Method of fast increasing glandular related virus mediating gene in expression of inside of retina cell

A retinal cell and adenovirus technology, used in gene therapy, genetic engineering, plant genetic improvement, etc., can solve problems such as disease-causing retinal cells and degeneration

Inactive Publication Date: 2006-09-20
上海交通大学附属第一人民医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Given that wild-type adenovirus is a helper virus for AAV replication, infection wit...

Method used

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  • Method of fast increasing glandular related virus mediating gene in expression of inside of retina cell
  • Method of fast increasing glandular related virus mediating gene in expression of inside of retina cell
  • Method of fast increasing glandular related virus mediating gene in expression of inside of retina cell

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Wild-type adenovirus accelerates and increases the transduction efficiency and gene expression level of AAV to retinal cells, but at the same time causes retinal cell lesions

[0070] 2×10 per hole 5 RPE (APRE-19, ATCC) cells were seeded in 6-well cell culture plates, and after 24 hours, the cells adhered to the wall, and 1×10 8 or 1×10 9 AAV 2 -GFP with 1 x 10 3 , 1×10 4 or 1×10 5 DL309 was added to the cultured cells, and then observed with a fluorescent microscope every day and photographed to record the brightness of GFP fluorescence, cell morphology and disease state of the infected cells. After 5-7 days, trypsinization was performed, the number of GFP-positive cells and GFP fluorescence intensity were detected by flow cytometry, and the copy number of GFP gene and the expression level of GFP were detected by PCR and Western blot.

[0071] The above test results show that when AAV 2 - The amount of GFP virus used was 1 × 10 8 When DL309 was not added, GFP e...

Embodiment 2

[0074] Conditionally replicating adenovirus accelerates and improves AAV transduction efficiency and gene expression level in retinal cells

[0075] The conditional replication adenovirus is different from the wild-type adenovirus DL309 in that although it has E1A and E1B genes, its expression is regulated by the TERT gene promoter or the HSP gene promoter, respectively. Therefore, its replication is regulated to a certain extent, with better safety and less toxic and side effects. The dosage of Ad-TERT-E1A / E1B and Ad-HSP-E1A / E1B in the in vitro experiment was 1×10 3 -1×10 6 pfu. AAV 2 -The amount of GFP was 1×10 8 and 1×10 9 Viral particles; for each RPE cell, the amount of adeno-associated virus is about 5 × 10 2 -5×10 3 virus particles, the amount of conditionally replicating adenovirus is about 0.005-5 pfu. The ratio of conditionally replicating adenovirus to adeno-associated virus was 1:1×10 2 -1:1×10 5 ; the dosage of each nerve cell is roughly 10 4 -10 5 For...

Embodiment 3

[0083] Replication-defective adenovirus accelerates and increases AAV transduction efficiency and gene expression levels in retinal cells

[0084] The amount of Ad-Luc used in the in vitro experiment was 1×10 3 , 1×10 4 , 1×10 5 and 1×10 6 pfu, AAV 2 The dosage is 1×10 8 or 1×10 9 virus particles. The same design as the conditional replicating adenovirus experiment, for each RPE cell, the amount of adeno-associated virus is about 5×10 2 -5×10 3 For each virus particle, the amount of replication-defective adenovirus Ad-Luc is about 0.005-5pfu; the ratio of Ad-Luc to adeno-associated virus is 1:10 2 -1:10 6 ; the dosage of each nerve cell is roughly 10 4 -10 5 For each virus particle, the dosage of Ad-Luc is about 0.05-5pfu. The ratio of Ad-Luc to Adeno-associated virus was 1:10 2 -1:10 5 .

[0085] 2×10 per hole 5 RPE (APRE-19, ATCC) cells were seeded in 6-well cell culture plates, and after 24 hours, the cells adhered to the wall, and 1×10 8 or 1×10 9 AAV 2 ...

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Abstract

This invention relates to the expression method of adeno-associated virus-mediated gene in retinal cells. This invention can significantly improve the transfer efficiency and gene expression level of adeno-associated virus-mediated gene in retinal cells by using a combination of different types of adeno-associated virus, and has no toxic or side effect. Experimental results show that at a certain dosage range, replication-competent adenovirus and replication-defective adenovirus can significantly accelerate AAV2-mediated gene expression, and no cell lesion is observed. This invention can be used for gene treatment of ocular fundus diseases, such as retinosis, maculopathy, diabetic retinopathy, uveitis, choroidal neovascularization, optic nerve degeneration and damage.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to a method for adeno-associated virus-mediated gene expression in retinal cells. Background technique [0002] The retina is composed of the neural retina and the retinal pigment epithelium (RPE for short). The neural retina is located on the innermost side of the eyeball wall, and the RPE is located on the outer side of the neural retina. There is a close relationship between the two in terms of histogenesis, structure and function. In the early stage of embryonic development, the neuroectoderm at the front of the forebrain protrudes to both sides to form eye vesicles, and soon the top of the eye vesicles is sunken to form eye cups, the inner layer of which develops into neural retina, and the outer layer develops into RPE. The two layers are closely adjacent, but there are A potential cavity, called the subretinal space. The neural retina is mainly composed of three different types of c...

Claims

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Application Information

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IPC IPC(8): C12N15/861C12N15/67A61K48/00A61P27/02
Inventor 黄倩吴继红吴小兵董小岩李川源韦芳刘新建陈霞芳李惠明王丰
Owner 上海交通大学附属第一人民医院
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