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ND4 gene recombinant adenovirus associated virus vector and preparation method and application thereof

A virus vector and gene technology, applied in the field of ND4 gene recombinant adeno-associated virus vector and its preparation, can solve the problems of slow expression and low transduction efficiency, achieve high expression, enhance effect, and ensure safety

Inactive Publication Date: 2020-12-22
WUHAN NEUROPHTH BIOTECHNOLOGY LTD CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is to provide a recombinant adeno-associated virus with ND4 gene in order to overcome defects such as low transduction efficiency and slow expression when the pAAV2 packaging NADH dehydrogenase subunit 4 (ND4) sequence is used as a gene drug in the prior art Carrier and its preparation method and application

Method used

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  • ND4 gene recombinant adenovirus associated virus vector and preparation method and application thereof
  • ND4 gene recombinant adenovirus associated virus vector and preparation method and application thereof
  • ND4 gene recombinant adenovirus associated virus vector and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Construction of recombinant adeno-associated virus vector of human NADH dehydrogenase subunit 4 gene and its virus packaging and purification method

[0042] (1) Construction of recombinant adeno-associated virus vector containing human NADH dehydrogenase subunit 4 gene

[0043] 1) Vector construction

[0044] The recombinant human NADH dehydrogenase subunit 4 (ND4) fusion gene sequence shown in SEQ ID NO: 1 and the optimized recombinant human NADH dehydrogenase subunit 4 (ND4) shown in SEQ ID NO: 2 The fusion gene sequence was synthesized by adding Kpn I and Sal I restriction sites respectively (the gene sequences were all synthesized by Chengdu Qingke Zixi Biotechnology Co., Ltd.), or primers were designed using the above-mentioned ND4 DNA sequence as a template (2 The primers contain Kpn I and Sal I restriction sites respectively) for PCR amplification, and the synthetic products or amplified products were combined with the plasmid vector pAAV-MCS (includi...

Embodiment 2

[0075] Example 2: Effect experiment of human NADH dehydrogenase subunit 4 gene recombinant adeno-associated virus on Leber's dominant hereditary optic neuropathy

[0076] (1) ND4 expression results detected in the vitreous cavity and retina of rabbit eyes

[0077] 1. Intravitreal injection of rabbit eyes

[0078] Rabbits were divided into groups according to the table below, with 2 rabbits in each group, and 50 μl of 1×10 12 Vg / mL of the above two different recombinant human NADH dehydrogenase subunit 4 gene adeno-associated viruses was injected into the vitreous cavity through the pars plana of the ciliary body 3 mm away from the limbus.

[0079]

[0080] In the present invention, unless otherwise specified, the "wild ND4" refers to human ND4, and the specific sequence corresponds to SEQ ID NO: 1; the "optimized ND4" refers to optimized ND4, and the specific sequence corresponds to SEQ ID NO: 2.

[0081] 2. Slit lamp, intraocular pressure, fundus photographic examinatio...

Embodiment 3

[0110] Example 3: AAV2 / 2-ND4 and AAV2 / 9-ND4 were injected into muscle tissue of mice respectively, and their expression differences were analyzed by qPCR.

[0111]

[0112]

[0113] Take 20 mice and divide them into 5 groups, and divide them into control groups according to the table above (vitreous injection of 5×10 10 vg rAAV-ZsGreen [Guangzhou Paizhen Biotechnology Co., Ltd.]), experimental groups A, B, C, and D were injected with the above four kinds of recombinant human NADH dehydrogenase subunit 4 gene recombinant adeno-associated virus (rAAV2 / 2-optimized ND4 and rAAV2 / 2-ND4 and rAAV2 / 9-optimized ND4 and rAAV2 / 9-ND4, 50 μl, 1 x 10 12 vg / mL), puncture injection in the inner thigh of mice. Observe the body weight change of mice within one month (results such as Figure 9 shown) and extract mouse thigh tissue mRNA. qPCR detects the ND4 gene, and compares the relative expression level of the ND4 gene between the experimental group and the control group (the results ...

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Abstract

The invention discloses a recombinant adenovirus associated virus vector containing an ND4 gene expression cassette. A skeleton of the recombinant adenovirus associated virus vector is pAAV9, and theND4 gene expression cassette contains a human ND4 gene or an optimized human ND4 gene. The invention also discloses a preparation method and application of the recombinant adenovirus associated virusvector. According to the preparation method, pAAV9 is used as a gene vector of ND4, and when ND4 is transduced into eyes of a patient, the expression amount on the retina is higher while the safety isensured, the higher transduction efficiency and efficient expression capability are achieved, and the optic nerve cell infection capability is stronger. When the recombinant adenovirus associated virus vector is prepared into a vitreous cavity injection medicine, the effect of the gene in Leber's gene therapy can be enhanced, the injection dosage can be reduced, and the healing time can be shortened.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular to an ND4 gene recombinant adeno-associated virus vector and its preparation method and application. Background technique [0002] Leber's hereditary optic neuropathy (LHON) is a mitochondrial hereditary disease that mainly affects the fibers of the macular papillary tract, leading to degeneration of the optic nerve. The disease usually occurs in young and middle-aged men, and the clinical manifestations are acute or subacute painless vision loss in both eyes at the same time or successively, accompanied by central visual field defect and color vision disturbance. [0003] LHON is caused by mutations in mitochondrial sites. Chinese scholars have initially confirmed that more than 95% of LHON patients in China are related to site mutations at 11778, 14484, and 3460 in mitochondrial DNA, among which site 11778 is the most common mutation, accounting for 89.2% of LHON in my country. ,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861C12N5/10C07K19/00A61K48/00A61K38/44A61P27/02
CPCA61K38/44A61K48/0025A61K48/005A61P27/02C07K2319/01C12N9/0036C12N15/86C12N2710/10343C12Y106/99003
Inventor 李斌
Owner WUHAN NEUROPHTH BIOTECHNOLOGY LTD CO
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