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Mesenchymal stem cells expressing brain-derived neurotrophic factor and use thereof

A neurotrophic factor and brain-derived technology, applied to genetically modified cells, cells modified by introducing foreign genetic material, animal cells, etc., can solve the problems of difficult mass production of MSCs, difficult to expect effects, limited proliferation, etc., and achieve Possibility of differentiation and proliferation is low, the formation of brain tissue is promoted, and the effect of high cell proliferation rate

Pending Publication Date: 2020-11-24
斯比根公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

First, the proliferation of MSCs is limited, so it is difficult to mass-produce the MSCs
Second, the obtained MSCs are mixed with a variety of cells, and it is difficult to maintain the same effect during production
Third, the therapeutic effect is not good when using only MSC
However, the mesenchymal stem cells used in the above studies are general mesenchymal stem cells obtained from tissues, and the proliferation of MSCs is limited, making it difficult to mass-produce them.
In addition, the degree of expression of the BDNF gene is not uniform, and it is difficult to expect the same effect at the time of production

Method used

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  • Mesenchymal stem cells expressing brain-derived neurotrophic factor and use thereof
  • Mesenchymal stem cells expressing brain-derived neurotrophic factor and use thereof
  • Mesenchymal stem cells expressing brain-derived neurotrophic factor and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0101] Example 1. Preparation of immortalized mesenchymal stem cells (MSCs)

Embodiment 11

[0102] Example 1.1. Preparation of a lentiviral vector comprising an immortalized gene

[0103] To immortalize MSCs, lentiviral vectors containing c-Myc and / or hTERT, respectively, as immortalization genes were prepared. At this time, in order to use the Tet-off system, a gene construct expressing tTA protein was inserted together.

[0104] First, in the expression cassette of the pWPT vector (Addgene, USA), the EF promoter was replaced with the CMV promoter, and the RSV promoter was additionally connected downstream to prepare the pBD lentiviral vector.

[0105] Using IRES, the c-Myc gene (SEQ ID NO: 6) and the thymidine kinase (TK) gene (SEQ ID NO: 4) were connected and inserted into the pBD lentiviral vector to regulate expression according to the CMV promoter. The vector prepared above was named pBD-1.

[0106] On the other hand, the hTERT gene (SEQ ID NO: 8) was inserted into the pBD lentiviral vector for regulated expression according to the CMV promoter. Among them, ...

Embodiment 12

[0108] Example 1.2. Production of Lentiviruses Comprising Immortalization Genes

[0109] Using the lentiviral vector prepared in the above Example 1.1., the lentivirus containing the immortalization gene was produced by the following method.

[0110] First, Lenti-X cells (Clontech Laboratories, USA) were cultured in 150 mm dishes (dish) using DMEM medium containing 10% fetal bovine serum. On the other hand, lentiviral vectors were extracted and quantified from DH5α E. coli cells using EndoFree PlasminMaxi Kit (Qiagen, USA).

[0111] After washing the above cultured Lenti-X cells with PBS, add 3ml of TrypLE TM Select CTS TM(Gibco, USA). After leaving the cells at 37° C. for about 5 minutes, it was confirmed that the cells were desorbed. The desorbed cells were added to DMEM medium containing 7 ml of 10% fetal calf serum for neutralization. The neutralized cells were aggregated in a 50ml test tube and centrifuged at 1500rpm for 5 minutes. The supernatant was removed, and ...

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Abstract

The present invention relates to a recombinant lentivirus comprising a gene encoding brain-derived neurotrophic factor (BDNF) protein, a mesenchymal stem cell transformed with the lentivirus, and a method for mass-producing a cell therapeutic agent expressing BDNF protein using the same. The mesenchymal stem cell transformed with the recombinant lentivirus of the present invention can regulate thetiming of expression of BDNF protein, exhibit high cell proliferation rate and high BDNF protein expression level, and are excellent in safety. Therefore, mesenchymal stem cells can be used for the treatment of various neurological diseases as a stem cell therapeutic agent capable of being mass-produced and maintaining the same effect.

Description

technical field [0001] This application claims the priority of Korean Patent Application No. 10-2018-0008569 filed on January 24, 2018, the full text of which is the reference of this application. [0002] The present invention relates to a recombinant lentiviral vector comprising a gene encoding brain-derived neurotrophic factor (BDNF) protein and cells transfected with the lentivirus prepared by using the above-mentioned vector. Background technique [0003] Brain-Derived Neurotrophic Factor (BDNF) is expressed in the brain, omentum, motor nerve cells, kidney, saliva, prostate, etc. in our body, and expressed in the hippocampus (hippocampus) and cerebral cortex ( cerebral cortex), hypothalamus, basal forebrain, etc. BDNF occupies an important part of long-term memory, and BDNF mRNA is expressed at a high level in the hippocampus of the brain in animals that learn things. Increased expression in this hippocampal region was also reported to be associated with learning leve...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/12C12N5/10A61K35/28A61K48/00A61K38/18A61P25/28A61P25/16A61P25/00A61P9/10
CPCA61K35/28C12N15/86C12N2740/16043A61P25/14A61P29/00A61P9/10A61P25/00A61K38/185C12N5/0663C12N2510/00C12N2510/04C07K14/475C12N5/0662C12Y207/01021C12N2740/15041A61K38/18A61K38/00C12N5/0668
Inventor 成永哲李纯旼金慧娟
Owner 斯比根公司
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