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Adeno-associated virus vector carrying C3 gene expression cassette and application thereof

A gene expression and virus technology, applied in the field of recombinant adeno-associated virus vectors, can solve the problems of CAM promoters not having cell expression specificity, side effects of corneal edema, etc., and achieve the effect of increasing permeability, less damage, and improving discharge efficiency

Active Publication Date: 2020-05-01
BEIJING GENECRADLE PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the scAAV2 virus can also infect corneal endothelial cells, the CAM promoter does not have cell expression specificity, and the infected corneal endothelial cells overexpress C3 protein, which brings side effects of corneal edema

Method used

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  • Adeno-associated virus vector carrying C3 gene expression cassette and application thereof
  • Adeno-associated virus vector carrying C3 gene expression cassette and application thereof
  • Adeno-associated virus vector carrying C3 gene expression cassette and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Embodiment 1 plasmid vector construction

[0076] Construct the AAV vector plasmid (pscAAV-CAM-C3 (pscAAV-CAM-C3 ( image 3 ), pscAAV-CAM-EGFP ( Figure 4 ), pscAAV-CAM-Gluc ( Figure 5 ), pscAAV-CH3L1-Gluc ( Figure 6 ), pscAAV-CH3L1-C3 ( Figure 7 ), pscAAV-MGP-Gluc ( Figure 8 ), pscAAV-MGP-C3 ( Figure 9 )) for packaging preparation to obtain the required recombinant AAV virus. These plasmid vectors are in the pscAAV-CAM vector ( figure 2 ), the precursor plasmid of the pscAAV-CAM vector is pAAV2neo ( figure 1 ).

[0077] (1) Construction of pscAAV-CAM vector

[0078]To construct a double-stranded AAV vector, we first constructed a general-purpose pscAAV-CAM plasmid based on pAAV2neo. The construction process is as follows. Based on the 3' ITR sequence in the AAV2 genome (GenBank No.AF043303), the trs sequence and the D sequence in the ITR sequence were deleted according to literature reports (Wang Z, et al. Gene Ther. 2003; 10: 2105-2111.), to obtain t...

Embodiment 2

[0091] Embodiment 2 recombinant AAV virus preparation and assay

[0092] References (XiaoX, etal .JVirol.1998;72(3):2224-2232.), using the three-plasmid packaging system to package the recombinant AAV virus, and using cesium chloride density gradient centrifugation to separate, purify and package the AAV virus. Briefly, AAV vector plasmids (pscAAV-CAM-C3, pscAAV-CAM-EGFP, pscAAV-CAM-Gluc, pscAAV-CH3L1-C3, pscAAV-CH3L1-Gluc, pscAAV-MGP-C3, or pscAAV-MGP-Gluc), The helper plasmid (pHelper) and the AAV Rep and Cap protein expression plasmid (pAAV-R2C2) were mixed according to the molar ratio of 1:1:1, and the HEK293 cells were transfected by the calcium phosphate method. After 48 hours of transfection, the cells were harvested and cultured. The supernatant was separated and purified by cesium chloride density gradient centrifugation. Seven recombinant viruses including scAAV2-C3, scAAV2-EGFP, scAAV2-CAM-Gluc, scAAV2-CH3L1-C3, scAAV2-CH3L1-Gluc, scAAV2-MGP-C3, scAAV2-MGP-Gluc w...

Embodiment 3

[0110] Example 3 scAAV2-C3 virus evaluation experiment in vivo and in vitro

[0111] In order to verify whether the scAAV2-C3 virus can effectively transduce trabecular meshwork cells, express and produce C3 protein, reduce the stability of actin in cells, change the morphological structure of trabecular meshwork cells, and increase the permeability of trabecular meshwork, It is beneficial to the discharge of aqueous humor and lower intraocular pressure, so as to achieve the purpose of treating glaucoma. We designed a series of experiments. First, using scAAV2-EGFP as a control, we transduced primary cultured human trabecular meshwork cells with scAAV2-C3 virus, and observed changes in cell morphology and actin to verify the ability of scAAV2-C3 virus to transduce trabecular meshwork cells. Does it work. Next, we injected scAAV2-C3 virus into the eyes of C57BL / 6J mice through the anterior chamber, and observed the reduction of intraocular pressure in mice. Then, using scAAV-...

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Abstract

The invention provides a recombinant adeno-associated virus-mediated drug for glaucoma treatment. A recombinant adeno-associated virus vector carries a clostridium botulinum C3 gene expression cassette. In-vivo experiments show that the recombinant adeno-associated virus vector can be efficiently introduced into trabecular meshwork cells via anterior chamber injection, continuously and stably express and produce C3 protein, increase the permeability of trabecular meshwork channels, reduce intraocular pressure, and provide a novel option for the glaucoma treatment.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant adeno-associated virus vector carrying a Clostridium botulinum C3 gene expression frame and its application in reducing intraocular pressure and treating glaucoma. Background technique [0002] Glaucoma, characterized by optic atrophy and visual field defect, is the second most common blindness in the world and the first among irreversible blindness (Mantravadi AV, Vadhar N. Primary care. 2015;42(3):437-449. ). The main risk factor of glaucoma comes from elevated intraocular pressure, that is, intraocular pressure exceeds the limit that the tissues in the eyeball, especially the retinal optic nerve, can bear, which will cause visual function damage. From the global epidemiological survey, it is found that glaucoma is related to age, the incidence rate of people over 75 years old is 7%, the prevalence rate of people over 40 years old is 2.4%, and the blinding rate is ab...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/864C12N7/01C07K14/33A61K48/00A61K38/16A61P27/06
CPCC07K14/33C12N15/86C12N7/00A61K48/005A61K38/164A61K9/0048A61K9/0019A61P27/06C12N2750/14121C12N2750/14143Y02A50/30
Inventor 田文洪马思思董小岩刘旭阳
Owner BEIJING GENECRADLE PHARM CO LTD
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