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Small interfering RNAs targeting atf5 and their uses

An ATF5, small interference technology, applied in the field of small interfering RNA, can solve the problem of not effectively inhibiting siRNA, etc., to achieve the effect of enhancing mitochondrial function, reducing expression level, and restoring osteogenic ability

Active Publication Date: 2022-07-19
上海市第四人民医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] ATF5 is a potential target for m.3243A>G mutation therapy, and there is currently no siRNA that effectively inhibits ATF5 gene expression

Method used

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  • Small interfering RNAs targeting atf5 and their uses
  • Small interfering RNAs targeting atf5 and their uses
  • Small interfering RNAs targeting atf5 and their uses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Monoclonal isolation and culture of urine stem cells from patients with mt.3243A>G mutation ( figure 1 )

[0028] 1) Add 5ml of the mixed solution of penicillin, streptomycin and amphotericin B (wherein the penicillin content is 10kU / ml, the streptomycin content is 10mg / ml, and the amphotericin B content is 25μg / ml) into the T75 bottle, and the T75 bottle mouth is Spray disinfectant around;

[0029] 2) Select the urine of the mitochondrial mt.3243A>G mutant population, and use the T75 bottle in step 1) to collect urine;

[0030] 3) Open the T75 bottle, add 50ml urine samples to each of the four 50ml centrifuge red tubes, and centrifuge with a centrifuge;

[0031] 4) Pipette the supernatant from each red tube to less than 5ml, add 15ml~20ml PBS to the first red tube, mix by pipetting, then transfer the resuspension to the next red tube, and mix by pipetting again Evenly, and so on, until finally the sediments of all tubes are collected in one tube, centrifug...

Embodiment 2

[0046] Example 2 Design and synthesis of RNA oligo

[0047] In order to realize the above-mentioned purpose of the present invention, we adopt the following technical scheme:

[0048] The gene sequence of ATF5 (Gene ID: 22809) was obtained by searching NCBI, and the targeted mRNA sequence (>NM_012068.6ATF5[organism=Homo sapiens][GeneID=22809][transcript=1] (SEQID NO.32) was downloaded from the website )). Based on this mRNA sequence, four siRNA sequences were designed, and the sense and antisense strands were both 21nt. Through Genbank BLAST query, it was ensured that the siRNA coding was not homologous to other genes.

[0049] Sequence 1 (forward primer gcgaguugauuucacagcutt, reverse primer agcugugaaaucaacucgctt),

[0050] Sequence 2 (forward primer cacggaaucgcgagcugaatt, reverse primer uucagcucgcgauuccgugtt),

[0051] Sequence 3 (forward primer caccugaccuggaagcuautt, reverse primer auagcuuccaggucaggugtt),

[0052] Sequence 4 (forward primer ccccugucuuggauacucutt, reverse...

Embodiment 3

[0054] Example 3 siRNA transfection knockdown experiment of ATF5

[0055] 1. Preparation of transfection mixture: 10D (optical density value) per tube of synthesized siRNA is prepared into a solution of 20umol / L, and 140pL of 0.1% DEPC-H20 needs to be added to each tube, 20uM siRNA=20umol / ul. Dilute 2.0, 3.0, 4.0, 5.0, 6.0 pL of siRNA with 100 uL of serum-free medium in 4 EP tubes, respectively. Each 50 uL medium contained 20, 30, 40, 50, 60 pmol of ATF5 siRNA, respectively. Dilute 5.0 and 7.5 uLlipofectamin with 250ul serum-free medium in 2 EP tubes respectively TM 2000 reagent, so that each 50ul medium contains 1.0, 1.5ul lipofectamine respectively TM 2000, gently mixed and incubated at room temperature for 5 min. Different content of lipofectamine TM 2000 was mixed with the corresponding diluted ATF5 siRNA in a new EP tube within 30 minutes after the dilution to prepare the corresponding transfection mixture, and left at room temperature for 20 minutes.

[0056] 2. ...

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Abstract

The present invention provides a small interfering RNA sequence targeting ATF5, the forward primer sequence is shown in SEQ ID NO.24, and the reverse primer sequence is shown in SEQ ID NO.25. The present invention also provides the use of the above-mentioned small interfering RNA sequence targeting ATF5 in the preparation of a medicament for treating mitochondrial m.3243A>G mutation-related diseases. The present invention provides a small interfering RNA targeting ATF5, which can effectively reduce the expression of the ATF5 gene, so as to be used for treating diseases related to mitochondrial m.3243A>G mutation.

Description

technical field [0001] The invention belongs to the field of biomedicine, and relates to the transcription factor ATF5 in the mitochondrial unfolded protein reaction, in particular to the small interfering RNA targeting ATF5 and the use thereof. Background technique [0002] Mitochondrial DNA 3243A>G mutation (hereinafter referred to as mt.3243A>G mutation) is the most common type of single-gene mutation diabetes mellitus. It is caused by A→G mutation at the 3243 nucleotide sequence. [0003] The mt.3243A>G mutation easily affects tissues with high ATP threshold, and the main clinical features are: early-onset diabetes onset before the age of 40. Most of the patients were emaciated, and pancreatic islet function progressively declined during the course of the disease. More than 75% of patients have sensorineural hearing loss. Other clinical symptoms include mitochondrial encephalomyopathy with hyperlactatemia and stroke-like episodes, retinitis pigmentosa, skelet...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12Q1/6883A61K31/713A61P19/08
CPCC12N15/113C12Q1/6883A61K31/713A61P19/08C12N2310/141C12Q2600/156
Inventor 王从容孙毅张宜男韦跃华李凤雯李佳璐刘鹏
Owner 上海市第四人民医院
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