Dual-RMCE-mediated (dual-recombinase mediated cassette exchange-mediated) TCR (T cell receptor) gene replacement system and method

A gene replacement, gene technology, applied in the field of molecular biology

Active Publication Date: 2016-02-10
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] There are some problems in TCR gene therapy: At present, anti-tumor TCR genes are usually transduced into T cells with retroviral vectors. Since viral transduction is a process of multi-copy and random integration, it may inactivate tumor suppressor genes and activate protumor cells. Genes induce leukemia, and the transduced TCR chains may randomly combine with endogenous TCR chains to form self-reactive TCR molecules, thereby inducing autoimmune diseases, such as: graft-versus-host disease (GVHD)

Method used

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  • Dual-RMCE-mediated (dual-recombinase mediated cassette exchange-mediated) TCR (T cell receptor) gene replacement system and method
  • Dual-RMCE-mediated (dual-recombinase mediated cassette exchange-mediated) TCR (T cell receptor) gene replacement system and method
  • Dual-RMCE-mediated (dual-recombinase mediated cassette exchange-mediated) TCR (T cell receptor) gene replacement system and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1TCR-RMCE replacement experiment

[0040] 1. Construction of the carrier

[0041] 1. Construction of the retroviral integration vector pMP71-LGFPF, which contains L oxp-E GFP neo- F RT gene replacement component, the construction method is: use not I-Loxp-F primer (5'-AATGCGGCCGCataacttcgtatagcatacattatacgaagttatcTACCGGGTAGGGGAGGCG-3') and FRT- Eco RI-R primer (5'-TGAATTCGAAGTTCCTATACTTTTCTAGAGAATAGGAACTTCTCCAGAAGAACTCGTCAAG-3'). Amplified 2.0 kb plasmid from pSR-GFP / Neo plasmid (VEC-PRT-0005 / 0006, purchased from Oligoengine Company) by PCR not I-Loxp-EFGPneo-FRT- Eco RI fragment. The PrimerStarMax PCR reaction system was used, and the reaction conditions were: 95°C, 2min; 95°C, 15s; 60°C, 30s; 72°C, 2min, 35 cycles; 72°C, 5min. After the PCR reaction, digest with NotI and EcoRI, and use glue to recover the PCR product, and connect it to the pMP71 vector.

[0042] 2. Construction of TCR replacement vector pDRAV-3-LTCRF: through NotI - TCR136...

Embodiment 2

[0059] Example 2 Application of PCR to detect RMCE-mediated gene replacement

[0060] 1. The PCR process is as follows:

[0061] Collect 1×10 6 Add 500 μL digestion solution (NaCl100mM, Tris-HCl50mM, EDTA25mM, SDS0.9%), add 5μL, 20mg / mL proteinase K, digest at 55℃ for 3h, mix well, add 500μL phenol chloroform, invert up and down 20 times, Centrifuge at 13,000 rpm for 10 min; take the supernatant into a new tube, add isopropanol (about 450 μL) at a ratio of 1:1, invert and let stand for 5 min (white flocculent DNA can be seen). Centrifuge at 13000rpm for 15min, discard the supernatant, and suck up the liquid with a gun. Add 500 μL of 75% ethanol, centrifuge at 13,000 rpm for 5 minutes; pour off the supernatant, and suck up the liquid with a gun (be careful not to pour or suck off the white precipitate), and repeat the alcohol wash. Dry, add 100 μL ddH 2 O (or TE), 55 degrees, 20min to dissolve.

[0062] Select the LTR region of the retrovirus, select P1 and P2 and P3 of ...

Embodiment 3

[0065] Example 3 Teratoma formation and in vivo differentiation of ES cells

[0066] Mouse TCR-ES cells 5×10 6, subcutaneously injected into NOD-Scid immunodeficient mice, and divided into four groups: (1) TCR-ES cell group; (2) TCR-ES+OP9 cell group; (3) TCR-ES+OP9-DL1 group; (4) TCR-ES+OP9+OP9-DL1 group; (5) ES cell group. All groups were simultaneously injected with SCF (200ng) and TPO (200ng) (Pepro-Tech) at the time of injection, and after 3 weeks of differentiation, hFLT3 (200ng) and IL-7 (200ng) were injected subcutaneously around the teratoma. After 7 weeks, the mice were sacrificed to detect the cell phenotypes of teratoma, spleen, bone marrow, and peripheral blood, and the tetramer staining and cytokine secretion of T cells were detected at the same time.

[0067] Collect 10 from Teratomas 5 cells (differentiated from TCR-ES cells), add 10 cells loaded with KVLEYVIKV-MAGEA1 polypeptide 5 a T 2 cells (10 -5 M), choose anti-CD3 (0.2mg / L) + anti-CD28 (1mg / L) as ...

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Abstract

The invention discloses a TCR (T cell receptor) gene replacement system and method mediated by retrovirus gene transfer combining with the Dual-RMCE (dual-recombinase mediated cassette exchange) technology. The TCR gene replacement system comprises a retrovirus integration vector pMP71-LGFPF containing a replacement component Loxp-EGFP-FRT, and a replacement vector pDRAV-3-LTCRF containing TCR target genes. The TCR gene replacement system has the advantages that TCR gene replacement can be performed fast in a single site of a genome, a large amount of tumor antigen specificity T cells can be produced by using the in-vivo and in-vitro cell differentiation technology, and one tumor antigen specificity TCR molecule can be expressed; the problems that retrovirus random integration causes side effects and exogenous TCRs and endogenous TCRs combine to form self-reactive TCRs of traditional TCR gene treatment are solved, and the safe and reliable new technology is provided for clinical anti-tumor T cell immunotherapy.

Description

technical field [0001] The invention relates to the technical field of molecular biology, and more specifically relates to a Dual-RMCE-mediated TCR gene replacement system and a method thereof. Background technique [0002] Tumor immunotherapy is to treat malignant tumors by enhancing anti-tumor immunity, and clinical trials in recent years have achieved remarkable results. Among them, the anti-tumor T cell immunotherapy using T cell receptor (TCR) targeting specific targets has made great progress, and has begun to enter clinical trials, and has obtained objective curative effect. Early clinical trials of T cell therapy mostly used anti-tumor tumor infiltrating lymphocytes (tumorinfiltratinglymphocytes, TIL), and the results showed that only in some tumors such as melanoma, there were a relatively large amount of TIL, which was expanded in vitro and returned to the patient. It has obvious tumor elimination effect. Due to immune tolerance and tumor microenvironment suppres...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N5/10
Inventor 李亮平龚英
Owner JINAN UNIVERSITY
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