12N15/66C12N5/10A61K35/17A61K31/7088A61P35/00
CPCC07K14/70521C12N15/1138C12N15/66C12N15/86C12N2310/14C12N2740/15043A61K39/4611A61K39/4631C12N5/0636A61K39/464417C12N2310/531C12N2320/32C12N2330/51C12N2740/16043A61P35/00C07K16/2878C12N2510/00C07K2319/03C07K2317/622A61K39/463A61K2121/00A61K2300/00C12N2740/15051
Inventor 祁伟俞磊康立清余宙
Owner SHANGHAI UNICAR THERAPY BIOPHARM TECH CO LTD
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