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Gene medicine for preventing and treating CNV (choroidal neovascularization)-associated eye diseases

A gene and virus technology, applied in the field of genetic medicine for the prevention and treatment of choroidal neovascularization-related eye diseases, can solve problems such as increased medical burden, risk of shedding, infectious endophthalmitis, retina, etc.

Active Publication Date: 2018-06-01
BEIJING GENECRADLE PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the one hand, continuous multiple administration increases the medical burden, on the other hand, multiple intravitreal injections also bring about an increased risk of serious complications such as infectious endophthalmitis and retinal detachment

Method used

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  • Gene medicine for preventing and treating CNV (choroidal neovascularization)-associated eye diseases
  • Gene medicine for preventing and treating CNV (choroidal neovascularization)-associated eye diseases
  • Gene medicine for preventing and treating CNV (choroidal neovascularization)-associated eye diseases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1. Construction of plasmid vectors

[0046] (1) Construction of different expression units carrying reporter gene Gluc

[0047] The promoter CASS (SEQ ID No.1), woodchuck hepatitis virus post-transcriptional regulatory element (WPRE, SEQ ID No.5) and UBC enhancer (SEQ ID No. .7), and introduced an XhoI restriction site at the 5' end of the CASS promoter, and an EcoRI restriction site at the 3' end. Through standard molecular biology operations, first replace the promoter on the AAV plasmid cloning vector pAAV2neo preserved in our laboratory with CASS, insert GFP and WPRE sequences between the EcoRI and BglII restriction sites of the multi-cloning site, and insert The UBC enhancer is introduced between the BamHI and NheI restriction sites downstream of BGH polyA, constituting the gene expression unit of the promoter-GFP-WPRE-poly A-UBC enhancer, named pAAV2neo-CASS-GFP, and its structure diagram is shown in figure 1 .

[0048] (2) Construction of AAV vector p...

Embodiment 2

[0050] Example 2. Comparison of expression levels of pAAV2neo-FPFab01, pAAV2neo-FPIG01, pAAV2neo-FPFab02 and pAAV2neo-FPIG02 in cells

[0051] Transfect 293T and ARPE19 cells with pAAV2neo-FPFab01, pAAV2neo-FPIG01, pAAV2neo-FPFab02, pAAV2neo-FPIG02 and pAAV2neo-CASS-GFP, respectively, collect the cell supernatant 96 hours after transfection, and detect FPFab and FPIG in the supernatant by ELISA level of expression. The specific operation is briefly described as follows: 293T or ARPE19 cells were divided into 5×10 4 cells / well were seeded into 48-well culture plates at 37°C in 5% CO 2 Culture in an incubator until the cells are 80-90% confluent, and use lipofectamine 2000 to transfect the plasmids pAAV2neo-FPFab01, pAAV2neo-FPIG01, pAAV2neo-FPFab02, pAAV2neo-FPIG02 or pAAV2neo-CASS-GFP respectively. Set up 4 replicate holes. The supernatant was taken 96 hours after transfection, and the expression level of FPFab or FPIG in the supernatant was detected by ELISA method.

[00...

Embodiment 3

[0056] Example 3. Preparation and assay of recombinant AAV virus

[0057] The three-plasmid packaging system was used to package and purify the recombinant AAV virus. For specific operations, refer to literature [17]. Briefly, AAV vector plasmids (pAAV2neo-CASS-GFP, pAAV2neo-FPFab01 or pAAV2neo-FPIG01), helper plasmids (pHelper) and AAV Rep and Cap protein expression plasmids (pAAV-R2C5 or pAAV-R2C8 or pAAV-R2C9) were prepared according to After mixing at a molar ratio of 1:1:1, HEK293 cells were transfected by the calcium phosphate method. After 48 hours of transfection, the cells and culture supernatant were harvested. According to the AAV vector purification method reported by Wu Xiaobing et al. [18], the virus titer was determined by dot blot method. For specific operations, refer to literature [19].

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Abstract

The invention provides composition and a method for preventing or treating CNV (choroidal neovascularization)-associated ocular diseases. The composition is a recombinant vector expression unit and arecombinant viral vector of a suitable AAV (adeno-associated virus) coat, wherein the recombinant vector expression unit contains a VEGF (vascular endothelial growth factor) antibody or a functional fragment gene of the VEGF antibody. The composition can prevent or treat the CNV-associated ocular diseases such as age-associated macular degeneration and diabetic retinopathy by intravitreal or subretinal injection.

Description

technical field [0001] The present invention relates to the application of recombinant adeno-associated virus (adeno-associated virus, AAV) vector expressing VEGF antibody in the prevention or treatment of eye diseases related to choroidal angiogenesis, such as age-related macular degeneration and diabetic retinopathy. Background technique [0002] Choroidal neovascularization (CNV) is a serious complication of various eye diseases. It can cause a series of pathological changes such as fundus tissue hemorrhage, exudation and hyperplasia, thus causing damage to the structure and function of the eyeball and seriously impairing vision. Features. This series of fundus lesions include age-related macular degeneration (age-related macular degeneration, AMD), diabetic retinopathy (diabetic retinopathy, DR) and retinopathy of prematurity, which seriously affect vision and even cause blindness. [0003] Age-related macular degeneration is a disease that increases in incidence with a...

Claims

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Application Information

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IPC IPC(8): C12N15/864A61K48/00A61P27/02A61P9/10A61P3/10
CPCA61K39/3955A61K48/0008A61K48/005A61K2039/505A61K2039/5256C12N15/86C12N2750/14143C12N2800/107
Inventor 余双庆董小岩田文洪马思思张著月
Owner BEIJING GENECRADLE PHARM CO LTD
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