Method for inducing transdifferentiation of glial cells into functional neurons and application of method

A glial cell, functional technology, applied in the field of biotechnology and gene therapy, can solve the problem of no neuron repair method, achieve induced neuronal electrophysiological maturation, high neuron induction efficiency, and ensure controllability Effect

Pending Publication Date: 2021-11-16
NEURAGEN BIOTHERAPEUTICS (SUZHOU) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In particular, currently, there is no neuronal re...

Method used

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  • Method for inducing transdifferentiation of glial cells into functional neurons and application of method
  • Method for inducing transdifferentiation of glial cells into functional neurons and application of method
  • Method for inducing transdifferentiation of glial cells into functional neurons and application of method

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preparation example Construction

[0089]For preparative culture of NG2 cells, cortical tissues from postnatal day 3-5 mice were removed and digested with 0.25% trypsin for 15 minutes. The blown cells were cultured in DMEM / F12 solution containing 10% serum for 7-9 days. The flask was then shaken and the supernatant collected, the cells resuspended by centrifugation and plated on poly-D-lysine (Sigma)-coated coverslips at 37°C in an incubator with 5% CO2 containing N2 supplements (STEMCELLTM), 10ng / ml platelet-derived growth factor (PDGF, Invitrogen), 10ng / mL epidermal growth factor (EGF, Invitrogen) and 10ng / mL fibroblast growth factor 2 (FGF2, Invitrogen) serum-free DMEM medium ( Cells were cultured in Gibco for 3 days.

[0090] immunochromogenic

[0091] The immunochromogenicity of cultured cells refers to "Direct conversion of fibroblasts to functional neurons by defined factors" (Vierbuchen, T. et al. Nature 463, 1035-1041 (2010)). Color double-labeling experiments were carried out according to published...

Embodiment 1

[0100] Example 1 Neurog2 transdifferentiates NG2 glial cells into functional neurons in vitro

[0101] (1) Plasmid construction and virus infection

[0102] Cloned on the carrier template of FUGW-IRES-EGFP (carrier information reference Efficient transfer, integration, and sustained long-term expression of the transgene in adult ratbrains injected with a lentiviral vector.Proc Natl Acad Sci U S A 93:11382–11388) The human NG2 promoter (SEQ ID No.:5) replaces the CAG promoter, and the CDS (SEQ ID No.:4) fragment derived from the human Neurog2 gene is constructed on the lentiviral vector to generate hNG2-hNeurog2-IRES -EGFP lentiviral plasmid. For the packaging of lentiviral vectors, refer to the literature "Production and purification of lentiviral vectors" (Tiscornia, G., Singer, O. & Verma, I.M. Nat. Protoc. 1, 241-245 (2006)).

[0103] 24 hours after NG2 cells were cultured, lentivirus was added, and the medium was replaced 24 hours after infection: DMEM / F12, B27, Glutamax...

Embodiment 2

[0109] Example 2 Neurog2 transdifferentiates adult dorsal midbrain astrocytes into neurons in vivo

[0110] (1) Adeno-associated virus plasmid construction and virus infection

[0111] Clone the GFAP promoter (SEQ IDNo.:5) on the vector template of AAV-FLEX-Arch-GFP (Addgene, #22222) to replace the CAG promoter and retain the CMV enhancer, and then replace GFP with the coding frame of mCherry to obtain AAV - mCherry plasmid (control group). The CDS (SEQ ID No.: 2, NCBI: NM_009718.3) derived from the mouse Neurog2 gene was cloned into the AAV-mCherry plasmid to obtain the AAV-mNeurog2 / mCherry plasmid. The target gene can be specifically targeted under the action of the GFAP promoter astrocytes.

[0112] (2) Neurog2 transdifferentiates astrocytes into neurons

[0113] The virus AAV-mCherry or AAV-mNeurog2 / mCherry was injected into one side of the tectum of adult wild-type mice, and then brain tissue samples were collected at different time points. After 3 days of virus injec...

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Abstract

The invention provides a method for inducing transdifferentiation of glial cells into functional neurons and application of the method. Specifically, the invention provides an application of a Neurog2 functional fragment, and the functional fragment can induce glial cells to form functional neuronal cells in vivo or in vitro, not only can play a transdifferentiation role in normal tissues, but also can promote nerve reconstruction of damaged nervous tissues, and is expected to become a new method for in-vivo nervous tissue repair.

Description

technical field [0001] The invention belongs to the fields of biotechnology and gene therapy, and specifically relates to a method and application for inducing transdifferentiation of glial cells into functional neuron cells, especially the application in spinal cord injury repair. Background technique [0002] The main pathological changes caused by mammalian central nervous system injury and various neurodegenerative diseases are irreversible neuron degeneration and necrosis and neural circuit destruction. How to replace dead and lost neurons and rebuild neural circuits in damaged and diseased brains or spinal cords is a key step in treatment. Since the central nervous system (brain and spinal cord) of adult mammals has a very limited ability to repair itself, it is difficult to compensate for the loss of neuronal cells by itself. [0003] Due to the low efficiency of transplantation of exogenous neurons or neurogenic cells, there are potential risks of tumorigenicity and...

Claims

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Application Information

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IPC IPC(8): C12N5/0793C12N15/86C12N5/10A61K48/00A61K38/17A61P25/00A61P25/08A61P25/16A61P25/28
CPCC12N5/0619C12N15/86A61K48/005A61K38/1709A61P25/00A61P25/08A61P25/16A61P25/28C12N2501/998C12N2506/08C12N2740/16043C12N2750/14143C12N2830/008C07K14/4702C12N2510/00C12N2502/086A61K35/30A61K2300/00C12N5/10
Inventor 陈如雷刘婷
Owner NEURAGEN BIOTHERAPEUTICS (SUZHOU) CO LTD
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