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Gene vector and gene therapy drug for treating retina ganglion cell denaturation

A technology of drugs and virus vectors, applied in the field of genetic engineering, can solve problems such as cell death

Active Publication Date: 2018-08-10
SHANGHAI FIRST PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

ER stress is critical for cell survival, but persistent ER stress leads to cell death

Method used

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  • Gene vector and gene therapy drug for treating retina ganglion cell denaturation
  • Gene vector and gene therapy drug for treating retina ganglion cell denaturation
  • Gene vector and gene therapy drug for treating retina ganglion cell denaturation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1: Construction and separation and purification of adenoviral vector

[0074] Plasmid AAV_FADD-DN was constructed as figure 1 , figure 2 As shown, the main elements include CMV enhancer / promoter and FADD-DN sequence. The CMV enhancer can enhance the expression of the transgene. Immediately after the target gene is hGHpA (SEQ ID NO: 7), and the expression cassette is flanked by inverted terminal repeats (TR), that is, the viral vector includes L-ITR (SEQ ID NO: 3), R-ITR (SEQ ID NO :8), viral vectors also include Ampicillin (SEQ ID NO: 9) and flori (SEQ ID NO: 11).

[0075]Viral vectors were obtained by plasmid co-transfection method. HEK 293T cells were co-transfected with the helper plasmid containing the AAV2 coat protein gene and the gene that can help AAV to replicate, and the AAV_FADD-DN plasmid to initially form a recombinant adeno-associated virus vector. After the initial purification of iodine butanol, the fast protein liquid chromatography with 5...

Embodiment 2

[0077] Example 2: Detection of Adenovirus Infection

[0078] Tissue treatment: Two weeks after intravitreal injection of HA-labeled AAV_FADD-DN in mice, the pretreatment of the eyeball was carried out according to the aforementioned tissue treatment method, and then the bottom of the eyeball was clamped and lifted up with micro forceps, and the conjunctiva and muscles around the eyeball were cut with scissors Tissue, keep the eyeball tissue intact, cut off the cornea, iris and lens to make the eye cup, and separate the retina and choroid complex.

[0079] Immunofluorescence staining: select the retina, block it in 20% goat serum solution for 1 hour, then incubate overnight with the primary antibody, the antibodies are TUJ1 and HA respectively; wash three times with PBS, 10 minutes each time, and incubate with the secondary antibody for 1 hour; wash three times with PBS, each time Cover the slides after ten minutes. Changes in retinal structure were observed and photographed u...

Embodiment 3

[0081] Example 3: Detection of RGC survival rate

[0082] Experimental method: 8-week-old mice were injected with AAV_FADD-DN (experimental group) and AAV-GFP (control group) into the vitreous cavity of one side of the eye, and at the same time injected anterior chamber magnetic beads into the injected eye. At 8 weeks, the eyeballs and retinas were processed according to the above method, and immunofluorescent staining was performed. Changes in retinal structure were observed and photographed using a confocal microscope. Five visual fields were randomly selected from each eyeball to count RGCs (scale bar: 20um), and the RGC survival rate was calculated.

[0083] Result: if Figure 4-5 As shown, quantitative analysis proves that RGCs gradually apoptotic at 2W, 4W, and 8W after injection of anterior chamber magnetic beads microparticles, and the number decreased significantly (experimental group); compared with this, RGC apoptosis in mice injected with AAV_FADD-DN was not sign...

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Abstract

The invention relates to a gene vector and a gene therapy drug for treating retina ganglion cell denaturation. The gene vector comprises dominant negative human Fas-associated protein with death domain (FADD-DN), an enhancer / promoter, a beta-globin intron capable of enhancing gene expression, a human growth hormone poly(A) tail sequence and the like. The invention further relates to application ofthe gene vector or a composition containing the gene vector to preparation of a drug for treating the retina ganglion cell denaturation. The gene vector provided by the invention has the advantages that the gene therapy vector provided by the invention can be used for treating or preventing retina ganglion cell pathological changes caused by glaucoma and the like.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a gene carrier and a gene therapy drug for treating degeneration of retinal ganglion cells. Background technique [0002] Retinal ganglion cell degeneration is the main cause of glaucoma and other diseases leading to visual impairment and even blindness, and pathological increased intraocular pressure is the main risk factor. The pathological mechanism of glaucoma is complex, involving genetic and environmental factors, but its exact molecular mechanism is still unclear. Some glaucoma patients present with high intraocular pressure. Symptomatic treatments such as lowering intraocular pressure can be used clinically, but the development of glaucoma cannot be reversed or delayed in the long run. It has been recognized that glaucoma is a neurodegenerative disease, and its pathological feature is the apoptosis of retinal ganglion cells and the degeneration of axons, as w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K38/17A61P27/06A61P27/02
Inventor 孙晓东罗学廷刘洋
Owner SHANGHAI FIRST PEOPLES HOSPITAL
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