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Functional fragments for reprogramming, combinations and uses thereof

A functional and functional protein technology, applied in the field of biotechnology and gene therapy, can solve the problems of low transformation efficiency, difficult clinical application, and limit the proliferation ability of glioma cells, so as to achieve the effect of improving efficiency

Pending Publication Date: 2022-03-25
NEURAGEN BIOTHERAPEUTICS (SUZHOU) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, some studies have found that some neurogenic transcription factors or combinations of transcription factors can transform glioma cells into neurons in vitro or in vivo, and limit the proliferation ability of glioma cells.
However, the existing transcription factors or combinations of transcription factors have only been proven to be used in vitro or in vivo with low transformation efficiency, making it difficult to achieve practical clinical application

Method used

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  • Functional fragments for reprogramming, combinations and uses thereof
  • Functional fragments for reprogramming, combinations and uses thereof
  • Functional fragments for reprogramming, combinations and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0163] Example 1 Functional fragments of single transcription factors promote the transdifferentiation of glial cells into neurons

[0164] First, we used the in vitro model of glial cell transdifferentiation to conduct preliminary screening and obtained a batch of transcription factors that can induce glial cell transdifferentiation into neurons. The coding sequences of the transcription factors used and the transformation efficiency are shown in Table 1. Show. The transformation efficiency in Table 1 is the number of positive cells with both co-localization of neuronal positive markers and detection of spontaneous postsynaptic currents. In the study, at least 20 NeuN were tested per transcription factor on average. positive transdifferentiated cells.

[0165] Table 1 In vitro glial cell transdifferentiation efficiency of single transcription factors

[0166] transcription factor Human (wild type) Transdifferentiation efficiency mouse (wild type) Transdiff...

Embodiment 2

[0169] Example 2 Functional fragments of single transcription factors promote transdifferentiation of glial cells in the dorsal midbrain

[0170] According to the in vivo model of glial cell transdifferentiation, we tried to use the transcription factors screened in Example 1 to induce glial cells in the dorsal midbrain, as shown in the following table (Table 2). Different transcription factors showed significantly different transformation efficiencies. .

[0171] Table 2. In vivo glial cell transdifferentiation efficiency of single transcription factor (sequence shown in Example 1)

[0172]

[0173]

[0174] On the basis of the in vivo model, we further conducted a detailed study on the expression elements of the AAV expression vector, and found at least three technical improvements that can significantly increase the transformation efficiency of glial cells.

[0175] (1) Insertion of VP16 fusion protein

[0176] VP16 is the activation domain (SEQ ID NO: 45) of the VP...

Embodiment 3

[0187] Example 3 The combination of functional fragments of transcription factors further increases the efficiency of glial cell transdifferentiation

[0188] According to the transdifferentiation model of glial cells in vitro and in vivo, combined with the vector transformation strategy described in Example 2, we firstly expanded the selected transcription factors into random combinations. Wherein, different transcription factors can be expressed in the same vector at the same time, or expressed in different expression vectors respectively, and the expression ratios described in the following table are the molar concentration ratios of the functional proteins expressed in the actual study. In NeuroD1, Brn2, Gsx1, Tbr1, Dlx2, Ptf1a, Pax6, Otx2 transcription factors, we unexpectedly obtained several groups of single transcription factors with low efficiency, but the combination can significantly improve the efficiency of transcription factors, and the expression of functional pr...

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Abstract

Functional fragments for reprogramming, combinations, and uses thereof. The invention provides a group of transcription factors and a transcription factor combination capable of synergistically promoting the transdifferentiation of glial cells and reprogramming into functional neurons or neuron-like cells, and the glial cells are reprogrammed into functional neurons or neuron-like cells by promoting the expression of the group of transcription factors in vivo or in vitro. The transcription factor can be effectively applied to repairing damage of a nervous system by transdifferentiation of glial cells or limiting deterioration of brain tumors derived from glial cells by utilizing the transdifferentiation capability of key transcription factors, and the application of the found transcription factor and the combination thereof in preparation of medicines for treating nervous system diseases.

Description

technical field [0001] The present invention belongs to the fields of biotechnology and gene therapy, in particular, the present invention relates to a method for transdifferentiation of glial cell-derived cells into neurons, and a method for applying this method to repair damage to the nervous system or to treat glial cell-derived tumors . Background technique [0002] The main pathological changes caused by mammalian central nervous system injury and various neurodegenerative diseases are irreversible neuronal degeneration and necrosis and neural circuit damage. How to replace dead and lost neurons in the damaged and diseased brain or spinal cord and rebuild neural circuits is a key step in treatment. Because 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 on its own. [0003] Due to the low efficiency of transplantation of exogenous neurons or ne...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0793A61K45/00A61P25/00A61P35/00
CPCC12N5/0619A61K45/00A61P25/00A61P35/00C12N2501/998C12N2506/08C12N15/86A61K48/005C12N2750/14143C12N2740/16043C12N2710/10343C07K14/4702C12N5/0622C12N2501/60C12N2510/00C12N2501/13C12N2506/30A61K38/00C07K14/4705C12N15/85A61K38/19
Inventor 刘月光陈如雷
Owner NEURAGEN BIOTHERAPEUTICS (SUZHOU) CO LTD
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