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

An oligonucleotide, viral vector technology, applied in the medical field

Active Publication Date: 2020-11-13
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 TGFBI mutation

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Luciferase reporter system screening for efficient RNAi drugs

[0054] 1. Mammalian cell (adherent) culture

[0055] 1. Cell Recovery

[0056] 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, so that they can be evenly heated until frozen. The cells in the storage tube are completely thawed;

[0057] 2) Sterilize the cryotube with alcohol;

[0058] 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;

[0059] 4) Close the cap of the cell bottle, place the cell bottle in the cell incubator, 37°C, 5% CO 2 Static cultivation;

[0060] 5) After about 6-8 hours (depending on different cells), replace the fres...

Embodiment 2

[0105] Example 2 RNAi drug treatment specifically reduces TGFBI MET619LYS gene expression

[0106] 1. Construction of 293-TGFBI wild-type and TGFBI MET619LYS mutant stable cells

[0107] 1. Construct LV-CAG-TGFBI (WT) and LV-CAG-TGFBI MET619LYS lentiviral vectors;

[0108] 2. Use the three-plasmid system to package the lentivirus and detect the virus titer;

[0109] 3. Infect 293 cells with a multiplicity of infection of MOI=100;

[0110] 4. Limiting dilution, plant 1 cell / well in a 96-well plate, identify whether the monoclonal cells express TGFBI after 2 weeks of culture, and expand the culture of positive cells.

[0111] 2. 293 cell transfection:

[0112] The method is the same as mentioned above.

[0113] 3. Detection of TGFBI RNA level by reverse transcription quantitative PCR

[0114] 1. The reverse transcription reaction system is as follows:

[0115]

[0116] Reverse transcription reaction conditions: 37°C for 1h, 75°C for 10min

[0117] 2. Real-time response...

Embodiment 3

[0150] Example 3 RNAi drugs can alleviate the cytotoxicity caused by TGFBI MET619LYS mutation

[0151] 1. AAV infection of 293 cells:

[0152] The method is the same as mentioned above.

[0153] 2. Detection of GRP78 / BiP RNA level by reverse transcription quantitative PCR

[0154] The method is the same as above, and the target gene detection primers:

[0155] GRP78 / BiP:5'-ATAGCATCTGAGCTGGCTCCT-3'(sense)

[0156] 5'-GCACATCTAGATCCCCGCATT-3'(antisense)

[0157] 3. TGFBI staining of extracellular matrix

[0158] 1. In the culture plate, soak the cell slide that has been covered with cells with PBS 3 times, 3 minutes each time;

[0159] 2. Fix the cell slides with 4% paraformaldehyde for 15 minutes, soak in PBS 3 times, 3 minutes each time;

[0160] 3. Permeabilize with 0.5% Tritonx-100 (prepared in PBS) at room temperature for 20 minutes;

[0161] 4. Wash the slides with PBS for 3 times, each time for 3 minutes, blot the PBS with absorbent paper, add normal goat serum to th...

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Abstract

The invention relates to the field of medicine, in particular to oligonucleotides, virus vectors, application of the oligonucleotides and the virus vectors and an RNAi medicinal preparation. The oligonucleotide is one sequence from SEQ ID NO: 3 to SEQ ID NO: 21; or a nucleic acid sequence having no less than 80% consistency with one of the sequences from SEQ ID NO: 3 to SEQ ID NO: 21. The oligonucleotide, the virus vector and the RNAi pharmaceutical preparation provided by the invention can be used for effectively treating and preventing corneal dysplasia or corneal dystrophy caused by TGFBI Met619Lys 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 dystrophies (Corneal Dystrophies, CD), also known as corneal dystrophy, is a general term for symmetrical, non-inflammatory corneal diseases. Its incidence rate is about 1 / 2000, and the ratio of male to female incidence is 1.7:1.0. Most of the patients are sporadic, and about 6%-10% of the patients have a clear positive family history, and the mode of inheritance includes recessive and dominant forms. Its onset is due to the progressive damage to the structure or function of the cells in the normal corneal tissue under the action of genetic abnormalities, resulting in the formation of deposits of various shapes in the corneal tissue, which gradually reduces vision, and causes erosion and photophobia. and other complications; the cornea of ​​advanced patie...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/867A61K31/7088A61P27/02
CPCC12N15/113C12N15/86A61P27/02C12N2310/14C12N2740/15043C12N2800/107C12N2320/32
Inventor 李斌任盛
Owner WUHAN NEUROPHTH BIOTECHNOLOGY LTD CO
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