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Composition containing Atoh7 and/or Pou4f, and preparation method and medical application of composition

A composition and drug technology, applied in the field of drugs for the treatment of eye diseases and biomedicine, can solve the problem of not participating in RGC, etc., and achieve reliable and representative results

Pending Publication Date: 2019-10-08
向孟清 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Isl1 inactivation also leads to the disappearance of most RGCs, but Isl1 may not be involved in the specification and fate determination of RGCs, but only in the regulation of their terminal differentiation and functional maintenance [38,39]

Method used

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  • Composition containing Atoh7 and/or Pou4f, and preparation method and medical application of composition
  • Composition containing Atoh7 and/or Pou4f, and preparation method and medical application of composition
  • Composition containing Atoh7 and/or Pou4f, and preparation method and medical application of composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0106] The specific process and details of the present invention will be described in detail below by taking "Atoh7 and Pou4f2 transdifferentiate Muller cells into RGCs in vivo" as an example.

[0107] (1) Construction of AAV virus vector (plasmid)

[0108] Mouse Atoh7 (MGI: 1355553, NCBI Gene: 53404) and Pou4f2 (MGI: 102524,

[0109] NCBI Gene: 18997) coding sequences were cloned into different AAV viral vectors respectively, and its expression was controlled by the GFAP promoter to obtain plasmid AAV-Atoh7 and plasmid AAV-Pou4f2.

[0110] Or use the P2A sequence to connect mouse Atoh7 and Pou4f2, and then clone it into the AAV virus vector to obtain the plasmid AAV-Atoh7-P2A-Pou4f2, which is verified by sequencing;

[0111] (2) Preparation of AAV virus

[0112] 1) 24 hours before the transfection of the plasmid (the AAV virus vector obtained in the preceding steps), spread 4.0×10 cells in a 10cm cell culture dish 6 HEK293T cells were cultured in DMEM medium containing 10%...

Embodiment 2

[0130] The use of monomers of Atoh7, Pou4f1, Pou4f2, or Pou4f3 also promotes the transdifferentiation of Müller cells into retinal ganglion cells in vivo. Those skilled in the art can understand the operation of this embodiment according to the operation process of Embodiment 1, and details are not repeated here. Experimental results such as image 3 It was shown that in the experiment using AAV virus carrying Pou4f3 monomer to achieve RGCs regeneration, a small number of Müller cells could differentiate into RGCs. Although most of the Müller cells remain undifferentiated, the Pou4f3 monomer is less effective in promoting the transdifferentiation of Müller cells into retinal ganglion cells than the Atoh7+Pou4f combination, but it is undeniable that the Müller cells can differentiate into retinal ganglion cells The role played by RGCs.

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Abstract

The invention discloses a composition containing Atoh7 and / or Pou4f, and a preparation method and medical application of the composition. A virus serves as a carrier, following transcription factors:(1) Atoh7+Pou4f2, or (2) Atoh7+Pou4f3, or (3) Atoh7+Pou4f1, or (4) any one of or other combinations except the combination of Atoh7, Pou4f1, Pou4f2, and Pou4f3 are subjected to overexpression in Muller cells of a human or animal retina, and thus the Muller cells are directionally differentiated into retinal ganglion cells (RGCs). The RGCs regenerated through the composition can be migrated to a ganglion cell layer and integrated to a local neutral network, have the normal RGC form and function, and are connected to different brain areas. The RGCs express various specific markers of normal ganglion cells and have activity such as electrophysiology similar to normal RGCs in vivo. The composition can make the RGCs in vivo be regenerated and can be applied to the aspects such as cell replacement therapy of neurologic diseases such as glaucoma.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to the field of medicine for treating eye diseases. Background technique [0002] Glaucoma (glaucoma) is one of the three major blindness diseases that lead to blindness in humans. It is a group of diseases characterized by optic disc atrophy, visual field defect and vision loss. Global data show that the incidence of glaucoma is as high as 3.54% in 40-80 years old, and it is on an upward trend, and may reach 112 million people by 2040 [1]. In China, the incidence rate of glaucoma in the general population is 2.59%, and the number of patients will reach 25 million in 2050 [2]. According to the cause of glaucoma, glaucoma can be divided into three categories: primary, secondary and congenital glaucoma. The most common cause is increased intraocular pressure caused by various factors that cause aqueous humor circulation disorders, but elevated intraocular pressure is not the only risk fact...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K38/17A61P27/06
CPCA61K38/1709A61K48/0025A61K48/005A61P27/06
Inventor 向孟清晋康新肖冬长
Owner 向孟清
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