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Genetic Medicines for Leber's Hereditary Optic Neuropathy

A gene drug, gene technology, applied in the direction of genetic material components, introduction of foreign genetic material using vectors, drug combination, etc., can solve the problem of low immunogenicity

Active Publication Date: 2022-05-10
BEIJING GENECRADLE PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

One, the AAV vector only retains the two ITR sequences required for packaging in the wild-type virus, and does not contain protein-coding genes in the wild-type virus genome (Salgenik M, et al . Microbiol Spectr. 2015; 3(4).), low immunogenicity

Method used

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  • Genetic Medicines for Leber's Hereditary Optic Neuropathy
  • Genetic Medicines for Leber's Hereditary Optic Neuropathy
  • Genetic Medicines for Leber's Hereditary Optic Neuropathy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Plasmid vector construction

[0053] First, we construct the AAV vector plasmid (pAAV-CMV-EGFP-COX10 ( image 3 ), pscAVV-CAM-EGFP-ATP5B ( Figure 4 ), pAAV-CMV-ND4-COX10 ( Figure 5 ), pAAV-CMV-ocND4-COX10 ( Figure 6 ), pscAAV-CAM-ocND4-ATP5B ( Figure 7 ), pscAAV-HSP-mND4 ( Figure 8 )), and then constructed the mitochondria-targeted AAV vector packaging envelope plasmid (pAAV-VP2A-COX8-EGFP ( Figure 9 ) and pAAV-VP1-VP3 ( Figure 10 )) and the AAV vector packaging envelope plasmid (pAAV-R2C2-3YF( Figure 12 ), pAAV-R2C2-4YF ( Figure 13 )). The single-chain AAV vector packaging vector plasmid (pAAV-CMV-EGFP-COX10 ( image 3 ), pAAV-CMV-ND4-COX10 ( Figure 5 ), pAAV-CMV-ocND4-COX10 ( Figure 6 )) pAAV2neo held in company ( figure 1 ) is the basic skeleton. Double-stranded AAV vector packaging plasmid (pscAVV-CAM-EGFP-ATP5B ( Figure 4 ), pscAAV-CAM-ocND4-ATP5B ( Figure 7 ), pscAAV-HSP-mND4 ( Figure 8 )) is in the pscAAV-CAM vector ( fi...

Embodiment 2

[0069] Example 2 Preparation and assay of recombinant AAV virus

[0070] References (Xiao X, et al . J Virol. 1998;72(3):2224-2232.), the three-plasmid packaging system was used to package the recombinant AAV virus, and the cesium chloride density gradient centrifugation method was used to separate, purify and package the AAV virus. Briefly, AAV vector plasmid (pAAV-CMV-EGFP-COX10, pscAAV-CAM-EGFP-ATP5B, pAAV-CMV-ND4-COX10, pAAV-CMV-ocND4-COX10, or pscAAV-CAM-ocND4-ATP5B), helper plasmid (pHelper) and AAV Rep and Cap protein expression plasmids (pAAV-DJ (for packaging AAVDJ virus), pAAV-R2C2, pAAV-R2C2-3YF or pAAV-R2C2-4YF) were mixed at a molar ratio of 1:1:1 After homogenization, HEK293 cells were transfected by the calcium phosphate method. After 48 hours of transfection, the cells and culture supernatant were harvested, and the recombinant AAV virus was isolated and purified by cesium chloride density gradient centrifugation. Packaged and purified to obtain AAVDJ-EGFP-CO...

Embodiment 3

[0094] Example 3 Carrying EGFP reporter gene AAV virus in vitro cell experiment

[0095] Using the three-plasmid packaging system, the pAAV-CMV-EGFP-COX10 and pscAAV-CAM-EGFP-ATP5B plasmids were packaged into AAVDJ-EGFP-COX10 and scAAVDJ-EGFP-ATP5B, respectively. The packaged AAVDJ-EGFP-COX10 and scAAVDJ-EGFP-ATP5B viruses were divided into 1×10 5 A dose of vg / cell was used to infect human BJ cells (purchased from ATCC (American Tissue Culture Collection, American Tissue Culture Collection), ATCC NO.CRL-2522). After the virus transduced the cells for 72 hours, the expression of green fluorescent protein and its distribution in the cells were observed with a confocal microscope (Leica TSC 4D). Localization of mitochondria in cells with the alpha subunit of ATP synthase. Under the same field of view, the overlapping area between the distribution of GFP in cells and the distribution of ATP synthase α subunit is the distribution area of ​​GFP in mitochondria. For the detection ...

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Abstract

The invention provides a medicine for treating Leber hereditary optic neuropathy mediated by recombinant adeno-associated virus. The recombinant adeno-associated virus vector carries the expression cassette for expressing ND4 protein in the nucleus. In vivo experiments show that the recombinant adeno-associated virus vector can be efficiently introduced into the retina through intravitreal injection, and the ND4 protein can be continuously and stably expressed, located in the mitochondria, and the adverse symptoms caused by ND4 gene mutation can be alleviated or restored. The results suggest that the recombinant adeno-associated virus vector may be developed into a new therapeutic drug for Leber's hereditary optic neuropathy.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to gene medicine for Leber hereditary optic neuropathy based on a recombinant adeno-associated virus vector. Background technique [0002] Leber's Hereditary Optic Neuropathy (LHON) (OMIM535000) is a maternally inherited disease that affects the retina and the anterior papillary macular tract fibers of the scleral cribriform plate, leading to degeneration of the optic nerve (that is, the maternal offspring is affected but the paternal offspring is not affected ) (Yu-Wai-Man P, et al. Prog RetinEye Res. 2011;30(2):81-114). In 1871, German ophthalmologist Leber T first reported the clinical symptoms, signs and genetic characteristics of the disease (Leber T. Graefe’s Arch Ophthalmol. 1871; 17:249-291.). LHON mainly affects young and middle-aged men, and the age of onset is usually 15-20 years old. Clinically, it mainly manifests as acute or subacute painless vision loss in both eyes at t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/864A61K48/00A61K38/17A61P27/02
CPCA61K38/1709A61K48/0058A61P27/02C12N15/85C12N15/86C12N2750/14143C12N2800/107
Inventor 田文洪马思思吴小兵董小岩
Owner BEIJING GENECRADLE PHARM CO LTD
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