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sgRNA and method for constructing gm-csf(-) cells using the same

A GM-CSF, cell technology, applied in the field of sgRNA and the construction of GM-CSF cells by using it, can solve the problem of lack of CRS treatment methods, and achieve the effect of preventing the occurrence of CRS and improving the curative effect

Active Publication Date: 2022-07-19
UTC THERAPEUTICS (SHANGHAI) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the problem of lack of effective treatment methods for preventing CRS in the prior art, the present invention provides a sgRNA and a method for constructing GM-CSF(-) cells using it

Method used

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  • sgRNA and method for constructing gm-csf(-) cells using the same
  • sgRNA and method for constructing gm-csf(-) cells using the same
  • sgRNA and method for constructing gm-csf(-) cells using the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: In vitro transcription (IVT) of CD19 FMC63 CAR mRNA

[0042] 1. The pDA-FMC63 CAR plasmid (NEB, Cat: R3133L) was linearized by SpeI digestion.

[0043] 2. The linearized vector was purified with a PCR cleanup kit (Qiagen) and eluted with RNase-free water.

[0044] 3. DNA concentration was measured by Nanodrop and checked by running an agarose DNA gel.

[0045] 4. In vitro transcription (IVT) was performed according to the manufacturer's standard operating procedure (Thermofisher, Cat. No. AMB13455). Briefly, 1 μg of template DNA, NTP / ARCA buffer, T7 buffer, GTP, T7 enzyme, and RNase-free H 2 O was added to a 0.2 mL PCR tube in a volume of 20 μl and incubated for 4 h at 37 °C.

[0046] After 5.4 hours, add 2 microliters of DNase I per reaction and react at 37°C for 15 minutes.

[0047] 6. The PolyA tailing procedure was then performed according to the manufacturer's standard operating procedure.

[0048] 7. IVT mRNA was purified using RNasy kit (Qiagen) (...

Embodiment 2

[0050] Example 2: Electroporation of Cas9 / GM-CSF gRNA to generate GM-CSF KO T cells

[0051] 1. On day 1, T cells were isolated from PBMCs and activated by anti-CD3 / CD28 magnetic beads (Thermofisher, catalog number: 402031 ) at a 1:3 ratio of T cells to magnetic beads.

[0052] 2. On day 4, the magnetic beads were removed from the T cells and washed twice with OPTI-MEM, and the cells were resuspended with OPTI-MEM at a concentration of 6e7 / ml.

[0053] 3. RNP complexes were prepared by mixing sgRNA and Cas9 protein (Thermofisher, Cat: A36499) at the desired ratio (GM-CSF gRNA 7.5 μg, Cas9 protein 15 μg / 100 μl electroporation) and incubated at room temperature for 10 min.

[0054] 4. Mix 100 microliters of cells with the RNP complex, then transfer the cell and RNP mixture into a 0.2 cm electroporation cup.

[0055] 5. Set parameters on the BTX ECM 830 machine: 360 voltage, 1 ms for electroporation, then transfer cells to pre-warmed medium and incubate at 37°C.

[0056] 6. Che...

Embodiment 3

[0058] Example 3: Electroporation of mRNA into A549-GFP tumor cells and T cells

[0059] 1. A549-GFP tumor cells and T cells were collected and washed 3 times with Opti-MEM medium. Among them, A549-GFP cells are in A549 cells (ATCC, CCL-185 TM , https: / / www.atcc.org / products / ccl-185), the specific method is: Infect A549 cells with GFP-expressing lentivirus, and then use flow cytometry to sort GFP-positive cells , and then obtain the A549-GFP cell line.

[0060] 2. Resuspend the cell pellet with Opti-MEM medium and adjust the cell concentration to 1×10e7 / ml.

[0061] 3. Add 5 micrograms of CD19 mRNA, 10 micrograms of anti-CD19 FMC63 CAR mRNA, etc. into 1.5 ml EP tubes, respectively, and then add 100 microliters of A549 cells or T cells, and mix well.

[0062] 4. Set parameters on BTX ECM 830 machine:

[0063] a) For T cells: 500 voltage, 0.7 ms;

[0064] b) For A549-GFP tumor cells: 300 voltage, 0.5ms;

[0065] 5. Add 100 microliters of cells mixed with RNA to the BTX ele...

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PUM

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Abstract

The invention discloses a sgRNA and a method for constructing GM-CSF(-) cells by using the same. The nucleotide sequence of the sgRNA is shown in SEQ ID NO:23. The method includes the following steps: (1) mixing sgRNA and Cas9 protein to prepare RNP complex; (2) mixing the RNP complex with cells and transforming or transfecting with virus or plasmid. The invention also discloses a kit comprising the sgRNA and their application in preparing GM-CSF(-) cells. The sgRNA of the present invention can effectively knock out GM-CSF in cells, provides a feasible method for reducing the expression of GM-CSF in activated CAR-T cells, and may help prevent the occurrence of CRS, thereby improving CAR-CSF Efficacy of T treatment.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a sgRNA and a method for constructing GM-CSF(-) cells by using the sgRNA. Background technique [0002] Chimeric antigen receptor T (chimeric antigen receptor-T, CAR-T) cell therapy has become a new and potentially revolutionary therapy for the treatment of cancer. However, the widespread application of CAR-T cell therapy has been limited due to the emergence of potentially lethal toxicity. These include the development of cytokine release syndrome (CRS) and neurotoxicity during CAR-T therapy. Grade 3 or higher CRS or neurotoxicity occurred in up to 50% of patients treated with CART19 cells, and several deaths were reported. [0003] Mechanistically, the development of CRS is associated with in vivo T cell expansion and massive production of T cell effector cytokines such as interleukin-6 [IL-6], interferon-γ [IFN-γ], monocyte chemotactic protein 1 [MCP-1] and granulocyt...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N5/10C07K19/00C12N15/62C12N15/87C12N15/27
CPCC12N15/1136C12N5/0636C07K16/2803C07K14/70517C07K14/70578C07K14/7051C12N15/87C07K14/535C12N2310/20C12N2510/00C07K2317/622C07K2317/56C07K2319/02C07K2319/03C07K2319/33C07K2319/74
Inventor 赵阳兵朱庚振
Owner UTC THERAPEUTICS (SHANGHAI) CO LTD
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