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Oligonucleotide, virus vector, application of oligonucleotide or virus vector, and RNAi medicinal preparation

A technology of oligonucleotides and viral vectors, applied in the medical field, can solve problems such as corneal dystrophy

Active Publication Date: 2020-11-17
WUHAN NEUROPHTH BIOTECHNOLOGY LTD CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But there is no RNAi drug for corneal dystrophy caused by mutation of COL8A2

Method used

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  • Oligonucleotide, virus vector, application of oligonucleotide or virus vector, and RNAi medicinal preparation
  • Oligonucleotide, virus vector, application of oligonucleotide or virus vector, and RNAi medicinal preparation
  • Oligonucleotide, virus vector, application of oligonucleotide or virus vector, and RNAi medicinal preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Luciferase reporter system screening for efficient RNAi drugs

[0058] 1. Mammalian cell (adherent) culture

[0059] 1. Cell Recovery

[0060] 1) Prepare warm water at 37°C-38°C in advance, take out the cells to be resuscitated from the liquid nitrogen tank, fix them with ophthalmic surgical tweezers, and quickly place them in the water to ensure that the cryopreservation tubes are completely submerged in the water to allow them to be evenly heated until frozen. The cells in the storage tube are completely thawed;

[0061] 2) Sterilize the cryotube with alcohol;

[0062] 3) Use a pipette to draw 5 mL of cell culture-based T25 cell culture flask in advance, and then use a new pipette to transfer the melted cells into the cell flask and blow it gently;

[0063] 4) Close the cap of the cell bottle, place the cell bottle in the cell incubator, 37°C, 5% CO 2 Static cultivation;

[0064] 5) After about 6-8 hours (depending on different cells), replace the fresh...

Embodiment 2

[0131] Example 2 RNAi Drug Treatment Inhibits Mutation-Specific COL8A2 Gene Expression

[0132] 1. 293 cell transfection:

[0133] The method is the same as mentioned above.

[0134] 2. Detection of COL8A2 RNA level by reverse transcription fluorescent quantitative PCR

[0135] 1. The reverse transcription reaction system is as follows:

[0136]

[0137] Reverse transcription reaction conditions: 37°C for 1h, 75°C for 10min.

[0138] 2. Real-time response system

[0139] 1) Detection primers and internal reference primers for target genes

[0140] COL8A2:5'-TCCGGCAGCCGCGAG-3'(sense)

[0141] 5'-GCATTTCCAGGTACTGGCCT-3'(antisense)

[0142] GAPDH:5'-GGAAGGTGAAGGTCGGAGTCAACGG-3'(sense)

[0143] 5'-CTCGCTCCTGGAAGATGGTGATGGG-3'(antisense)

[0144] 2) Reaction system

[0145]

[0146] 3) Reaction procedure:

[0147]

[0148] 3. AAV infection of 293 cells

[0149] 1. Prepare AAV RNAi control virus and RNAi drug virus.

[0150] 2. The MOI of the recombinant virus w...

Embodiment 3

[0171] Example 3 RNAi drugs can treat and prevent corneal dystrophy in humanized mutant COL8A2 mice

[0172] 1. AAV-RNAi virus infection and analysis of mice

[0173] 1. Construct humanized COL8A2 mutant transgenic mice.

[0174] 2. Prepare 5*10E12 vg / mL AAV RNAi control virus and RNAi drug virus.

[0175] 3. Inject 1 μL / eye of the control RNAi virus or the RNAi virus of the drug group into the eyes of 1-month-old or 6-month-old mice through anterior chamber injection.

[0176] 4. When the mice were 12 months old, the mice were killed, and the corneal tissues of the mice were separated, and stained to detect the number of corneal endothelial cells and the total protein content of COL8A2.

[0177] 2. Test results

[0178] The 1-month-old mice have not yet developed the disease, and the morphology of corneal tissue cells is normal. The 6-month-old mice have already seen corneal descemet's descemet's vegetation and loss of endothelial cells. In this example, 5*10E9 vg / eye of ...

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PUM

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Abstract

The invention relates to the field of medicine, in particular to oligonucleotide, a virus vector, application of the oligonucleotide or the virus vector, and an RNAi medicinal preparation. The oligonucleotide is one of sequences from SEQ ID NO: 4 to SEQ ID NO: 40; or a nucleic acid sequence having no less than 80% consistency with one of the sequences from SEQ ID NO: 4 to SEQ ID NO: 40. The oligonucleotide, the virus vector and the RNAi medicinal preparation provided by the invention can effectively treat and prevent corneal dystrophy caused by COL8A2 mutation.

Description

technical field [0001] The invention relates to the field of medicine, in particular to oligonucleotides, viral vectors and applications thereof, and RNAi pharmaceutical preparations. Background technique [0002] Corneal dystrophy (Corneal dystrophy, referred to as CD) is a group of hereditary, usually progressive ophthalmic diseases that originate in the cornea and only invade a certain layer of the cornea at the beginning; in the late stage, it can spread to adjacent layers, and even affect the full thickness of the cornea; drug treatment invalid. Those who affect vision can be treated with corneal transplantation. According to the anatomical location, it is divided into three categories: anterior, stromal, and posterior. Usually the posterior aspect is more severe and accounts for the greatest proportion of patients (endothelial corneal dystrophy). The vast majority of CDs have corneal opacities of various shapes. CD has been studied for many years, but the mechanism...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/864A61K31/7105A61P27/02
CPCC12N15/113C12N15/86A61P27/02C12N2310/14C12N2310/531C12N2750/14143C12N2800/107C12N2320/32
Inventor 李斌任盛
Owner WUHAN NEUROPHTH BIOTECHNOLOGY LTD CO
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