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Application of genetically modified oligodendrocyte progenitor cells in multiple sclerosis

A gene modification and progenitor cell technology, applied in the field of biomedicine, can solve the problems of inability to reduce inflammatory response and autoimmune damage, high recurrence rate of MS patients, and insignificant symptom effect, so as to reduce autoimmune damage and achieve significant overall improvement effect. , good therapeutic effect

Active Publication Date: 2021-11-26
ALLIFE MEDICINE(BEIJING) LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Due to the high recurrence rate of MS patients, it is difficult to cure, and the disability rate is high. Therefore, current researchers have made clinical explorations on stem cell therapy for MS, including bone marrow-derived mesenchymal stem cells (Mesenchymal stem cells, MSC), Adipose-derived MSCs, umbilical cord-derived MSCs, neural stem cells (Neural stem cells, NSCs), MSC-differentiated neural precursor cells (Neural precursors, NPs), hematopoietic stem cells (Haematopoietic stem cells, HSCs) and other related stem cell transplantation treatments, Although the use of stem cells promotes the production of myelin sheath to a certain extent, it cannot reduce the inflammatory response and autoimmune damage. Therefore, the overall improvement of symptoms of MS related to stem cell transplantation in current research is not obvious
In addition, since OPC cannot be isolated from the human body, although OPC can repair myelin sheath, it still cannot be promoted clinically.

Method used

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  • Application of genetically modified oligodendrocyte progenitor cells in multiple sclerosis
  • Application of genetically modified oligodendrocyte progenitor cells in multiple sclerosis
  • Application of genetically modified oligodendrocyte progenitor cells in multiple sclerosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0123] Example 1 Construction of lentiviral structure

[0124] 1. Experimental materials

[0125] The backbone vector pCW57.1 was purchased from addgene (article number: plasmamid-41393);

[0126] Gene synthesis company: Anhui General Biotechnology Co., Ltd.

[0127] 2. Construction method

[0128] The lentiviral vectors constructed in this example were named TEToff-CXCL-puromycin, TEToff-IL10-T2A-IL27-Zeo, and TEToff-IL3-hygroR, respectively.

[0129] (1) TEToff-CXCL-puromycin vector construction

[0130] Through gene synthesis, the gene CXCL11 is synthesized, and the sequence of the gene CXCL11 is shown in SEQ ID NO:1;

[0131] Such as figure 1 As shown, in the NheI (3' end) and AgeI (5' end) segments of the backbone vector pCW57.1, the gene CXCL11 was inserted to obtain TEToff-CXCL-puromycin;

[0132] (2) TEToff-IL10-T2A-IL27-Zeo vector construction

[0133] By gene synthesis, synthesis such as figure 2 In the structure shown, the sequences of the core structural g...

Embodiment 2

[0138] Example 2 Transfection and screening of iPSC cell lines

[0139] 1. Experimental materials

[0140]The iPSC cells used in this example are from Beijing Chengnuo Medical Technology Co., Ltd. The lentiviral vector used in this example is the lentiviral vector constructed in Example 1. Other experimental materials used in this example are shown in the table 1.

[0141] Table 1 Experimental materials

[0142]

[0143]

[0144] 2. Cell culture

[0145] (1) Passaging begins when the cells expand to 75-85% degree of polymerization. Take the T25 culture dish as an example, suck off the old culture medium, wash it twice with DPBS at room temperature, then add 3mL Accutase preheated at 37°C, and place at 37°C, 5% CO 2 In the cell culture incubator for 5 minutes, observe the gaps between individual cells under the microscope;

[0146] (2) Discard Accutase, add 3 mL of TeSR-E8 complete medium to stop digestion, transfer to a 15 mL centrifuge tube, and centrifuge at 1000 ...

Embodiment 3

[0172] Example 3 Preparation and identification of iPSC-derived OPC cells

[0173] 1. Experimental materials

[0174] The super-iPSC cells constructed by transfection screening in Example 2 of the present invention, and other experimental materials used in this example are shown in Table 3.

[0175] Table 3 Experimental materials

[0176]

[0177]

[0178] 2. Preparation method of iPSC-derived OPC cells

[0179] 2.1 Prepare the following medium for later use

[0180] (1) Complete medium for neural induction: 98% DMEM / F-12 medium, 1% non-essential amino acids, 1% GlutaMAX-I, 0.1 mM 2-Mercaptoethanol, 10 μM SB431542, 0.25 μM LDN193189 and 100 μM vitamin A acid, 25 μg / mL insulin;

[0181] (2) N2 medium: 97% DMEM / F-12 medium, 1% non-essential amino acid, 1% GlutaMAX-I, 0.1 mM 2-Mercaptoethano, 1% N2 supplement, 1 μM SAG and 100 μM vitamin A acid;

[0182] (3) B27 medium: 95% DMEM / F-12 medium, 1% non-essential amino acid, 1% GlutaMAX-I, 0.1mM 2-Mercaptoethanol, 1% N2 su...

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Abstract

The invention discloses genetically modified oligodendrocyte progenitor cells and a preparation method and application thereof. The invention provides a method capable of simultaneously repairing a myelin sheath, promoting the generation of the myelin sheath and reducing inflammatory response and the autoimmune injury. In the method, the genetically modified oligodendrocyte progenitor cells are adopted; through transplantation of the genetically modified oligodendrocyte progenitor cells, direct repair of the myelin sheath is implemented, inflammatory response of nerves is relieved, and neurological functions are improved. The genetically modified oligodendrocyte progenitor cells have very good application prospect in clinical treatment of multiple sclerosis.

Description

technical field [0001] The invention belongs to the field of biomedicine, specifically, the invention relates to the application of a genetically modified oligodendrocyte progenitor cell in multiple sclerosis, more specifically, the present invention relates to a genetically modified oligodendrocyte Glial progenitor cells and methods for their preparation and use. Background technique [0002] Multiple sclerosis (MS) is the most common immune-mediated chronic demyelinating disease of the central nervous system (CNS), mainly occurring in young patients, and is the most common cause of disability in young people except trauma s reason. In the acute active stage of the disease, there are multiple inflammatory demyelinating plaques in the white matter of the central nervous system, and the old lesions form calcified plaques due to the proliferation of glial fibers. Dry, frequently occurs in young and middle-aged, women are more common than men. The median age of onset is 29 y...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85A61K35/28A61P17/00A61P37/02
CPCC12N5/0622C12N15/85C07K14/54C07K14/521A61K35/28A61P17/00A61P37/02C12N2510/02C12N2506/45C12N15/86C07K14/5403A61K38/00A61K35/30C12N5/0018C07K14/5428A61P25/28C12N2740/15043C12N2510/00C12N2500/44C12N2500/38C12N2500/32C12N2501/33C12N2501/999C12N2501/01C12N2501/12C12N2501/135C12N2501/15C12N2501/155C12N2501/165C12N2501/41
Inventor 顾雨春吴理达
Owner ALLIFE MEDICINE(BEIJING) LTD
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