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Recombinant adeno-associated virus-nadh dehydrogenase subunit 4 gene full-length and agent for the treatment of leber hereditary optic neuropathy

A technology for optic neuropathy and dehydrogenase subunit, which is applied in the field of recombinant adeno-associated virus-NADH dehydrogenase subunit 4 gene full-length and pharmaceuticals, which can solve the problems of unreachable treatment, limited injection volume, and AAV2-ND4 transfection efficiency Low-level problems, to achieve the effect of increasing expression and improving therapeutic effect

Active Publication Date: 2018-02-09
WUHAN NEUROPHTH BIOTECHNOLOGY LTD CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But in practice, we found that the transfection efficiency of AAV2-ND4 in the previous study was low and could not achieve the purpose of treatment: since AAV2-ND4 needs to be injected into the vitreous cavity of the eye, it can be transfected to the optic nerve, but the vitreous cavity of the eye The amount of injection is limited, 0.1ml can be injected at most, and the dose is very small, resulting in very little normal ND4 transfected to the optic nerve. If the concentration of AAV-ND4 is increased, the body’s immune response will be caused, and repeated administration will also cause immune response. effect

Method used

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  • Recombinant adeno-associated virus-nadh dehydrogenase subunit 4 gene full-length and agent for the treatment of leber hereditary optic neuropathy
  • Recombinant adeno-associated virus-nadh dehydrogenase subunit 4 gene full-length and agent for the treatment of leber hereditary optic neuropathy
  • Recombinant adeno-associated virus-nadh dehydrogenase subunit 4 gene full-length and agent for the treatment of leber hereditary optic neuropathy

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] 1. Test materials

[0042] Table 1

[0043]

[0044] 2. Experimental steps

[0045] The full-length gene sequence of the recombinant human NADH dehydrogenase subunit 4 (ND4) of the present invention is redesigned according to the ND4 gene sequence and the cox10 gene sequence provided by the genbank database. The gene sequence of the present invention is designed in three parts ( figure 1 ). The first part includes the CAG promoter, and the second part is the coding sequence of ND4 with the mitochondrial localization sequence of cox10. The third part is the 3' non-coding region (Untranslated Regions UTR) of cox10. We reduced the length of the 3' UTR to 625bp.

[0046] A method for constructing an adeno-associated virus vector for the recombinant application of the recombinant human NADH dehydrogenase subunit 4 gene, the steps comprising:

[0047] (1) Construction of recombinant adeno-associated virus vector containing human NADH dehydrogenase subunit 4 gene

...

Embodiment 2

[0087] Example 2: Effect experiment of AAV2-CAG-ND4 recombinant adeno-associated virus on Leber hereditary optic neuropathy

[0088] 1. In vitro experiments to observe the expression of AAV-CAG-ND4

[0089] 293 cells were cultured in vitro, divided into two groups, transfected with AAV2-ND4 and AAV2-CAG-ND4 respectively, and the expression of ND4 was observed by immunofluorescence. The results showed that the expression of ND4 in the AAV2-CAG-ND4 group was significantly higher than that in the AAV2-ND4 group. Sex (P=0.001; Figure 5 ).

[0090] 2. Intravitreal Injection of Mouse Eyes

[0091] Take 24 mice and divide them into two groups, divide them into experimental group and control group, use 0.1% AAV2-ND4 and 0.1% AAV2-CAG-ND4 to puncture the pars plana of the ciliary body outside the limbus into the vitreous cavity, and carry out Intravitreal injection.

[0092] 3. Slit lamp, intraocular pressure, fundus photography examination

[0093] The rats in the two groups wer...

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Abstract

The invention discloses a recombinant adeno-associated virus-NADH dehydrogenase subunit 4 gene full length and a medicament for treating Leber hereditary optic neuropathy. The full length of the gene is shown in the nucleotide sequence of SEQ ID NO: 1. Wherein, the nucleotide sequence has a full length of 3824bp, which consists of a CAG promoter sequence, a coding sequence of ND4 with a Cox10 mitochondrial localization sequence and a UTR with a length of 625bp. The medicament is injected into the vitreous cavity of the eye for the treatment of Leber's hereditary optic neuropathy. The medicament of the present invention is injected into the vitreous cavity, can maintain vitality in the vitreous cavity, and can be efficiently transfected into optic nerve cells. The signal peptide at the front end of the protein N, Orientation guides the protein into the mitochondria, and the mature ND4 protein enters the mitochondria to play a role. Therefore, drugs are effective in the treatment of Leber hereditary optic neuropathy.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a recombinant adeno-associated virus-NADH dehydrogenase subunit 4 gene full-length and a medicament for treating Leber hereditary optic neuropathy. Background technique [0002] Leber's hereditary optic neuropathy (Leber's hereditary optic neuropathy, LHON) is a disease caused by mitochondrial gene mutations. Leber's hereditary optic neuropathy is one of the world's recognized blindness in adolescents. It manifests as acute or subacute painless vision loss in both eyes at the same time or successively, and may be accompanied by central visual field defect and color vision disturbance. [0003] Scholars have confirmed that more than 20 mitochondrial mutation sites lead to the occurrence of LHON, including 11778, 14484, 3460, 3490 and so on. Among them, the 111778 site mutation is the most common, accounting for 89.2% of LHON in my country, and the prognosis is the wor...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N15/861C12N15/10A61K48/00A61P25/02A61P27/02
Inventor 李斌
Owner WUHAN NEUROPHTH BIOTECHNOLOGY LTD CO
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